Development of Reagentless Fluorescence Immunosensors for Continuous Analyte Monitoring

Abstract

Fluorescence-based immunodiagnostics are an emerging field in biosensor development and exhibit several advantages over traditional detection methods. While various affinity biosensors have been developed to generate a fluorescence signal upon sensing varying concentrations of analytes, reagentless, reversible and continuous monitoring of complex biological samples remains challenging. Here, we aimed to genetically engineer biosensors based on single-chain antibody fragments (scFv) that are site-specifically labelled with environmentally sensitive fluorescent unnatural amino acids (UAA). A rational design approach resulted in quantifiable analyte-dependent changes in peak fluorescence emission wavelength and enabled antigen detection in vitro. Incorporation of a polarity indicator in proximity of the antigen binding interface generated a titratable wavelength blueshift with nanomolar detection limits.In order to ensure continuous analyte monitoring, scFv candidates with fast binding and dissociation kinetics were selected from a genetic library employing a high-throughput display and affinity screening approach. Initial rankings were further refined towards rapid dissociation kinetics using bio-layer interferometry (BLI) and surface plasmon resonance (SPR). The most promising candidates were expressed, purified to homogeneity, and tested for their potential to detect varying concentrations of the target antigen in a continuous microfluidics-based assay. Variations of dissociation kinetics within an order of magnitude were achieved without compromising specificity of the antibody fragments. This approach is generally applicable to numerous antibody/antigen combinations and currently awaits integration in a wide range of assay platforms for one-step protein quantification. Figure 1