Improved Electrochemical Biosensors using Engineered Binding Proteins - Project Summary The goal of this project is to develop a new class of electrochemical biosensors that use engineered binding proteins to simplify fabrication and improve performance. Electrochemical sensors have the potential to provide inexpensive, sensitive, quantitative diagnostic assays. However, there are few success stories in commercial electrochemical sensors outside enzymatic systems (e.g. glucometers), particularly in electrochemical immunosensors. Immunoassays provide actionable information about health and a wide array of diseases. Immunosensors use pairs of antibodies rather than an enzyme to recognize targets, and targets are typically present at much lower concentrations than glucose, making the analytical problem more challenging. Impactful, widely used immunoassays such as pregnancy and covid tests have had optical rather than electrochemical readouts. Electrochemical immunosensors would be advantageous, as they can be sensitive and quantitative, avoiding subjective interpretation, and can connect with affordable consumer electronics for longitudinal monitoring of conditions. A major obstacle to progress has been the development of a reproducible electrode modification strategy that can be readily adapted to large- scale manufacturing while maintaining sensitivity and specificity. Current methods for antibody immobilization rely on adsorption, electrostatic deposition/entrapment, and/or covalent tethering. Each method has limitations, such as significant loss of antibody function, difficulty achieving reproducible coatings, and reduced sensitivity due to thick coating layers that diminish electrical signals. In recent years, small, engineered binding proteins have been developed that contain material-binding domains on one side and target binding domains on the opposite side. This approach can generate better sensitivity in shorter assay times, higher stability, and easier fabrication than systems using antibodies and traditional deposition methods. However, the vast majority of these efforts have focused on paper tests with optical detection methods. We hypothesize that engineered binding proteins can be used to enhance the performance of electrochemical biosensors relative to traditional electrode modification procedures for carbon composite and laser-induced graphene electrodes. To test this hypothesis, we will 1) develop immunosensors made using engineered binding proteins that target polystyrene in the electrode on one side and protein A on the other to generate surfaces that properly orient antibodies and 2) develop immunosensors with proteins that bind laser-induced graphene on one side and the target analytes on the other end. The outcome of this project will be a new approach to creating electrochemical immunosensors that is faster and easier than current approaches while generating higher performance.
