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 diluted canine serum indicate that the sensor maintains high specificity and sensitivity in a complex biological medium with small amounts of nonspecific 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.