Utilizing Engineered Monobodies for the Electrochemical Quantification of Lysozyme

Monobody binding proteins are derived from the Fn3 domain of human fibronectin. These robust proteins can be engineered to bind to a wide range of small molecule-, nucleotide-, or large biomolecule-based analytes with high specificity and stability, thus making them ideal for biosensing applications. Here, we demonstrate an electrochemical biosensor utilizing an engineered monobody as a biorecognition element for the detection of lysozyme as a model biomarker. An engineered monobody binding protein was immobilized onto glassy carbon electrodes through a process of electrochemical grafting to create the sensing interface, while a water-soluble ferrocene derivative was used as an electrochemical indicator. Square wave voltammetry of resulting monobody-modified electrodes revealed a significant decrease in peak current density upon incubation with 24 µM lysozyme (250 ± 20 µA cm-2 decrease in peak current compared to 20 ± 9 µA cm-2 decrease upon incubation with 24 µM bovine serum albumin as a negative control), and the sensor exhibited a linear detection range up to 1 µM lysozyme (with a sensitivity of 129 µA cm-2 µM-1, a limit of quantification of 290 nM and a limit of detection of 87 nM). Measurements taken from lysozyme samples in canine serum indicate that the sensor maintains high specificity and sensitivity in a complex biological medium with small amounts of non-specific adsorption. This work demonstrates significant potential for monobodies to expand the existing toolkit of electrochemical biorecognition elements while enhancing the performance and reliability of portable diagnostic devices.