(61c) Novel Electrochemical Biosensor Using Small Binding Proteins for Early Detection of Aggressive Disease

Detecting aggressive disease in preliminary stages can improve the chances of survival of patients. Combining early detection with continuously monitoring the level of the relevant biomarkers plays a pivotal role in establishing personalization in the future of medical treatment. Typical concentrations of prostate-specific antigen (PSA) biomarker in a healthy person are found to be within 14 - 60 pM and between 300 pM - 30 nM in advanced stages of prostate cancer. With the current sensing or diagnostic tools, it is extremely difficult to detect such biomarkers concentrations in the initial stages which lie in the range of 10 pM to 100 pM. Though biomarker detection provides a dynamic and powerful approach for diagnosis especially due to its reliability and the insight it provides for studying the disease mechanism, it is coupled with limitations related to cost-effectiveness, proper storage infrastructure, and time sensitivity. Prevalent biosensors use aptamers, antibodies, or enzymes as the bio-recognition element to target relevant biomarkers. These serve as the predominant point-of-care diagnostic tools, yet are often limited by factors such as complexity, stability, and reproducibility. Advances in proteomics and computational protein engineering have highlighted the potential of improving the early diagnosis of complex disease by using integrated data from a range of biomarker detections. For instance, if levels of multiple relevant biomarkers are monitored and taken into consideration for determining the stage and trajectory of the disease, the confidence in prediction would be greater than separately relying on individual biomarkers. The need to develop an electrochemical biosensing platform that will overcome these barriers and enable monitoring of various biomarkers with remarkable sensitivity in the lower picomolar range are the driving factors for this project.

The chief components of the novel biosensor include two small engineered binding proteins, an electrochemically active enzyme, and an electrode. The tandem binding proteins are connected via a long, flexible peptide linker which allows them to simultaneously bind to a biomarker of interest with high affinity. The reactive enzyme is covalently attached to the C-terminus of the tandem binders, while the electrode surface anchors the N-terminus of this protein assembly (tandem binders and enzyme). The design is such that only when a target biomarker is simultaneously engaged by both binding proteins, the reactive enzyme is pulled into close proximity to the surface of the electrode, triggering an electrochemical response that can be converted to a readable signal using voltammetry. To reduce false-positive signals, the size of the linker is optimized such that the reactive enzyme remains distant from the electrode surface while the target is not being engaged. Our method employs small protein binders that are predicted to tightly bind the target at multiple, non-competing sites from a library of known human fibronectin variants using a combination of computational tools for homology modeling (Swiss-Model) and high-resolution docking (PyRosetta). The gene fragments of the selected variants of fibronectin were used for protein production followed by characterizing the binding interaction using flow cytometry and chromatography. Two engineered binders, verified to simultaneously bind the target, are fused using a long flexible linker whose length is optimized to minimize background signal without compromising the sensitivity towards binding interactions. Future directions of the project will aim in creating an array of microelectrodes capable of detecting several unique biomarkers simultaneously, thus rendering a non-invasive, real-time, advanced diagnostic tool capable of constant monitoring and treatment.