The burden of poor prognosis and high mortality rates associated with complex and aggressive diseases can be reduced with early detection. Biomarker sensing provides a dynamic approach to early diagnosis. However, the lack of a single diagnostic biomarker that can be correlated to a specific disease, there is a need to create a biosensing platform that can detect multiple targets which vary in size and complexity. To address the need to use stable biorecognition elements for sensing, we have explored the utility of synthetic binding proteins, which are like antibodies in function, except much smaller in size. Small synthetic proteins derived from human fibronectin, also known as monobodies, can act as powerful and highly modular biorecognition elements. Using computational tools such as homology modeling and protein-protein docking, we have identified monobodies with a unique chemistry that have strong binding affinity for specific targets of interest. In this work, we have developed an innovative electrochemical biosensor that harnesses the modularity of monobodies for the detection of large biomolecules. We used lysozyme as our model target due to its clinical relevance, cost efficiency, and ease of availability. As these monobodies cannot inherently generate any signal on binding with the target, we have functionalized them using NHS-EDC chemistry and electrochemically grafted them on the surface of the electrode. These modifications help generate a readable signal when the biosensor comes into contact with the target of interest. Immobilization of the monobodies on the surface of the electrode has created a non-conductive layer that impedes electron transfer, thus enabling the selective detection of target molecules. Our findings indicate that this biosensor exhibits high specificity, negligible non-specific adsorption, and exceptional electrical stability, making it a promising tool for accurate biomolecule detection in complex physiological fluids like serum. This method offers the potential for multiplexing, enabling the creation of a versatile, adjustable biosensor that can support more accurate prognosis through detecting a range of disease-related biomarkers. The development of this novel protein-electrode interface opens exciting possibilities for improving the performance and reliability of portable diagnostic devices, with significant implications for clinical and analytical applications.
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