Electrochemical biosensors are analytical devices that detect analytes by transforming a biochemical reaction into a quantitative, electrical signal. This class of biosensors has proven valuable in research, quality control, food safety, medical diagnosis, and monitoring of therapeutic efficacy. Electrochemical biosensors integrate the specificity of biological recognition molecules (e.g., antibodies) with the advantages of electrochemical detection techniques (reproducible, quantitative electrical output) to provide sensitive and specific analytical devices. Miniaturized amperometric biosensors that use redox enzymes to generate an electric current in response to the voltage applied at a working electrode have been successfully commercialized.Mechanistic mathematical models that describe the multiple mass-transfer and chemical-reaction steps that give rise to the electrical output are needed to help design, optimize, and validate electrochemical biosensors for medical and environmental applications.In this work, experimental and theoretical studies of two types of multistep electrochemical biosensors were performed. An electrochemical immunosensor (EI) was fabricated on screen-printed electrodes (SPEs) for detection of a model protein (mouse IgG) by integrating principles of an enzyme-labled immunosorbent assay (ELISA) using horseradish peroxidase (HRP) as the labeling enzyme and an electrochemical transducer. Experimental conditions such as substrates concentration, pH, and applied voltage were optimized using a fractional factorial design. A mathematical model was developed to simulate the EI's steady-state signal by solving the non-linear ordinary differential equations including enzyme kinetics and diffusion-based mass transfer rates for all the reactants. A new concept, current-control coefficient, was introduced to measure the extend to each reaction step limited the current density. The model allows the rate limiting step to be identified and experimental conditions that optimize detection sensitivity to be determined.In addition, experimental and theoretical studies of an inhibition-based bi-enzyme electrochemical biosensor (IBE) for a model inhibitor of acetylcholinesterase (AChE), phenylmethylsulfonyl fluoride (PMSF), were conducted. The IBE was fabricated by co-immobilization of AChE and tyrosinase (Tyr) on the gold working electrode of a SPE. The inclusion of a hydrolase enzyme (AChE) and an oxidase enzyme (Tyr) provided an amplification system which improved the biosensor's sensitivity significantly. A comprehensive mathematical model was developed to simulate the time-dependent electrochemical signal in the IBE. The unsteady-state model was developed by solving a system of non-linear partial differential equations including enzymatic reactions, inhibition kinetics of AChE by an inhibitor (PMSF), and diffusion-based mass transfer steps. The model successfully simulated the IBE's response to the substrate (phenylacetate) and the inhibitor. Using the model along with the current-control coefficient and sensitivity parameters, the effect of the governing factors on the IBE's performance was studied. The model allowed to optimize the governing factors to achieve optimum sensitivity for detection of the inhibitor and design the biosensor to achieve specific performance criteria.
Read
