«1.1. .
1';"1’31"1"'I..I;1:113“‘I.I.I....t.1
”14.. ”1.11.1.1 . .‘.
1.1"‘:I1&:.1.1‘1'I.'1.‘;.H..I 4:144!
11 I.,1.I'.3.I.~. I.“ 4......“ 4.4
"1):“ “‘i:'1"'1‘1'1“1'11,1 1.111.1.1.1I.I,1, 1.
I?“ 1'11}... 1,3,5 .44; 1 I .
L ..
III I‘III-I '11.:
‘I’W 21‘ :1 W .I.I. 115,131 11" 1'3”
‘I 1.1:. “I. .,.,
““1" W
9:: Ex.‘

'1‘
11““!

.IIE;""‘I}‘I‘. 1. 2": .‘.:
IIII“~§I;;III

I»... >4.
. . um
:23. ‘I

I 3‘ "I 1W; "“2"“ ""21“ :1"
1:13.11); 1,, ‘W‘ ,1, 5"} 11 {1.1.21 11),}.
11.3.: . EV}:
- 1...}. 11" -. 1..

l ‘ .‘
I‘Ii‘IIEE'I

$3,;

4

. vs

:4' .
.,II
"1:1‘1I .
1'11 {:3‘I. 1‘ . I‘I:.,. ...‘~:‘.‘:' "11‘1“‘1'3‘ l.“"L 3:14.: ‘ ‘.'l.
t K
.. . . I; III-III. .‘. .11...
"1.4.1.... . ”‘13.: $3191.11 ' "“1191!“
1.." 1'1” L‘ .. . .
.I,'I .,.I,'. 113;.I.1II‘.L _. '13.4_1'.1:1‘1. .1”. .4?
“‘ IW‘I- ""1 “ I1.3.‘..1._. 1‘13“?
‘“"‘f‘."“ “V >11 ‘ ‘1 .. 1"1:"" ”11‘1‘1' "I.
, . IIII'III . .. 1.11.1111”.
4' .313 'II"‘1'1.ItI.1II'KI
II‘H".'.‘1
1111., .1.3. 1""?
1.3.1;
$1181

X
I...

\L 1.
1U1.)‘,I.II '
.. 44 L‘

4...?
““111
UK” 1

V
3:41.

I. I; .,
.‘.. “III-III"...
‘91.?“ . "|"“'1gt

 

1I‘-I
115:"; 11:31“
"1‘“ :34“ (41444
.,)'.1. 1111.11"
III'IM I I‘I,"I ‘3".
. 1'3. ““1
4';K"1

... .

.1: 41 v» 11 ‘21'..“|1“'3i"11:"“"‘

“I331... I. . 4, ' ..111 ”.4“ ‘1,\ 'I. "'1 .13311‘1‘11

In. IIII . . .I‘..V - ‘1""111 1"y:‘-’ ‘I 11".3 “I.

12"” .1 . ‘ .1. v ‘1 ““35“ 1314:. EI‘I'JS‘L ' 1,.
L "'4

I
‘921' ~

.. :II'I‘I in!” ‘ “if“. 1¢t
,I..'":111;'I1.71I., .,.,I.‘ «1‘!
“'31,.m

, .
L 4'va”, 4:31:33?
1;~.I‘14I,‘t.1‘.\1-.;1.“x_7..1n ‘I’I‘I. dd"
1.11.1.9“; #1..
Y

.6.
3".“

‘1‘" "1‘ 1‘" III " 1 , 1:(~I{‘§I1‘1‘““'1.1.I.' ’
1‘. ‘:,I( "I"~‘"‘"‘1"I‘I‘131 "13.,1‘LI1'2111‘"I‘:':'\;U 1.1.. 1‘51} ‘1‘ ":11" .'. .43:
,4. .1 '11._I).'.l .‘ , "‘1‘ 84:28.”... '3...‘I4....13411','.11::~11
1,:I' ‘ .1 . 11'1-1,..I'.'I‘I"'1,'”‘ ‘ 11.1}1.
1'11“!” ,I 4 ,.4:. Wu" 9.391.143: 4 .111... “-1,. 1,, ,1.II '31 w.‘ Wig, .34."...
51.. I I“ 44.11 1W: H. “(11,” ”1‘4‘ .'.”...4 1“ .1434. “1"'1".'““ ”up:
Ll‘L11.1.”1;‘1"":'1"' 111 " j“1“‘1 I 1.3311..."
1 )1 4.131%. 44 ()1. ,1.1‘.3.".i"1‘fl ‘44. 1., .Q"1_1“V"5““11)')""‘:'1‘11 . {4:4, I
I ' ' . . .I 'I ‘ . NU.
... 111.11.41.41... .1 .4111,1,|,I,I.}. fl...“ . ... 441.1 ,1.I 33: W 14.14.: 42‘. .; ‘45:. '11")
‘11‘1‘1‘111.14:3:"“I'I.'§"1‘.'11:4:m(§41""
I.“ , ““1“‘ 1 .1: #3454 . ‘11

. 14"" ILN“. , , 1'" "1‘13.
. $1., ' I.1,4I11 I1“‘L 1:: ‘1 ; ‘1‘" '11:".‘1'1.1“‘1'1 31.1.1. :I'...;,...
131......” . II 1'a""'I‘1 4.. 591'}: 1‘111911' 1 ." 1 ”1'1" 11 11
.3144,‘ . 11.11 I . . 4.4..4 I. ;..I~.1:1‘i.‘1.141,4'4 . . [.‘.4414Wn44
-I'II "31.1.13 ' 1"!‘1 ' 31313.1" ‘.‘:Wi‘1‘ '1 "1., .9.“
‘1 I .11" ":11?" V1" '" 11:3
’1'5‘1 I1 1

""."
I1 1.I

4‘1'1'1.1..""““‘ 9:11.41, ' ' 1‘1

'31}
‘ "€113; 55,
1'11“). 4"1‘1'17 ‘11.... 4.4”..111": “11.11.11.
'1 1"

Q31 , ,

. .r‘IIl 1 .'. 1.1.1:“:
‘1'1'1-‘I'1I‘L M‘)‘

1!:-I'- 1'1‘1‘1

. 1 I ,

“'13 b

21,1). I ‘II
"1‘1“"14‘3331.
.9 .
I

u-ru'r.
"Y . CL“.- .

. .. ....I.I,...I

. .., .
Z 1 '111‘.
.411.)

13:11.“
‘ I11

.312 .
,i
4“ 1'1 1'11“" 1
I")! ‘}1':‘

H.

.233}

1.",
‘ 'I’II‘I‘I‘I‘III
. "531$" 4 65.1312; “

1194111512115?
11.1%‘1‘44444.§&

3.39:3"

1“".1 " ....,II'
I 1'1
III: " “"“'
1.1

“’14:”
I'I, 1"1
.III.II~,.I1 “""“I .1.
1.” 11-31.
"-11 I IN“)
.11111,I{¢‘.':" ‘1
1%:

r—

:I
131
I"

‘:.II ."I

.1”:
. I

....._.—'£—£.a=‘““
‘:

A --”,1€§W:4:; .'5:_.=‘72§-‘.=“~ 35:.
15.44%: “

,.._.'

1 . '3:

.34":

1_Z;"~:4.~v:-r:;

”LA...

$252.23:

.2313... 2;
4.1.1" :—--—-

£24:
.M~‘*—;~.

-«n
M»

’27..
“a
._.._‘..x-—$
.1

”n:

.. _...,'
J

‘ 1%

1;}:

”.4..quLG

‘. .. "b—
m1

mg I .. j..

‘ w

”m ”‘2.
m

...
“11‘1““ .

iI

11.’ 1..'II

gym“... -
as:

 

 

Date

0-7 839

This is to certify that the

thesis entitled
A MICROCOMF'lJT'EZR~-CUNTRULI...EII Ei'mF'F'EIIIMI‘:LK'JN
CLINICAL ANALYZER IN NHICH IMMOBILIZEH
ENZYME REQCTIUN LUDF'ES (WEE US‘SEH
presented by
MARTIN [MAUIIJ JUSEIF‘H

has been accepted towards fulfillment

of the requirements for
P h . n . CHEM I STRY

degree in

flaw

Major professor

JU L. Y 18 5: 19398

 
 
 
   
    

 
 

LIB‘Ka.‘ ‘ Y .
Michigan State ~
University A

 

A MICROCDMPUTER-CONTROLLED STOPPED-FLOW CLINICAL ANALYZER

IN “HIGH IMMOBILIZED ENZYME REACTION LOOPS ARE USED

139

Martin David Joseph

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements

for the degree of
DOCTOR OF PHILOSOPHY
Department of Chemistrg

197B

ex“

ad

bite

ABSTRACT

A MICROCOMPUTER-CONTROLLED STOPPED-FLOW CLINICAL ANALYZER

IN WHICH IMMOBILIZED ENZYME REACTION LOOPS ARE USED

By

Martin David Joseph

A microcomputer—controlled stopped-flow clinical
analyzer (SFCA) has been developed for application in
clinical analysis. The analyzer uses an immobilized
enzyme to catalyze the conversion of a specific analyte to a
specific product. The concentration of the product is
determined spectrophotometrically in a follow—up indicator
reaction. which takes place in the observation cell of the
stopped-flow module. The separation of these two reactions
permits the selection of optimum conditions for each. and
also the use of either a reaction—rate method or an
equilibrium method to monitor each reaction.

A procedure was developed for the immobilization of
enzymes onto the interior surface of nylon tubing. Glucose
oxidase was immobilized by this procedure: and showed only
moderate loss of activity after several months of use.
Immobilized alcohol dehydrogenase was used routinely for
over a month.

A commercial stopped-flow instrument was modified to

Martin David Joseph

incorporate the immobilized enzyme reaction loops into the
stopped-flow mixing system. The sequencing of the SPCA and
the acquisition of analog spectrophotometric data were placed
under the control of a microcomputer. The interfaces were
designed to function in harmony with software to allow a
variety of sequencing schemes to be implemented. A mini-
microcomputer hierarchical system was designed to facilitate
modification of microcomputer software: and to permit
sharing of the facilities of a minicomputer system among
several microcomputers. The hierarchical system was used to
divide computational tasks between the microcomputer and a
more powerful minicomputer system.
The kinetics of the immobilized enzymes were studied.

The Michaelis-Menten model of soluble enzyme kinetics was
applied: and modifications to the model which attempt to
account for diffusional effects were incorporated. The
performance of the instrument in clinical analysis was
evaluated. Adequate sensitivity was obtained by allowing
the sample to react with the enzyme for as short a time as 2
min. Excellent agreement with accepted methods was obtained

in analysis of serum samples.

To Betty

Matt and Meg

ii

ACKNOWLEDGEMENTS

My sincere thanks and appreciation go to Professor
Stanley R. Crouch. my research director. As advisor and
friend he has been both inspirational and invaluable. I am
also very grateful to Dr. Andrew Timnick. who has served as
second reader. It has been a pleasure to teach for Andy and
I have enJoyed his friendship.

I would like to thank Dr. Gene Pals for many
imaginative discussions on an endless variety of topics. and
for being a good friend. I am indebted to Dr. Dave Baxter
for his help in solving a great many problems. and to Dr.
Eric Johnson for sharing his software expertise and for
being a patient tutor. Much appreciation is also due to Roy
Gall for help in the stapped-flow studies. and to Rytis
Balciunas for slaving over the PROM monitor board with me
for what often seemed to be an eternity. I am also thankful
to Dr. Floyd Holler for his assistance in initial stopped-
flow work and for helpful discussions.

My parents and family have my thanks and appreciation
for their support. especially Joe. without whose friendship
and advice this work would have been so much less enJoyable.
Finally. I am eternally indebted to my wife Betty. for
giving me the opportunity. and for keeping my sights set far
and my spirits high. and to the kids. for greeting me at the

door.

iii

Chapter

LIST OF
LIST OF
CHAPTER

CHAPTER

CHAPTER

TABLE OF CONTENTS

TABLES.
FIGURES .
I. INTRODUCTION.

II. IMMOBILIZED ENZYMES IN
ANALYTICAL CHEMISTRY .

Methods of Immobilizing Enzymes

1. Adsorption.

{'J

Cross-Linking
3. Physical Entrapment
4 Covalent Attachment .

Analytical Applications of
Immobilized Enzymes .

1. Electrochemical Detection
a. Amperometric.
b. Potentiometric.

pa

Spectrophotometric and
Fluorometric Detection.

3. Enthalpimetric Detection.

Commercial Systems Which Emplby
Immobilized Enzymes .

III. IMMOBILIZATION OF ENZYMES
ON NYLON.

Initial Preparation and Hydrolysis
of the Nylon. . . . . .

Attachment of Glutaraldehyde.
Immobilization of the Enzyme.
Specific Procedure for Enzyme

Immobilization.

iv

Page

ix

11

13

16

18

21

22
24

28

31

 

Chapter

CHAPTER

A.

CHAPTER
A.

B.

IV. FLOW SYSTEMS .
Technicon Auto Analyzer
1. Description .

2. Design for Immobilized Enzymatic
Analysis of Glucose

3. Characteristics .
Stopped-Flow Clinical Analyzer.
1. Description
2. Characteristics
V. MICROCOMPUTER INSTRUMENTATION .
Overview.
The 6100 Microcomputer.
1. Description
2. PROM Monitor Software
a. Transparent Mode.
b. Binary Loader
c. RAM Monitor
3. Operating Procedures.
4. PROM Monitor Logic.
SFCA Interfaces
1. General Considerations
2. Control Interface.

a. The Open and Close Sample
Valve Instructions.

b. The Syringe Control
Instructions. .

c. The Skip on Flow Stopped
Instruction . . .

d. The Read Switch Register
Instruction . . .

Page

33
33

33

34
3e.
39
39
43
46
4.5
so
so
51
53
55
56
57
59
61
61

63

63

64

66

66

 

"I

Chapter Page

3. Sequencing and Timing . . . . . . . . . . . 7O
4. Analog Interface. . . . . . . . . . . . . . 71
a. The Convert Command . . . . . . . . . . 72

b. The Skip on Conversion Done
Command . . . . . . . . . . . . . . . . 73
c. The Read ADC Command. . . . . . . . . . 73
d. The Set DAC Command . . . . . . . . . . 76
D. SFCA Software . . . . . . . . . . . . . . . . . 76
1. Microcomputer Software. . . . . . . . . . . 78
2. Data Transfer Subroutines . . . . . . . . . 83
3. Minicomputer Software . . . . . . . . . . . 83

CHAPTER VI. EVALUATION OF THE STOPPED-FLOW

CLINICAL ANALYZER. . . . . . . . . . . . . 85

A. Solution Volumes. . . . . . . . . . . . . . . . 85
B. Sequencing. . . . . . . . . . . . . . . . . . . 87
C. Indicator Reaction. . . . . . . . . . . . . . . 88
D. Sources of Error. . . . . . . . . . . . . . . . 9O
1. Sampling. . . . . . . . . . . . . . . . . . 91

2 Enzymatic Reaction. . . . . . . . . . . . . 92

3. Indicator Reaction. . . .'. . . . . . . . . 93

4 Measurement of Absorbance . . . . . . . . . 95

a. Light Source Characteristics. . . . . . 95

b. Physical Interferences. . . . . . . . . 96

c. Spectral Interferences. . . . . . . . . 97

d. Detector Noise. . . . . . . . . . . . . 98

e. Photocurrent Amplifier. . . . . . . . . 99

f. Digitization. . . . . . . . . . . . . . 99

5. Summary of Error Sources. . . . . . . . . . 100

vi

 

Chapter ‘ Page

CHAPTER VII. CHARACTERIZATION OF THE

IMMOBILIZED ENZYMES. . . . . . . . . . . . 102

A. Kinetics of the Immobilized Enzymes. . . . . . . 102

1. Michaelis-Menten Model . . . . . . . . . . . 102

a. Glucose Oxidase. . . . . . . . . . . . . 105

b. Alcohol Dehydrogenase. . . . . . . . . . 111

2. Diffusion Effects. . . . . . . . . . . . . . 114

3. Induction Period . . . . . . . . . . . . . . 116

B. pH Profiles. . . . . . . . . . . . . . . . . . . 117

C. Long Term Stability and
Storage Conditions . . . . . . . . . . . . . . . 119
CHAPTER VIII. PERFORMANCE OF THE INSTRUMENT

AS A CLINICAL ANALYZER. . . . . . . . . . 124

A. Optimum Analytical Parameters. . . . . . . . . . 124

1. Reagent Concentrations . . . . . . . . . . . 125

a. Glucose Determinations . . . . . . . . . 125

b. Ethanol Determinations . . . . . . . . . 127

2. Mode of the Enzymatic Reaction . . . . . . . 130

3. Mode of the Indicator Reaction . . . . . . . 131

B. Linearity of Calibration Curves. . . . . . . . . 132

1. Glucose Determinations . . . . . . . . . . . 132

2. Ethanol Determinations . . . . . . . . . . . 132

C. Precision. . . . . . . . . . . . . . . . . . . . 135

D. Interferences. . . . . . . . . . . . . . . . . . 135

E. Serum Preparation. . . . . . . . . . . . . . . . 136

F. Proposed Multi-Reaction-Loop SFCA. . . . . . . . 137
G. Initial Study of Cholesterol

Determinations . . . . . . . . . . . . . . . . 139

CHAPTER IX. SUMMARY AND FUTURE PROSPECTS. . . . . . . . 145

vii

 

Chapter Page

REFERENCES . . . . . . . . . . . . . . . . . . . . . . 149
APPENDIX A: Selected Program Listings . . . . . . . . 155
APPENDIX B: A Stopped-Flow Clinical Analyzer

in Which Immobilized Enzyme Reaction
Loops are Used. . . . . . . . . . . . . . 193

viii

Table

I!)

LIST OF TABLES

Software Commands to the Control Interface .

Control Signals for the GOA-McPherson
Stopped-Flow Module.
Software Commands to the Analog Interface.

Manual Mode Commands and Their Function.

ix

Page

63

64

72

81

Figure

0~

<1 (I) \i

10

11

12

13

14

LIST OF FIGURES

Scanning Electron Microscope Photographs
of Virgin and Hydrolyzed Nylon Surface .
Attachment of Glutaraldehyde .
Proposed Aqueous Forms of
Glutaraldehyde .

Attachment of the Enzyme .

Auto Analyzer Configuration for
Glucose Determinations .
Stopped-Flow Clinical Analyzer .
Point-to-Point Hierarchical Network.
SFCA-Micro-Minicomputer System .
Schematic Diagram of Instruction
Decoding Circuitry

Schematic Diagram of Sample Valve
Control Circuitry.

Schematic Diagram of Syringe
Control Circuitry.

Schematic Diagram of Skip on

Flow Stopped Circuitry

Schematic Diagram of Switch
Register Circuitry

Schematic Diagram of Skip on

Conversion Done Circuitry.

Page

25

27

29

3O

35

4O

48

52

62

65

67

68

69

74

Figure

15

16

17

18

19

20
21
22

23

24

25

26

Schematic Diagram of Read ADC
Circuitry.

Schematic Diagram of Set DAC
Circuitry.

Computer Simulated Initial
Rate Curve .

Glucose Progress Curve
Lineweaver-Burke Plot for
Glucose Oxidase.

Ethanol Progress Curve

pH Profile of Glucose Oxidase.

pH Profile of Alcohol Dehyrogenase .

Calibration Curve for Glucose
Determinations

Calibration Curve for Ethanol
Determinations

Proposed Multi-Reaction-Loop SFCA.
Effect of Azide on Initial dA/dT

for Iodide-Peroxide Reaction .

xi

Page

75

77

104

108

110

113

118

120

133

134

140

144

CHAPTER I

INTRODUCTION

Analytical chemistry has played an important role in
the field of medicine for a number of years. As man’s
knowledge of the relationship between the levels of certain
substances in the body and disease increases. there is an
ever increasing demand for accurate information about a
patient’s body chemistry. Also. with the greater focus on
preventative medicine. the physician may soon be able to
predict disease. based on body chemistry information. before
the appearance of normal physical symptoms. In order to
keep up with this ever increasing demand in the clinical
laboratory. analyses must be carried out faster. more
accurately. and at lower cost. Thus there is a real need
for automation at every stage of clinical analysis. from
sample preparation and introduction to data acquisition and
the calculation of a final result.

Immobilized enzymes should play a key role in meeting
the challenge. Enzymes. by their nature. have the ability
to catalyze a particular reaction of a specific substance.
even when the substance occurs in a complex matrix such as
plasma or serum. when attached to an inert support. the
enzyme can be reused. often hundreds or even thousands of
times. The increased number of determinations per unit

amount of enzyme can reduce greatly the cost of routine

enzymatic analysis. Problems of instability are often
diminished when an enzyme is immobilized. Hence.
immobilized enzymes are far superior in many aspects to
their soluble forms.

This thesis describes the development of an automated
stopped-flow clinical analyzer (SFCA). which makes use of an
immobilized enzyme to catalyze a specific reaction of a
specific substrate (analyte). The concentration of the
product of the enzymatic reaction is determined in a
stopped-flow spectrophotometer. The operation of the SPCA
and data handling are under the control of a microcomputer.
A hierarchical network was developed to allow fast and
efficient communication between a more powerful minicomputer
system and this and several other microcomputers.

Because the overall prOJect involved work in several
different areas of analytical chemistry. this thesis is
generally divided according to the various sub-prOJects.
Each chapter presents a particular aspect of the entire
prOJect as a complete unit. which can be read independently
of the other chapters.

Chapter II presents a history of immobilized enzymes
in analytical chemistry and includes a summary of current
applications in the clinical laboratory. The enzyme
immobilization procedure developed in this work for
attaching glucose oxidase and alcohol dehydrogenase to nylon
tubing is described in chapter III. These immobilized

enzymes were employed in two different flow systems. the

 

 

 

commercial Technicon Auto Analyzer and the stopped-flow
clinical analyzer. These flow systems are described in
chapter IV.

Microcomputer instrumentation. for the control of the
stopped—flow clinical analyzer and for the acquisition and
processing of the spectrophotometric data. is described in
chapter V. A hierarchical network system is also described.
In chapter VI. instrumental aspects of the SPCA are
evaluated. both from a theoretical basis and from actual
observed characteristics. Sources of error are discussed.

Immobilized glucose oxidase and alcohol dehydrogenase
are characterized in chapter VII. The traditional
Michaelis-Menten model of enzyme kinetics is applied. and
modifications to the model are included to describe
diffusional effects. In chapter VIII. the potential of the
SPCA in clinical analysis is evaluated. A proposed system
which would contain multiple immobilized enzymes is
discussed. and initial work on the immobilized enzymatic
analysis of serum cholesterol is presented. Finally. a
summary of this work and recommendations for future work are

presented in chapter IX.

 

 

 

 

 

CHAPTER II

IMMOBILIZED ENZYMES IN ANALYTICAL CHEMISTRY

The development of immobilized enzymes is reviewed in
this chapter in relation to the field of analytical
chemistry. The first section reviews the methods of
immobilizing enzymes. In the next section. an overview of
analytical applications of immobilized enzymes is presented.
Finally. some commercially available analytical devices

which employ immobilized enzymes are presented.

A. Methods of Immobilizing Enzymes

Several methods have been developed for immobilizing
enzymes onto a wide variety of inert supports. Several
reviews (1—8) have recently appeared covering the topic in
detail. The methods can be classified into four main
categories: (1) adsorption. (2) cross-linking. (3) physical
entrapment. and (4) covalent attachment. Some methods
involve combinations of these four. and within each category
many variations have been reported. The enzyme to be
immobilized as well as the application should be considered

when choosing a method.

 

 

 

1. Adsorption

Adsorption of the enzyme to an inert support is by
far the simplest method of immobilization and usually does
not involve the use of harsh reagents or conditions which
might be harmful to the enzyme. Adsorption methods have
been reviewed in depth by McLaren and Parker (9).

The earliest reported enzyme immobilization appeared
in 1916 by Nelson and Griffin (10). The enzyme invertase
was adsorbed onto charcoal. and its activity detected by
measuring changes in optical rotation of solutions of cane
sugar when the immobilized enzyme was added.

Adsorption methods have not become prevalent due to
the ease with which the enzymes are desorbed. particularly

in the presence of substrate (11.12).

2. Cross-Linking

Enzymes can be immobilized by cross-linking with low
molecular weight multifunctional reagents. Covalent bonds
between the enzyme and the reagent form intermolecular
cross-links. Multifunctional reagents commonly used include
diazobenzidine and its derivatives and glutaraldehyde.
Reactive groups in the enzyme (4) include terminal amino and
carboxyl groups and the substituents of some amino acid
residues such as arginine (guanidyl substituent). lysine

(amino). hystidine (imidazole). cysteine (sulphydryl).

 

 

 

 

serine (hydroxyl). tyrosine (phenol). aspartic acid
(carboxyl). and glutamic acid (carboxyl).

When attempting an immobilization procedure which
involves the formation of covalent bonds to the enzyme.
bonds to a group in the enzyme’s active site should be
avoided. The amino acid residues located in the active
sites of many enzymes have been compiled (13).

To avoid bonding to the active site in trypsin
(14.15). the enzyme has been first polymerized with the N-
carboxyl-anhydride of L—tyrosine before it is to react with
various immobilizing polymers. The polytyrosyl chain which
forms SUpplies an alternative site to the essential residues
in the enzyme. Other approaches to protecting the active
site involve performing the immobilization in the presence
of the substrate or a competitive inhibitor (18.17). The
presence of a species such as substrate which selectively
binds to the active site may also result in enhanced
activity of the immobilized enzyme due to stabilization in
its active conformation (19). A review of cross-linking

immobilization methods can be found in Melrose (4).

3. Physical Entrapment

When a polymer is formed in the presence of an
enzyme. the enzyme can become physically entrapped within
the polymer lattice. The polymer lattice can be so designed
that the large enzyme molecules are not able to diffuse out.

but the smaller substrate and product molecules are able to

 

 

 

 

diffuse freely in and out of the matrix. Reagents used for
enzyme entrapment include polyacrylamides. silicone rubber.
silica gel. and starch.

Cholinesterase (19) has been immobilized in a starch
matrix and placed on a polyurethane foam pad. Enzyme
activity was retained for at least 12 months. Hicks and
Updike (20) immobilized glucose oxidase. catalase. lactate
dehydrogenase. amino acid oxidase. and glutamic
dehydgogenase by entrapment in polyacrylamide gels. These
enzymes showed little loss of activity after three months.

The principal advantages of physical entrapment

methods are the ease of preparation and the fact that the

enzyme is left unperturbed. Entrapped in a cavity of the
prOper size. the enzyme is able to take on conformational
changes which may occur during the catalysis. Enzymes

entrapped within semipermeable microcapsules are considered
to mimic best the environment of a living cell. An obvious
disadvantage is that entrapment of enzymes which catalyze
reactions of large substrate molecules will often result in

little or no observed activity.

4. Covalent Attachment

Enzymes have been covalently attached to a wide
variety of water-insoluble supports by numerous methods.
Covalent attachment is the most common method of enzyme
immobilization. since theoretically it offers the most

stable and most versatile method (2). A representative

 

 

 

 

 

 

 

sampling of reports of covalent attachment is presented in
this section.

Lactate dehydrogenase (24) has been attached to anion
exchange cellulose sheets. Enzymes have been diazotized to
cellulose particles (25) and to polyaminostyrene beads (26).
Other supports include water-insoluble derivatives of
trypsin (27) and carboxymethylcellulose (28). Weetall and
Hersh (29-33) have described procedures for the covalent
coupling of enzymes to inorganic materials with the aid of
an intermediate coupling agent. They note that inorganic
carriers have several advantages including immunity to
microbial attack and immunity to changes in configuration
over a wide pH range or under various solvent conditions.

Porous glass is a popular support for covalent
attachment of enzymes. Using silane as a coupling agent.
alkaline phosphatase (29). urease (30). trypsin. and papain
(31) were covalently coupled to silica glass. The products
were used in a packed column continuously for long periods
of time without significant loss of activity. Lactate
dehydrogenase and pyruvate kinase (34) were immobilized on
glass beads by diazotization and used in packed bed
reactors. Glucose oxidase has been covalently attached to
controlled-pore glass and to NiO on a Ni screen through a
silane coupling reagent (32).

Glucose oxidase (35). urease. and urate oxidase (39)
have been chemically attached to polystyrene tubes and used

in automated analysis. Goldstein (36) has developed a

 

 

 

 

 

synthesis of polyanionic and polycationic resins as supports
for trypsin. chymotrypsin. subtilisin Novo. subtilisin
Carlsberg. and papain.

L-Aspariginase has been attached to nylon tubing
(38). Nylon has also been used as the support for
immobilization of lactate dehydrogenase. maltate
dehydrogenase. alcohol dehydrogenase (40). urease (41).
trypsin (42). and glucose oxidase (43) for use in continuous

flow analysis.

B. Analytical Applications of Immobilized Enzymes

Immobilized enzymes have tremendous potential as
reagents in chemical analysis. Their maJor advantage over
non-enzymatic methods is their specificity. As such. in

many cases they can be used in conJunction with non-

selective detectors such as pH. gas or ion-specific
electrodes. thermistor probes. or certain non-specific
photometric methods. This section describes some

representative examples of analytical uses of immobilized
enzymes. and is divided according to the type of detector

employed.

1. Electrochemical Detection

a. Amperometric

One of the most common electrochemical detectors used

in combination with immobilized enzymes is the oxygen

 

 

 

 

 

 

 

10

electrode. The so-called enzyme electrode. first reported

by Updike and Hicks (43). is an elegant chemical transducer.

Glucose oxidase is polymerized in a gelatinous membrane over
a Clark type oxygen electrode and hence combines the
specificity of the immobilized glucose oxidase with the
electrochemical transducer to produce a portable probe for
glucose determinations. Oxygen consumption during the
conversion of glucose to gluconic acid is monitored by the
current output of the electrode. held at a fixed polarizing
voltage. About 30 s to 3 min is required for the diffusion
processes to reach equilibrium.

Total serum cholesterol has been determined by
immobilized cholesterol oxidase and cholesterol esterase
(44). The enzymes were immobilized onto alkylamine glass
beads and placed in a rotating porous stirrer near the
platinum electrode. The hydrogen peroxide produced in the
enzymatic reaction was measured amperometrically at +0.60 V
vs. SCE. The reaction was monitored either by a rate
method. by measuring the initial current change over a 2 min
period. or after equilibrium had been attained. by measuring
the total current change about 10 min after the start of the
reaction. A precision of 1-3% relative standard deviation
was obtained in equilibrium mode determinations. The
immobilized enzymes were used in 200-300 analyses with only
moderate loss in activity.

Immobilized glucose oxidase has been used in

combination with an oxygen electrode in the continuous flow

 

 

 

 

 

 

 

 

11

analysis of glucose (37). Both equilibrium and reaction-
rate methods were evaluated. The enzyme appeared to be
stable for 200 days while analyzing over 1000 samples. and
had a storage life in excess of one year.

Williams. D019. and Korosi (48) have used a platinum
electrode to monitor reactions of both glucose oxidase and
lactate dehydrogenase entrapped between the electrode and a

dialysis membrane.

b. Potentiometric

The ammonium ion electrode has been used extensively
as a detector for the enzymatic conversion of urea to
ammonium. Guibault and Montalvo (45) have prepared a urea
electrode by immobilizing urease in a polyacrylamide matrix
on a thin synthetic net. The net is placed over an ammonium
ion~selective electrode. When the electrode is placed in a
soluton containing urea. the urea diffuses to the urease
membrane where it is hydrolyzed to ammonium ion. At pH 7.
the ratio of ammonium ion to ammonia is approximately 100.
The response is somewhat slow; after approximately 100 s.
the potential is measured and found to be proportional to
the urea concentration in the range 10 to 300 ppm. The urea
electrode has been improved and further described by these
workers (46.47).

Urea has also been immobilized over the gas diffusion

membrane of an ammonia probe. Anfalt. Granelli. and Jagner

 

 

 

 

 

 

12

(49) used glutaraldehyde as a cross-linking agent to
immobilize the enzyme. Papasthathopoulas and Rechnitz (50)
measured urea by this technique directly in whole blood
samples by standard addition. The electrode potential was
measured approximately 5-6 min after immersion of the
electrode and found to be proportional to the urea
concentration.

Since the ammonia gas electrode responds to ammonia
and not ammonium ion. the pH of the monitored solution must
be basic enough to ensure that all ammonium ion is converted
to ammonia. The urease-catalyzed reaction is however
optimized at a somewhat neutral pH. Hence the separation of
these two components permits selection of the optimum pH for
each. Watson and Keyes (51) immobilized urease on a porous
alumina support. Samples containing urea were inJected into
a continuously flowing buffer stream passing through the
immobilized enzyme column. A sodium hydroxide solution was
continuously added to the reagent stream as it left the
column. which converted ammonium ion to ammonia (pH 11).
The solution then came into contact with an ammonia gas
electrode which was located downstream. Total conversion of
urea took place in the enzyme column. and as such the method
is immune to imprecisions arising from fluctuations in the
rate of the enzymatic reaction caused by changes in solution
composition and temperature.

In a similar technique. urease (52) was immobilized

on controlled—pore glass using glutaraldehyde and used in

 

 

 

13

the continuous flow analysis of urea with an ammonia gas
sensor as the detector.

An extensive mathematical treatment of the use of
potentiometric sensors to monitor reactions of enzymes
immobilized in semi-permeable membranes has been presented
by Blaedel. Kissel. and Boguslaski (53).

Changes in solution pH that occur during certain
enzymatic reactions has also been used as an indicator. May
and Li (54) monitored the enzymatic hydrolysis of urea with
a commercial pH stat. Urease was immobilized in
hydrocarbon-based liquid-surfactant membranes by adding as
aqueous urease solution dropwise to the membrane-forming
solution. The enzyme-containing emulsion was added to a
solution of urea. and the reaction monitored at pH 6.7 by
recording the rate of addition of HCl.

A pH stat was also used to monitor reactions
involving immobilized trypsin. chymotrypsin. subtilisin
Novo. subtilisin Carlsberg. and papain (immobilized as

described earlier) by Goldstein (36).

2. Spectrophotometric and Fluorometric Detection

Spectrophotometric and fluorometric detectors are
commonly used to monitor immobilized enzymatic reactions. as
the required instrumentation is found in almost all clinical
laboratories. The measurement can be either direct or

indirect. Direct measurement involves monitoring a

 

 

 

 

14
chromophore or fluorophore whose concentration changes
during the reaction. Indirect measurement involves the

conversion of a species (whose concentration is changing)
into a chromophore or fluorophore. Both methods can be used
to monitor the reaction continuously. or monitor the species
after the reaction has proceeded for a specified time.
Newirth. et al. (34) monitored solutions of pyruvate
(PYR). phosphoenolpyruvate (PEP). and adenosine-5‘-
diphosphate (ADP) with immobilized lactate dehydrogenase
(LDH) and pyruvate kinase (PK). The LDH-PK assay system
utilizes the change of absorbance at 340 nm due to the

disappearance of reduced nicotinamide adenine dinucleotide

(NADH).

PK
PEP3"+ ADP3’+ H+-——-> PYB' + ATP‘t'

PYR" + NADH + HIE-e. lactate- + NAD+
In a continuous flow system. solutions were run through
either one or both of two packed bed reactors containing the
immobilized LDH and PK and on to a 'spectrophotometric flow
cell. PYR solutions (0.01—O.1 mM). PEP and PEP-PYR
solutions (0.01-0.1 mM in PEP). and ADP solutions (0.01—O.1
mM) were analyzed by the method.

The increase or decrease in NADH absorbance was used
to monitor the immobilized enzyme catalyzed reactions of
pyruvate. oxalacetate. and ethanol (40). The enzymes were

immobilized onto nylon tubes and used in an automated

 

 

 

 

 

l5

continuous flow analyzer. Substrate solutions pass through
the immobilized enzyme tube and on to the flow cell for the
determination of the NADH present. Each immobilized enzyme
tube was used for at least 1000 analyses over a period of 20
days without any loss in activity.

The hydrogen peroxide produced in the enzymatic
conversion of uric acid was monitored indirectly by
Filippusson. et al (39). Urate oxidase was immobilized on
nylon powder which was then packed into a column and used in
an automated continuous flow analyzer. Samples were mixed
with buffer and passed through the packed column where a
certain fraction of the uric acid was converted to allantoin
and hydrogen peroxide. After the sample left the column.
HCl and KI were added to the reagent stream. The peroxide
converted the iodide to triiodide. The absorbance at 349 nm
due to triiodide was measured in a flow cell downstream. and
was found to be pr0portional to the uric acid concentration
in the sample. Calibration curve linearity extended from
0.01 to 0.10 mM uric acid. Over 400 analyses were performed
with the immobilized enzyme over a 4 month period.

The hydrogen peroxide-iodide reaction has also been
used to monitor the enzymatic reaction of glucose with
glucose oxidase (35). The enzyme was covalently attached to
a polystyrene tube and used in the continuous flow analysis
of glucose solutions ranging from 0.5 to 10 mM.

Nessler’s reagent was used to monitor ammonia

production in the enzymatic conversion of L-asparagine (38).

 

 

 

 

16

The enzyme. L—asparaginase. was attached to a nylon tube.
Solutions of the substrate passed through the tube. reagents
were added to the departing stream. and passed through a
flow cell. The flow kinetics of the system were studied.
and it was found that at low flow rates the enzymatic
reaction is largely diffusion-controlled.

The activity of trypsin aattached to nylon tubing has
been determined spectrophotometrically (42). The hydrolysis
of solutions of N-benzoyl-L-arginine ethyl ester (BAEE) was
monitored by measuring the absorbance at 255 nm of the
effluent relative to that of the solution entering the tube.
The kinetics of the immobilized trypsin were studied by
varying the flow rate of solution through the tube (and
hence the residence time) and monitoring the extent of

reaction.

3. Enthalpimetric Detection

The enzyme thermistor combines the specificity of an
enzyme-catalyzed reaction with the generally non-specific
detection of small changes in reaction solution temperature.
Mosbach and Danielsson (63) developed the first enzyme
thermistor by immobilizing trypsin and apyrase and
monitoring. with a thermistor probe. the temperature changes
which occurred during the enzymatic reaction. The change in
thermistor resistance was recorded as a function of time.

Linear calibration curves were obtained when the substrate

 

 

 

 

17

concentration was related to either the peak height. peak
area. or the gradient at the inflection point. The system.
however. required approximately 10 min to reach baseline
after each sample.

Bowers and Carr (56) have presented an excellent

treatment of the theory of immobilized enzyme thermochemical

reactors. Samples containing urea were inJected into a
flowing buffer stream which passes through a reactor
containing urease attached to porous glass. The temperature

of the solution leaving the column was monitored with a fast
thermistor and related to substrate concentration. Peak
height. area. and width were studied as a function of sample
concentration. Linearity in all three relationships were
observed for urea concentrations up to 200 mM. above which
complete conversion of substrate was not obtained in the
column.

Urease immobilized on controlled-pore glass was also

monitored enthalpimetrically in other reports (57.58).

Cholesterol. glucose. lactose. and uric acid (59) were
analyzed with immobilized enzymes in combination with
thermal detecton. Similarly. hexokinase (59) was
immobilized on controlled-pore glass and used in

thermochemical analysis.

Enthalpimetric monitoring of the enzymatic reacton
has the advantage of being free from several interferences
which plague other detectors. The detector is. however.

completely non—specific in that it responds to any

 

 

 

 

 

 

18

temperature change in the monitored solution. Another
drawback to their use has been the somewhat slow analysis
rate possible (due to slow response of the thermistor) and
the large solution volume requirements. The use of non-
compressable supports for the enzymes and the use of fast

thermistors (56) has dramatically improved the situation.

C. Commercial Systems Which Employ Immobilized

Enzymes

Immobilized enzyme reactors as well as complete
analytical instruments which employ immobilized enzymes are
rapidly becoming commercially available. and more are to
appear. A review of commercially—available immobilized
enzyme products has been prepared by Burns (60).

Owens-Illinois. Inc. (Toledo. OH) has patented a
blood urea nitrogen (BUN) analyzer. The BUN analyzer has
been evaluated by Watson and Keyes (51). Urease immobilized
on a porous alumina support is used to convert the urea to
ammonium ion. Samples are inJected into a continuously
flowing buffer stream which passes through the enzyme
column. Sodium hydroxide is added to the reagent stream as
it leaves the column and converts the ammonium ion to
ammonia. which is then detected with an ammonia gas
electrode located downstream. The column has been used in
over 1000 analyses over a 3 month period.

Kimble produces a similar instrument. which has been

 

19

evaluated by Hanson and Bretz (61). A two-point calibration
is recommended every 10 samples. Standard deviations of 1-32
were obtained on serum and plasma samples.

The "glucose oxidase reactor" can be purchased from
Northgate Laboratories (Hamden. CT). The immobilized enzyme

reactor has been incorporated in a Technicon AutoAnalyzer-II

 

continuous flow system and evaluated by Leon et al (62).
Over 25.000 samples were analyzed for glucose over a 74 day
period with the reactor.

Technicon Corp. (Tarrytown. NY) is offerring

"Autozyme" tubes for use with their AutoAnalyzer. Tubes

 

containing immobilized enzymes (glucose oxidase. glycerol
kinase. and co-immobilized hexokinase/glucose~6~phosphate
dehydrogenase) are available. Analytical aspects of these
products have been evaluated by Leon. et al (64). The
immobilized glucose oxidase tube was used in the analysis of

20.000 samples over a 4 month period. during which time the

 

activity decreased by only 30%. The shelf life (O‘C) is
estimated to be over one year. The immobilized glycerol
kinase. which is a very unstable enzyme in its soluble form.
was used to determine serum triglycerides. Serum ATP was
determined by the co-immobilized hexokinase/glucose-6-
phosphate dehydrogenase.

Chua and Tan (65) evaluated the Glucose Analyzer
marketed by Yellow Springs Instrument Co. (Yellow. Springs.
OH). Immobilized glucose oxidase on a membrane is placed

over a platinum electrode. which senses hydrogen peroxide

 

 

 

 

20

produced in the enzymatic reaction. The current output is
measured over a fixed time (approx. 45 5) early in the
reaction. and related to glucose concentration in the
sample. A digital readout displays the concentration in
mg/dl. Approximately 300 analyses can be performed with

each membrane.

 

 

 

 

 

 

 

CHAPTER III

IMMOBILIZATION OF ENZYMES ON NYLON

Glucose oxidase and alcohol dehydrogenase were
immobilized onto the interior surface of nylon tubing for
use in automated clinical analysis. Nylon tubing was chosen
as the support because solution containing the analyte
(substrate) can be quickly and easily brought into contact
with the immobilized enzyme for conversion to product and
then removed to determine the amount of product formed.
Nylon tubing containing immobilized enzyme was used in both
a continuous flow type arrangement and also in the discrete
sampling stopped-flow clinical analyzer.

Development of the immobilization procedure involved
repeated synthetic attempts at obtaining adequate activity
of the immobilized enzyme. During the development of the
procedure. substrate samples were analyzed by the
immobilized enzyme on the Technicon Auto Analyzer continuous
flow system described in Chapter IV. Each immobilization
attempt was Judged by its potential in a clinical laboratory
environment. where large numbers of samples must be
processed daily. A procedure reported by Inman and Hornby
(66) was attempted first. but proved unsuccessful for
reasons discussed below. This chapter describes the

development of an immobilization procedure which is believed

 

 

 

 

 

 

22

to be applicable to a variety of enzymes.

Briefly. the enzymes were immobilized on nylon tubing
of 1.5 cm and 0.87 mm inner diameters as follows. After
removing amorphous nylon. the interior surface of the tubing
was partially hydrolyzed with mild acid. Terminal carbons
of glutaraldehyde molecules were then attached to the
partially hydrolyzed nylon. after which the remaining
terminal carbon bonded to primary amine groups of the
enzyme.

This procedure results in enzyme molecules becoming
attached to the support by a six carbon cross-link. which
allows flexibility in the molecule and hence no steric
hindrance to activity. Enzymes in which primary amine
groups are not present at or near the active center could

be immobilized by this procedure without loss of activity.

A. Initial Preparation and Hydrolysis of the Nylon

Amorphous nylon was first removed from the nylon tube
by incubation in a 20% solution of calcium chloride in 80%
methanol/20% water (v/v) solvent at 509C for 20 min. When
the tube was then rinsed with distilled water. a stringy
white precipitate was purged from the tube. Apparently the
CaCl solution acts to remove sections of the nylon surface
which are not totally a part of the polymeric structure.
This should then result in a somewhat increased surface area

for enzyme attachment as well as a more permanent support.

 

 

23

The remaining steps in the procedure were done by
pumping the particular reagent through the tube with a
peristaltic pump. By choosing pump tubing of appropriate
inner diameter. the flow rate of reagent through the nylon
tube can be selected from 0.015 to 4 ml/min.

Hydrolysis of the interior surface of the nylon tube
was carried out by treating it with a mildly acidic
solution. The surface hydrolysis is complete after 20
minutes of pumping 0.5 M hydrochloric acid through the tube
at room temperature.

Early attempts at immobilization involved incubating
the hydrolysis reaction at 40°C and using 4 M HCl. This
invariably resulted in excess hydrolysis. The tube became
so clogged that solution flow could not be maintained.
Hydrolysis conditions were then made less severe by allowing
the reaction to proceed at room temperature. after which
solution could be easily pumped through the tube.

However. as shown in Figure 1. scanning electron
microscope photographs of the interior surface of both
virgin nylon tubing and tubing hydrolyzed under these
conditions (4 M acid. room temperature) showed that the
hydrolysis was so severe that sections of the tubing had
been hydrolyzed nearly halfway through the tube wall. The
extent of hydrolysis was also shown to be quite different in
various sections of the tube wall.

Although the resulting structure could support

immobilized enzymes. it proved to be too porous to be of any

 

 

 

24

value in enzymatic analysis. Substrate and/or product
molecules became trapped in the matrix and at times could
not be removed by eluting for 30 minutes or more. which
resulted in serious carry—over of these substances from one
sample to the next and precluded its use in routine clinical
analysis.

When the hydrolysis conditions were changed so 0.5 M
HCl was used at room temperature. the problem of carry—over
was completely eliminated. All traces of a previous sample
could be completely removed by rinsing the enzyme loop with
small portions of buffer solution. Presumably. the surface
hydrolysis could be carried out even under milder conditions

to attain good enzymatic activity.

B. Attachment of Glutaraldehyde

The bifunctional reagent glutaraldehyde is an ideal
reagent for enzyme immobilization because both of its
aldehydic carbons can be utilized in the formation of a
cross-link between the support and the enzyme.

Substrate molecules must have free access to the
active site on the enzyme molecule. and once they reach the
active site. the enzyme molecule often must be able to take
on conformational changes during the conversion of substrate
to product. The product molecule must then be able to
diffuse back into the bulk solution so that its

concentration can be determined. Each of these steps may be

 

 

 

 

Figure l.

25

 

Inner Surface of Hydrolyzed Nylon (5000 diameters)

Scanning Electron Microscope Photographs of Virgin and
Hydrolyzed Nylon Surface

 

 

 

26

hindered if the enzyme is directly bound to the support.
Also. electrostatic interactions between the support and
either the substrate or product species may slow the overall
conversion rate.

The use of glutaraldehyde as a cross-linking agent
leaves the enzyme somewhat less "immobilized" than if
directly bonded to the support. and thus it can move
somewhat in the near vicinity of the support wall.

In the procedure deveIOped. a 12.5% solution of
glutaraldehyde is slowly pumped through the partially
hydrolyzed nylon tube. during which terminal carbons are
covalently bonded to free nitrogens on the nylon wall.
according to Figure 2. The remaining aldehyde groups on
each attached glutaraldehyde molecule are then available for
covalent bond formation with the enzyme. It is certainly
possible that a significant fraction of the attached
glutaraldehyde molecules will bond both aldehydic grOUps to
the support and hence be unavailable to bond to the enzyme.

In the development of this procedure. it was found
that the solution pH during the glutaraldehyde attachment is
very critical. and must be maintained at 9.2. The
glutaraldehyde solution in this procedure was buffered in
0.1 M tris(hydroxymethy1)methylamine. Final enzymatic
activity was not detected when this reacton was performed at
pH values of 8.5. 8.8. or 9.1. Apparently at a pH lower
than 9.2. nitrogens on the hydrolyzed nylon are protonated

and their reaction with the glutaraldehyde carbonyl is

 

 

 

2?

:2
O: O
:2;
O
N
I
2
D
+

O
11‘.

°~>.
a:

Figure 2. Attachment of Glutaraldehyde

 

 

 

 

 

 

28

hindered.

Glutaraldehyde is the most prevalent multifunctional
reagent used in enzyme immobilization. yet its structure and
chemistry are not well understood (3). It has been proposed
that aqueous solutions of glutaraldehyde are largely
polymeric (67). and also to the contrary that it exists
mainly as the three hydrates shown in Figure 3 (68). The
mechanism of the reaction of glutaraldyhyde with enzymes is

similarly unresolved.

C. Immobilization of the Enzyme

After the glutaraldehyde attachment. the nylon tube
is thoroughly rinsed with the tris buffer for 20 minutes. A
solution of the enzyme (approx. 4 mg/ml) buffered at pH 6.2
is then slowly pumped through the tube in a closed loop at
0°C (ice-water bath) for 4 h. During this time. primary
amine groups on the enzyme. presumably. react with free
aldehyde groups of the attached glutaraldehyde to form the
structure shown in Figure 4. As can be seen. this structure
allows the enzyme molecule flexibility and a certain degree

of mobility.

The mechanisms involved in glutaraldehyde attachment
and subsequent enzyme immobilization described here are only
. proposed mechanisms. The actual covalent linkages described

have been proposed elsewhere in the literature (3). although

 

 

N
x

29

 

oohsodaanapsao we ulnom 260:5 comononm .m onswam

can.

 

:.\

«ESE 9.0-:

C

UTNE (N55615:
a __
o c

I)

 

30

0
ll
fiWc-NH)Wcoz-Nat
CH
6:
+

Figure 4. Attachment of the Enzyme

 

31

no definitive evidence has been put forth. It was not
within the scope of this work to determine the mechanism of
the immobilization procedure. Rather. this work involved
the development of a somewhat empirical and functional
procedure. Certainly. though. if the mechanism were better
understood. reaction conditions could be intelligently
chosen to maximize not only the amount of enzyme immobilized

but also the resulting activity

D. Specific Procedure for Enzyme Immobilization

The following is a detailed description of the
immobilization procedure developed. Steps 2—9 were carried
out by using a peristaltic pump to bring a constant stream

of fresh reagent through the nylon tube.

1. Fill a 1.0 m length of the nylon tubing with a solution

containing 20% CaCl in 80% methanol/20% water (v/v).
2. Rinse with 500 ml of dist. water at 8 ml/min.

3. Hydrolyze the surface by pumping a 0.5M HCl solution

through the tube at room temperature for 40 min (8 ml/min).
4. Rinse with 500 m1 of dist. water at 8 ml/min.

5. Pump a 12.5% solution of glutaraldehyde in 0.1M
tris(hydroxymethyl)methylamine buffer. pH 9.2.

through the tube at 2 ml/min for 20 minutes at 0°C.

32
Rinse with the 0.1M tris buffer at O'C for 20 minutes.

In a closed loop. pump a solution containing

4 mg/ml of the enzyme per ml of 0.2M phosphate buffer at
0°C at 2 ml/min for 4 hours. The pH of the phosphate
buffer should be chosen according to the particular

enzyme to avoid possible denaturization.

Rinse with 0.1M NaCl at room temperature at 8 ml/min

for 1 hour to remove any physically adsorbed enzyme.

Rinse and fill with buffer of appropriate pH. and

store refrigerated (5°C) when not in use.

 

CHAPTER IV

FLOW SYSTEMS

Two types of flow systems have been employed in the
implementation of the immobilized enzymes. and these flow
systems are described in this chapter. The first system. a
Technicon Auto Analyzer. uses continuoulsy flowing reagent
streams to mix the sample with the desired reagents and then
pass the solution through a transparent flow cell for
spectrophotometric observation. Secondly. the stopped-flow
clinical analyzer which was developed in this work is
described. This system is a discrete sample analyzer which
uses a commercial stopped-flow mixing system for reagent and

sample mixing and spectrophotometric monitoring.

A. Technicon AutoAnalyzer

1. Description

The design concept of the Auto Analyzer was developed
by Skeggs (69) in 1957. Solutions are pumped through
flexible tubing at selected flow rates by a peristaltic
pump. The flow rate for each reagent line is determined by
the inner diameter of the tubing over which the pump rollers

are driven at a constant rate. The reagent lines are Joined

33

 

f _ __~,._-' .5..._._ g, , ~-- q '-'~—"— -__~ - ..u- ‘-—..nc.fi , .-.——.—-

34

where desired by glass connectors to produce the desired
configuration. A non-adJustable thermostatted heating bath
can be used to incubate solutions only at 42°C as they pass

through the bath.

2. Design for Immobilized Enzymatic Analysis of

Glucose

The Auto Analyzer flow system was used in early
studies of immobilized glucose oxidase. The enzyme was
attached (as described in chapter III) to the inner surface
of a coiled nylon tube. 2 meters in length and 1.5 cm inner
diameter. and inserted into the flow system. For the
analysis of glucose samples. the flow system components were
arranged as shown in Figure 5.

In this configuration. the sample or blank solution
was first mixed with the buffer solution and air bubbles
were added to the flow stream. The solution then passed
through the immobilized glucose oxidase tube which was
immersed in the heating bath at 42°C. As the solution
passed through the column. a fraction of the glucose was
converted to gluconic acid and hydrogen peroxide. After the
reacted solution left the column. a buffered solution
containing potassium iodide and Mo(VI) was added to the flow
stream. which then passed through a mixing coil. In the
mixing coil. the hydrogen peroxide was completely reduced by

the excess iodide. and a corresponding amount of triiodide

 

 

 

35

 

A,X§amnle
V _

 

nAir _
V

 

 

 

l 7

HB Immobilized Enzyme
Loon

 

A Buffer
U

Q

 

r
J AKI

v

 

 

A Mow )

‘T L

 

Mixer 3 r—awaste

 

 

g;waste

 

 

 

 

 

 

Spectrophotometer

 

 

 

Figure 5. Auto Analyzer Configuration for
Glucose Determinations

 

“we; a :b.‘ _ g ':.-_,':;0_‘ . u, .—.—. a f_--".-— ' . _ .

 

 

 

 

—i—' _ " " ”TVS-T:_ m' 'V '»%~--»'4—44-v:31'ifim‘4*5' — " ““"“"’~" ‘-

36

was produced. The Mo(VI) catalyzes the peroxide-iodide
reaction. The air bubbles were then removed from the flow
stream. as described later in this section. and the stream
then passed through a quartz flow cell housed in a Beckman
DB-G spectrophotometer. The absorbance of triiodide at
365 nm was recorded on a strip chart recorder as the
triiodide solution passed through the flow cell. Either the
peak height or the peak area of the absorbance vs time curve

was related to the concentration of glucose in the sample.

3. Characteristics

Air bubbles are added at regular intervals to the
flow stream in the AutoAnalyzer to isolate segments of the
stream. which permits efficient mixing within each segment
and also results in less axial dispersion of each sample
plug as it flows through the system. The bubbles are
removed from the stream JUSt before it passes through the
spectrophotometer to avoid large spikes in the response.

A maJor disadvantage of the AutoAnalyzer encountered
in the study of the kinetics of the immobilized enzymes was
the fact that it was difficult and time consuming to vary
the enzymatic reaction time. even over a moderate range.
The duration of the enzymatic reaction is controlled by both
the volume of the column. which was fixed. and the flow rate
through the column. However. by altering only the flow rate

of the sample. other problems can result. The relative

 

a: _-'-~= 3": - .-

 

 

 

 

 

37

amounts of each solution added to the flow stream (sample.
buffer. air. iodide. and molybdate) are determined by their
relative flow rates. Hence. although varying the flow rate
of the sample line could have been used to vary the
enzymatic reaction time. this would have also varied the
relative concentrations of all other reagents. Since the
iodide and Mo(VI) gave a small blank absorbance. the
baseline would have to be reset each time the flow rate was
changed. More importantly. the effectiveness of the buffer
in pH control would have to be checked as its concentration
was also changed. Similarly. the adequacy of the final
iodide and Mo(VI) concentrations would have to be verified
as well.

The mixing coil was very effective in mixing the
column effluent with the indicator reagent. The mixer
consists of 10 turns of glass tubing. so that each solution
segment is inverted 10 times as it passes through the coil.
The time spent in the mixer of course depends on its inner
diameter and the flow rate through its in the flow design
used in these studies. the mixing time was typically 30 s.

The debubbler consists of an inverted tee inserted
into the flow stream. The air bubbles and a small amount of
liquid are drawn out the top of the tee by connecting the
top to the pump. The proper flow rate for the debubbler was
selected such that the bubbles were completely removed from
the lstream while at the same time only a small amount of

liquid was removed. The debubbler was placed as near as

 

 

 

——fiW“Wfip~-vfio . --..--~~m.a-.L~u~--. u. 4 . --...... -... -- a
__ ,.,-__. .- _-.

38

possible to the flow cell to avoid axial dispersion of the
sample plug after the air bubbles had been removed.

In theory. the triiodide produced in the indicator
reaction should pass through the quartz flow cell as a plug.
and hence should then give a response curve in the shape of
a gaussian peak. The flow rate of solution through the flow
cell was typically 3 ml/min. The dimensions of the flow
cell are 1 cm X1 cm X 2 mm. with a total volume of 200 ul.
Hence. if the chromophore moved into and out of the flow
cell as a plug. each cross-sectional slice of the plug would
reside in the cell for approximately 4 s. In practice.
however. the sample plug was observed to require about two
to three times that expected to completely pass through the
cell. mainly due to inefficient design of the flow cell. A
relatively large dead volume exists in the flow cell.
especially in the corners. where solution can become
trapped.

The slow passage of a plug of the chromophore through
the flow cell made it difficult. if not impossible. to
calculate the actual extent of the enzymatic reaction in the
column. The peak area of the observed response could not be
related to the integral of the chromophore concentration in
the plug.

The peak height of the absorbance response curve.
however. could be used to generate a linear calibration
curve for glucose. However. due to the degree of axial

dispersion and subsequent decrease in peak height of the

 

 

 

 

was-man... .. .-.-. AMM, -‘...ms—M— _

39

sample plug as it travelled through the flow system. the
sensitivity was low. This is a fundamental disadvantage af
the Auto Analyzer flow system when the peak height is the
measured quantity. In clinical laboratories. the flow
system is often designed so that the sample plug must flow
over a long distance before reaching the flow cell. and
often requires several minutes to complete the Journey.
Axial dispersion also limits the analysis rate. since
samples must be introduced far enough apart to avoid overlap

of the responses.

B. Stopped-Flow Clinical Analyzer

1. Description

The design of the stopped-flow clinical analyzer is
illustrated in Figure 6. The components enclosed within the

dashed line are part of a GOA-McPherson stopped-flow module.

This particular module is well. suited for automated
operation. since no manual manipulation of valves is
required. A single cycle can be initiated either from the

front panel or with TTL-compatable signals to a plug on the
rear panel.

The drive syringes (081 and D52) are replaceable
glass syringes. and the volume driven with each push is
manually adJustable from 200 to 500 ul/syringe. The

plungers are driven by a pneumatic cylinder which is driven

 

 

 

 

‘ ' 3H.- ”Wu-W '

 

“ ".'-‘.7-K_.' 1.'~' -

u .

 

#0

Housmana Haoasaao scam-aodqeum .m enemas

I | ‘ l I l I l i I i l l l I l l

 

 

_ :98: A

noon :oapodom

olhugm OONHHADOIIH

QOZQM" WV

aca—mm—EDE)

 

 

 

 

 

 

 

 

l
lllllillliilil

 

 

41

with 35-45 psi pressure supplied by an external air
cylinder. The mixing chamber (MC) and observation cell (DC)
are housed together in a Kel-F block mounted near the front
of the module in the optical path. Solution is directed
from the drive syringes to the mixing chamber/observation
cell inlet through narrow diameter plastic tubing. and the
connections are made with threaded fittings. The quartz
observation cell is cylindrical in shape. measures 2 cm in
length and 2 mm in diameter. and has a volume of 62.8 ul
The stop syringe (SS) is also replaceable glass. and the
volume taken into the syringe before the flow stops is
adJustable from 400 to 1000 ul. The dead time of the system
is ~~5 ms (70).

The GOA-McPherson stopped—flow module was designed to
be used with the Heath series modular spectrophotometer
system. A complete stopped-flow spectrophotometer can be
easily assembled by mounting the stepped-flow module with a
light source module. monochromator. photomultiplier module.
and a data collection system.

The GOA-McPherson module was modified to incorporate
the immobilized enzyme reaction loop. As shown in Figure 6.
one of the reagent lines was brought outside the module.
This was done by installing HPLC tubing couplers (Altex) on
the front panel of the module. and. on the inside the
module. using short pieces of HPLC tubing to connect 082 to
one of the couplers. and the mixing chamber/observation cell

entrance port to the other coupler. Outside the module.

 

 

 

short pieces of tubing were also used to connect the other
ends of the two couplers to a pair of 3~way slider valves
(SV) (Altex). The immobilized enzyme reaction loop was
inserted between the valves. so that. when the 3-way valves
were switched to the push position. the reaction loop was
connected to the stopped-flow system. When the syringes
were then driven. solution from DSZ forced the solution from
the reaction loop into the mixing chamber for analysis.

The S-way slider valves consist of a rectangular
Teflon slider block mounted within the valve which directs
the solution flow from the common line to either of the
secondary lines. These valves were automatically switched
in tandem by a pneumatic actuator/spring return mechamism.
Tubing is connected to the valve ports by threaded fittings
so that connections can be easily made.

The Operation of the SPCA can be best described by
the following typical sequence of events for the analysis of
a substrate sample. Referring to Figure 6. the stopped-flow
mixing system is first rinsed with the desired reagents.
D81 is filled with the indicator reagent and 052 is filled
with the push liquid (buffer). When this cycle is
completed. the sample valves (SV) are switched to the fill
position and the buffered sample is drawn into the reaction
loop by the action of the applied vaccuum. When the 100p
has been thoroughly rinsed and filled. the sample valves are
switched back to the push position. which stops the sampling

process and marks the beginning of the enzymatic reaction.

 

 

 

 

43

After the enzymatic reaction has been allowed to proceed for
a preset time. the stopped—flow syringes are driven. The
push liquid in D32 forces the reacted solution from the
reaction loop through the connecting tubing into the mixing
chamber. where it is rapidly mixed with the indicator
reagent and driven into the observation cell. The indicator
reaction then takes place in the observation cell. The
indicator reagent converts one of the enzymatic reaction
products into a chromophore. whose absorbance is monitored
as an indicator of the amount of product produced in the

enzymatic reaction.

2. Characteristics

The HPLC tubing and sample valves are designed to
withstand a pressure of 500 psi. which is more than adequate
for this system. These components are made of Teflon and
are therefore chemically inert to the reagents used in these
analyses. The sample valves are designed with zero dead
volume.

When the immobilized enzyme loops are fitted with the
threaded and fittings. they can be quickly and easily
inserted or removed from the system. which simplifies
replacement the reaction loop.

The design of the SPCA greatly facilitates a
fundamental study of the kinetics of the immobilized

enzymes. The volume contained within the reaction loop was

 

 

 

44

designed to be much greater than the combined volumes of the
connecting tubing. mixing chamber. and observation cell.
The push volume was adJusted so that the solution initially
inthe reaction loop completely replaced the solution that
was previously in the observation cell. Thus. the reacted
solution in the reaction loop could be accurately sampled
and monitored in the observation cell. without the problem
of axial dispersion prevalent in the Auto Analyzer flow
system. The concentration of the enzymatic reaction product
could be calculated from the measured absorbance. the molar
absorptivity of the chromophore. and a knowledge of the
stoichiometry of the indicator reaction.

The design of the SPCA allows one to monitor the
enzymatic reaction and the indicator reaction separately.
each by either a reaction—rate method or by an equilibrium
method. This choice allows the use of an indicator reaction
which may. for example. be slow. or have an unfavorable
equilibrium constant. or undergo side reactions. Also.
since the two reactions are physically separated. the
reaction conditions can be drastically different in each.
and can be chosen to yield the optimum sensitivity.
accuracy. and precision from each reaction.

Another fundamental advantage of the SFCA design is
that several immobilized enzyme reaction loops containing
the same or different enzymes can be multiplexed into the
stopped-flow mixing system. This could tremendously improve

sample throughput.

 

 

 

45

The SFCA also is efficient in its use of reagents.
since the volume of the stopped-flow syringes is small. and
since reagents are consumed only when neccessary rather than
continuously flowing.

A disadvantage with the present design of the SFCA is
the lack of precise thermostatting. The entire system is
held at room temperature. Any increase in the temperature
of the reaction loop. mixing chamber. etc. must be
dissipated into the surrounding air. In the stopped-flow
module. thermostatted water is circulated in copper tubing
which is in contact with a cast aluminum base.
Thermostatting within the module is therefore dependent on
heat transfer to and from the aluminum base via thermal
conduction. Holler (71) showed that this design is somewhat
inefficient. His studies demonstrated that temperature
equilibrium of the solution in the observation cell is slow.
and does not accurately track the temperature of the

thermostatting bath

 

 

 

CHAPTER V

MICROCOMPUTER INSTRUMENTATION

Computer technology. specifically microprocessors and
microcomputers. has had a dramatic impact on analytical
chemistry. With the aid of a computer. chemists are able to
perform long and tedious experiments which were previously
infeasible or highly prone to human error because of
monotonous sequencing or massive data collection and
processing. The high speed data acqusition and almost
instantaneous feedback control offerred by computers have
increased the amount of information that can be extracted
from a measurement process. The use of a mini—and
microcomputer as an aid to clinical analysis and the
networking of computers in our laboratories are described in

this chapter.

A. Overview

The microprocessor offers an inexpensive method of
process control and data acquisition. It is highly flexible
in application since it can be programmed to follow any
sequence. With sufficient memory. the microcomputer can
perform the entire analysis. including instrument set up.

calibration. sampling. data collection. and calculation of a

46

 

 

 

47

final result (concentration. rate constant. absorption
spectrum. etc.).

At the other extreme. microcomputers can be slaved to
a larger computer (e.g. mini) and relegated to perform
specific tasks. In this configuration. the minicomputer
commands the slave micros to perform a task or to begin a
certain sequence. receives information from the micros about
the status of the experiment. and receives raw data (e.g.
pH. temperature. etc.) collected by the slaves. The data
are processed by the mini and displayed in a variety of
formats such as lists. plots. etc.

In this work. a microcomputer was relegated to the
tasks of instrument sequencing. acquisition of raw data
corresponding to transmitted radiant power. and performance
of simple calculations on the raw data. The partially
worked-up data were then transmitted to a more powerful
minicomputer for more complex calculations. display. and
magnetic storage. A network was devised for allowing a user
terminal and several microcomputers to share the facilities
of the minicomputer.

Several network configurations (72) are possible for
interconnecting the various devices (microcomputers and
terminals) in our research laboratories to the minicomputer.
Different networks involve varying levels of communication
between the devices and varying degrees of hardware and
software requirements. The point-to-point network shown in

Figure 7 was implemented. In this configuration. access to

 

 

 

 

 

 

(:jPlotter

 

<::Printer

 

Dre
369

i

 

PCM-lz
or

pop 8/E 16—

Minicomouter

 

 

‘V/

'\

 

Console
Interface

 

\.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 7.

 

Mass
Storage
Device(s)

 

 

 

 

 

 

 

 

 

 

 

 

 

Network Console
Switch
Interface Interface ... Interface Interface
Micro Micro o.. Micro Micro

 

 

Point-to-Point Hierarchical Network

 

 

 

49

the minicomputer is controlled by a network switch. so that
the facilities of the mini are time—sliced. When a
particular device is switched to the mini. it is able to
retain complete control for as long as desired. and releases
control when its Job is completed. The network switch then
sequentially samples each device until it finds one which

needs access to the mini.

The point-to-point configuration was chosen for
several reasons. Because of the wide spectrum of
experiments performed in our laboratories and the

independence of each. communication between microcomputers
is at present unneccessary. Since mini-to-micro
communication is made through an asynchronous interface. no
restrictions are made on the internal configuration of each
microcomputer. The time-sliced sharing of the minicomputer
can be implemented because of the typically short time
requirements ‘of each microcomputer. Once software has been
developed. the mini-to-micro link needs to be made only to
down-load the software into micro memory; a relatively much
longer time is then consumed performing the experiment.
during which the mini is accessable to other devices.
Finally. this configuration is the simplest to implement
both in hardware and software.

At present. either of two minicomputers can serve as
the head of the point-to-point network. A PDP 8/e
minicomputer (Digital Equipment Corp.) is configured with

16k of read/write memory. extended arithmetic element. real

 

 

 

50

time clock. RKOS cartridge disk drive. dual floppy disk
drive. printer. plotter. and console. A second system is
configured around a PCMr12 minicomputer (Pacific
Cyber/Metrics) and includes 16k of RAM. dual floppy disk
drive. printer. and console. The POM—12 utilizes an
Intersil 6100 microprocessor as the CPU. This processor
uses virtually the same instruction set as the PDP B/e. so
the two minicomputers are software compatible. Software
which does not make use of hardware unique to either system
can be developed on either mini. Both Operate under OS/B.

the operating system developed by DEC for the PDP 8’s.

B. The 6100 Microcomputer

1. Description

Control of the sequencing of the stopped-flow
clinical analyzer and data acquisition from the photocurrent
amplifier are accomplished with a .twelve-bit microcomputer
which utilizes an Intersil 6100 microprocessor. The
central processor board. 4K RAM board. and the two serial
interface boards were purchased (Pacific Cyber/Metrics. San
Ramon. CA) and constructed from a kit. This particular
microcomputer was chosen because the 6100 MPU uses virtually
the same instruction set as the PDP file in our laboratory.
and. as such. software for the microcomputer can be created

and developed on either the PDP B/e or POM-12 minicomputer

 

 

———

51

under 08/8 and then down-loaded into the microcomputer’s
memory for execution.

Software for SFCA execution is written either in
FORTRAN-ll/SABR or PALB (the PDP-8 assembly language) on the
minicomputer. stored on a mass storage device (floppy disk
or cartridge disk). and compiled and assembled under 08/8.
The absolute binary program thus generated can then be downw
loaded without further modification. via the asynchronous
serial interface directly from the mini to the micro. This
procedure greatly facilitates debugging the SFCA software
since it can be quickly edited. assembled. and reloaded. It
also has versatility in the SFCA operation. as a variety of
routines for a variety of SFCA operating procedures can be
quickly called up from mass storage and downloaded.

The SFCA micro-mini computer system is shown in
Figure 8. Direct communication between the mini and micro
is required only to transmit the desired binary program down
to the micro. after which the link can be broken and the
microcomputer operated in a stand-alone configuration.
Optionally the link is later remade to transmit raw data
acquired during an experiment back to the mini. where

higherrlevel language routines can work up the data. list.

 

or plot.

2. PROM Monitor Software

A general purpose routine to handle the down-loading
of binary programs from the mini into micro memory was

designed to operate in PROM in the microcomputer. This PROM

 

 

 

52

 

 

 

Plotter

Printer::>$—4>

”W l

 

 

 

 

 

 

( Printer#::>t
§[Tlnterface

 

(::Plotter#::¥‘

Figure 8.

 

 

 

 

 

 

 

 

 

 

 

  
  
  

 

 

 

PCM-lZ
or ' Mass
Storage
pop B/E Device(s)
Minicomputer
Console
Interface
In
Control #
6100 vw_lnterface
Microcomputer Analog
Interface

 

'1'—

 

T

 

 

 

 

  
    
   

Interface
Stopped-Flow
Clinical
Terminal Analyzer

 

 

 

 

SFCA-Micro-Minicomputer System

 

 

 

53

Monitor routine serves three basic functions for the micro:
(1) a transparent mode which serves as a software link
between the microcomputer terminal and the minicomputer’s
main terminal ports (2) a binary loader for loading an
incoming binary program into the micros mainframe memory.
and (3) a RAM Monitor which allows the user. via commands
typed at the keyboard. to examine and/or modify locations in
mainframe memory. and to begin execution at a specified

location.

a. Transparent Mode

The transparent mode provides a link between the user
terminal and the minicomputer; in this mode the
microcomputer is essentially transparent to the user. The
transparent mode is entered automatically after power—up and
initialization of the PROM Monitor. The program then
monitors both of the micro’s serial interfaces. Ordinary
characters input at one port are immediately output to the
other. This procedure continues until a special character
is received at either port. These special characters cause
the Monitor to chain to either the binary loader or RAM
Monitor. The user can cause chaining to the RAM Monitor by
typing CNTRL-A at the terminal. while the minicomputer can
cause chaining to the binary loader by issuing leader—
trailer code (octal 200).

A standard feature of the 08/8 Peripheral Interchange

 

 

 

 

 

54

Program (PIP). when outputting a binary program. is to begin
and end the actual binary data with leader~trailer. This
character is not otherwise normally output by 08/8 software.
and hence the transparent mode is not unneccessarily
interrupted unless the minicomputer is about to start
transmitting a binary program. Similarly. CNTRL-A is not
normally typed by the user when operating OS/S software.

An important consideration in designing software for
mini—micro communication (specifically with the operating
system) was to be able to use the O/B software without
modification. In the particular case Of the transparent
mode routine. this meant that characters had to be input
from the minicomputer and then output to the terminal at a
fast enough rate that no characters would be missed. This
was readily accomplished by operating the terminal interface
at a higher baud rate than the minicomputer interface.
Normally. for example. the terminal is Operated at 2400 baud
(240 characters/s) while the minicomputer interface is
Operated at 1200 baud (120 characters/s). As such. each
transmission Of a character to the terminal requires 4.167
ms. while character transmissions from the minicomputer
require 8.333 ms. Once a character is received from the
minicomputer. the next one cannot be completely transmitted
before 8.333 ms has elapsed. and the micro software then has
the additional 4.167 ms to check the input for a special
character. which is more than ample time.

As expected. operation of both interfaces at the same

 

 

 

 

55

baud rate results in occasional missed characters.

The asynchronous interface between the micro and mini
has the additional advantage Of allowing the micro to run
batch processes by communicating directly with 08/8 software
without operator intervention. The micro can be viewed as a
"virtual terminal" to the mini since the 08/8 software
receives characters as if a terminal were present. Micro
software can mimic the action of a user entering commands at
a terminal by transmitting a sequence of characters. The
routine needs only to wait for each character to be echoed
by the mini before sending the next character. Commonly
used command sequences can be stored in micro memory and

later transmitted in this fasion.

b. Binary Loader

The binary loader secton of the PROM Monitor is
automatically chained to when leader-trailer code is input
from the minicomputer. a signal that the transmission of an
absolute binary program is beginning. Approximately 60-70
frames of leader-trailer precede the actual binary code. so
no binary data are missed during chaining.

The binary loader routine was adapted from a software
front panel routine developed at Pacific Cyber/Metrics for
their POM-12 minicomputer. Their loader was modified by
eliminating several unneccessary features so that it would
fit in the 256-word PROM space along with the transparent

mode and RAM Monitor.

 

 

 

 

56

First. the loader input device number was changed
to 13 (the minicomputer interface). Second. extended memory
loading was eliminated. since the micro at present has only
one field (4K) of read/write memory. Finally. the action
taken upon completion of the loading process was changed.
The last two binary words Of the transmission comprise the
checksum of the transmission. which provides a crude check
on the entire transmission. The loader calculates the
checksum as the program is input and loaded. and then
compares this with the input checksum. If the two are not
identical. the loader halts. A halt indicates that an error
has occurred. If the two are identical. the program is
assumed to have been accurately input and loaded. and the

loader then chains to the RAM Monitor.

c. RAM Monitor

This section of the PROM Monitor is chained to either
after the successful completion of a binary load. or when
the operator types a CNTRL-A at the terminal while in the
transparent mode. The RAM Monitor serves the functions of
examining and/or modifying microcomputer read/write memory.
and beginning execution at a specified RAM address. The
Monitor begins by typing a prompting dollar sign. this is
repeated each time the Monitor is ready for terminal input.

After prompting the user. the Monitor expects to
receive from the terminal four octal numbers followed by a

control character; any other sequence is ignored. The

 

 

 

57

control characters are (1) "G" which instructs the Monitor
to chain to the RAM address specified by the four octal
numbers. and (2) "D" which instructs the Monitor to deposit
these data at the last specified RAM address. Any other
control character causes the contents of the specified RAM
address to be displayed on the terminal. Examples of each

are given below.

$1000/7000 RAM location 1000 now contains a 7000.

$76000 The contents of location 1000 is
changed to 7600.
$02000 RAM execution begins at location 200.
When chaining to mainframe memory. the RAM Monitor

first clears the CPU accumulator and link.
3. Operating Procedures

The combined functions of the PROM Monitor and its
harmonious interaction with minicomputer software is best
demonstrated by following a . typical sequence for
microcomputer execution of predeveloped software.

After initialization of the PROM Monitor. the
asynchronous interface between the mini and micro is
connected and 08/8 bootstrapped on the minicomputer.

A typical sequence for execution of a FORTRAN-II

program (stored on the mass storage device) is then as

follows:

 

 

(1)

(2)

(3)

(4)

(5)

(6)

 

58

FROM

COMMAND ROUTINE OPERATION

COM PROGI.FT TM The FORTRAN-II source file
is compiled. assembled. and
loaded into microcomputer memory.

SAVE 8Y8 PROGI TM A CORE-IMAGE file of the program
is created.

MAP PROGI TM The user finds out the actual
entry point of the program.

R BIN4SV TM

*PROGI<PR001 TM This creates an absolute binary

file from the CORE—IMAGE file.

DOWNLOAD PROGl TM.BL The absolute binary program is
transmitted (with leader—trailer)
and loaded into microcomputer
mainframe memory.

$10020 RM Execution is started at RAM address
1002. the starting address
determined in step 3.

To simplify the procedure of executing software on
the micro. the 08/8 Concise Command Language (CCL) program
was modified to reCOgnize the MAP and DOWNLOAD commands.
MAP runs LODMAP. a program developed in our research group
for determining the memory requirements and starting address
of core-image files. DOWNLOAD runs PIP with the papertape
punch (PTP) as the output device and the /B option to
indicate that a binary file is to be transferred.

The sequence for executing a program written in PALS

is somewhat simpler:

 

 

 

 

59
PROM
COMMAND ROUTINE OPERATION
(1) COM PR00.PA TM The PAL8 source file is assembled

into absolute binary and saved
on mass storage.

(2) DOWNLOAD PR002 TM.BL The binary file is transmitted

(3)

and loaded.

$02000 RM Execution is begun at RAM
address 200.

4. FROM Monitor Logic

A maJor portion of the design logic for the PROM
Monitor board was modelled after the POM—12 front panel
logic. The circuitry consists of the neccessary logic and
memory to provide the functions described above. The
Monitor itself resides in three 256-word PROM’s. and three
16-word RAM’s are used for scratch-pad memory.

After power-up. the Monitor routine is initiated by
first RESETting the CPU and then asserting a Control—Panel
(CP) interrupt to the processor. The RESET is done by
grounding the RESET line on the bus thruogh a momentary
action switch on the board; this causes the 6100. after
completing the current cycle. to clear the accumulator and
link. to Jam-set the program counter to 7777. and to HALT.
A CP interrupt can then be asserted by grounding the CPINT
line on the bus. The CP interrupt actually can be asserted
even if the 6100 is RUNning. and therefore is logically not
allowed if either an interrupt request or DMA request has

been granted by the CPU.

 

 

 

so

The CP interrupt forces the 6100 into the RUN state
(if HALTed). During instruction-fetch cycles. the CPU
asserts the CPSEL bus line rather than MEMSEL so that
instructions are fetched from the PROM Monitor memory rather
than mainframe memory. the low 7 address bits are then
decoded by the PROM’s. PROM space extends from 7000 to
7377. while RAM space occupies addresses 0000 to 0017. hence
if the most significant bit of the requested address is HIGH
the PROM’s are enabled and if LOW the RAM’s are enabled.

The PROM Monitor routine chains to mainframe memory
by executing the sequence ION (which does not actually
enable interrupts) followed by a JMP I 0. The program
counter is hence loaded with the contents of Monitor memory
location 0 and. provided the CPU RUN/HALT flip-flop is in
the RUN state. execution then begins at that address in
mainframe memory. This RUN/HALT flip—flop is always forced
into the HALT state after a RESET. executed Just prior to
the CP interrupt; the flip-flop is then toggled into the RUN
state by Monitor logic when the CP interrupt switch is
asserted. The CP interrupt switch triggers a 2 5 pulse from
a monostable to the RUN/HALT line on the bus. which toggles
the flip-flop into the RUN state. The monostable pulse is
used rather than the switch itself to debounce any switching

transients.

 

 

 

61
B. SFCA Interfaces
1. General Considerations

The stOpped-flow clinical analyzer is interfaced to
the microcomputer through two wire-wrapped printed circuit
boards; one is dedicated to the control functions and the
other as a general purpose analog interface for the
acquisition of analog data. The two basic functions were
separated to minimize noise contributions from the control
logic to the analog signals. Through a series of software
commands. the interfaces can cause the instrument to open
and close the sample valves. empty. fill. and push syringes
in the stopped-flow module. digitize an analog voltage from
the photocurrent amplifier. and output an Offset voltage to
the amplifier.

The interfaces latch each instruction from the data
bus on the trailing edge Of the LXMAR pulse. As shown in
Figure 9. bits 3—8 of the instruction are then decoded as
the device numbers device 50 on the control interface and 51
on the analog interface cause the proper decoder outputs to
be asserted LOW. This device number is combined with a
DEVSEL LOW to produce COM50 and COM51 at the interfaces.
The COM5O (control) and COM51 (analog) are then combined
with bits 9-11 (the assignment bits) of the instruction to

produce a specific action.

 

 

 

 

 

62

 

 

 

>Luwaoswu acetoocc ccauoacumch we Emccawc owumEmgom .c wcsuwm

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

mesa
ama...lxn+...... e
. clllllilIAlllJ C
. .unlxnv...... a aa_ea meme“
o IIIIIIIIIIOA Tl . .
. llnoATllll A z (I. :2ch
. .1:....xnu.... n. a a ... .
. (LAW EE- c .
emaililnslxn+.... < .u. .
a L r. .
e a .
a a . . .1. .
a
LA) «(53
L. n .
ac gnu. e 000 mu: 1...Amxav
a a a “Hg .z. .
Aoausuav m m w ... U
m.. a a c .a..Acxav
pmzca
«a... .n.
828 u. an.

 

 

 

 

 

63

2. Control Interface

The control interface handles the operation of the
stopped-flow and sample valves. A summary of software
commands to the control interface are given in Table 1. The
folowing sections give details of the action of each

instruction.

Table 1. Software Commands to the Control

Interface

 

Command Operation

6500 Open sample valves

6501 Close sample valves

6502 Empty stopping syringe

6503 Fill drive syringes

6504 Push drive syringes

6505 Skip the next instruction if
flow has stopped

6506 RESET

6507 Read the 6-bit switch register

a. The Open and Close Sample Valve Instructions

The actual opening and Closing of the sample valves

is done by activating a solid state relay which then

IIIIIIIIIIIIIIIIIII-llll--————______,_

64

switches AC line current to a solenoid valve controlling air
pressure to the pneumatic actuator/ spring return mechanism
on the sample valves themselves. Hence. the OPEN and CLOSE
commands need only apply TTL HIGH and LOW levels across the
control pins Of the solid state relay. As shown in Figure
10. one half of a 7474 d-K type flip-flop is employed. the 0
output being set HIGH by the 6500 command and LOW by the
6501. This 0 output is cleared (set LOW - valves closed) by
a RESET command (6506) or by the assertion of the RESET bus

line.

b. The Syringe Control Instructions

The GOA-McPherson stopped—flow module contains the
neccessary internal control logic to convert TTL-level
signals from either the front panel or from a control port
to a specific action in the module. The input signals are
named CYCLE and DELAY and a summary of the function of these

control signals is given in Table 2.

Table 2. Control Signals for the GOA-McPherson

Stopped-Flow Module.

 

CYCLE DELAY FUNCTION

l-éO any Empty stopping syringe
0-91 0 Fill drive syringes

1 1—90 Push drive syringes

 

 

65

 

scowaocwu _ccocou m>Pa> OPEEwm so sesame: owpaewzom .cp weaned

«apex

 

: zmao m>._<> (J,

 

ago
x40

 

hmmmm . pm<

 

omzoo

 

 

 

 

we

m+

om<
.omZOQ

IIIIIIIIIIIIIIIII-llll---————_______

66

J-K type flip-flops are used to generate the CYCLE
and DELAY signals required for each Of the three functions
as shown in Figure 11. The actual inputs to the stopped-
flow control logic are outputs of Open-collector NAND gates.
as the module operates on open-collector logic. A RESET
command (6506) sets both CYCLE and DELAY signals HIGH which
causes the stopped-flow module to complete any current

cycle.

c. The Skip on Flow Stopped Instruction

An optical interrupter in the stopped~flow module
generates a flow-stopped signal to the control interface.
The OI is triggered when the drive syringes reach the ends
Of their travel. and the actual trigger point is adJustable.
As shown in Figure 12. if this signal is TRUE and the 6505
instruction is executed. the interface directs the CPU to
SKIP the next instruction by asserting the proper CONTROL
and SKIP bus lines. The skip-on-flowrstopped instruction is
used by the SFCA routines to determine when to begin data

acqusition.

 

d. The Read Switch Register Instruction

A six-bit switch register located on the control
interface board can be read into bits 0-5 of the CPU

accumulator by the 6507 instruction. As shown in Figure 13.

 

6?

 

4 >5“.—

.. “in;

 

 

 

 

 

 

szoesaceu _ozoesa aeeczsm to Satanic gossameum ._F azsaea

 

 

 

 

 

 

 

 

 

85K
.6
W
n%.
jbzf. m+
ago,
V30
0 Stu.
ales... m+

 

 

 

... sumo“

 

¢m<

 

: comm omzou

 

mm<

 

 

 

 

 

 

 

 

 

 

= name amzoa

 

FT.
Nae
: Nome amzoa

 

 

 

68

71(365

 

+5JWin

 

- *r-(CO)
com5o __..., 6j05 L
A55 , ._....(ci)

Flow Stoppguéfi :}
. _E-'J DIS 'HSKP)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 12. Schematic Diagram of Skip on Flow
Stopped Circuitry

 

69

 

 

>cpmzocmu cmumwcma supwzm tame to Encamwc oeumEosom .mP mosses

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

m+
mamas
. mm.
Ammcw11a nnnu
. .111. "HHHU
. L A linu
. Illll U
» 111. nHHu
. nnnu
Acxcv11L .1
nVAHH$11.AuexV
do. , llL<<<1m+ nma
r. n1. A m... ._ acme 5:8
:81!
€81],

 

 

 

 

 

 

 

 

70

this instruction causes the interface to drive the contents
of the switch register into the accumulator. During that
part of the IOT cycle when XTC is LOW. the CPU samples the
CONTROL lines to determine what action (if any) is to be
taken. The combination of CONTROL lines shown in Figure 13
(CO LOW. C1 LOW. C2 HIGH) causes the contents Of the data
bus to be Jam transferred into the accumulator (i.e. the AC
is first cleared). The data present at the switch register
is driven onto the data bus during the last half of the IOT
cycle (XTC LOW) and the required CONTROL lines asserted by
enabling tri-state buffers.

I The SFCA routines periodically sample this switch

register to direct program flow.

3. Sequencing and Timimg

With the control interface instruction set. the
operator can easily select any type of sequencing for the
SFCA. The actual sequence of events as well as the time
lapse between each event can be customized to the type of
experiment being performed simply by changing the software.
or even by designing software which allows user
modifications during run time.

An Obvious application of this versatility is in a
case where the immobilized enzyme reaction rate is being
studied. Here the time between sample valve closing
(essentially the start of the enzymatic reaction) and

syringe drive (the end) is controlled by a variable.

 

 

 

 

71

calibrated software timing lOOp. Even the time during which
the sample valves are Open can be selected at run time.
since the actual sample volume drawn depends on this and the
vaccuum pressure applied.

When making changes in the solution such as pH. type
Of buffer. ionic strength. or other parameters. it is
desirable to rinse the reaction loop for an extended period
of time. Ionic species and large molecular weight species
may become trapped in the enzyme/nylon matrix. These can be
eluted from the loop by drawing large volumes of the new
solution through the loop.

It has been found that when filling the drive
syringes with the sample valves closed (i.e. Open to the
flow system) solution in the reaction loop is perturbed due
to the valve design in the stopped-flow module. It is
usually possible to avoid this by filling the drive syringes
during sample loop fill time. as the reaction loop is then
isolated from the rest of the flow system. It may also be
desirable to allow temperature equilibration of the reagents
in the drive syringes after filling: this can be
accomplished by extending the time between filling and

pushing the syringes to suit the particular experiment.

4. Analog Interface

The analog interface serves the function of acquiring

analog voltage data from the photocurrent amplifier.

 

 

 

Software commands recognized by this interface are

summarized in Table 3.

Table 3. Software Commands to the Analog Interface

 

Command Operation

6510 Initialize ADC conversion

6511 Skip the next instruction if
conversion done

6512 Read the ADC output into the AC

6513 Set the DAC with the 12*bit word in AC

Instructions are latched and decoded as described
earlier. with device number 51 being active. Each command

is described in detail in the following sections.

a. The Convert Command

The twelve-bit analog-to-digital converter (ADC)
begins a conversion after receiving a convert pulse Of

duration 100 ns to 2 us. Upon decoding a 6510 instruction.

 

the interface triggers a 1 us pulse from a 74121 monostable
to the A/D convert pin. Analog signals are brought to the
interface by a coaxial shielded cable. and the shielding is
connected to a large ground plane on the interface board to

minimize noise.

 

 

 

 

73

b. The Skip on Conversion Done Command

Analog-to-digital conversions require typically 25
us. after which the ADC STATUS flag is asserted LOW to
indicate the conversion is complete. As shown in Figure 14.
if this flag is LOW (done) and a 6511 instruction is
executed. the interface signals the CPU to skip the next
instruction by asserting the SKIP bus line LOW and the
CONTROL bus lines HIGH during the last half of the IOT
cycle. The bus lines are asserted by enabling the
appropriate logic levels through a tri-state buffer.

If the ADC STATUS flag is HIGH (busy) when the 6511
instruction is executed. the 74365 tri-state buffer is
disabled. and the program flow falls through to the next
instruction (usually a JMP back to continue testing the

flag).

c. The Read ADC Command

After the ADC conversion is complete. the resulting
12-bit data word is read into the accumulator by execution
Of the 6512 command. As shown in Figure 15. this command
causes the interface logic to enable three 74365 tri-state
buffers. driving the ADC output onto the data bus at the
Proper time by asserting the CONTROL lines to the
appropriate states. The combination of CONTROL line states

shown in Figure 15 (CO LOW. C1 LOW. C2 HIGH) causes the CPU

 

 

 

7L.

COMSI
ASl

ADC
STATUS
(x'rc)

 

 

 

 

Figure 14. Schematic Diagram of Skip on

Conversion Dene Circuitry

75

maven

 

 

>auwaocwu uc< came so Emacmwc awpaEmsom

Pruw muwm
uc<

 

 

 

 

 

 

 

 

 

mac mpwm

TIIII.
ill uc<
fl

 

 

 

 

 

4

 

 

 

Aaav.11,

AFUV.11

ES].

 

11s>>1.m+

 

 

 

 

 

4 N—mm

.m_ acacia

Aaexv

mm<
szcu

76

to Jam transfer the contents of the data bus (in this case

the A/D output) into the accumulator (i e. the AC is first

cleared).

d. The Set DAC Command

The digital inputs to the twelve-bit digital-to—
analog converter (DAC) on the interface are set from the
accumulator by executing the 6513 instruction. Upon
decoding this instruction. the contents of the accumulator
are latched by a pair of 74174 hex latches. the outputs of
which then set the DAC as seen in Figure 16.

The DAC is typically used to output an offset voltage
to the photocurrent amplifier; it can also be used to
display digital data on a strip chart recorder.

oscilloscope. etc

C. SFCA Software

Software for the SFCA was designed to serve several
functions. First. experimental parameters need to be
determined according to the type of experiment to be
performed: during the experiment the instrument is sequenced
and raw data acquired. the operator occasionally needs to be
notified when. for example. to change sample solutions. open

or close the PM shutter. and so on; and finally evaluation

 

77

 

__-u more
cam

m-c were
uaa

>cpmaocwu uqc now so Ecccmwc oeumEmgom

 

 

Hill}

 

 

 

 

 

LILJJI

 

e~_ee
baaxcv
Fl
.11. .
you .
.11 Aexcv
coo Amxav
111
111 m
,11.A excv

 

 

Aoexv
mas
Peace

.ep misuse

 

 

 

78

of the data involving calculation of average absorbances and
statistics. plotting and/or printing the data. and storage
on a mass storage device.

As these functions involve various degrees Of
complexity. they were divided into two groups: the more
complicated. core-consuming routines were written in

FORTRAN-IV for operation on either the POM-12 or PDP 8/E

minicomputer. while the simpler. less core-consuming
routines were written in PALS for Operation on the
microcomputer. A few short testing programs were written in

FORTRAN-lI/SABR and executed on the micro to test
individually some of the individual functions of the
interfaces.

Data communication between the mini and micro are
accomplished through a set of complementary subroutines
written for that purpose. Actual listings of most of the
programs are given in Appendix A. but a brief description Of

the software is presented here.

1. Microcomputer Software

The 6100 microcomputer used in these experiments has
at present only 4096 words of read/write memory. which must
accommodate both the software for controlling the SFCA and
data acquisition as well as the raw data itself. Attempts
were made to develop the software in FORTRAN-II/SABR. but

were unsuccessful due to the relatively large memory

 

 

 

 

 

 

 

79

requirements of the language; even seemingly short routines
quickly exceeded the available 4K. Therefore. a general
purpose set Of software modules were developed in PALS.
which allow the flexibility of executing a variety of
experiments with only minor alterations. These routines
will execute in much less than the available memory.

The 4K of memory (octal addresses 0000 through 7777)
were allocated as follows. Page zero (000 through 177) is
used to store certain experimental parameters and pointers
to the commonly called subroutines. since data stored on
page zero can be directly accessed from any other memory
location. The executive routine occupies the next several
core pages. typically locations 200 through 1777. The
executive routine handles such functions as keeping track of
which particular sample is currently being analyzed.
requesting Operator intervention if required. and sequencing
the experiment by executing CALLS to the general purpose
subroutines.

Most of the actual functions are carried out by these
subroutines. called with or without arguments. which then
RETURN to the executive. The general purpose subroutines
occupy the 7 core pages following the executive. or 1400
locations. The remainder of the 4096 locations (typically
more than 2K) are available for raw data. which consists of
averaged ADC conversions for each sample stored during the
experiment. These data can be Optionally printed on the

terminal or. at the conclusion of the experiment.

 

 

 

 

80

transmitted to the minicomputer for the more complex
calculations. Actual program flow is described below.

The working parameters for a particular experiment
are input either from the terminal or minicomputer. These
include the total number of samples and the number of runs
for each sample. the enzymatic reaction time for each. the
delay time from flow stoppage to data acqusition. the data
acquisition rate. and whether the indicator reaction is to
be monitored continuously (for reaction rate analyses) or
after equilibrium has been attained.

When the experimental parameters have been input. the
dark current and 100% transmittance are sampled after the
stopped-flow Observation cell is flushed with the reference
or blank solution. Zero and 100% T can then Optionally be
again sampled and updated after the completion of each run;
program flow is directed to the scaling routines by setting
a bit on the switch register.

Manual control Of the SFCA can be obtained by typing
an ALTMODE at the terminal any time the software is either
waiting for terminal 1/0 or in a software timing loop.
After gaining manual control the operator can cause all
hardware functions to be performed be typing key letters at

the terminal. A summary of these commands is given in Table

4.

 

 

 

 

81

Table 4. Manual Mode Commands and their Function.

 

Command Function
0 Open sample valves
C Close sample valves
E Empty stopping syringe
F Fill drive syringes
P Push drive syringes
R RESET
D Set DAC from keyboard
A Sample ADC

CNTRL-C Exit to Monitor (transparent mode)

CNTRL-R Return to calling program

When ready for each new sample. the executive routine
prints the current sample and run number on the terminal and
waits for any character to be typed before Opening the
sample valves. This feature can be disabled by setting a
hit an the switch register.

The sequencing for each sample is then as follows.
After opening the sample valves. a software timing loop is
entered for the duration of the sample fill time while
sample solution is drawn into the reaction loop. This
sampling time is preset according to the solution uptake
rate and the reaction loop volume and is determined
empirically. The stop syringe is emptied and the drive

syringes filled during sampling. The sample valves are then

 

 

 

 

closed. and the timing of the enzymatic reaction begun.

After the enzymatic reaction has proceeded for a
preset time. the drive syringes are pushed. The program
waits for the flow-stopped signal. and then delays the
preset time before beginning data acqusition. If the
indicator reaction is to be monitored after equilibrium.
this delay is to the attainment of equilibrium. after which
the ADC is sampled in rapid succession. averaged. and
stored. The number of conversions to be averaged is preset
by the operator.

When monitoring the rate of the indicator reaction.
the delay after flow is stopped is to allow settling of the
observed solution; incomplete mixing is often observed in
the GCA HcPherson module when mixing reagents of widely
different concentrations. The ADC is then rapidly sampled.
averaged. and stored as the first data point of a two—point
fixed-time reaction-rate measurement. After delaying the
preset time. the ADC is then again rapidly sampled.
averaged. and stored as the second. data point. The time
lapse between the two data points is pre-determined
according to the rate of the indicator reaction being
monitored.

The entire data set can optionally be listed on the
terminal at the end of the experiment. and finally

transmitted to the minicomputer for further data analysis.

 

 

 

83

2. Data Transfer Subroutines

A protocol had to be developed for accurately
transmitting data between the mini and micro so that the
overall software scheme outlined above could be implemented.
Simple FORTRAN READ and WRITE statements were tried first.
but proved to be unreliable due to timing difficulties;
typically the WRITE statement resulted in a very short time
lapse between character transmissions. while the
complimentary READ statememt involved a much longer time
lapse between reading each character. The result was that
characters were occasionally missed. since one or more
characters had been sent before the previous one had been
read.

The most succesful procedure developed requires that
the receiving program indicate to the sending program that
it is ready to accept a character by transmitting a special
character. The sending program then merely waits until it
receives this special character before transmitting each

character. resulting in no loss of data.

3. Minicomputer Software

Complex calculations such as the use of logarithms
and evaluations of standard deviations are more efficiently
handled by a higher level language such as FORTRAN-IV

(requiring much more memory than is available on the micro).

 

 

 

84

The FDRTRAN~IV software which operates on the minicomputer

accepts the raw data from the micro (averaged ADC
conversions for each sample along with the 100%
transmittance for each). The routines will then calculate

the average absorbance and standard deviation for each
sample and then display the information in a variety of
formats and/or store the data on floppy disk or cartridge
disk for later use. Calibration curve data can be called Up
from mass storage if calculation of an unknown concentration
is desired.

The parameters for an experiment can be optionally
set Up by the minicomputer software. For example. if the
kinetics of the immobilized enzymatic reaction are to be
studied. the reaction time for each sample is varied between
specified extremes while all other parameters remain
constant; a calibration curve experiment requires that only
the substrate concentration in each sample vary. The
minicomputer software is able to set up the desired

parameters and then transmit them to the microcomputer.

 

 

 

 

 

CHAPTER VI

EVALUATION OF THE STOPPEDrFLDW CLINICAL ANALYZER

The potential of the SFCA as a useful analytical
instrument was evaluated both by separate characterization
of several of the individual elements of the system and also
as an integral unit. This chapter describes the evaluation
of the instrumuntal aspects of the unit and its fundamental
potential in enzyme-catalyzed analysis. The two chapters
following describe the characterization of the immobilized
enzymes and an evaluation of the analytical capabilities of

the SPCA.

A. Solution Volumes

A complete purging of solution from the immobilized
enzyme reaction loop and filling with new solution requires
drawing a volume equal to approximately three times the
reaction loop volume. The volume contained in a 1.0 m
length of the nylon tubing was determined by filling the
tube several times with water and then weighing the combined
fillings. The loop volume was thus determined to be 585 ul.
Hence. during most of this study. 2-3 ml volumes of each new
solution were drawn to ensure replacement of the old
solution with the new.

Once the enzymatic reaction in the reaction loop has

85

 

 

 

 

 

86

proceeded to the desired extent. the solution is forced by
the push liquid through connecting tubing into the stopped-
flow mixing chamber and observation cell. The volume
contained in the connecting tubing is kept small
(approximately 80 ul) by using very narrow diameter tubing
(0.8 mm i.d.). The volume of the observation cell is
reported to be 63 ul (73). Hence. the push volume of each
syringe must be enough to replace completely the total
volume of 143 ul to ensure that solution in the observation
cell is completely replaced by new solution with each push.
The push volume must be not so large as to allow dilution of
the observed solution by the oncoming push liquid itself.

The reagent syringes were adJusted to deliver 300 ul
per syringe per push. The effectiveness of using this
volume was tested by filling a duplicate reaction loop (of
identical dimensions) with a dye (p-Nitrophenol) and
monitoring the transmittance of a series of alternating
distilled water and dye solutions pushed from the reaction
loop.

A determination of the expected transmittance of the
dye solution was made by successively pushing the dye from
the reaction loop into the observation cell. Alternating
then between the blank and dye solutions showed a
transmittance reproducibly alternating between sample and
reference. which verified that the 300 ul push volume was
adequate.

The volume of solution taken up by the step syringe

 

 

 

 

 

87

before flow is stopped is adJustable in the GOA-McPherson
module. This volume was set so that the drive syringe
plungers stopped JUSt before reaching the ends of the

syringes.

B. Sequencing

The best sequence of events involved for each sample
or blank to be analyzed was empirically determined by
observing each action of the sequence. It was noted that
some solution is drawn from the reagent reservoirs through
the observation cell by suction each time the stOp syringe
is emptied. Solution in the reaction loop is also disturbed
when filling the drive syringes. As a result. it was
decided that the syringes be operated while the reaction
loop is being filled; switching the sample valves to the
fill position isolates the reaction loop from the rest of
the flow system. so possible contamination of the
immobilized enzyme with harsh reagents in the drive syringes
could be avoided.

Since all of these studies were made with all
solutions at room temperature. it was not neccessary to
await temperature equilibration of reagents in the drive
syringes. The GCA-McPherson module relies on ambient air
within the enclosed module to transfer heat to or from the
glass syringes to thermostatted water circulating within

copper tubing below the syringes. If temperature

 

 

 

 

88

equilibration of reagents in the syringes is desired.
sequencing can be delayed after filling the syringes to
allow for that.

As the 2-3 ml of solution (sample or blank) is drawn
through the reaction loop by the vacuum. the stop syringe is
emptied. and the drive syringes are filled. The sample

valves are then switched to the push position. and timing of

the enzymatic reaction is begun. At the end of the
specified incubation period. the reagent syringes are
driven. The push liquid forces the reacted solution from
the 100p into the stopped-flow mixing chamber and
observation cell for analysis. After data acqusition. the

cycle is repeated.

C. Indicator Reaction

An important feature of the SFCA is that the

indicator reaction takes place in the stoppedfflow
observation cell. This allows the option of monitoring this
reaction either by reaction-rate methods or after

equilibrium has been attained. Another advantage of this
arrangement is that. since the indicator reaction takes
place outside of the immobilized enzyme reaction 100p.
drastically different reaction conditions can be used in
each of the two reactions. An indicator reaction which may
involve a strongly acidic or basic pH or other species which

could otherwise interfere with the enzymatic reaction (in

 

 

 

 

89

fact even destroy its catalytic ability) can be utilized.

In choosing whether to use a rate or equilibrium
method for the indicator reaction. some of the following
points should be considered. Equilibrium methods have the
advantage of simpler data acqusition and data analysis. One
needs only to wait for equilibrium to be established.
measure the absorbance of the chromophore. and compare it to
the reference. The simple two—point fixed-time kinetic
method requires two transmittance measurements a precise
time interval apart and a more accurate measure of the
reaction rate requires even more data and more data
analysis. Reaction-rate methods are generally more
sensitive to variations in the solution pH and temperature
than corresponding equilibrium methods. Reaction-rate
methods have the advantage of involving a relative
measurement. the rate of change of the transmittance or
absorbance with time. Problems such as light source drift
and other spectral interferences which do not change during
the measurement process do not affect a rate method. while
they can cause serious errors in an equilibrium method.

Chemical interferences can also cause errors in an
equilibrium measurement. For example. in enzymatic-based
determinations which involve the monitoring of hydrogen
peroxide produced in the reaction. sodium azide is often
added to the reaction medium to inhibit the consumption of
the peroxide by catalase (an impurity often present in

commercial enzyme preparations). The iodide-peroxide

 

 

 

9O
reaction then suffers from interference by the azide. The
triiodide formed in the indicator reaction is slowly

consumed. so the absorbance due to triiodide first quickly
increases and then gradually decreases over the next few
minutes.

By measuring the initial rate of formaton of the
triiodide. errors due to the azide interference can be
avoided. A two-point fixed-time rate method with two
absorbance measurements a short time (1-5 s) after the flow
stops should result in an accurate determination of the

hydrogen peroxide present.

D. Sources of Error

Several factors play a role in the overall accuracy
and precision of substrate determinations by the SFCA. The
substrate in both sample and standard solutions must follow
the same kinetics in the reaction 100p. The product
produced in both sample and standard must then react with
the indicator reagents in an identical fashion. Similarly.
all other elements of the detection system must behave
identically for both solutions. The accuracy and precision
in each of the transductions involved in the analysis are

evaluated in this section. in the order that they occur.

 

 

 

91
1. Sampling

The introduction of the sample solution into the
immobilized enzyme reaction loop can be complicated by
several factors. The most important of these is the
achievement of complete removal of the previous solution.
As previously noted over-extensive hydrolysis of the inner
surface of the loop in the immobilization process results in
a strong affinity between the support—enzyme matrix and the
substrate and/or product. This affinity is considered to be
ionic in nature. although the addition of strong’
electrolytes to solution in one of these extensively
hydrolyzed loops did not significantly improve the elution
of substrate/product from the 100p.

When the much milder hydrolysis conditions are used
in the immobilization process. the problem of affinity
disappears almost completely. Flushing the loop with 2-3 ml
of the buffer solution (blank) results in no detection of
product in the subsequent indicator reaction. It must be
kept in mind however that the presence of certain species in
the sample may inadvertently interact to enhance the
affinity of the matrix toward the substrate/product.
Frequent sampling of a reference or blank solution should be
carried out to monitor the complete replacement of solution
in the sampling process.

Fluctuations in the vacuum pressure used to draw
solution into the reaction loop cause variations in the

actual volume of solution taken during the fixedrtime

 

 

 

 

 

92

sampling period. Normally the time delay between opening
and closing the sample valves is set at 5 s. which was
previously shown to be adequate for complete solution
replacement. The effectiveness of a 5 s sampling period
should be periodically verified. Longer sampling times do
not improve solution replacement but only add to the total

solution volume requirements.

2. Enzymatic Reaction

Due to the relatively small amount of enzyme (i.e.
activity) present in the reaction loop. the enzymatic
reaction has been used in a fixed-time reaction-rate mode in
all of these studies. That is. the enzymatic reaction is
allowed to proceed in the reaction loop for a preset time.
identical for both the sample and all standards. If this
reaction time is kept short enough so that only a negligible
fraction of the substrate is converted to product. the
reaction will be psuedo-first order in substrate (see
Chapter VII for a more detailed description of the
immobilized enzyme kinetics).

Under these conditions it can be assumed that an
equal fraction of the substrate in both sample and standard
solutions will be converted. This assumption is critical if
the amount of substrate in the sample is to be determined
from a calibration curve. etc. The rate of the enzymatic

reaction is. however. very sensitive to such variables as

 

—7

93

solution pH. type of buffer. temperature. ionic strength.
the presence of interferences. and others.

These parameters must be carefully monitored and
controlled to be as nearly identical in both standards and
sample as possible. Typically standard solutions are
aqueous preparations of pure material. while the sample
itself is extracted from a complex matrix such as blood or
serum.

The magnitude of the error introduced by such changes
as pH. temperature. and others can range from negligible to
intolerable. The use of commercially available control

samples in a variety of matricies is very helpful in

 

 

evaluating and correcting for matrix effects.

3. Indicator Reaction

The amount of product formed in the enzymatic

 

reaction is determined spectrophotometrically either by a
reaction with a chromophore or with a reagent to produce a
chromophore. By monitoring either the rate of appearance or
disappearance of the chromophore or the total amount
produced. and by knowing the stoichiometry of the indicator
reaction. one can calculate the amount of product initially
present. The transduction from enzymatic reaction product
to chromophore is yet another step in the entire analysis
scheme. and as such its contribution to the total accuracy

and precision must be considered.

 

94

As previously described. the indicator reaction can
be monitored in either a rate or an equilibrium mode.
Errors introduced by variations in the rate of the indicator
reaction will obviously affect a reaction—rate measurement
much more than an equilibrium measurement. While
differences in the solution pH. temperature. or other
parameters can seriously alter the rate. the final position
of the equilibrium may be slightly affected or even
unchanged. It has been noted. however. that species may be
present which can shift the position of the equilibrium. In
this case reaction—rate monitoring will certainly be
superior to equilibrium monitoring.

Other elements of the detection system (i.e. those
involved in the absorbance measurement) must also be
considered in evaluating the overall performance of each
mode. These factors are described in the next section.

In this work. the hydrogen peroxide produced in the
enzymatic reaction was determined almost exclusively by the

peroxide—iodide redox reaction.

_ + MOM/I)

H202 + 31 + 2H -————————€> 2HzO + 13
The absorbance of triiodide is measured after
equilibrium is attained; experimental data were actually
acquired 30-40 5 after the flow stops to improve precision

(see Section D.4.b.L

The accuracy of determining hydrogen peroxide by this

 

95

method was evaluated by filling the reaction loop with
hydrogen peroxide solutions of known concentrations.
sequencing the stopped-flow to initiate the indicator
reaction. and measuring the equilibrium absorbance. The
absorbance was linear for concentrations of hydrogen
peroxide ranging from 4.00 UN to 100 UM. which encompasses
the range of peroxide to be determined in the enzymatic

analysis of glucose

4. Measurement of Absorbance

As previously mentioned. a variety of photometric
errors can also contribute to the overall accuracy and
precision. In this section the most important of these

error sources are discussed

a. Light Source Characteristics

Short term fluctuatons and long term drift are the
maJor sources of error from a non-ideal light source (74).
Most of this work involved monitoring the absorbance
of triiodide at 365 nm. where both tungsten and deuterium
continuum lamps are candidates as the source. The long and
Short term characteristics of each were evaluated

The spectral output of each lamp at 365 nm
(monochromator bandpass 3 nm) was recorded both by a strip

chart recorder and by sampling the signal through the ADC

 

 

 

96

and averaging in the microcomputer. As expected. both lamps
showed comparable long term characteristics. drifting on the
order of 1-2% with a frequency of approximately 0.001 Hz.
The short term characteristics proved to be significantly
less serious for the deuterium source at 365 nm. In the
frequency range 0.5 to 10 Hz. the deuterium lamp fluctuated
less than 1%. the tungsten lamp operated in the optical
feedback mode showed average deviations about the mean of 2—
32. As a result. the deuterium lamp was used almost
exclusively in this work.

Errors due to the short term fluctuations were
minimized by averaging multiple A/D conversions (typically
100) for each intensity measurement. Long term drift was
compensated for by frequently running a reference or blank.
and also by ensemble averaging multiple runs of each
solution. Low frequency fluctuations of the source are not
a serious problem in a reaction—rate measurement of the
indicator reaction. since multiple intensity measurements

are made over typically l-lO s.

b. Physical lnterferences

Variations in light throughput can be caused by
particulates or air bubbles in the observation cell.
Incomplete removal of precipitates formed during serum
Preparation causes erroneously low transmittance for the

sample. Precipitates may also be formed during the

 

 

 

 

97

indicator reaction due to impurities in the sample or
indicator reagents. Air bubbles may be inadvertently drawn
into the drive syringes during filling and then be driven
into the observation cell. which also causes decreased
transmittance and erroneous results.

Incomplete mixing of reagents in the stOpped-flow
mixing chamber can create fluctuations in transmitted
radiant power. This can be a serious problem in the SCA-
NcPherson module. especially when mixing two solutions of
drastically different composition (e.g. different pH or salt
concentrations). In this case mixing continues in the
observation cell after the flow has been stopped and has

been observed to continue for up to 30 s.

Incomplete mixing causes serious errors if the
indicator reaction is monitored by a rate method; in an
equilibrium determination. the measurement of the solution

transmittance is usually delayed for 30~40 s after the flow

stops to improve both accuracy and precision.

c. Spectral Interferences

The triiodide produced in the indicator reaction is
monitored at 365 nm. The absorbance maximum for triiodide
is actually at a somewhat shorter wavelength (349 nm);
maximum sensitivity in this particular reaction is however
obtained at 365 nm. since the Mo(VI) catalyst present has an

absorbance which steadily decreases from 349 to 365 nm.

 

 

IIIIIIIIIIIIIIIIII-llll---———_______f

98

The spectrum of triiodide in the presence of Mo(VI)
was recorded on a Cary~14 spectrophotometer. using a
solution of Mo(VI) of equal concentration as the reference.
The difference between triiodide in the presence of Mo(Vl)
and the Mo(VI) showed an absorbance maximum at 365 nm. The
absorption band was found to be quite wide. so the
monochromator slit widths of the SFCA subsequently were set
to 1500 um (3 nm bandpass).

Stray light entering the stoppedrflow module can be a
serious problem. contributing 5-10Z to the total light
passing through the module under normal room lighting. This

potential source of error was minimized by placing the

 

 

stopped-flow module before the monochromator in the light
path. In this configuration only stray light of wavelengths
within the bandpass of the monochromator will be passed on

to the detector.

d. Detector Noise

The mayor sources of imprecision arising from a

 

photomultiplier tube are phtotcurrent shot noise. dark
current noise. and Johnson noise (74). Photocurrent shot
noise. arising from the random time distribution of photons
striking the photocathode and the random intensity
distribution of electrons reaching the photoanode. is
PTOportional to the square root of the light intensity

reaching the PM tube. Dark current noise and Johnson noise

 

 

 

 

 

99

are not a function of the light intensity. Since the signal
is proportional to the light intensity. best signal-to-noise
ratio is obtained. under shot-noise limited conditions. when
the light intensity is a high as possible.

Detector noise was also minimized by sampling the
photocurrent voltage repeatedly in rapid succession and
averaging typically 100 samples for each data point. This
essentially reduces the noise equivalent bandwidth of the

measurement system for improved signal-to-noise ratio.

e. Photocurrent Amplifier

The response linearity and precision of the Keithley
current amplifier were evaluated by using a precision
current source as the input and monitoring the response with
a digital voltmeter. The accuracy and precision were found

to be better than 0.1% over a wide range of input currents

(l uA to 100 uA). Temperature variations may cause minor
fluctuations in the transfer function. However. errors in
the photocurrent amplification should not contribute

significantly to the overall accuracy.

f. Digitization

The transfer function of the ADC was evaluated' by

using a precision voltage reference source as the input

 

 

 

 

100

analog voltage and monitoring the digital output over the
complete range of +5 to -5 volts.

With the analog voltage held constant at near full
scale (+5 V). the digital output of the ADC had an standard
deviation of 0.018%. A variation of l in the least
significant bit of the ADC would cause a deviation of 0.025%
of full scale.

The ADC was then sampled over its entire dynamic
range by averaging 100 conversions at each of 11 input
voltages (measured with a digital voltmeter). A plot of the
transfer function (digital output vs. analog input) had a
slope of 409.37 (standard deviation = 0.19) and a Y~
intercept of 2049.3 (standard deviation = 1.1). The
expected slope is 409.5 and the Y-intercept 2047.

Long term drift of the ADC transfer function due to
supply voltage drift and/or temperature variations is
compensated for by frequent sampling of the reference
photocurrent.

Individual digital data point acquisitions in this
study were carried out by averaging 100 A/D conversions.
Under these conditions. imprecision due to digitization

errors should be negligible.

5. Summary of Error Sources

The relative contribution of each of these potential

sources of error to the overall accuracy and precision of

 

if

101

the method are summarized in this section.

Probably the most important source of error is
variation in the rate of the enzymatic reaction from sample
to sample due to fluctuations in temperature. pH. the
presence of activators and inhibitors. and other conditions.

Carefully prepared aqueous standards can result in excellent

precision. typically less than 1% relative standard
deviation. Serum samples. however. can contain impurities
which can seriously affect the accuracy of the

determination.

The effect of carry over from one sample to the next
can best be minimized by controlling the surface hydrolysis
during the immobilization of the enzyme. Effective purging
of each sample from the reaction loop can be easily checked
from time to time by running a blank.

The indicator reaction used in these studies (iodide-
peroxide) has been shown to be an accurate and precise
monitor of the peroxide produced in the enzymatic reaction.

Photometric errors have ,been minimized both by
frequent sampling of the dark and 100% transmittance and by

averaging many ADC conversions for each data point. Errors

 

caused by long term drift in the light source. current~to~
voltage conversion. and analog-to-digital conversion were
minimized by periodic sampling of the dark and reference
photocurrents. Higher frequncy fluctuations in these
elements were minimized by averaging typically 100 ADC

samples for each data point.

 

 

 

 

CHAPTER VII

CHARACTERIZATION OF THE IMMOBILIZED ENZYMES

Glucose oxidase (GO) and alcohol dehydrogenase (ADH)
have been immobilized on nylon tubing by the procedure
described in Chapter III. A fundamental characterization of
these immobilized enzymes is presented in this chapter. The
Michaelis-Menten model of enzyme kinetics is applied to the
system. and modifications to the model are included to take
into account diffusional effects in the reaction loop. Data
on the relative enzymatic activity as a function of pH are
presented. Finally. the long term stability of the

immobilized enzymes is considered.

A. Kinetics of the Immobilized Enzymes

1. Michaelis-Menten Model

The model of enzyme kinetics developed by Michaelis
and Menten (75). although a simplified description of an
often complex mechanism. can be applied to the kinetics
observed in these studies. The model. as described in the
following equations. assumes the reversible formation of an
intermediate enzyme-substrate complex (ES). which then
dissociates into the product (P) and the regenerated enzyme

(E).

102

 

 

103

I
E + S:;:::::ES

k'2.
ES-——-—$>E + P

The rate equation which describes the rate of appearance of

the product with time is then

RI = 1‘32 [E]G [S] -_- Ru! [3]
K- + [3] K- "’ [ST

 

 

where Km is called the Michaelis constant of the enzyme. and
is a rough measure of the dissociation constant of ES. Here
[E30 is the initial enzyme concentration. Several
assumptions are made in deriving the initial rate equation.

The substrate concentration is assumed to be greater than

the enzyme. The rate of the reaction is measured early in
the reaction. before the build-up of product can cause any
significant back reaction. Finally. the intermediate

complex is assumed to be in rapid equilibrium with S and E
or that ES is at steady state at any instant (Briggs-Haldane
assumption (76)).

The initial rate equation derived under these
conditions is illustrated in Figure 17 as a plot of the
initial rate vs the initial substrate concentration. This
curve was computer generated from the Michaelis-Menten
equation. It can be seen that the initial rate is linear
with S when the initial substrate concentration is much less
than Km. and reaches a maximum when S is much greater than

Km.

 

 

 

 

 

104

ossso opcm Acapaea vopaaseam nonsense
Am<uozzv we<memm3m eHzH

wnH.0h5wdm

S

H.
:8
:;G>

 

5 .Lb .Ilrr_lb _ FLL ptrh —
— "finalld'd—fi — _

‘1_
d—
-—

a+i
ex
_
_
_
_
_
_
.
_
_
_
_
_

 

 

.—
A-

mmwo. 0

301 X
(NIN/HVTONN) LP/JP TVIIINI

F+FFF+.""hggLuhLLLthhhhpkkbL&&bhkbbkLkkbbkhbht

Ham.o

 

 

105
a. Glucose Oxidase

In the specific case of immobilized glucose oxidase.
the reaction is
GO
Glucose + Oz-————€rGluconic Acid + H202
If the oxygen is present in excess and the glucose
concentration is much less than the Michaelis constant. then
the initial rate of appearance of H202 will be proportional
to the initial glucose concentration.

A more detailed description of this reaction has been
presented by Gibson. et al (77). Their work demonstrated
that the enzyme first converts glucose to gluconic acid and
is itself reduced in the process. In a second step. oxygen
reacts with the reduced form of the enzyme to produce
hydrogen peroxide and regenrate the oxidized form of the
enzyme. This mechanism is illustrated below.

k, k2
on + Glucose-———-€>Ered P1-——-—€>Ered + Gluconic Acid

Ered + Oz—kie on PQ—Lon + H2O2
In this study. early experiments with immobilized
glucose oxidase on the Auto Analyzer flow system (as
described in Chapter IV). the inJection of a 4 g/l glucose
sample (30 s plug) through the GO column resulted in an
extremely prolonged elution of hydrogen peroxide from the

column. typically 30 min or more. On the basis of the

 

 

106

mechanism proposed by Gibson. some observations can be made.
If the column had contained a large amount of active enzyme.
enough in fact to convert nearly all of the glucose to
gluconic acid. the first step would have then converted a
fraction of the enzyme to its reduced form. Dissolved
oxygen passing through the column would then react with the
reduced enzyme. converting it back to its oxidized form as
the oxygen itself was converted to hydrogen peroxide.
Dissolved oxygen in a saturated solution (under ambient air
pressure) is about 0.25 mM (78). The 4 g/l glucose solution
was. however. 22.2 mM. or roughly 100 times as concentrated
as the dissolved oxygen. It can then be concluded that. as
the substrate plug passed through the column. a large
fraction of the immobilized glucose oxidase was converted to
its reduced form. and was then slowly converted back to its
oxidized form as sufficient oxygen came into contact with
the enzyme. This would give rise to the observed prolonged
elution of hydrogen peroxide from the column. which would be
expected to extend long after the substrate plug had passed
through.

When air bubbles in the segmented flow stream were
replaced with pure oxygen bubbles. the observed response.
for the same 30 s plug of 4 g/l glucose. had a slightly
greater peak height (0.29 vs 0.27 with air bubbles). The
general shape of the response curve as well as the length of
time required to reach the baseline was. however. not

significantly different. A saturated solution under pure

 

 

 

107

oxygen (760 torr) is 1.275 mM in oxygen (78). which is still
only approximately 5% of the glucose concentration in this
sample; hence the same behavior is predicted.

Hornby. et a1 (35) employed immobilized glucose
oxidase in a similar flow system. They noted an approximate
two-fold increase in the maximum rate (Rmax. Figure 17) and
over a two-fold increase in Km when air bubbles were
replaced with pure oxygen. This behavior was not observed
in the glucose oxidase column employed in this study.
Hence. the column may have actually contained such a high
degree of activity that the dissolved oxygen limited the
extent of the reaction even during the short time of contact
between the glucose and the enzyme.

The dissolved oxygen under ambient air (0.25 mM) may
be rate-limiting in an enclosed reactor such as the
immobilized glucose oxidase column. In the stopped-flow
clinical analyzer. where the sample solution is incubated in
the enclosed reaction loop. the dissolved oxygen restricts
the total amount of hydrogen peroxide that can be produced.
In this environment. only the oxygen initially present in
the solution as it is drawn into the reaction loop is
available to convert the enzyme from reduced to oxidized
form.

The extent of reaction was studied as a function of
the incubation time in the reaction loop. The results are
shown in Figure 18 for a 500 ppm glucose solution at pH 6.30

and at room temperature. The initial rate of the reaction

 

 

 

108

O

w>c=o mmwcaoca mmouzpc .m— weaned

ESE m2; ZOEU/Dm
o. .m

m.

In

P
.1 b
J.

O

O

0
O

C)
C)

mm<uH~OZ

to...

C)

 

 

 

 

 

109

was determined from the slope along the linear portion of
the curve. early in the reaction. and was found to be

3.56 uM/min.

Lineweaver and Burke (79) rearranged the Michaelis-

Menten rate equation to give

l/Ri = (Km/Rmax)x(1/Si) + l/Rmax

By plotting the inverse of the initial rate of reaction as a

function of the inverse of the initial substrate
concentration. Km and Rmax for the enzyme can be determined
from the slope (Km/Rmax) and the y—intercept (l/Rmax). The

Lineweaver-Burke plot for immobilized glucose oxidase is
shown in Figure 19. From the plot. Km was determined to be
19.0 mM (standard deviation 1.0) and Rmax was 333 uM/min
(standard deviation 10). These Michaelis parameters were
also determined for glucose oxidase immobilized on another
column; Km was found to be 15.8 mM (standard deviation 1.0)
and Rmax was 44.1 uM/min (standard deviation 1.5). The
determined value of the maximum rate depends on the amount
of active enzyme present. which is known to vary widely from
column to column. The determined Km. however. is
characteristic of the enzyme and should remain constant for
the same enzyme in the same environment. Hornby. et al (35)
reported a Km value of 15.0 mM for glucose oxidase
immobilized on polystyrene tubes. and the value of 15 mM has
also been reported for the soluble enzyme.

The value of Km corresponds to that substrate

 

 

 

 

 

110

 

mmmtpxc mmoozpc LOL. pow; wazmle>w¢2owq .mp wLJUmu

_
$210.2. .0; w) m

e. do...

:21
. \EEV

_m\—

L

1 $9.0

 

 

 

 

 

 

111

concentration which gives an initial rate equal to half the
maximum initial rate. Km also gives an indication of the
range of substrate concentration over which the enzyme is
analytically useful. as demonstrated by the linearity of the
calibration curve obtained over that range. A typical upper
limit of the substrate concentration equal to one tenth Km
is usually accepted. For glucose in this system. this limit

is approximately 1.5 mM (270 ppm).

b. Alcohol Dehydrogenase

Immobilized alcohol dehydgogenase catalyzes~ the

reaction
ADH

CH 3CHon + NAD+;=> CH3CHO + NADH + H"
If the NAD is present in excess and the ethanol
concentration is much less than Km for the enzyme. the rate
of appearance of NADH in the initial stages of the reaction
will be proportional to the ethanol concentration. The
equilibrium lies far to the left (12); semicarbazide
hydrochloride is added to the solutions to consume the
acetaldehyde as it is formed. In the stopped-flow clinical
analyzer.’ the enzymatic reaction was allowed to proceed in
the reaction loop for a fixed time interval. This time

interval was kept short enough that only a small fraction

(typically 1-2%) of the ethanol was converted to product.

 

 

 

112

The absorbance at 340 nm due to NADH was then measured and
related to the ethanol concentration.

The kinetics of the ADH catalysis are illustrated in
Figure 20 as a plot of the observed percent reaction as a
function of the reaction time for a 150 ppm ethanol sample
at pH 7.7 and at room temperature. It can be seen from the
plot that the slope is linear for approximately the first 8
minutes of the reaction. during which 1-2% of the ethanol is
consumed. The small positive y-intercept is caused by NAD
absorbance which was not present in the blank solution. The
initial rate of the reaction. determined from the linear
slope. was found to be 11.2 uM/min.

Some interesting facts about this reaction were
revealed in deuterium- labelling experiments carried out by
Fischer. et al (82). They discovered that the relative
orientation of the ethanol and NAD molecules during the
catalysis is always the same. and that it involves a direct
transfer of a hydrogen atom from the ethanol to NAD. The
reaction was thus shown to be stereochemically specific.
always occurring with the same face of the NAD ring.

A Lineweaver-Burke plot for the immobilized ADH was
carried out at a pH of 7.7. and yielded a maximum rate of
18.9 uM/min (standard deviation 1.2) and a Km value of
3.21 mM (standard deviation 0.11). Hayes and Velick (83)

reported Km as 18 mM for soluble ADH at a pH of 7.9.

113

AEEE
Nu

¢>L3c wwwcccso poccxpm

mifw ZOrHU<mm
. a

.CN 925*...

 

j

 

.. m6

mm<UF*OZ

114

2. Diffusion Effects

In the stoppedrflow clinical analyzer. the transport
of reactants to the immobilized enzyme surface and the
transport of products back into the bulk solution is totally
dependent on diffusion. Although the inner diameter of the
loop is only 0.86 mm. its diameter is significantly greater
than the thickness of the immobilized enzyme layer. This
results in a diffusion layer between the bulk solution and
the surface if the rate of diffusion to the surface is less
than the rate of the enzymatic reaction. The rate of the
enzymatic reaction may in certain cases be much greater than
the diffusion rate. in which case the overall conversion
rate will be equal to the diffusion rate. As described by
the Nernst-Plank equations (84). the diffusion rate across a
diffusion boundary is proportional to the bulk solution
concentration. Hence even if the reaction is diffusion-
limited. the method will still be analytically useful since
the observed rate will be proportioanal to the analyte
concentration. ‘

Hornby. Lilly. and Crook (80) have applied the
Nernstian diffusion model to enzyme kinetics at a surface.
The model accounts for both electrostatic and diffusion
effects. The equation they derived is

an; 31
RT

= (Kn +£lr54‘LWRT-leigrud‘r) + 31

 

 

115
where
R = observed rate
51 = initial concentration of substrate

Km = Michaelis constant

x = thickness of quasi—Nernst diffusion layer
Rmax = maximum rate

D = diffusion coefficient

R = gas constant

T = absolute temperature

2 = charge on the mobile species

F = Faraday constant

grad», = electric field gradient

Note that the equation is similar in form to the Michaelis—
Menten equation. If either the substrate or the support is
neutral (z=0). as in the case of both glucose and ethanol.

the apparent Michaelis constant (Km‘) can be defined as
XRnox
Km. = Km +‘T

This equation demonstrates the effect of substrate transport
on the observed Michaelis constant. From the equation. it
can be seen that Km is enhanced with either increased Rmax
(i.e. increased activity) or increased diffusion layer
thickness (i.e. decreased diffusion rate). With Rmax
constant. an increase in Km can be interpreted as a decrease
in the initial rate of the reaction. Referring again to
Figure 17. for Km to occur at a higher substrate

concentration and Rmax to remain constant. the initial slope

116

must be decreased. It is also intuitively obvious that as
the rate of diffusion becomes slower than the rate of
enzymatic conversion. the overall rate will decrease.

From experiments performed in this study. it is not
apparent which process is actually rate determining. The
slope of the percent reaction vs substrate concentration
curves showed a bending toward the concentration axis at
high substrate concentrations. as predicted from the
Michaelis-Menten model. However. the diffusion rate also
increased as the bulk solution concentration was increased.
and may have eventually become greater than the rate of the
enzymatic reaction. When diffusion was no longer rate-
limiting. the observed rate would then be determined by the
enzyme kinetics. and the rate curve would still be predicted

to take on the observed shape.

3. Induction Period

Enzymatic reactions usually often show an induction
period between the initial contact of reagents and the
appearance of the product species. In the case of soluble
glucose oxidase. the production of hydrogen peroxide is
often not detected for approximately 30 s after mixing. The

duration of the induction period may be dependent upon

several factors. and is known to vary inversely with the

substrate concentration (85). If the induction period is

caused by an orientation process between enzyme and

 

117

substrate (1 e. a build up of ES). it would follow that at
higher concentrations the induction period should be
shorter.

This phenomenon has been observed in the kinetics of
the immobilized glucose oxidase reaction. In studies of the
hydrogen peroxide produced as a function of reaction time
(at a fixed substrate concentration). the observed curve
does not pass through the origin but has a negative y-
intercept (cf. Figure 18). The curve passes through the
time axis (zero H202 detected) at approximately 20-30 5.
which corresponds to the induction period. The magnitude of
the x-intercepts (observed induction periods) for several
different immobilized glucose oxidase columns were found to
be quite varied. however. so no conclusions could be drawn.

The fact that the induction period is inversely
related to the substrate concentration resulted in glucose
calibration curves which had a small negative y-intercept
(see Chapter VIII). For the more concentrated samples. the
production of hydrogen peroxide actually occurred over a
larger fraction of the fixed incubation period. since the
induction period was shorter for these samples. This

behavior was not observed in the immobilized ADH reaction.

B. pH Profiles

Figure 21 illustrates the pH profile of the

immobilized glucose oxidase reaction. For this study. a

 

118

AVA

ID

".3

IQ

amatexc mmoozpc so mpmwcca :6

on. mum

-
d

.Pw misuse

.god

<<EICICJC/)

alwumxvgu

 

119

0.2 M phosphate buffer was adJusted to the desired pH by the
addition of either NaOH or HCl and the final pH measured
with a pH meter. This buffer was then used to dilute a
stock solution of glucose to a final glucose concentration
of 1.20 g/l.

The pH profile shows a typically broad. flat curve
characteristic of glucose oxidase. The extent of reaction
varied by only 8% from pH 5 to 7. and decreased dramatically
above pH 7.5. Similar data have been presented by Bright
and Appleby (86) for soluble glucose oxidase. which showed a
pH maximum at 5.6.

The pH profile of the immobilized alcohol
dehydrogenase reaction is shown in Figure 22. A
pyrophosphate buffer (0.075 M) was adJusted to the desired
pH and used to dilute a stock ethanol solution to a final
ethanol concentration of 300 ppm. The solutions were
incubated in the ADH reaction loop for 2.0 min at room
temperature. after which the absorbance due to NADH was

measured. Maximum activity was observed at a pH of 9.05

C. Long Term Stability and Storage Conditions

An important theoretical advantage of immobilized
enzymes over their soluble forms is improved stability. The
instability of enzymes in solution has been a maJor reason
why enzymatic analysis has not become more prevalent in

clinical laboratories. Glucose oxidase is an exception.

 

120

mfi

0.».

wmwcwcoctsgmo Pocoupa u: mewoca In
.m,p Awe mla

“ 1.

.mm mcsmwu

l

. .Jru.o

<<[:E]:|(/)

l

' JOOO

 

 

 

 

 

being one of the most stable enzymes in solution. Certain
enzymes. however. denature so rapidly in solution that they
have little value as analytical reagents.

In order to get an accurate evaluation of the
stability of an immobilized enzyme. it should be subJected

to continuous use. under the actual conditions of the

analysis. This sort of rigorous testing was not possible in
this work; however. an estimate of the stability of these
immobilized enzymes under analysis conditions can be

inferred from the stabilities observed here. The shelf life
of these immobilized enzymes has also been evaluated.

When in use. the immobilized glucose oxidase and
alcohol dehydrogenase loops were held at room temperature.
All solutions drawn into the reaction loops were buffered at
a mild pH to prolong the life of the enzymes. When the use
of a loop was not anticipated for a few days or more. it was
filled with a buffer solution and stored under
refrigeration. However. if use was anticipated the
following day. it was not refrigerated. As a result. the
enzymes were exposed to room temperature for approximately
half of their total lifetime.

For the immobilized glucose oxidase. a small decrease
in activity was usually observed when the loop was left at
room temperature overnight. Occasionally. the activity was
observed to have remained constant or even slightly
increased the following day. probably due to a slight

increase in the room temperature. When left unused for one

week at

4%.

oxidase
During
were 31
conser

contin

glucos
soluti
store.

910cc

less
the
buff!
Unde1
COM;
day,
week
DH.

the

le
the

mar

122

peek at room'temperature. the activity had decreased by only
4%.

When used in this manner. the immobilized glucose
axidase retained significant activity for over 6 months.
)uring that time. an estimated 500 aqueous glucose samples
nere analyzed with the loop. Based on these observations. a
:onservative estimate of the life of the enzyme under
:ontinuous use would be about 3 months.

No significant loss of activity was detected in a
ilucose oxidase loop which had been filled with a buffer
solution and stored refrigerated for nearly 4 months. When
stored under these conditions. the shelf life of immobilized

glucose oxidase is estimated to be in excess of one year.

Immobilized alcohol dehydrogenase was found to be far
less stable than the glucose oxidase. In early studies of
the ADH reaction. the sample and wash solutions were
luffered at a pH of 8.7. near the observed pH optimum.
Jnder these conditions. when the enzyme was subJected to
:ontinuous use. the activity steadily decreased from day to
lay. and activity could not be detected at all after one
ueek. Either the enzyme had become slowly denatured at that
3H. or possibly the enzyme had slowly become detached from
the support.

Subsequently. ethanol analyses were carried out at a
3H of 7.7. At this pH. the activity was only about 50% of
the observed optimum. but the enzyme was found to be far

bore stable. Also. to further increase the life of the ADH.

4_____1L.

the loop
not in .
the acti
and the
one mont
l
immobil:
that pH
covalen
These b

PT‘ESERC

123

the loop was filled with a buffer solution of pH 6.5 when
not in use. When used continuously under these conditions.
the activity decreased by only about 3-4% from day to day.
and the total lifetime of each loop was increased to about
one month.

In choosing the pH and type of storage buffer for
immobilized enzymes. one should consider both the effect of
that pH on the enzyme itself and also its effect on the
covalent bonds involved in its attachment to the support.
These bonds may slowly break at certain pH values or in the

presence of certain reactive species.

PERT

evalua
clinic
tool.

clinic
withsi
infer:

Ethan:

evalu
react
react
the

and p
The

Final
is e\
and j

PIES!

dite

 

CHAPTER VIII

PERFORMANCE OF THE INSTRUMENT AS A CLINICAL ANALYZER

Immobilized enzymatic analysis with the SFCA is
evaluated in this chapter from the perspective of the
clinical chemist. In order to be an effective clinical
tool. especially in the demanding environment of a large
clinical laboratory. the instrument must be able to
withstand continuous use and still produce accurate analysis
information. Data on the analysis of both glucose and
ethanol samples are presented from this perspective.

First. the Optimum analytical parameters are
evaluated. and the clinical Chemist’s choice of either a
reaction-rate or equilibrium method for both the enzymatic
reaction and the indicator reaction is considered. Next.
the linearity and precision of the response are evaluated.
and possible interferences to the analyses are considered.
The method of serum preparation is also considered. In the
final sections. a proposed multi-reaction-loop configuration
is evaluated in terms of its potential analysis throughput.
and initial work on the enzymatic analysis of ChOlEStETOl 15

Presented.

A. Optimum Analytical Parameters

The optimum working parameters used in the

determination of both glucose and ethanol are presented in

124

this sec
as the 1

been prn

immobi
dilute
profil
Figurq
obser'
6.30.
bu fl
PhOSp

the 1

this
date
to

COM

has
69.

by;

this section. An evaluation of instrumental variables. such

as the optical parameters and the data acquisition rate. has

been presented in chapter VI.

1. Reagent Concentrations
a. Glucose Determinations

For the determination 0? glucose. the solution in the
immobilized enzyme reaction loop consisted of the sample
diluted with a phosphate buffer. As was shown in the pH
profile 0? immobilized glucose oxidase (see chapter 7.
Figure 21). the extent of the enzgmatic reaction was
observed to varg about 1-2% For a pH change of 0.1 around pH
6.30. As a result. in order to minimize imprecision caused
bg Fluctuations in the rate a? the enzymatic reaction. the
Phosphate bu$fer must control the pH at 6.30 +/— 0.01 during
the reaction.

To calculate the buFFer capacity required to achieve
this control. a worst case example is considered. In
determining glucose samples. we allow the enzymatic reaction
to proceed to approximately 1-22 completion. IF the most
concentrated glucose sample is to be 250 ppm glucose
(1.39 mM). the maximum expected hydrogen ion consumption.
based on a 5% reaction in the loop. is calculated to be
69.4 uM. (More concentrated samles were diluted with the

buffer before analgsis.) A calculation of the pH change of

 

 

a 0.1 M
and KH:
hqdr0921
Note th.
reactio
to at m
be.alte

0.1 M ;

produc

the pr

The 1

the u]

In or
Drape
react
EQUij
Pequ-
Agai
250

huar
FOur

0.0!

a 0.1 M phosphate buffer. consisting o? a mixture oF KZHPUQ
and KHZPOu at pH 6.30. upon the consumption of 69.4 uM
hydrogen ion shows the change to be only 0.001 pH units.
Note that this expected pH change is based on a 5% enzymatic
reaction. In practice the reaction was allowed to proceed
to at most 2% completion. Hence. the pH is not expected to
be altered significantly during the enzymatic reaction. in a
3.1 M phosphate buFFer oF pH 6.30.

In the indicator reaction. the hydrogen peroxide
produced in the reaction loop reacts with excess iodide in

the presence 0? the Mo(VI) catalyst to produce triiodide:
2H+ + H202 + 31‘-————e>l3' + 2H20 (1)

The triiodide Formed is also in equilibrium with iodine and

the unreacted iodide:
12 + l':;:::l3’ (Keq = 710) (2)

In order For the equilibrium absorbance oF triiodide to be
Proportional to the hydrogen peroxide produced in the
reaction loop. reaction (1) above must go to completion. and
equilibrium (2) must lie Far to the right. The latter
requirement is met by the presence 0? an excess of iodide.
Again using the worst case example of a 5% conversion 0? a
250 ppm glucose sample in the reaction loop. the maximum
hgdrogen peroxide concentration in the column effluent is

Found to be 69.4 UN. 1? the indicator reagent is made

0.05 M in iodide. the iodide concentration would essentially

 

remain 0.
peroxide.
thousand
produced

1
indicatc
from He
catalys

triiodi

0.1 M p
indicai
the No‘
condit
mixer

observ

mOdera

enigma
PhOSp
1‘tact

ammon

(Ont

remain 0.05 M after a complete reaction with the 69.4 um
peroxide. This concentration of iodide is nearly a
thousand-fold greater than the concentration of triiodide
produced.

The concentration of ammonium molybdate in the
indicator reagent was 0.102 M. as in a similar procedure
from Malmstadt (88). Increasing the concentration of this
catalyst had no effect on the equilibrium absorbance of the
triiodide.

The indicator reagent was buffered at pH 6.30 with a
0.1 M phosphate solution for several reasons. First. the
indicator reaction is somewhat pH dependent (1). Second.
the Mo(VI) can undergo side reactions under certain pH
conditions. Third. as noted previously. the GCA-McPherson
mixer is not very efficient. and schlieren effects are
observed in the observation cell when two solutions of
moderately different concentrations are mixed.

In summary. for the determination of glucose. the
enzymatic reaction was buffered at a pH 0F 6.30 With 0.1 M
Phosphate. The indicator reagent. which was mixed with the
reaction loop effluent. consisted of 0.05 M K1: 0-102 M

ammonium molybdate. and 0.1 M phosphate. pH 6.30.

b. Ethanol Determinations

In the determination of ethanol. the buffered sample.

containing ethanol and NAD. was allowed to react in the

 

reacti
the co
of NAE
as a
of NA]
340 ‘
flow

solut

cont.

Fact

tOTW
at“

The

who
cor
in

to

Fe

128

reaction loop for a preset time. during which a fraction of
the coenzyme NAD was converted to NADH. The concentration
of NADH present at the end of the incubation period was used
as a monitor of the reaction rate. Since the concentration
of NADH could be determined directly by its absorbance at
340 nm. no indicator reaction was necessary. The stopped-
flow system in this application served only to move the
solution from the reaction loop to the observation cell for
the absorbance measurement. The reagent drive syringe
contained a buffer solution. which diluted the NAD by a
factor of two in the stopped-flow.

From the stoichiometry of the ADH reaction. each
conversion of an ethanol molecule to acetaldehyde is
accompanied by the conversion of a molecule of NAD to NADH.

The rate equation for the reaction is

RmaxCS
R = (Kc + C)(Km + S)

where S is the concentration of ethanol and C is the
concentration of NAD (83). If the concentration of NAD is
in excess and the reaction time is short enough so that its
concentration remains essentially unchanged during the

reaction. the rate equation simplifies to

Rmaxs

R=Km+s

where C and Kc have been included in the constant Rmax.
Hence. under these conditions. the initial rate will be

Proportional to the initial concentration of ethanol.

 

 

reactiI
reacti
increa
incree
7.70
optim

pH.

loll:

To
pH t
5%
ton
rea
Put
hut
oh
22

MU

129

A pyrophosphate buffer was used for the the ADH
reaction. As described in chapter 7. the pH chosen for this
reaction was the result of a compromise between the
increased activity observed as the pH approaches 9. and the
increased stability observed at lower pH values. A pH of
7.70 was chosen. Although the activity was only 50% of the
optimum. the enzyme was stable for at least a month at this
pH.

Hydrogen ions are produced in the ADH reaction by the
following equation:

ADH
CH3CH20H + NAD+————->CH3CHO + NADH + H+
To evaluate the efficiency of the buffer in controlling the
pH during the enzymatic reaction. a worst case example of a
5% conversion of the most concentrated ethanol sample is
considered. For a 150 ppm (3.26 mM) ethanol sample. a 5%
reaction would produce 0.163 mM H4: In a 0.075 N
PUPOphosphate buffer at pH 7.70. the addition of 0.163 mM of
hydrogen ion will result in a shift of the pH to 7.69.
which. according to the pH profile of ADH (chapter 7. Figure
22). should increase the rate of the enzymatic reaction by

much less than 1%. Since in practice the enzymatic reaction

was allowed to proceed to only 1-2% completion. the 0.075 M

PUrophosphate buffer is quite adequate for pH control. as
long as the ethanol samples are diluted to the 0-150 ppm

range.

In summary. the sample [solution consisted of a

 

 

COM

Sifl

hui

th

130

composite reagent of ethanol (0-150 ppm; more concentrated
samples were diluted). NAD (0.50 mM). and pyrophosphate
buffer (0.075 M. pH 7.70). Semicarbazide hydrochloride
(0.075 M) was also present in the sample solution to consume

the acetaldehyde as it formed.

2. Mode of the Enzymatic Reaction

The design of the stopped-flow clinical analyzer
permits the enzymatic reaction to be monitored in either a
reaction-rate mode or an equilibrium mode. A discussion of
the theoretical advantages and disadvantages of each mode
has been presented in Chapter VI. In this section. the
alternatives are considered for applications in clinical
determinations.

In evaluating the potential of this instrument in a
clinical environment. one must consider the time involved in
each analysis. It has been shown in these studies that a
2 min incubation period in the reaction loop resulted in
approximately 2% conversion of substrate to product. for
both the glucose oxidase and alcohol dehydrogenase catalyzed
reactions. As predicted by the Michaelis—Menten model.
reaction to completion would require a prohibitive amount of
time. when the analysis time is crucial. the equilibrium
mode is no longer a viable choice for enzymes immobilized on
nglon tubing. unless an immobilization procedure which

produces much more activity is developed.

 

 

 

131

Also. in the case of the glucose oxidase reaction.
another restriction on monitoring the enzymatic reaction
after equilibrium is the fact that dissolved oxygen in the
sample solution enclosed in the reaction loop will limit the

extent of the reaction.

3. Mode of the Indicator Reaction

Since the indicator reaction in the determination of
glucose takes place in the observation cell of the stOpped~
flow module. it also can be monitored either continuously

for reaction-rate analysis. or after equilibrium has been

reached. Monitoring the reaction after equilibrium has
several advantages. especially when considering analyses
performed in a clinical lab. First. since the indicator

reaction (iodide-peroxide) is fast (half life of a few
seconds). the equilibrium method requires little or no more
analysis time than the rate method. Also. the equilibrium
method involves simpler data acquisition and calculation of
the final result. although when done on a computer the
difference is minimal. Finally. a reaction-rate method on
this short a time scale is not feasible with the GCA-
McPherson stopped-flow module as it is currently designed.
Incomplete mixing in the mixing chamber results in the
continuation of mixing in the observation cell after the

flow has stopped. For slower indicator reactions. with half

 

 

 

lives of 30 s or more. reaction-rate monitoring may be
superior. However. for the very rapid iodide-peroxide
reaction used in this scheme. the equilibrium mode is better

suited in a clinical application.

B. Linearity of Calibration Curves

1. Glucose Determinations

A calibration curve obtained for glucose using a
2.0 min incubation time is shown in Figure 23. A linear
least-squares analysis of the data yielded a slope of
1.60x10(-3) A/ppm. with a relative standard deviation of
0.24%. The y-intercept was found to be -6.39x10(-3) with a
relative standard deviation of 8.3%. The correlation
constant was .099990. The linearity is shown to extend to
250 ppm glucose. which encompasses the expected

concentration range in clinical samples.

2. Ethanol Determinations

The response linearity obtained for ethanol is

illustrated in Figure 24. The absorbance data were obtained

after the samples had been incubated for 2.0 min in the

reaction loop. A linear least squares analysis of the data

yielded a slope of 6.77x10(-4) A/ppm (relative standard

 

133

 

mCOWuwCVEccuwc mmouzpc com m>c=u :owumcpwpmo .mm wcsoww
AEQE uzou mmouauo
can 3. of on or
c c c c 1.. .
.136

(CDLD

1

Idaho

 

 

13o

mcoeumcwEprmc Pccmzum com m>c=o cowpmccwpmo

Start 0200 402.41.;
ea. or as on

Tier: m .ley. .21.

h

A

.em messed

Imod

<mm

I 2.0

 

 

135

deviation 6.64%). and a y-intercept of 0.0273 (relative
standard deviation 0.1%). The correlation coefficient was
found to be 0.9942. Excellent linearity was obtained to
about 100 ppm ethanol. above which the slope decreased
slightly. This curvature at high substrate concentrations
may be due to the fact that the substrate concentration was
approaching the apparent Km for the immobilized enzyme. and
the reaction was no longer pseudo-first order in ethanol.

These data correspond to approximately 1% conversion.

C. Precision

To evaluate the precision of glucose determinations.
a 200 ppm aqueous solution of glucose was analyzed 10 times.
The relative standard deviation of the absorbance was found
to be 2.24% (S/N = 45). For the determination of ethanol.
the relative standard deviation of 6 determinations was
2.95% (S/N = 39). The precision of these methods is limited
mainly by fluctuations in the rate of the enzymatic reaction

caused by variations in room temperature.

D. Interferences

In the determination of serum glucose with

immobilized glucose oxidase. Kunz and Stastny (37) evaluated

some interferences which may hinder the analysis when a

reaction-rate method is used. They found that oxalate and

 

 

 

136

fluoride ions. in concentrations up to 20 g/l. did not
interfere. and also that albumin did not interfere in
concentrations up to 100 g/l. Halmstadt and HadJiioannou
(88) found that oxalate. citrate. fluoride. and heparin did
not interfere with a rate method for glucose which used
soluble glucose oxidase. Similarly. willis and Hill (89)
noted that heparin. oxalate. and fluoride ions do not
interfere with the ADH reaction. Thus. these substances are

not expected to interfere in these determinations.

E. Serum Preparation

Since the protein substituent of serum samples
absorbs at the monitoring wavelength and since other
substituents in serum may interfere in the enzymatic

'reaction. it is necessary to deproteinize the serum samples
before analysis.

First attempts at deproteinization involved the
precipitation of the protein with a 10% trichloroacetic acid
solution. followed by adJustment of the solution pH by the
dropwise addition of concentrated NaOH and dilution with the
buffer. It was found that the pH of the solution could not

be accurately controlled or measured. since the Volume was

so small. As a result the final pH was imprecise. as
demonstrated by large fluctuations in the observed
absorbances.

The method of coprecipitation of the enzyme proved to

 

 

 

 

 

137

be most effective. In this method. 0.5 ml of serum was
pipetted into a centrifuge tube. and chemically equivalent
amounts of barium hydroxide and zinc sulfate (approximately
0.125 M each) were added. The required volume ratio of
these two reagents was determined by titrating the Ba(0H)2
with the ZnSOu to pH 7.0. The protein precipitated as the
insoluble BaSOu and Zn(0H)2 formed. After centrifugation.
the supernatant was a clear solution. and was assumed to be
free of the ions added. One ml of the supernatant was then
pipetted into 3.0 ml of the phosphate buffer. mixed. and
taken for analysis.

This method is attractive in that essentially all of
the Ba(DH)2 and ZnSDu_ are removed from solution via
precipitation. which results in deproteinization without the
addition of possible interferences and without any change in
pH. Multiple analyses of serum samples prepared by this
method showed excellent accuracy and precision. as shown in

Table 2 of Appendix B.

F. Proposed Multi-Reaction-Loop SFCA

From a clinical standpoint. a maJor disadvantage of
the SFCA as it is currently designed is that a great deal of
the analysis time is spent waiting for the enzymatic
reaction to proceed to the desired degree. It has been
shown that excellent sensitivity can be obtained with these

immobilized enzymes by allowing the sample to react for as

 

 

 

138

short a time as 2 min. In certain applications. the
resulting throughput rate of 20—30 samples/hr is adequate.
However. the throughput rate can be vastly improved if the
stopped flow mixing system could be utilized to monitor one
completed reaction while others were in progress.

The proposed multi-reaction-loop SFCA would optimize
the use of the detection system by multiplexing several
reaction loops into the stopped-flow mixing system. Two
different configurations are possible. In one. the reaction
loOps would all contain the same immobilized enzyme. and
would be used in the determination of one particular
substrate. In another configuration. each loop would
contain a different enzyme. and would be used to analyze
rapidly for several different substituents of each serum
sample. In either configuration. the initiation of the
enzymatic reaction in each loop would be staggered. so that
the detection system (stopped-flow spectrophotometer) would
be available as each reaction was terminated.

The operation of the multi-lOOp system is described
here for a system which contains six reaction loops. For
the example. it is assumed that each enzymatic reaction is
to be allowed to proceed for 2 min. and that the
spectrophotometric detction of the reaction product requires
10 s. In this case. the reaction loops would be filled with
sample at intervals of 20 s. If the first enzymatic
reaction is started at t=0 s. the second would be started at

t=20 s. and so on. At t=120 s. the reaction in the first

 

 

 

 

 

 

139

loop would be terminated by driving its contents into the
stepped-flow observation cell. Then. at t=130 s. the first
100p would again be filled with the next sample. At
t=140 s. the reaction product in the second loop would be
measured. and so on. This alternating sequence of push and
fill would then continue at 10 5 intervals. With this
sequencing. samples could be analyzed at the rate of 180 per
hr.

The proposed design for this six-loop SFCA is shown
in Figure 25. The configuration is essentially the same as
the single loop system with the exception of a pair of 6-to-
1 valves located on each side on the reaction loops. The
automatic switching of these 6-to-l valves can be initiated
by TTL-compatible signals generated by the microcomputer
interface.

The sequencing of the 3-way sample valves and the
stopped-flow module is exactly as previuosly described. The
only addition to the entire sequence involves switching the
6-to-1 valves to the proper position before either filling a
loop with sample or driving the reacted solution into the

stopped-flow module to initiate the indicator reaction.

9. Initial Study of Cholesterol Determinations

The determination of serum cholesterol has become
increasingly important because of the implied relationship

between high cholesterol levels and cardiovascular disease

 

 

 

 

 

 

 

 

140

 

 

 

 

 

 

 

      

    

 

 

.25? A D .r

I

A?
m PE,
28.. 8:085 m m m H. m 783 NWO
9.52... 5552587 U H H H H

pt»
1‘

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

141

(90.91). and also because abnormal cholesterol levels are of
diagnostic value in several other diseases (92.93). Non-
enzymatic determinations of serum cholesterol are in general
use in clinical laboratories. but suffer from poor
specificity. difficulty in standardization. unstable or
corrosive reagents. and many interferences (94-97).
Recently. the specificity of certain enzymes in the

determination of cholesterol has been studied (98-100).
In the enzymatic determination of total serum

cholesterol. the following sequence of reactions is used:

cholesterol

 

 

cholesterol ester + H20 4~$>cholesterol + fatty acid
esterase
cholesterol
cholesterol + 02 ffaecholest-4-en-3-one + H 02
oxidase 2

The total cholesterol (cholesterol and cholesterol esters)
has been indicated by the comsumption of oxygen (101). or by
the reaction of the peroxide to produce a chromophore
(100.102) or fluorophore (103). -

In this study. the peroxide was monitored in the
stopped-flow apparatus by allowing it to react with iodide
in the presence of a Mo(VI) catalyst. Cholesterol oxidase
(Miles Labs. Elkhart. IN) was immobilized on a nylon tube.
by the procedure described in chapter III. However. the
immobilized enzyme proved to be either highly unstable or

had become detached. since activity lasted for only a few

 

 

 

142

days. Perhaps the immobilization at a different pH. or in
the presence of cholesterol may improve the stability.

Studies of the peroxide-iodide indicator reaction in

the cholesterol determination scheme demonstrated the
versatility of the SFCA design. In reported procedures for
the enzymatic determination of serum cholesterol. the

reagents included sodium azide (approximately 1 g/l) to
inhibit the consumption of peroxide by the enzyme catalase.
which is an impurity in cholesterol oxidase preparations.
Unfortunately. N is a serious interferent in the peroxide-
iodide reaction. When N was present. the absorbance of
triiodide in the indicator reaction was observed to increase
rapidly over the first 5-10 5 of the reaction. and then to
decrease slowly over the next 5 min to approximately half
the absorbance maximum.

The peroxide-iodide reaction was studied with and
without the presence of azide. with the use of a stopped—
flow spectrophotometer developed by Beckwith (104). and
later modified by Notz (105). Holler (71). and Call (106).
This system was used because its mixing system is superior
to that of the GCA-McPherson stopped-flow. and because fast
data acquisition and analysis are computer controlled.
Buffered solutions of hydrogen peroxide of known
concentrations. with and without 0.015 M sodium azide. were
placed in one reagent syringe. and a buffered solution of
0.5 M KI and 0.102 M Mo(VI) was placed in the other syringe.

The buffer in both cases was 0.1 M phosphate. adJusted to a

 

 

143

pH of 7.50. After driving the reagents through the mixer
into the observation cell. the reaction was monitored at
365 nm for 5 min. The data were then displayed. and initial
slopes were calculated.

Plots of the observed initial slope (dA/dT over the
first few seconds) with and without azide present vs the
concentration of hydrogen peroxide are shown in Figure 26.
The slope of the curve is only slightly decreased when azide
is present. which demonstrates that the potential
interference due to azide can be eliminated by the use of a
reaction-rate method. In both cases. the linearity extends
to 25 UN peroxide. which encompasses the range of peroxide
concentrations to be determined in the SFCA. The peak
height of the absorbance vs time curve could also be used to
generate a linear calibration curve. Thus. by being able to
observe the time dependence of the absorbance in the
indicator reaction. the interference from azide can be
eliminated. The microcomputer can either calculate the
initial slope of the observed absorbance vs time curve. or
simply on-line determine the peak absorbance. which occurred
12-15 5 after the flow stopped

This initial study demostrates the feasibility of

serum cholesterol determinations with the stopped-flow
clinical analyzer. In order to develop the procedure
completely. the immobilization of cholesterol oxidase and

cholesterol esterase would have to be studied further. in

order to improve stability.

 

 

 

 

corpocwz mthcgwcuwtmtcH Low ”Ii/E $.52: co 2»ch me Home:

SEQ: 0200 mom:
m.

o—
“

ON
L .
‘ I

dim

1M4

   

-umeEgo muc—med u mccpmv 2:2 5.5.1 1| .Il

Ssumescc mscfixmnd u 253 wENc ozllllnll

.mm mchwu

I mod

:0me

.EU\<_U

1.2.0

 

 

CHAPTER IX

SUMMARY AND FUTURE PROSPECTS

The maJor goal of this work was to demonstrate the
potential of a stopped-flow analyzer in clinical analyses.
and to this end the prOJect was entirely successful. Rapid.
accurate determinations of both glucose and ethanol were
carried out on the SFCA with as little as 100 ul of serum.
The technique uses microliter quantities of inexpensive
reagents for each analysis. while the enzyme itself can be
used in hundreds or thousands of determinations. Thus the
main clinical obJectives of speed. accuracy. and low cost
have been realized.

Another important aspect of this research has been
the application of computers to analytical chemistry. The
6100 microcomputer has great potential for completely
automating the analysis scheme. The ability of the micro to
share the Facilities of a powerful minicomputer has been
made possible by the development of a hierarchical network.

The development of the enzyme immobilization
procedure was geared toward the discovery of a practical
procedure. During this development and subsequent
characterizations of the immobilized enzymes. some important
aspects of the procedure were discovered. such as the effect
of the hydrolysis conditions and the pH during the
glutaraldehyde attachment. Certainly. if the entire

immobilization sequence were better characterized.

145

 

 

 

 

146

conditions for each step could be intelligently chosen for
each different enzyme.

The initial hydrolysis of nylon may be accomplished
under very mild conditions. with comparable resultant
activity. In these studies. nylon tubes were reused as
supports after an enzyme had become inactive. The
immobilization was started with the hydrolysis step. This
hydrolysis presumably detached the previous enzyme and
cross-link from the nylon wall. but did not significantly
further hydrolyze the surface. since comparable activity
could be attained. These tubes could possibly be reused
indefinitely.

The presence of substrate or product. or a
competitive inhibitor. during enzyme attachment may result
in enhanced activity. Also. the time required to complete
the entire procedure might be shortened tremendously by
reacting the enzyme in the final step for a much shorter
time. This reaction may actually be complete in an hour or
less. It might also be instructive to determine the
efficiency of this step. both in terms of the fraction of
soluble enzyme actually attached. and the fraction of
attached enzyme which is actually active. A similar study
was carried out in our lab (107) for the immobilization of
glucose oxidase on controlled-pore glass. where it was found
that 44% of the enzyme in solution had been attached.

Since the rate of conversion in the reaction loop

could be controlled by diffusion of substrate to the active

 

 

 

 

147

surface. a means of agitating the solution during this
reaction might result in improved sensitivity

The multi-reaction-loop system proposed in chapter
VIII is expected eventually to burden the operator. since
samples must be delivered to the loops frequently. An
automated sample delivery system. such as a rotating turret.
could be developed. and also placed under the control of the
microcomputer. The potential throughput rate of the multi—
loop system is also limited by the existing 4K of memory in
the microcomputer. By expanding the memory. software for
the micro could be written in FORTRAN-II. and be designed to
carry out more. if not all, of the calculations.
Calibration curve data for each loop could be stored in
memory and recalled to calculate final concentration data.
which then could be printed immediately on the console or
lineprinter.

Another valuable addition to the 6100 microcomputer
would be a real time clock. With the use of interrupts to
signal a clock overflow condition. the CPU would be free to
perform calculations or input/output operations during the
execution of a timed operation.

The precision of the enzymatic reaction could be
improved by thermostatting the reaction loop. For the
glucose oxidase—catalyzed reaction. thermostatting the
reaction at a temperature above room temperature (e g. 37°C)
would also result in enhanced activity (87). If the

indicator reaction is monitored after equilibrium. the

 

 

 

148

temperature in the stOpped—flow module is not critical.

The use of different indicator reactions warrants
investigation. Deproteinization of serum samples could
possibly be eliminated if the monitoring wavelength were
such that serum substituents did not absorb. Fluorescence
monitoring of the indicator reaction could also be
implemented. Bostick and Hercules (108) have employed the
chemiluminescence of luminol to detect hydrogen peroxide
production in the glucose oxidase reaction.

Rapid characterization of a patient’s blood chemistry
could be carried out in a hospital emergency room or in a
physician’s office if the entire instrument could be
miniaturized. The stopped-flow mixing system and detection
system would be designed specifically for this application.
The size could be drastically reduced. since a simple
tungsten lamp. filters. and a PM tube would suffice. The
dead time of the stopped-flow system would not be critical.
so the syringe drive pressures and flow rates could be
diminished to decrease size. The software for complete
instrument sequencing. calibration. and calculation of the
final results could be stored permanently in memory. and the
microcomputer itself could also be miniaturized. If the
instrument were made portable. it could be used in

neighborhood mass screening programs.

 

 

 

 

 

 

REFERENCES

 

 

12.

13.

14.

References

L. D. Bowers and P. N. Carr. Anal. Chem.. fig. 544A
(1976).

H. H. Weetall. Anal. Chem.. 39. 602A (1974L

0. Zaborsky. "Immobilized Enzymes“. CRC Press.
Cleveland. 1973.

G. J. H. Melrose. Rev. Pure Appl. Chem.. 21. 83 (1971).

D. N. Gray. M. H. Keyes. and B. Watson. Anal. Chem.. 52.
1067A (1977).

K. J. Skinner. Chem. and Eng. News. Aug. 18. 1975.

G. R. Stark. “Biochemical Aspects of Reactions on
Solid Supports". Academic Press. New York. 197L

H. H. Weetall. Separ. Purification Methods. g. 199 (1973).

A. D. McLaren and L. Packer. Advan. Enzymol. Relat
Areas Mol. Biol.. gg. 245 (197OL

J. N. Nelson and E. G. Griffin. J. Amer. Chem. Soc.. 35.
1109 (1916).

H. L. Green and G. Crutchfield. Biochem. J.. 115.
183 (1969).

I. Langmuir and V. J. Schaefer. J. Amer. Chem. Soc.. 99.
1351 (1938).

M. O. Dayhoff. "Atlas of Protein Sequence and
Structure". National Biomedical Research Foundation.
Maryland. 1969.

A. Bar-Eli and E. Katchalski. J. Biol. Chem...235.
1690 (1963).

A. Bar-Eli and E. katchalski. Nature. JflB. 856 (1960L

H. I. Silman. D. Hellner. and E. Katchalski. Isr.
J. Chem.. 1. 65 (1963L

H. Ozawa. J. Biochem.. fig. 419 (1967).

S. A. Bernhard and G. L. Rossi. in "Structural
Chemistry and Molecular Biology". (A. Rich and

N. Davidson. eds.). Freeman Press. San Francisco and
London. 1968.

1h9

 

 

20.

2L

22.

30.

BL

32.

33.

34.

35.

36.
37.

38.

39.

150
E. K. Bauman. L. H. Goodson. G. G. Guibault. and D. N.
Kramer. Anal. Chem.. 31. 1378 (1965)

G. P. Hicks and S. J. Updike. Anal. Chem.. 38.
726 (1966).

T. Wieland. H. Determan. and K. Buenning. Z. Naturforsch.
21. 1003 (1966)

A. D. McLaren and G. H. Peterson. Soil Sci. Amer. Proc..
22. 239 (1958).

P. Bernfeld and J. Wan. Science. 142. 678 (1963).
C. Wilson. Anal. Biochem.. 31. 506 (1969).
M. A. Mitz and L. J. Summaria. Nature. 189. 576 (1961L

N. Grubhofer and L. Schleith. Naturwissenschaften.
32. 508 (1953L

A. Habeeb. Arch. Biochim. Biophys.. 119. 264 (1967L

H. Weetall and N. Weliky. Jet Propulsion Laboratory
Programs Summary No. 37-26. 3. 160 (1965).

H. H. Weetall. Nature. 223. 1969.

H. H. Weetall and L. S. Hersh. Biochim. Biophys. Acta.
185. 464 (1969).

H. H. Weetall. Science. 166. 615 (1969).

H. H. Weetall and L. S. Hersh. Biochim. Biophys. Acta.
206. 54 (1970).

L. S. Hersh. H. H. Weetall. and I. W. Brown. J. Biomed
Mater. Res.. 3. 471 (1969L

T. L. Newirth. M. A. Diegelman. E. K. Pye. and R. G.
Kallen. Biotech. and Bioeng..‘;§. 1089 (1973).

W. E. Hornby. H. Filippusson. and A. McDonald. FEBS
Latt.l 2: 8 (1970).

L. Goldstein. Biochim. Biophys. Acta. 315. 1 (1973L
H. J. Kunz and M. Stastny. Clin. Chem...gg. 1018 (1974).

P. S. Bunting and K. J. Laidler. Biotech. Bioeng.._;6.
119 (1974).

H. Filippusson. W. E. Hornby. and A. McDonald. FEBS
Lett.. 22, 291 (1972).

 

 

40.

4L

42.

43.

44

45

46.

47.

4B.

49.

50.

5L

52.

53.

54.

55

56.

57.

58.

151

W. E. Hornby. D. J. Inman. and A. McDonald. FEBS
Lett.. 23. 114 (1972).

P. V. Sundram and W. E. Hornby. FEBS Lett.. 10. 325
(1970). ‘—

W. E. Hornby and H. Filippusson. Biochim. Biophys
Acta. 220. 343 (1970).

S. J. Updike and G. P. Hicks. Nature. 214. 986 (1967).

H. Huang. S. S. Kuan. and G. G. Guibault. Clin. Chem..
gg. 671 (1977).

G. G. Guibault and J. Montalvo. J. Amer. Chem. Soc..
jg. 2164 (1969).

G. G. Guibault and J. Montalvo. Anal. Lett.. 2. 283
(1969). T

G. G. Guibault and J. Montalvo. J. Amer. Chem. Soc..
23. 2533 (1969).

D. Williams. A. Doig. and A. Korosi. Anal. Chem..
42. 118 (1970).

T. Anfalt. A. Granelli. and D. Jagner. Anal. Lett..
2. 713 (1969).

D. S. Papasthathopoulas and G. A. Rechnitz. Anal.
Chim. Acta. 22. 17 (1975).

B. Watson and M. H. Keyes. Anal. Lett.. 9. 713 (1969).

G. Johansson and L. Ogren. Anal. Chim. Acta. 84. 23
(1976). ‘—

W. J. Blaedel. T. R. Kissel. and R. C. Boguslaski.
Anal. Chem.. 41. 2030 (1972L

S. W. May and N. N. Li. Biochem. and Biophys. Res
Com.. 41. 1179 (1972).

R. S. Schifreen. D. A. Hanna. L. D. Bowers. and P. W.
Carr. Anal. Chem.. 42. 1929 (1977L

L. D. Bowers and P. W. Carr. Anal. Chem.. 48. 544A
(1976). “7

L. D. Bowers. L. M. Canning. C. N. Sayers. and P. W.
Carr. Clin. Chem...§g. 1314 (1976).

B. Mattiasson. B. Danielsson. and K. Mosbach. Anal.
Lett..-3. 217 (1976).

 

 

59

60.

6L

62.

63.

64.

65

66.

67.

68.

69.

70.

71

72.

73.

74.

75

76.

77.

 

152

L. D. Bowers and P. W. Carr. Clin. Chem.. 22. 1427
(1976).

J. A. Burns. Cereal Foods World. 21. 594 (1976).

D. J. Hanson and N. S. Bretz. Clin. Chem.. 22. 477
(1977).

L. P. Leon. et .31., Clin. Chem.. g. 1017 (1975).

K. Mosbach and B. Danielsson. Biochim. Biophys. Acta.
364. 140 (1974).

L. P. Leon. M. Sansur. L. D. Snyder. and C. Horvath.
Clin. Chem.. 23, 1556 (1977).

K. S. Chua and I. K. Tan. Clin. Chem.. 21. 150 (197BL

D. J. Inman and W. E. Hornby. Biochem. J.. 129. 255
(1972).

F. M. Richards and J. R. Knowles. J. Mol. Biol.. 32.
231 (1968).

P. M. Hardy. A. C. Nicholls. and H. N. Rydon. J. Chem.
Soc. D.. 12, 565 (1969L

Skeggs. Am. J. Clin. Pathol.. gg. 311 (1957)

F. J. Holler. S. R. Crouch. and C. G. Enke. Anal.
Chem.. 38. 1429 (1976).

F. J. Holler. Ph.D. Thesis. Michigan State University.
East Lansing. MI. 1977.

R. E. Dessey. Anal. Chem.. 49. 1100A (1977L

GCA McPherson Stopped-Flow Module Preliminary Operators
Manual. GCA McPherson Co.

J. D. Ingle. Ph.D. Thesis. Michigan State University.
East Lansing. MI. 1971.

L. Michaelis and M. L. Menten. Biochem. 2.. 52.
333 (1913).

G. E. Briggs and J. B. S. Haldane. Biochem. J.. 33.
338 (1925).

G. H. Gibson. B. E. P. Swoboda. and V. Massey. J. Biol
Chem.. 239. 3927 (1964).

J. Livingston. R. Morgan. and A. H. Richardson.
J. Phys. Chem.. 23. 2356 (1961L

 

 

 

153

79. H. Lineweaver and D. Burke. J. Amer. Chem. Soc.. 56.
658 (1934). '—

80. W. E. Hornby. M. D. Lilly. and E. M. Crook. Biochem.
J.. 107. 669 (1968).

 

81. K. Kusai. Ann. Rept. Sci. Works. Fac. Sci. Osaka
Univ.. 8. 43 (1960).

82. W. H. Fischer. A. Roedig. and K. Rauch. Liebigs Ann..
552. 203 (1942).

83. Hayes and Velick. J. Biol. Chem.. 207. 225 (1954).

B4. W. J. Moore. "Physical Chemistry". 4th Ed..
Prentice-Hall. Englewood Cliffs. NJ. 1972.

85. H. V. Malmstadt and 8. R. Crouch. J. Chem. Educ..
fig. 340 (1966).

86. H. J. Bright and M. Appleby. J. Biol. Chem.. 244.
3625 (1969).

87. R. H. Smith and J. Kilford. Int. J. Chem. Kinetics.
8. 1 (1976).

88. H. V. Malmstadt and S. I. HadJiioannou. Anal. Chem..
34. 452 (1962).

89. F. u. Willis and J. B. Hill. Clin. Chem . 12. 91
(1969).

90. G. Pincherle. J. Chron. Dis.. 23. 289 (1971).
91. D. Zilversmit. Am. J. Cardiol.. 35. 449 (1975).

92. R. C. Neerhout. Clin. Chem. Acta. 4;. 347 (1972).

93. C. Hirayama and T. Isira. Clin. Chem. Acta. 21.
21 (1976).

94. L. L. Abell and B. B. Levy. Stand. Methods Clin.
Chem.. 2. 26 (1958).

95. D. B. Tonks. Clin. Biochem.. 1. 12 (1967).

96. W. D. Brown. Aust. J. Exp. Biol. Med. Sci.. 22:
209 (1961).

97. B. Zak. Clin. Chem.. E2, 1201 (1977).
98. W. Richmond. Clin. Chem.. 123 1350 (1973).

99. H. Flegg. Ann. Clin. Biochem.. 12. 79 (1973).

 

100.

101.

102.

103.

104.

105.

106.
107.

108.

154
C. C. Allain. L. S. Poon. and C. S. G. Chan. Clin.

Chem.. 29. 470 (1974).

A. Noma and K. Nakayama. Clin. Chem...22. 336 (1976).

M. A. Pearse and S. H. Bodourian. Clin. Chem . 23
280 (1977).

H. Huang. J. W. Kuan. and G. G. Guibault. Clin. Chem..
21. 1605 (1975).

P. M. Beckwith. Ph.D. Thesis. Michigan State University.
East Lansing. MI. 1972.

P. K. Notz. Ph.D. Thesis. Michigan State University.
East Lansing. MI. 1976.

R. S.-Gall. Current Research (1978).

P. Roth. unpublished results.
D. Bostick and D. Hercules. Anal. 01'1”!” 32. Mi? 11975)-

 

APPENDIX A

Selected Program Listings

155‘

 

 

/+2

156

PARAMETER DEFINITIONS FOB. SFCA PALS-V1011 12-JUL-78 PAGE 2

/POIN'I'ERS T0 FREQUENTLY CALLED SUBROUTINES

*20
DEC I N . XDEC IN
TXOUT‘, XTXOUT
TYPE , XTYPE
RE . XREAD
NOUI‘. XNO‘U'I‘

SAMPLE , XSAI‘EPL
C

AVCLfi, XAVCLR
AVADD, XAVADD

ODATA, XODXTA
LST, XLST

PARAMETER DEFINITIONS FOR SFCA PALS-VIOA 12-JUL—78 PAGE 1

/+2 PARAMETER DEFINITIONS FOR SFCA
/

/STOPPED-FLOW CLINICAL ANALYZER
/SUBROUT I NE ONE

/SFCA. S1 CONTAINS PARAMETER DEFINITIONS
AND PAGE ZERO P0 I NTERS

/SFCA.S2 CONTAINS FREQUENTLY CALLED SUBROUTINES
/ WHICH LOAD BEGINNING AT THE PAGE FOLLOWING THE
/ EXECUTIVE ROUTINE. AND OCCUPY 7 PAGES.

/
/DATA STORAGE BEGINS AT THE PAGE FOLLOWING SFCA.SZ
/ AND EXTEND T0 LOCATION 7777.

/

/PGR= M. D. JOSEPH
/ DATE : 27-MY— 7.8
/VER= 1B

/FILE: SFCA.SI

/

/

 

 

157
/ EQUILIBRIUM MODE SFCA PALS—V10A lZ-JUL-78 PAGE 3
/ EQUILIBRIUM MODE SFCA
/
/PGR: M.D. JOSEPH
/DATE: 29-MAY-73
/VER= 1C
/FILE: SFCAEQ.EX
/
/PAGE ZERO DATA
0150 *150
00150 0000 TSANP, 0 /TOTAL NO. SAMPLES
00151 0000 CSAMP, 0 /-(CURBENT SAMPLE NO.)
00152 0000 TRUNS. 0 /TOTAL RUNS EA SAMPLE
00153 0000 CRUN, 0 l-(CURRENT)
00154 036 RTIME, 0 /POINTER T0 REACTION TIMES
00155 0000 EQTIME, 0 /DELAY TO EQUILIBRIUH
00156 0300 DA A, 0 /P01NTER T0 DATA TABLE
00157 0000 MDARK, 0 f-(DARK)
00160 0300 REF. 0 /REF-DAZK
00161 0000 IFLAG, 0 /=0 FOR TTY SETUP
00162 3000 DATAB. DATAT /START OF DATA TABLE
0170 $170
00170 0000 ZBLOCK l0 /REACTION TIMES
DECIMAL
1750 1SAMP=1000 /NO. AVERAGES/DATA POINT
0005 ISANPT=5 /SAMPLE VALVE OPEN TIME (SEC)
0002 IFILLTEZ /SYRINGE EQUILIBRIUL TIME

OCTAL

 

 

/ EQUILIBRIUM.MODE SFCA

00200
00201
00202
00203
00204
00205
00206
00207
00210
00211
00212
00213
00214
00215
00216
00217
00220
00221
00222
00223
00224
00 25
00226
00227
00230
00231
00232
00235
00234
00235
00236
00237
00240
00241
00242
00243

‘ 00244

00245
00246
00247
00250
00251
00252
00253
00254
00255
00256
00257
00260
00261
00262
00263

0200

7300
4434
4434
4421
0305
0321
0325
0311
0314
0311
0302
0322
0311
0325
0315
0240
0315
0317
0304
0305
0240
0323
0306
0303
0301
0240
0326
0264
0301
0000
4434
4434
4421
0323
0305
0324
0240
0325
0320
0240
0306
0322
0317
0315
0240
0324
0324
0331
0277
0240
0000
4423

STARIZ

'III
158
PALS-VIGA 12-JUL-78 PAGE 4
$200
CLA CLL KRESTART ENTRY
CR;CR

‘N'BI'I‘E; “E; "Q; “U;CII; "L; "I; "B; UR; "13“U; "M; II gun; "O; I'D; "E

' :“S;'F;"C;“A;” :‘V;"4;”A;0

cn.ca
WRITE' as; "E; "T; a 3 BU; up; u ‘an IR; "03 um; a ;"T3 "T; "Y; 11?; u :0

 

 

CALL READ

 

159

/ EQUILIBRIUM MODE SFCA PALS—VIOA 12-JUL-78 PAGE 4-1
00264 1377 TAD (-"N

00265 7650 SNA CLA

00266 7001 IAC

00267 3161 DCA IFLAG /=0 FOR.TTY SETUP

00270 1161 TAD IFLAG

00271 764 SZA CLA

00272 4441 CALL MONITR /SET UP FROM MINI

00273 4434 CR

00274 4421 WEI ‘3“N;“O;".;" :“S;“A;"M;"P;"L;"E;“S;" ;"(;"l;“-;"8;");0
00275 0316

00276 0317
00277 0256
00300 0240
00301 032

00317 1161 TAD IFLAG

0032. 7640 SZA CLA; JMP A1

00321 5327

00322 4420 CALL DECIN; 0 /GET NUMBER OF SAMPLES
00323 0300

00324 1323 TAD .-1

00325 3150 DCA TEAM?

00326 5331 Jl‘fl’ Ad’l-I'SAII‘IP

00327 1376 A1 TAD ( .

00330 44.45 ’ CALL IDATA /1NPUT FROM HINI "

00331 44.21 A2, WRITE; IIN;NO;II.;I| :"R;"U;"N;"S;" ;IIE; A30

00332 0316

00333 0317

00334 0256

00335 0240

00336 0322

00337 0325

00340 0316

00341 0323

00342 0240

00343 0305

00344 0301

00345 0000

00346 1161 TAD IFLAG

00347 7640 SZA CLA; JMP A3

0035 5 56 .
0035? 4320 CALL DECIN; 0 /NUMBER RUNS EACH SAMPLE

00352 0000

_ — — 2
/ EQUILIBRIUM MODE SFCA PALS VIOA 12 JUL 78 PAGE 4"

00353 1352 TAD .~1

00354 3152 DCA TRUNS

00355 5775' #gg ¥$33NS

00356 1374 A3, .

00357 4445 CALL IDATA légggTEggggEMINI
00360 5775' JMP P400 /

00374 0152
00375 0400
00376 0150
00377 7462
0400' PAGE

 

 

160

/ EQUILIBRIUM MODE SFCA

00400 7300 P400, CLA CLL

00401 1377 TAD (170

00402 3154 DCA RTIME
00403 1150 A4, TAD TSAHP
00404 7041 CIA; DCA CSAMP

PALS-VIOA 12-JUL~78 PAGE 5

/INITIALIZE DATA

1
00406 4421 GETSEC, WRITE;"I;"E;" ;"T;“I;“H;"E;"(;0

004-20 1150 TAD TSAP'IP
TAD csarr; IAC

CALL DECOUT

00424 4421 WRITE;");0
00425 025

00426 0300

00427 1161 TAD IFLAG

SZA CLA; JMP A5
CALL DECIN;0

TAD .—1
DCA I BTIME
0043C 5241 JR? A6

00437 1154 A5, TAD RTIME
00440 4445 CALL IDATA
00441 2154 A6, 182 RTIME
00442 2151 ISZ CSAfiP

JNP GETSEC

TAD IFLAG
SZA CLA; JMP A?

00460 4420 CALL DECIN:0

TAD .-1

DCA EQTIME
00464 5267 JHP OPER
00465 1376 A7. TAD (EQTEME
CALL IDATA

/ENZYMATIC INC TIME

/INPUT FROM MINI

WRITE; “E; "a; " z "'1'; "I: "u; “E;0

/DELAY TD FIRST DATA PT

/FEOH MINI

 

 

/ EQUILIBRIUM MODE SFCA

00467

00553
00554
00555

 

OPER,

SSCALE,
SAMP,

161

PALS-V10A 12-JUL-78 PAGE 6

CLA CLL /OPERATE
TAD TSAMP
CIA; DCA CSAMP

TAD (170
DCA HTIME
WRITE;"H;"E;"—;"I;“N;"I;"T;" ;'D;"A:"T;"A;"?;0

CALL READ

TAD (-“N

SNA CLA

JNP SSCALE

TAD DATAB /T0 DESTROY OLD DATA
DCA DATA

CALL SCALE /SET 0,100
/LOOP HERE FOR EA SAMPLE

R
TAD TRUNS
CIA; DCA CRUN

/EERE FOR EA RUN

CALL SCALE /OPTIONALLY RESET 0.100% T
WRITE; "S: "A; "M: "P; " :0

TAD TSAMP
TAD CSAMP; IAC

CALL DECOUT /WRITE CURRENT SAM? NO
WRITE:“ ;" :“R:“U;“N;" :0.

 

 

 

/ EQUILIBRIUM MODE SFCA

00556
00557
00560
00561
00562
00563
00564
00565
00566
09567
00570
00371

00572
00573
00574
00575
00576
005??

1152
1153
7001
4426
6507
0373
7650
4423
7200
444

0002
5772

0600
(Ar.

go"-
2000
7462
0355
0170
0600

TAD TRUNS
TAD CRUN; IAC

CALL DECOUT
CETSR

AND (4000
SNA CLA
CALL READ
CLA

CALL DELAY;2

Jfi? I (CYC

PAGE

162

PALS-VIOA 12-JUL-78 PAGE 6-1

 

/AND RUN N0

/WAIT KB BEFORE OPEN SAN? VALVES

 

 

/ EQUILIBRIUM MODE SFCA

00600
00601
00602
00603
00604
00605
00606
00607
00610
00611
00612
00613
00614
00615
00616
00617
00620
00621
00622
00623
00624
00625
00626
00627
00630
00631
00632
00633
00634
00635
00636
00637
00640
00641
00642
00643
00644
00645
00646
00647
00650
00651
00652
00653
00654
00655
00656
00657
00660
00661
00662
00663
00664
00665
00666

4444 CYC.

1155
3204
4440
0000
4433
1750
1157
3556
6507
0377
7640
5227
4421
0240
0311
0255
0304
0275
0240
0000
1556
4426
4434
2156
7410
5776
1160
3556
2156
7410
5776
2153
5775’
2154
2151
5000
4434
4434
4421
0314
0311
0323
0324
0240
0304
0301
0324
0301
0277
0000
4423
1374
7650
5777

A81

ALIST,

163

PALS-V10A 12-JUL-78 PAGE 7
CALL CYCLE /COMPLETE CYCLE
TAD EQTIHE
DCA .+2

CALL DELAYz0 /WAIT FOR THINGS TO SETTLE

CALL SAMPLE; ISAMP /SAMPLE AND AVERAGE ADC

TAD MDARK /SUBRT DARK CURRENT
DCA I DATA /STORE IN THE TABLE
GETSR

AND (1000

SZA CLA /IHMEDIATE OUTPUT?

JMP A8 /NO

WRITE; II ;"I: 11...; "D; II: 3 ll ;@

TAD I DATA

CALL DECOUT
CR; ISZ DATA

SKP; JNP I (DAOVFL /CHECK DATA TAB OVFL

TAD REF

DCA I DATA

ISZ DATA

SKP; JNP I (DAOVFL

/STORE CURRENT REF

/MORE RUNS THIS SAHP?

ISZ CRUN

JNP RUN /YES M‘
ISZ RTIME /ADV POINLER
ISZ CSAMP /HORE SAMPLES?
JMP /YES

CR;CR

WRITE;”L;"I:"S;"T;“ :"D;"A:"T;"A;"?;0

CALL READ

TAD (_IIN

SNA CL
JNP I (NOLIST

A

 

 

 

 

161+

I’EQUILIBRIUM MODE SFCA PALS—VIOA 12-JUL-78 PAGE 7-1

00667
00670
00671
00672
00673
00674
00675
00676
00677
00700
00701
00702
00703
00704
00705
00706
00707
00710
00711
00712
00713
00714
00715
00716

4434
4434
4421
0323
0301
0315
0320
0240
0240
0311
0255
0304
0257
0322
0255
0304
0000
4434
4%34
1162
3156
1150
7041
3151

CR; CR
WRITE;'S;'A;“H;"P;"

CR;CR

TAD DATAB
DCA DATA
TAD T823 NP
CIA; DCA CSAHP

I!

; "I‘ll—zflD; n/;01R31I_; IID;0

/RESET DATA PTR

 

165
/ EQUILIBRIUM MODE SFCA PALB-VIOA 12-JUL—78 PAGE 8
00717 1373 SAMPL, TAD {-6
00720 337 DCA M6 /SET COUNT
00721 1152 TAD THUNS
00722 7061 CIA; DCA CBUN
00723 3153
00724 4434 CR
00725 1150 TAD TSAMP
00726 1151 TAD CSAMP; IAC
00727 7001
00730 4426 CALL DECOUT
00731 4421 BURL, WRITEg" ;" :0
00782 @240
00733 0940
00734 0000 =
00735 1556 TAD I DATA .
00736 2156 ISZ DATA 1
00737 4426 CALL DECOUT '
007é0 6421 WRITE;9/;0
00741 025
00762 0000 1
00743 1556 TAD 1 DATA
00746 2156 ISZ DATA
00705 4426 CALL DECOUT
00746 2153 ISZ CRUN
00747 7410 SKP
00750 5365 JHP All
00751 237 ISZ M6
00752 331 JHP RUNL
00753 373 TAD (-6
00754 3370 DCA M6
00755 4434 CR
00756 4421 WRITEz" :" 3" 3" 39

00757 0200
00760 0260
00761 02€0
00762 0240
00763 0030

00764 5331 JMP BUNL
00765 2151 All, ISZ CSAMP
00766 5317 JHP SAMPL
00767 5245 JNP ALIST
00770 0000 N6. 0

00773 7772

00774 7462
00775 0527
00776 1053
00777 1000
1000 PAGE

 

/ EQUILIBRIUM MODE SFCA

01000
01001
01002
01003
010 6
01005
01066
01007
01010
01011
01012
01013
01010
01015
01016
01017
01020
01021
01022
01023
01026
01025
01026
01027
01030
01031
01032
01033
01034
01035
01036
01037
01040
01041
01062
01043
01044
01045
01046
01047
01050
01051
01052

01053
01054
01055
01056

01175
01176
01177

4434 NOLIST,

4434
4421
0380
0315
0311
0324
0240
0304
0801
0324
0301
027
0000
4423
1377
7640
5776
4461
1162
3156
1150
7061
3151
1150
4446
1152
4446
1152
7041
3153
1556
2156
4046
1556
2156
4406
2153
5237
2151
23
4441
5776

7300
1375
4441
5776

7775
0200
7447
1200

SAMPX.

BUNK,

DAOVFL.

166

CR: CR
WRITE;WX;'M;"I;"T}" ;"D;“A;"T;"A:”?;0

CALL READ

TAD {-“Y

SZA CLA /XJ‘IIT DATA TO MINI?
JHP I (START /N0; RESTART

CALL MONITR ASET UP MINI

TAD DATAB

DCA DATA

TAD TSAMP

CIA; DCA CSAMP

TAD TSAMP
CALL ODATA
TAD TRUNS
CALL ODATA
TAD TRUNS
CIA; DCA CRUN

TAD I DATA
ISZ DATA
CALL ODATA
TAD 1 DATA
ISZ DATA
CALL ODATA /REF-DABK
ISZ CRUN

JHP RUNX

ISZ CSAMP
JMP SAHPX
CALL MONITR
JHP I (START

CLA CLL /DATA TABLE FULL (TERU 7777)

TAD (-3
CALL MDNITR
JNP I (START

/SANPLE-DARK

/TBANS MODE T0 INIT MINI

PAGE

PALS-VIOA 12-JUL-78 PAGE 9

 

 

 

 

167

/+3 FREQUENTLY CALLED SUBBOUTINES PALS-VIOA 12-JUh-78 PAGE 10
/+3 FREQUENTLY CALLED SUBROUTINES
/ ,

/STOPPED-FLOW CLINICAL ANALYZER

/SUBROUTINE TWO

/

/SFCA.SI CONTAINS PARAMETER DEFINITIONS

/ AND PAGE ZERO POINTEHS.

/

/SFCA.82 CONTAINS FREQUENTLY CALLED SUBROUTINES

/ WRICH LOAD BEGINNING AT THE PAGE FOLLOWING THE
/ EXECUTIVE ROUTINE, AND OCCUPY 7 CORE PAGES.

/

/DATA STORAGE BEGINS AT THE PAGE FOLLOWING SFCA.S2
/ AND EXTEND TO THE END OF MEMORY.

/

/PGR: M.D. JOSEPH

/DATE: 28-MAY‘78

/VER= IB
/FILE: SFCA.82
/

/

 

FREQUENTLY CALLED SUBBOUTINES PALS-VIOA l2-JUL-78 PAGE 11

/GENERAL PURPOSE SURROUTLNES
/
/SUMMARY OF SUBBOUTINE CALLS

DECIN - KB INPUT A DECINAL NUMBER IN THE RANGE 0-3999,
RETURN THE OCTAL EQUIVALENT AN CALL+1

CALL DECIN
OCTAL
< RE’I Ur‘ E '1)

\\\\\\\\\\

TXOUT ~ TYPE ASCII TEXT, PACKED ONE B-BIT CHAR/WORD,
TERMINATED WITH A ZERO

CALL TXOUT
TEXT...

O
(RETURN)
TYPE — TYPES CHAR IN AC. LEAVES AC UNAFFECTED

CALL TYPE (AC=CHARACTER)
(RETURN)

KB INPUT AN 8’BIT‘CHARACTER, RETURNS 1N AC
AC CAUSES JMS MONITR
ALTHODE CAUSES JMS MANUAL

\\\\\\\\\\\\\

N
3

CALL READ
(RETURN) AC=B-BIT CHARACTER

TYPES CHARACTER IN AC AS AN ASCII NUMERIC
CHARACTER SHOULD BE IN THE RANGE O-ll(8)
AC IS CLEARED BEFORE RETURN

CALL NOUT (AC=CHARACTER)
(RETURN)

OCTOUT - TYPES NUMERIC IN AC AS OCTAL
RETURNS AC CLEARED

\\\\\\\é\\\\\\
1%
I

CALL OCTOUT (AC=OCTAL NUMERIC)
(RETURN)

DECOUT - TYPES CHARACTER IN AC AS DECIHAL NUMERIC
RETURNS AC CLEARED

CALL DECOUT (AC=NURERIC)
(RETURN)

\\\\\\\\\\

 

 

 

 

169

/+3 FREQUENTLY CALLED SUBROUTINES PALS-VIOA l2-JUL-78 PAGE 12
/SUMMARY OF SUBROUTINE CALLS, CONT

SAHPLE — SAMPLES AND AVERAGES A/D UP TO 4096 TIMES
RETURNS AVERAGE IN AC

CALL SAMPLE
NO. POINTS TO AVERAGE
(RETURN) (AC=AVERACE)

O

RLF - OUTPUTS CR,LF
RETURNS AC CLEARED

CALL CRLF
(RETURN)

AVCLR - INITIALIZE DOUBLE PRECISION AVERAGER
RETURNS AC CLEAREU

CALL AVCLR
(RETURN)

AVADD ~ ADD AC TO ONGOING SUM
CLEARS AC
CALL AVADD (AC=INTEGER)
(RETURN)
VER - CALCULATE AVERAGE. RETURN AVG IN AC

:9

VER
ERROR = AVERAGE > 7777
(RETURN) (AC=AVERAGE)
DELAY - DELAY N SECONDS WHILE MONTTORING KB
CALL DELAY
SECONDS TO DELAY (OCTAL)
(RETURN)

IANUAL - MANUAL OPERATION OF SFCA
AR FORCES RETURN

H

CALL MANUAL
(RETURN)

MONITR.- TRANSPARENT MODE AND ERROR HANDLING
AA CHAINS TO *209
“R FORCES RETURN

CALL MONITR
(RETURN)

\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\

 

170

/+3 FREQUENTLY CALLED SUBROUTINES PALS-VIDA l2-JUL-78 PAGE 13
/SUMHARY OF SUBROUTINE CALLS, CONT

----—-—c.--—n-——-—--—--—--—~——-—-~
--—-—--------—-------------———---

SCALE - OPTIONALLY SET DARK AND REFERENCE
SAMPLES A/D ISAMP TIMES, AVERAGES, AND
STORES AT MDAEK, REF (PAGE ZERO)

CALL SCALE
(RETURN)

CYCLE - CYCLES SFCA THROUGH EMPTY, OPEN, FILL,
DELAY<SAHP), CL‘SE, DELAY(HEACTION),
WAIT FOR FLOW STOFPED.

CALL CYCLE
(SAI’IP TIME = ISAMPT; REACT TIME = RTII-IE)
(RETURN)

IDATA - INPUT ONE 12-BIT WORD FROM NINI,
STORE AT POINTER PASSED.
AC RETURNED CLEARED

CALL IDATA (AC=POINTER)
<RETURL>

ODATA - OUTPUT'GNE lQ-BIT WORD TO.HINI
DATA IN AC; AC CLEARED

CALL ODATA (AC=DATA)
< RETUIU‘I)

\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\

 

 

 

/+3

01200
01201
01202
01203
01204
01205
01206
01207
01210
01211
01212
01213
01214

1215
01216
01217
01220
01221
01222
01223
01224
01225
01226
01227
01230
01231
01232
01233
01234
01235
01236
01237
01240
01241
01242
01243
01244

FREQUENTLY CALLED SUBROUTINES

0000
7200
1377
3327
1376
3223
3331
3332
3333
3334
3330
4421
0240
0275

240
0000
4423
4422
4274
3331
2223
2327
5220
4423
1375
7640
5322
4434
1331
7450
5245
1374
3331
1373
1330
3330
5234

171

PALS-V10A 12-JUL-78 PAGE 14

/DECIMAL INPUT

/DECIMAL INTEGER (0-9999);

/

XDECIN,

GETD,

STO,

CVRT,
L19

RETURN OCTAL EQUIV
(4096=0, 4097=1, ETC.)
0 /CALL DECIN; OCTAL; RETURN
CLA

TAD (-4

DCA N4

TAD (DCA DIGI

DCA STO

DCA DICl;DCA DIG2;DCA DIGS;DCA DIG4

DCA OCT
CALL TXQUT; “ ;"=;" ;0

CALL READ
CALL TYPE
JMS CHK
DCA DIGI
ISZ STO
182 N4
JflP GETD
CALL READ
TAD {-215
SZA CLA;

KMAKE SURE IT’S A NUEERIC
/WILL CHANGE

/DO ONLY 4
/NOW ALLOW.A CR

JMP IERR /NONE OF THE ABOVE

CALL CRLF
TAD DIGI
SNA; JDIP L2

TAD (-1
DCA DIGl
TAD (1750
TAD OCT
DCA OCT'
JMP L1

KDECRENENT

/ADD 1000(10)

 

 

172
/+3 FREQUENTLY CALLED SUBROUTINES PALB-V10A 12-JUL-78 PAGE 15
01245 1332 L2, TAD DIC2
01246 7450 SNA; JMP L3
01247 5256
01250 1374 TAD (-1
01251 3332 DCA D102
01252 1372 AD (144
01253 1330 TAD OCT
01254 3330 DCA OCT /ADD 100(10)
01255 5245 JR? L2
01256 1333 L3, TAD D163
01257 7450 SNA; JHP L4
01260 5267
01261 1374 TAD (-1
012’? 333 DCA DI‘3
01263 1371 TAD (12
01264 1330 TAD OPT
01265 3330 DCA OCT /ADD 10(10)
01266 5256 JR? L3 1
01267 1334 L4. TAD D104
01270 1330 TAD OCT 1
01271 3500 DCA I XDECIN /PASS BACK TO CALLER
01272 2200 ISZ XDECIN /POINT TO RETURN
01273 5600 JfiP I XDECIN
01274 0000 CHK, 0
01275 1375 TAD (-215 /CR TERMINATES
01276 7450 SNA: JR? XCV
01277 5310
01300 1370 TAD ('43 /260 = LOWEST OCTAL
01301 7510 SPA; JHP IERR

01303 1367 TAD (-12 /271 = HIGHEST

01304 7500 SMA; JHP IERR

01305 5322

01305 1371 TAD (12 /RESTORE AND STRIP ASCII
01307 5674 J“? I CHK

 

/+3

01310
01311
01312
01313
01314
01315
01316
01317
01320
01321
01322
01323
01324
01325
01326

0132?
01330
01331
01332
01333

0133-

01335
01336
01337
01340
01341
01342
01343
01344

01345
01346
01347
01350
01351

01367
01370
01371
01372
01373
01374
01375
01376
01377

FREQUENTLY CALLED SUBROUTINES

1333 XCV,

3334
1332
3333
1331
3332
3331
2327
5310
5233
4421
0277
0000
4434
5201

0000
0030
0000
0000
0000
0000

0000
7200
1735
2335
7450
5735
4422
5336

0000
6146
6141
5347
5745

7766
7735

0012 .

0144
1750
7777
7563
3331
7774
1400

IERR,

N4,
OCT,
D101,
D102,
D103,
D104,

173

PALS-V10A 12-JUL-78 PAGE 16

/HOVE DIGITS ACCORDING TO
/HOW MANY WERE INPUT

TAD D103
DCA D104
TAD D102
DCA D103
TAD D101
DCA D102
DCA D101
ISZ N4
JMP XCV
JHP CVRT
CALL TXDUT;"?;0

CALL CRLF
JHP XDECIN+1

660906

/ASCII TEXT OUTPUT
/ONE B-BIT CHAR/WORD; 0 TERMINATES

XTXOUT,

0 /CALL TXOUT} TEXT; 0; BET
CLA

TAD I XTXOUT

ISZ XTXOUT

SNA; JD? 1 XTXOUT‘ /0 TERM

CALL TYPE
JHP XTXOUT+1

/LST - OUTPUTS CHARACTER 1N AC T0 LPT (DEV 14);

LEAVES AC UN CHANGED

0 /CALL LIST (AC=CHAR); RETURN

6146

6141

JP]? .-1
JMP I XLST

PAGE

 

 

 

/+3

1714

FREQUENTLY CALLED SUBROUTINES PALS—V10A 12-JUL—78 PAGE 17

/TYPEOUT; CHAR IN AC; AC NOT CLEARED

XTYPE. 0
TL

KS /CHECK KB
DCA XNOUT fSAVE Tim?
TAD (—223 /“S MEANS WAIT

CLA CLL
TAD XFGUT
JNP 1 XFYPE
/NOUT OUTPUTS AC AS ASCII NUHERIC

XNOUT} 0 /CALL FOUT (NUM IN AC); RETURN
TAD (260
CALL TYPE

CLA
JNP 1 XNOUT
/OCTOUT OUTTUTS NUMERICS IN OCTAL
XOCTOU, 0 /CALL OCTOUT (OCTAL IN AC); BET
DCA X0
TAD X0
RTL;RTL

AHD (7
CALL NOUT
JMP I XOCTOU

X0. 0

 

/+3

01450
01451
01452
01453
01454
01455
01456
01457
01460
01461
01462
01463
01464
01465
01466
01467
014?0
01471
01472
014?3
01474
01475
01476
0147?
01500
01501
01502
01508
01504
0 1505
01506
01507
01510
01511
01512
01513
01314
01515
01516
01517
01520
01521
01522
01523
01524

01525
01526

FREQUENTLY CALLED SUBROUTINES

0000
3325
3326
1325
1374
7510
4311
2326
5254
1373
3325
4317
1325
1372
7510
5272
2326
5265
1371
3325
4317
1325
13?0
7510
5303
2326
52?6
136?
3325
4317
1325
4424
5650
0000
1373
7500
5262
13?4
5711
0300
1326
4424
7200
3326
5717

0000
0000

175

PAL8-V10A lZ-JUL-78 PAGE 18

/DECOUT CONVEBTS OCTAL-DECINAL AND OUTPUTS

XDECOU, 0

XDOD,

XDOC,

TEMP,
DIG,

DCA
DCA
TAD
TAD
SPA
JNS
ISZ
JMP
TAD
DCA
JHS
TAD
TAD
SPA
JNP
ISZ
JMP
TAD
DCA
JNS
TAD
TAD
SPA
JMP
ISZ
JMP
TAD
DCA
JNS
TAD

CALL NOUT
JMP I XDECOU

0

TAD (1750

SEA; JHP XDOD
TAD (-1750
JMP I XDOC

0
TAD

CALL NOUT

CLA
DCAA

JMP I TNUM

0
0

TEMP
DIG
TEMP

(-1750

XDOC
DIG

. --4
(1750
TEMP
TNUH
TEI'IP
(-144

.+3
DIG
.-4
(144
TEE?
TN U M
TEMP
(~12

.+3
DIG
.-4
(12
TEMP
TNUM
TEMP

DIG

DIG

/CALL DECOUT (OCTAL IN AC); RET

/SUBTR 1000(10)

/INCB THOUS

/RESTORE

ASUBTR 100(10)

/INCR HUNDREDS

/RESTORE

KSUBTR 10(10)

/DON’T SKIP IF ALREADY NEG

 

 

/+3

0152?
01530
0153!
01532
01533
01534
01535
01536
015;?
61540
01541
01542
01503
01544
01545
01546
01547
@1559
01551
025513
01553
01534
@1555
01556
01557

01566
0156?
01570
01571
01572
01573
01574
01575
01576
01577

FREQUENTLY CALLED SUBROUTINES

176

PALS-V10A 12-JUL~78 PAGE 19

/HDIV - DIVISION WHERE QUOT<1
/RETURNS QUOT*1000

XHDIV,

XHLOOP.

XFDRET,

XHDC,

0

CLA

TAD (—1750
DCA XHDC
CALL AVCLR
TAD I XHDIV
RTR;RAR

AND (77?
CALL AVADD
JM? XHDHLT
ISZ XEIDC
JMP XHLOOP
ISZ XHDIV
TAD I XHDIV
RTH;RAR

AND (777
DCA I XAVCT
ISZ IQ'IDIV

CALL AVER
SKP
ISZ XHDIV

JHP I XHDIV
0

PAGE

/CALL HDIV;DIVIDEND;DIVISOR;OVFLzflET

/CET DIVIDEND

/ADD TO SUNS 1000 TIMES

/GET DIVISOR

/ERROR RETURN
/NORMAL RETURN

 

/+3

01600
01601
01602
01603
01604
01605
01606
01607
01610
01611
01612
01613
01614
01615
01616
01617
01620
01621
01622
01628
0 1624-
01625
01626
01627
01680

FREQUENTLY CALLED SUBBOUTINES

0000
6031
5201
6036
1377
7450
5214
1376
7450
5222
1375
5600
1200
3230
4441
1280
3200
5201
1200
3230
$442
1230
3200
5201
0000

177

PALB—VIOA 12-JUL-78 PAGE 20

/READ; B-BIT CHAR RETURNED IN AC
/‘C CHAINS TO MONITOR (JHS)
/ALTMODE CHAINS TO MANUAL MODE (JMS)

XREAD,

XMON,

XRRET,

0

KSF

JMP .-1

KRB

TAD {-203
SNA; JMP XMON

TAD (-30
SNA; JMP XMAN

TAD (233
JMP I XREAD
TAD XHEAD
DCA XHRET
CALL MONITR
TAD XRRET
DCA XREAD
JNP XHEAD+1
TAD XREAD
DCA XRRET
CALL MANUAL
TAD XRRET
DCA XREAD
JMP XBEAD+1
0

/“C CHAINS TO MONITOR

/ALTMODE CHAINS TO MANUAL

/RESTORE

/RETURN FROM MONITOR

 

/+3

01631
01632
01633
01634
01635
01636
01637
01640
01641
01642
01643
01644
01645
01646
01647
01650
01651
01652
01653
01656
01655
01656
01657
01660
01661
01662
01663
01664
01665
01666
01667
01670
01671
01672
01673
01674
01675
01676
01677
01700
01701
01702
01703
01704
01705
01706

FREQUENTLY CALLED SUBROUTINES

0000
3266
6131
5302
6136
1374
7640
5233
1373
6146
6141
5243
7200
4270
7002
3267
4270
1267
3666
4421
0240
0275
0240
0250
0060
1666
4426
4434
5631
0000
0300
0000
6131
5271
6136
1374
7450
5271
1373
0372
5670
6031
5233
4423
7200
5233

/IDATA -

178

PALB-V10A 12-JUL-78 PAGE 21

INPUT 12 BITS FROM MINI

XIDATA, 0

XIDWI,

XIDP,

XIDTEM,
XIDIN.
XIDWZ.

XIKB,

DCA XIDP
6131

JMP XIKB
6136

TAD (-100
SZA CLA
JMP XIDWI
TAD (100
6146

6141

JMP .-1
CLA

JMS XIDIN
BSW

DCA XIDTEM
JMS XIDIN
TAD XIDTEM
DCA 1 XIDP
WRITE; I! g 11:; fl

TAD I XIDP
CALL DECOUT
CR

JNP I XIDATA
0

0

0

6131

JMP .‘1
6136

TAD (’100
SNA

JHP XIDWQ
TAD (100
AND (77

JMP I XIDIN
KSF
JHP'XIDWI
CALL READ

CLA
JI‘IP XIDWI

/CALL IDATA (AC=POINTER); RETURN
/STORE POINTER

/WAIT FOR SPECIAL CHARACTER
/ALSO WATCH.EB

/READ MINI

/SPECIAL?
/NOPE

/SEND IT BACK

/GET 6 BIT BYTE

/COMBINE BYTES
/STORE

”(:0 / ( INDICATES INPUT FROM MINI

KECEO TO TERMINAL

/READ MINI
/ANOTHER SPECIAL

/YEP; LO0K.AGAIN
/NUST BE DATA

[HE TYPED SOMETHING

 

 

/+3

01707
01710
01711
01712
01713
01714
01715
01716
01717
01720
01721
01722
01723
01724
01725
01726
01727
01739
01731
0 732
01733
01734
01735
01736
01737
01740
01741
01742
01743
01744
01743
91746
01747
01750
01751

01752

01770
01771
01772
01773
01774
01775
01776
01777

FREQUENTLY CALLED SUBROUTINES

0000
3352
6131
5345
6136
1371
7640
5311
1370
4337
6131
5317
6136
1371
7640
5317
1352
7802
0372
4337
1352
0372
4337
5707

'000
6146
6141
5341
7200
5737
6031
5311
4423
7200
5311

0000

0101
7677
0077
0100
7700
0233
7750
7575
2000

 

179

/0DATA - SEND 12 BITS TO MINI

PAL8-V10A 12-JUL-78 PAGE 22

XODATA, 0

XODWI,

XOPR,

XOOUT,

XOKB.

XODTEM,

DCA XODTEH
6131

JNP XOKB
6136

TAD (“101
SZA CLA
JNP XODWL
TAD (101
JNS XOOUT
6131

JMP XOPR
6136

TAD (’101
SZA CLA
JNP XOPR
TAD XODTEH
BSW

AND (77
JMS XOOUT
TAD XODTEM
AND (77
JNS XUGUT
JNP 1 XQDATA
0

6146

6141

J11? .’1
CLA

JNP I XUOUT
KSF

J11? XODWI
CALL READ
CLA

JMP XDDW1

0

PAGE

/CALL ODATA (AC=DATA); RETURN

/WAIT FOR SPECIAL
/ALSO WATCH KB

/SPECIAL?

/NOPE

/SEND IT BACK
/ANY RESPONSE YET?

/NGT YET
/HERE IT IS

[SPECIAL AGAIN?
/NOT YET BUT HE WILL
/OK - GET THE GOOD STUFF

/SEND FIRST BYTE

/SECOND BYTE
/RETURN

[HE TYPED SOMETHING

 

/+3

02000
02001
02032
@2003
62004
02035
02066
02007
02010
02011
62012
02013

@2032
02633
02024
@2025
62026
G232?
0203

@2031
@2032
@2033
02934
@2035
@2636
02037
02010
02041
02042
026é3
02044

02045
02046
02047
02050
02051
02052
02058
02054
02055
02056
02057
02060
02061
02062
02063

FREQUENTLY CALLED SUBROUTINES

0000
4421
0322
0305
0323
@305
@324
@240
I364
@891
0322
0313
0277
@300
4428
1377
7650
5245
@434
4921
0303
-314
0317
0323
0305
C?“
4423
4434
448.
1700
7041
3157
1157
7041
4426
4434
5201

4434
$421
0322
0305
0323
0305
0324
0240
082

0305
0306
0240
0277
0000
4623

180

PALS-VIOA 12-JUL‘78 PAGE 23

/SCALE - OPTIONALLY SAMPLE 0, 100%

XSCALE, 0
XDARK, WRITE;

XREF,

fCALL SCALE; RETURN
"R3 HE; "S; "E; IT; ll :11D;IA;IIR;HK;u?;o

CALL READ

TAD (-“N

SNA CLA

JNP XREF

CR

WRITE; "C;"L:“0;"S;“E;0

CALL READ; CR

CALL SAflPLE; ISAHP /AVEEAGE ISAMP READINGS

CIA; DCA “DARK /STORE -(DARK)
TAD HDARK; CIA
CALL DECOUT
CR

JNP XDABK

CR

WRITE; :"730

“R; "E; "S; IKE: ET; I zlfl; HE; "F; I!

CALL READ

 

 

 

 

181
/+3 FREQUENTLY CALLED SUBROUTINES PALB-VlOA 12-JUL-78 PAGE 23-1
@2064 1377 TAD (~"N
02065 7650 SHA CLA
02066 5316 JNP XSCRET
02067 4434 CR
@2670 4421 WRITE;“O;"P;"E:"N;0
02071 0317
02072 0320
020?3 030:
020?é 0a16
£2055 060
02076 4423 CALL READ
020?? 4444 CALL CYCLE
0210' 1155 TAD EQTEY
02101 3303 DCA .+2
02102 4440 CALL DELAY;0
02103 6390
0210é 4433 CALL SAMPLE; ISAMP
62133 1?5
02 03 3160 DCA REF
0210? 1160 TAD REF
02110 4426 CALL DECOUT
02111 4934 OR
02112 1160 TAD BET: TAD MDARK /STDRE REF-DARK
02113 1157
02114 3160 DCA REF
02115 5245 JMP XBEF
02116 4434 XSCRET. CR
02117 5600 JNP I XSCALE

/CYCLE - ONE CYCLE OF SFCA

02120 0000 XCYCLE, 0 /CALL CYCLE; RETURN
02121 6502 EMPTY

02122 6500 PEN

02123 6003 FILL

02124 4%40 CALL DELAY; ISAMPT

02123 0905

02126 6301 CLOSE

02127 1554 TAD I RTIME fGET REACTION TIME
02130 3892 DCA .+2

02131 4440 CALL DELAY; 0

02132 0300

02133 6504 PUSH

02134 6505 SFS

02135 5834 JMP .-1

02136 5720 JMP I XCYCLE

2200 PAGE

 

 

/+3

02200
02201
02202
02203
02204
02205
02206
02207
02210
02211
02212
02213
02214
02215
02216
02217
02220
02221
02222
02223
02220
02225
02226
02227
02230

02231
02232
02233
02234
02235
02236
02237
02240
02241

FREQUENTLY CALLED SUBROUTINES

UP TO 4096 TIMES AND
AVERAGE IN AC

0000
7200
1600
7041
3231
2200
4435
6510
6511
5210
6512
7040
4436
5223
2231
5207
443

5226
5600
7240
4441
5600
7344
4441
5600

0000
7200
1377
4022
7200
1376
4422
7200
5631

/SANPLE A/D
/RETURN THE

XSAMPL. 0

SLOOP,

TAD
CIA

JMP I

CLA

DCA XCRLF
ISZ XSAMPL
CALL AVCLR
CONVBT
SCD

JMP .-1
CETAD

CNA

CALL AVADD
JIIP SERRI
ISZ XCRLF
JI‘fi’ SL017?
CALL AVER
JI‘IP SEEM

JMP I XSAMPL

CLA CMA
CALL

/CRLF OUTPUTS CR,LF

XCRLF.

0

CLA

TAD (215
CALL TYPE
CLA

TAD (212
CALL TYPE
CLA

JMP I XCRLF

I XSAMPL

sONITR
XSAHPL
CLA CLL CHA HAL
CALL HONITR
JMP I XSAMPL

182

PALS-V10A 12-JUL-78 PAGE 24

/CALL SAMPLE; NPTS AVG; BET (AVG IN AC)
/GET NPTS T0 AVERAGE

/CLEAR TEE.AVERAGER
/START A/D

/EAIT FOR DONE FLAG
/READ A/D

/ADD TO AVERAGE
/AVADD OVFL

/CALC THE AVERAGE
/AVER OVERFLOW

/CALL CBLF; BET (AC CLEAR)

 

 

/+3

@2242
62243
02244
02245
@2245
99947

~..

@2250
02251
@2252
02253
02256
02255

2236
0223?
02260
02261

02262
02263
02264
02263
02266
02267
02270
02271
02272
02273
02274
02275
02276
02277
02800
02301
02302
02303

FREQUENTLY CALLED SUBROUTINES

0000
7200
3322
3323
3324
5642

0000
7100
132.

3323
7034
1322
3322
2324
2250
5650

0000
7200
1322

run-'3:-

cc;
3326
1324

703
3320
328
3321
1321
1820
7510
5304
3321
2326
5274
5662

1113

PALB-VIOA 12-JUL-78 PAGE 25

/DOUBLE PRECISION AVERAGEB
/MAXZ4095 INTEGEHS (0-4095 EACH)

XAVCLR, 0
CLA
DCA
DCA
DCA
JMP

XAVADD, 0
CLL
TAD
DCA
RAL
TAD
DCA
ISZ
ISZ
JNP

XAVER. 0
CLA
TAD
DCA
DCA
TAD

CIA; DCA MDIVS

TAD
DCA
AVLOOP. TAD
TAD
SPA
JHP
DCA
XAVRET, ISZ
JM?
JHP

HSUI‘I
LSUI‘I
AVCNT
I XAVCLR

LS UE‘I
LSUX

HSUM
ESUfi
AVCNT
XAVADD

I XAVADD

BSUM
HSUH2
QUOT
AVCNT

LSUM
DIVD
DIVD
MD I VS

BORROW
DIVD
QUOT
AVLOOP
I XAVER

/CALL AVCLR; RET

/CALL AVADD (INTEGER IN AC); OVFL ERR; BET

/UPDATE LOW SUM
/CARRY TO LINK

/UPDATE HIGH SUM
NORMAL RETURN

/CALL AVEB; ERR (OVFL);

KSUBTR DIVISOR
/EMY NEED TO BORROW

/0VERFLOW (QUOT>4095)

RET (QUOT IN AC)

 

 

 

/+3

02304
02305
02306
02307
02310
02311
02312
02313
02314
02315
02316
02317

02320
02321
02322
02323
02324
02325
02326

02327
02330
02331
02332

FREQUENTLY CALLED SUBBOUTINES

3321
1320
7041
1321
7710
5301
1325
7450
5327
1375
3325
5301

0000
0000
0300
0000
0000
0000
0000

300
1326
2262
5662

BORROW,

MDIVS,
DIVD,
HSUN.
LSUM,
AVCNT,
HSUM2.
QUOT,

AVDONE,

DCA
TAD
CIA
TAD
SPA
JNP
TAD
SNA
JNP
TAD
DCA
JMP

®®®®®®®

CLA

ISZ
JHP

DIVD
MDIYS

DIVD
CLA
XAVRET
HSUM2

AVDONE
{-1
H8531?

XAVRET

CLL
QUOT
XAVER

I XQVER

184

PALB—VIQA l2-JUL~78 PAGE 26

/DON’T BORROW IF DIVD WAS ALREADY NEG

/ANYIHINC LEFT TO BORROW?
/NOPE

/DECREMENT HIGH SUM
/CONTINUE

/RETURN

 

 

/+3

02833
02334
02335
02336
02887
02340
02341
02342
02343
0234%
02345
02346
0234?
02350
02351
02352
02353
02354
02355
0 356
0235?
02360
02301
02362
02363

02364

02370

02374
023?5
02376
0237?

FREQUENTLY CALLED SUBROUTINES

0000
7200

XDELAY. 0
CLA
TAD I XDELAY
ISZ XDELAY
SNA:

CIA; DCA XNSEC

KS?

JMP XCOUNT
CALL READ

c—222

TAD

185

PALS-V10A 12-JUL—78 PAGE 27

/DELAY DELAYS N SECONDS WHILE MONITORING KB

/CALL DELAY; NSEC; RETURN
/GET N SECOFDS

JMP I XDELAY' /0=N0 DELAY

CL
I

XHC
XDCZ
FEE]
FDCI
XDCI
.—l

XDCB
.-5

XNSEC

XSEC

I XDELAY

PAGE

A
XDELAY
2

/CHECK KB
/NO
/“R FORCES REIURN

/WAIT A SEC

lENOUGH?

 

186

/+3 FREQUENTLY CALLED SUBROUTINES PALB—VIOA lZ-JUL-?8 PAGE 28
/MANUAL CONTROL
/
/COMMAND FUNCTION
/ ______________
/
/ 0 OPEN SAMPLE VALVES
/ C CLOSE
/ E EMPTY STOPPING SYR
/ F PILL DRIVE SYRINGES
/ P PUSH
/ R RESET
/ D SET D/A
/ A SAMPLE A/D
/ "R RETURN
02400 0000 XMANUA, 0 /CALL MANUAL; RETURN
02401 4434 CALL CRL?
@2492 4&2 CALL TROUT; "M;":;O
02403 @315
02402 6272
02435 0390
02406 442- CALL READ
0243? 13?? TAD (~215 /CR = CRLF
G2é10 ?45 SNA; JMP XNMNUA+1
624:1 l 5501
02413 13?6 TAD {—5 /“R FORCES RETURN
@2é13 744 SZA; JMP XMAQ
02414 5224
02413 .421 CALL TXOUT;"R;"E;"T;O
02416 0822
02417 6305
62420 0324
02621 6300
02é22 443 CALL CRLF
62623 5600 JN? I XMANUA
0242A 1375 XNAZ, TAD (—61 /CLOSE
02425 7440 SZA; JMP XMAS
92426 5240
0242? 4421 CALL TXOUT;"C;"Lz”0;"S:"E;0
O2é30 0303
@2431 0314
02432 OBI?
02438 0323
@243% @365
02433 098
@2436 @501 CLOSE
@243? 52G1 JMP XMANUA+1

 

/+3 FREQUENTLY CALLED SUBROUTINES

BEL-‘83 ,

XI'iA4 ,

XMAS .

XI‘fAé .

XI'IA'? ,

 

025§s 0323
02526 OSLO

187

TAD (“'1
SZA; JI‘E’ KIM-’3‘

CALL TXOUT; “D: "/; "A;0

CALL DEC IN; 0
TAD . - 1

SETDA

J PIP Y‘mNUAfi 1
TAD (- l

SZA; J11? XMAS

CALL TXOUT; "E: “ 1', "P; "T; "Y;0

EMPTY

JMP XI‘L’XNUA+1
TAD -1 /F
SZA; JI'i? X11111}

CALL TXOUT; "F; "I; "L; "L;0

SZA; JI‘IP XI’UL7
CALL TXOUT; "O; “P; “E; “N:0

OPEN

JI‘IP XMANUA+1
TAD (-1
SZA;JI‘1P XMAS

CALL TXOUT; "P: 'U; "S: "H;0

PALS-VIGA 12—JUL—78 PAGE 29

 

/+3

02527
02530
02531
02532
02533
02534
02535
02536
02537
02540
02541
02542
02543
02544
02545
02546
02547
02550
02551
02552
02553
02554
02555
02556
02557
02560
02561
02562
02563
02564
02565
02566
02567

02571
02572
02573
02574
02575
02576
02577

FREQUENTLY CALLED SUBHOUTINES

0000
6504
5201
1372
7440
5346
4421
0322
0305
0323
0305
0324
0000
6506
5201
1371
7440
5201
442

0301
0257
0804
0240
0275
0240
0000
6510
6511
5362
6512
7040
4426
5201

@021
7776
7767
7777
7717
7773
7563
2600

XMAS,

XMA9.

 

188

PUSH

JHP XMANUA+1

TAD (-2 /R
SZA; JMP XHA9

CALL TXDUT;"R;"E;"S;"E;"T;0

RESET

JHP XMANUA+1

TAD (21 [A
SZA; JHP XNANUA+1

WRI'I‘E; "A; ll/; "D; II ;ll=;ll :0

CONVRT

SCD

JMP .-1
GETAD

CHA

CALL DECOUT
JNP XMANUA+1

PAGE

PALS-V10A 12-JUL-78 PAGE 29-1

“_.._1_.________.

 

/+3

02600
02601
02602
02603
02604
02605
02606
0260?
02610
02611
02612
02613
02614
02615
02616
02617
02620
02621
02622
02623
02624
02625
02626
0262?
02630
02631
02632
02633
02634
02635
02636
02637
02640
02641
02642
02643
02644

FREQUENTLY CALLED SUBROUTINES

0000
7440
5245
4434
4421
0207
0317
0316
0240
0314
0311
0316
0305
0000
4434
7200
6031
5236
6036
1377
7450
5776
1375
7450
5600
1374
6146
6141
5233
5217
6131
5217
6136
6046
6041

242
5217

1139

/MONITOR
/TRANSPABENT MODE AND ERROR HANDLING

XMONIT. 0
SZA; JMP ERROR

XMKB,

XMINI.

CR
CALL TXOUT:207;"0;«N;u

CALL CRLP
CLA

KSF

JMP XMINI
KRB
TAD
SNA
JMP I (200
TAD (-21
SEA
JMP
TAD
6146
6141
JHP ."l
JHP XMKB
6131

JHP XNKB
6136

TLS

TSF

.113" ."l
J”? XNKB

(~201

I XMONIT

(222 /BESTORE CHAR

PALS-V10A

/CALL MONITR (AC<>0 MEANS ERROR);

12-JUL-78 PAGE 30

BET

;IIL: "I; "N; "Ego

/“A FROM KB CHAINS TO $200

/“R FROM KB FORCES RETURN

 

/+3

@2645
02646
92647
02650
@2651
02632
02653
02634
02655
02656
62057
02660

02661
02662
02663
@2664
02665
@2666
0266?

02670
02671
92672
@2673
026?4
02675
026?6
@267?
02700
@2701
02702
02703
02704
02703
02766
02?0?
02710
02711
02712
02713
02714
02715
02716
02717

FREQUENTLY CALLED SUBBOUTINES

7901
7450
5270
7§Ol
7450
539?
7961
7450
5326
7031
7430
5346

4421
0325
03?5
0249
@399
@090

5'9’3
H'U’

4421
=323
0325
0315
@240
@317
032
896
631%
@240
0300
0000
IZQO
4425
5203
4421
0301
6326
0397
0249
@317
0826
0366
0314

/ERROR CODES:

-l
-2
~3
_4.

\\\\\\

ERROR,

IAC
SNA;

IAC
SNA;

IAC
SKA;

IAC
SNA;

19o

PAL8-Vl@A lZ-JUL-78 PAGE 31

TOO MANY ARCS TO AVEHACER
AVERAGE OVERFLOH

DATA TABLE OVERFLOW

SYNC ERROR.ON TRANSMISSION

JHP XADOVF
JMP XAVOVF
J U? EAO‘V'

JMP XSYFC

/
/CHECK FOR OTHER ERROR ENTRIES HERE

/

XADOVF.

CALL TXOUT;"U;”E;"

;"@;0

JMP XMDNIT+3

CALL TXOUT; "S;"U; "31;" 3 "0; "V; n ‘ 3 "L: n ;n@;0

TAD XMDNIT

CALL OCTOUT

JHP XMONIT+3

XAVOVF, CALL TXDUT;“A;"V;“G;“ ;"O:“V;"F;"L;" 3"@;9

 

/+3

02720
02721
02722
02723
02724
02725
02726
02727
02730
02731

02732

02733
02734
02735
02736
02737
02740
02741
02742
02743
02744
02745
02746
02747
02750
02751
02752
02753
02754
02755
02756
02757
02760
02761

02774
02775
02776
02777

191

FREQUENTLY CALLED SUBROUTINES PALB-V10A 12-JUL-78 PAGE 31-1

0240
0300
0000
1200
4425
5203
4421
0304
0301
0324
0301
0240
0317
0326
0306
0314
0240
0300
0000
1200
4425
5203
4421
0323
0331
0316
0303
0240
0305
0322
0322
0240
0300
0000

0222
7757
0200
7577
3000

3000

XDAOVF,

XSYNC,

TAD XMONET

CALL OCTOUT

JNP XHDNIT+3

WRITE; ”D;”A:"T;"A;' :‘O;"V;"F;"L;" ;"@;0

TAD XMONET

CALL OCTOUT

JHP XHONIT%3

WHITE; ".8; "Y; "N; VICg n ; "E; "R; "FL; II ;Iv@;0

PAGE
DATAT:.

 

 

 

/+3

ALIST
AVADD
AVCLR
AVCNT
AVDDNE
AVER
AVLOOP
A1

A11

A2

A3

A4

A5

A6

A7

A8

BORROW -

CALL
}SZ
CLOSE
CCHSVIYT
CR
CHLF
CHUE‘I
CSAEP
C V} LT
C YC
CYCLE
DAOYF
DAT
DATAB
DATA’I‘
DECIN
DECOUT
DELAY
DIG
DIGI
D102
D103
D1G4
DIVD
EMPTY
EQTIHE
ERROR
FILL
GETAD
GETD
GETSEC
GETSR
HDIV
HSUM
11517122
IDATA
IEER
IFILLT

192

FREQUENTLY CALLED SUBBOUTINES

0645
0036
0035
2324
2327
0037
2274

1274
6301
6510
4434
0384
0153
0151
1233
0600
0044
1053
0156
0162
3000
0020
0026
0040
1526
1331
1332
1333
1334
2321
6502
0155
2645
6503
6512
1220
0406
6507
0027
2322
2325
0045
1322
0002

IFLAG
ISAHP
ISAMPT
LST
LSUM
L1
L2
L3
L4
MANUAL
HDARK
NDIVS
MONITR
M6
NOLIST
N OUT
N4
OCT
OCTOUT
ODA’I‘A
OPEN
PER
PUSH
P400
QUOT
READ
REF
RESET
RTEME
RUN
RUNL
BUNK
SAM?
SAMPL
SAHPLE
SANPX
SCALE
SCD
SERRI
SEPBZ
SETDA
SFS
SLOOP
SSCALE
START
STO
TEMP
TNUM
TRUNS
TSAMP
TXOUT
TYPE
WRITE
XADOVF
XAVADD

0161
1750
0005
0047
2323
1234
1245
1256
1267
0042
0157
2320
0041
0770
1000
0024
1327
1330
0025

DA
\I‘?

6500
0467
6504
0400
2326
0023
0160
6506
0154
0527
0731
1037
0523
0717
0033
1084
0043
6511
2223
2226
6513
6505
2207
0522
0200
1223
1525
1517
0152
0150
0021
0022
4421
2670
2250

XAVCLR
XAVCT
XAVER
XAVOVF
XAVEET
XCOUNT
XC BL!“
XCV
XCYCLE
XDAOVF
XDARK
XDCI
X582
XDECIN
XDECGU
XDELAY
X338
EDGE
REDS
XHDIV
XHDEET
XHLBOP
"SUH
XIDATA
XIDIH
XIUP
XIDTEM
XIDWI
XIDVZ
HIKE
XLST
XLSUH
KHAN
iANUA
XHAZ
EMA?
Xflié
XMflS
XNAG
XNfi7
XMAS
XHA9
XHCI
XNCZ
XHINI
XMKB
XHON
XHDNTT
XNOUT
XNSEC
X0
XOCTOU
XODATA
XODTEM
XODW l

PAL8-V10A

2242
0033
2362
2707
2301
2351
2231
1310
2120
2726
2301
2366
2370
1200
1450
2(33
1511
1462
1557
152?
1556
1534
0930
1631
1670
1660
1667
1633
1671
1702
1345
0031
1622
2430
2424
2440
2455
2471
2504
2517
2532
2545
2355
2367
2636
2617
1614
2600
1416
2364
1447
1423
1707
1752
1711

X01
XOOUT
XOPR
XREAD
XREF
XRRET
XSAYPL
XSCALE
XSCRET
XSEC
XSYNC
XTXOUT
XTYPE

12-JUL-78 PAGE

1745
1737
1717
1600
2645
1630
2200
2600
12116
2843
2?46
1335
1463

32

 

 

APPENDIX B

A Stopped-Flow Clinical Analyzer in Which
Innobilized Enzyme Reaction Loops are Usod

Reprinted with permission from Clinical Chemistrz.
Vol. 23, Page 1033, June. 1977. Copyright 1977
by the American Association of Clinical Chenists.

 

 

Reprinted from CLINICAL CHEMISTRY. 23, 1033 (1977).] . ‘
Copyright 1 77 by the American Association ofClinical Chemists and reprinted by permissron ol the co

193

pyright owner.

A Stopped-Flow Clinical Analyzer in Which Immobilized-Enzyme

Reaction Loops Are Used

Martin 0. Joseph. Daniel J. Kasprzak, and S. R. Crouch‘

A stepped-flow clinical analyzer is described that makes
use of a reaction loop containing immobilized enzyme(s)
tor the determination of the analyte/substrate. The analyzer
has been evaluated by determining glucose with immo-
bilized glucose oxidase. The stopped-flow mixing system
was constructed at a current cost of less than $500. The
analyzer separates the enzymatic reaction from a followup.
spectrOphotometric indicator reaction. This separation
allows the enzymatic reaction to be used in either a
fixed-time. kinetic mode or in an equilibrium mode. Like-
wise. the indicator reaction can be used in either mode.
Results lOr glucose in blood serum indicate that good
precision and accuracy can be obtained.

In recent years immobilized enzymes have become
increasingly useful as specific catalysts for determining
clinically important substrates (1-4). Immobilized en~
zymes are attractive as reagents in the clinical labora~
tory because they possess the usual specificity and
sensitivity of soluble enzymes and can be re-used, often
in hundreds or thousands of determinations, thus
greatly reducing the cost of routine use of an enzymatic
kinetic or equilibrium method. In addition, immobilized
enzymes are often more stable than they are in solution;
calibration is thus simpler and less frequently re-
quired.

In this report, a stopped-flow clinical analyzer is de-
scribed that combines the advantages of immobilized
enzymes with the speed, mixing efficiency, and ease of
automation of the stopped-flow mixing technique. The
system utilizes a simple. low-cost. stopped-flow sam-
pling and mixing unit together with a reaction loop that
contains the immobilized enzyme. The reaction 100p is
similar in principle to the sample loop described by
Pardue e‘. a). (5, 6) except that the enzyme-catalyzed
reaction occurs in the loop. After a suitable incubation
period in the loop. the sample solution is rapidly mixed
by the stopped-flow unit with any desired reagent(s)
and sent to an observation cell for spectrophotometric
monitoring of the appropriate indicator reaction. The
separation of the enzyme~catalyzed reaction from the

Department of Chemistry. Michigan State University, East Lan—
sing. Mich. 48824

' To whom reprint requests should be addressed.

Received Feb. 28. 1 77; accepted Mar. 30, 1977.

 

indicator reaction provides extreme versatility. The
enzyme-catalyzed reaction can be allowed to go to
completion (equilibrium method) or it can be incubated
for only a short. fixed time interval (kinetic method).
Likewise. either the reaction rate or the equilibrium
absorbance of the spectrophotometric indicator reaction
can be used in the final measurement. Because of
stopped-flow mixing. measurements can be made on
time scales ranging from milliseconds to hours.

The semi-automated stopped-flow clinical analyzer
described below illustrates the measurement principle
and chemical flexibility of the system. We evaluated the
analyzer by determining glucose in blood serum with a
reaction loop containing immobilized glucose oxidase
(EC 1.1.3.4). Possible extensions ofthe system to mul-
tiple determinations of the same substrate or to near-
simultaneous determinations of multiple substrates are
discussed.

Materials and Methods
Instrumentation

The instrumentation we used to determine glucose
is schematically represented in Figure 1. This system,
except for the enzyme reaction loop. was constructed
completely from commercially available items. Two
l-ml gas syringes with threaded ends, A and B in Figure
1 (from Glenco Scientific, Houston, Tex. 77007), are
used to deliver indicator reagent and push liquid, re-
spectively. Valves 1 through 4 are 0.060-inch bore,
three-way slider valves complete with accuator-return
mechanisms C and D and solenoid controllers 82 and
83 (from Altex, Berkeley, Calif. 94710). The syringes are
driven by a 2-inch stroke pneumatic cylinder, PC, with
solenoid controller 8, (Scovill, Wake Forest, N. C.
27587). Solutions are mixed by forcing them through E,
a KEL-F tee (Altex). The indicator reaction is moni-
tored in a 250-ul micro flow cell (F) with lO-mm light
path (Thomas, Philadelphia, Pa. 19105). The flow cell
is fitted into a single-beam uv-visible spectrophotom-
eter (GCA McPherson EU-700, Acton, Mass. 01720). All
tubing except for the enzyme reaction loop is 1.5 mm
id, 3 mm o.d. Teflon with apprOpriate connector fit-
tings (Altex).

CLINICAL CHEMISTRY. Vol. 23. No. 6. 1977 1033

 

 

 

 

 

 

 

  
 

 

 

 

 

 

 

<9 SAMPLE
ENZYME
REACTION
@ -------------- LOOP
= VACUUM
s
__ wasrs
G J
F
seecraoeaoromerea

 

 

 

Fig. 1. Diagram of stOpped-tiow clinical analyzer

The heart of the system is the enzyme reaction loop,
prepared as described below and fitted with plastic
high-pressure liquid chromatography connectors. The
enzymatic reaction that occurs in the loop for the de-
termination of glucose is:

1
Glucose + 02 + H20 555;: gluconic acid + H202

The extent of this reaction is determined outside the
loop by allowing the H202 produced to mix with and
rapidly oxidize iodide to triiodide in the presence of a
molybdate catalyst (7). The indicator reaction is:

M (V1)
14202 + 31~ + 2H+ —°—» 121-120 H;

Triiodide, measured spectrophotometrically at 365 nm,
indicates the concentration of glucose in the sample.
The indicator reaction is followed by an equilibrium
method in this case, because the absorbance of triiodide
is measured at equilibrium. The enzymatic reaction,
however, is of the fixed-time reaction-rate type. The
enzymatic reaction time is fixed by the incubation time
of the sample in the loop. This time is kept short (2 min)
to ensure that the enzymatic reaction is pseudo-zero
order. This combination of reaction-monitoring meth-
ods exemplifies the flexibility of the system.

The actual function of the enzyme reaction loop in the
system is best described in terms of a typical sequence
of events for the determination of glucose in a sam—
p e.

First, syringes A and B are filled with KI/molybdate

1034 CLINICAL Cl‘EMlSTRY, Vol. 23. No. 6, 1977

 

194

reagent and a push liquid (phosphate buffer), respec-
tively, by simultaneous switching of valves 1 and 2 to
the fill position and retraction of PC. Valves 1 and 2 are
then switched to the “push-ready” position, as are
valves 3 and 4 (positions shown in Figure 1). Next, PC
is caused to push the contents of A and B through the
system lines and out to the waste. This is repeated
several times to remove air bubbles and fill all lines with
solution. With A and B filled and valves 1 and 2 ready,
valves 3 and 4 are simultaneously switched to the fill
position, and a buffered glucose sample is aspirated into
the enzyme reaction loop by a gentle negative pressure.
Then valves 3 and 4 are switched to the “push-ready"
position, which terminates sample introduction. The
sample is incubated for a controlled time until PC is
caused to push the contents of the enzyme loop through

the mixer. Here the H202 produced by the enzymatic
reaction is rapidly mixed with the KI/molybdate re-

agent, and the mixture is transported to the flow cell for

observation and detection of 13". The flow is stopped

upstream when the drive syringes reach the ends of their

travel.

The system described above was constructed at a cost
of less than $500, not including the specific flow cell and
spectrophotometer mentioned here, for which there are
suitable substitutes. The instrument can be assembled
within a day or two due to the ease with which the valves
and tubing can be connected via plastic fittings rated
at 3450 kPa (500 psi). The simplicity, low cost, and
versatility of this system make it an attractive analyzer
for clinical determinations.

Reagents

Buffer. A 0.2 mol/liter solution of dibasic potassium
phosphate in de-ionized water was adjusted to pH 6.30
:i: 0.02 by use of a Heath pH meter (Model EU-302A)
by adding concentrated hydrochloric acid. This buffer
was then used to prepare all other solutions except
where noted.

Indicator reagents. A sodium molybdate stock so-
lution, 90 g/liter, was prepared and allowed to equili-
brate for 48 h. The iodide/molybdate indicator reagent
was then prepared daily by adding a fresh solution of
potassium iodide to molybdate stock, resulting in a so-
lution containing, per liter, 18 g of sodium molybdate
and 0.5 mol of iodide.

Glucose standards. A glucose stock solution was
prepared by dissolving 1.00 g of anhydrous granular
D-glucose and diluting to 1.0 liter with de-ionized water.
This solution was allowed to stand at room temperature
for 24 h to ensure complete mutarotation, and was then
stored at 5 °C. Aqueous glucose standards were then
prepared by appropriately diluting this stock with the
buffer.

Deproteinization reagents. We deproteinized the
serum samples by adding chemically equivalent
amounts of barium hydroxide and zinc sulfate. A bari-
um hydroxide solution (20 g/liter) was titrated with zinc
sulfate (20 g/liter) to a pH of 7 to determine the required
volume ratio of the two reagents.

 

Preparation of serum samples. A control serum
(“Montrol”; Dade Division, American Hospital Supply
Corp., Miami, Fla. 33152) was reconstituted according
to the manufacturer’s instructions, and 0.5 ml of the
solution was pipetted into a centrifuge tube. The
chemically equivalent amounts of barium hydroxide
and zinc sulfate were added, and the contents of the
tube were thoroughly mixed, centrifuged for 3 min, and
1.0 ml of the supernatant fluid was pipetted into 3 ml
of the phosphate buffer. This solution was then drawn
into the enzyme reaction loop for analysis.

Immobilization of glucose oxidase. A procedure re-
ported by lnman and Hornby (8) was modified for im-
mobilizing glucose oxidase on the inner surface of nylon
tubing. 100 cm long and 0.86 mm id. Most of the im-
mobilization steps were accomplished by slowly
pumping the required reagent through the tube with the
use of a Technicon AutoAnalyzer pump to provide a
constant stream of fresh reagent. Amorphous nylon was
first removed by filling the tube with a 200 g/kg solution
of CaClg in methanol and incubating at 50 °C. The
nylon was then mildly hydrolyzed by pumping 1.0
mol/liter HCI through the tube at room temperature,
followed by rinsing with water. Glutaraldehyde, 125
ml/liter in 0.1 mol/liter tris(hydroxymethyl)methyl-
amine buffer, pH 9.2, was then attached to the hydro-
lyzed nylon at 0 °C, followed by rinsing with the buffer.
An 8 g/liter solution of glucose oxidase (Sigma, Type II)
in 0.1 mol/liter phosphate buffer, pH 6.3, was then
pumped through the tube at 0 °C for 4 h in a closed loop,
during which time covalent linkages were formed be-
tween the glutaraldehyde and free amino groups on the
enzyme (8). Finally, the tube was rinsed with 0.1 mol/
liter sodium chloride to remove any physically adsorbed
enzyme. The enzyme reaction loop was filled with buffer
and stored at 5 °C when not in use.

Results and Discussion

The analytical capabilities of the analyzer system
were evaluated by determining glucose in aqueous
samples and in blood serum, with use of a reaction loop
containing immobilized glucose oxidase. The first
generation analyzer we used in these studies was man-
ually sequenced. That is, the solenoid controllers for
each of the valves in Figure 1 were actuated in the ap-
propriate sequence by a series of manual toggle
switches, which applied or disconnected the ac line
voltage from the controllers. The spectrophotometric
readout was obtained from a strip-chart recorder, linear
in transmittance. Thus, the final absorbance values
recorded for 13“ were obtained by converting chart-
recorder readings from transmittance to absorbance.
The temperature of the enzyme reaction loop was not
controlled.

The optimum push volume of the stopped-flow sys-
tem was determined to be 0.78 ml per syringe by filling
a dummy reaction loop with dye (p-nitrophenol). This
volume ensured thorough purging and refilling of the
observation cell, but prevented significant dilution of
the observed solution by the push liquid. The incuba-

195

 

Table 1. Results for Aqueous Glucose Standards

mucosa Absorbance, CV %

conch, Sag/liter 0.00549 2:0
40 0.0610 3.0

60 0.0960 2.1

80 0.127 4.7

100 0.162 1.8

150 0.240 0.8

200 0.314 0-1

250 0.388 1.3

Slope = 1.60 X 10-3 A per mg/liter (CV, 0.24%)
intercept = -6.39 X 10‘3 A
Correl. coelt. (r) = 0.99990

 

tion time for the enzymatic reaction was fixed at 2 min.
Shorter incubation times did not provide adequate
precision because of decreased response and also be-
cause of imprecision in manual sequencing; longer in-
cubation times only added to analysis time without
greatly enhancing the signal-to-noise ratio. Hence,
transmittance was directly read out slightly over 2 min
after the sample was introduced into the reaction
loop.

A calibration curve for glucose was prepared in the
range 20-250 mg/liter by running aqueous standards in
triplicate. Table 1 shows the results. The observed
negative intercept is expected in such a fixed-time
method, because the enzymatic reaction is known to
exhibit an induction period that is inversely propor-
tional to glucose concentration (9), and hence a two-
point calibration should be done periodically. The re-
sults in Table 1 indicate the excellent linearity and
precision which may be obtained with this simple ana-
lyzer. Each determination requires only 1 ml of sample
(about 100 pl of blood serum). The major sources of
imprecision in this simple system appear to be varia-
tions in incubation time due to manual control, fluctu-
ations in reaction rate due to variations in room tem-
perature, and imprecision in reading the transmittance
from the chart recorder.

For determining glucose in blood -serum samples, we
prepared a two-point calibration curve, using the 20 and
200 mg/liter aqueous glucose standards. Two reconsti-
tuted Monitrol samples were analyzed, and the results
(Table 2) agree well with the reported values. Note that
the first two values reported for sample 1 were obtained
on a different day from the second two values, although
the same two-point calibration was used. Clearly, be-
cause of the high stability of the immobilized enzyme
l00p, the same calibration curve may be useful for sev-
eral days. A reaction loop prepared earlier than the one

used in these determinations showed excellent activity
for six months before the enzyme was de-activated in-
advertently by incubation with a highly acidic solution.
Research is underway to determine more precisely the
activity loss of immobilized enzyme loops with time
under various conditions of storage and use.

CLINICAL Cl'EMISTRY. Vol. 23. No. 6. 1977 1035

 

 

 

196

 

Table 2. Results of Determination of Glucose in

Serum
Glucoee conch. rug/liter
Determined Reported Difference ‘3

Sample 1

690 2.8

725 2.1

703 690. ° 730° 0.9

673 5.2
Sample 2

2006 2090 b 4.0

2078 0.6

' % difference from the av reported value
° Beckman "Glucose Analyzer“
c American Monitor "Programachem"

 

In this study we have demonstrated the potential of
the analyzer for the determination of glucose in serum
”samples. Although the system was evaluated by using
one particular enzyme/substrate couple, many other
clinically useful enzyme/substrate couples are obviously
applicable. Versatility is inherent, not only in the en-
zyme chosen for the reaction loop, but also in the re-
agent chosen for the indicator reaction. Furthermore,
any of several combinations of methods (kinetic or
equilibrium) may be used to follow the separate enzy-
matic and indicator reactions. We are now undertaking
investigations on the capabilities of this new system in
the clinical laboratory. New immobilization techniques
are being studied for bonding other, more expensive
enzymes to various inert supports. The multiplexing
potential inherent in the system will be used, to allow
several reaction loops containing the same or different

1030 CLINICAL CI'EMISTRY. Vol. 23. No. 6, 1977

 

enzymes to be used nearly simultaneously, thereby
minimizing the time lost in incubation and maximizing
the use of one detection system. The entire system wrll
then be automated by interfacing it to a minicomputer
or microprocessor for sample handling, instrument
control, and data acquisition and subsequent process-
ing. A more extensive paper describing this work will be
submitted at a later date.

We thank Mr. Robert Lantz for his initial work on immobilization
procedures and Mr. F. J. Holler for his assistance in constructing the
stopped-flow apparatus. This work was supported in part by National
Science Foundation grant No. CHE76-81203.

References

l. Weetall, H. H., Immobilized enzymes: Analytical applications.
Anal. Chem. 46. 602A (1974).

2. Bowers, L. D.. and Carr, P. W.. Applications of immobilized en-
zymes in analytical chemistry. Anal. Chem. 48, 544A (1976).

3. Leon. L. P., Narayanan. S., Dellenbach. R., and Horvath. C.. [.m-
mobilized glucose oxidase used in the continuous-flow determination
of serum glucose. Clin. Chem. 22, 1017 (1976).

4. Bowers, L. D.. and Carr, P. W.. An immobilized-enzyme flow-
enthalpimetric analyzer: Application to glucose determination by
direct phosphorylation catalyzed by hexokinase. Clin. Chem. 2, 1427
(1976).

5. Sanderson, D.. Bittikofer, J. A., and Pardue, H. 1... Computer
controlled stopped-flow studies—application to simultaneous kinetic
analyses. Anal. Chem. 44,1934 (1972).

6. Mieling, G. 13., Taylor, R. W.. Hargis, L. D.. et al.. Fully automated

stopped-flow studies with a hierarchical computer-controlled system.

Anal. Chem. 48,1686(1976).

7. Malmstadt, H. V.. and Pardue, H. L.. Quantitative analysis by an

automatic potentiometric reaction rate method. Anal. Chem. 33, 1040

(1961).

8. Inman, D. J., and Hornby, W. F... The immobilization of enzymes

on nylon structures and their use in automated analysis. Biochem.

J. 129, 255 (1972).

9. Malmstadt, H. V.. and Crouch. S. R.. Systems for automatic direct

readout of rate data. J. Chem. Educ. 43, 340 (1966).

 

     

 

RIES

Ill

Lilli)illlllllllll

   

“"‘liliii