ENHANCED KINETICS ANALYSIS OF PROTEINS AND LARGE BIOMOLECULES USING NOVEL HIGH SENSITIVITY PROBE

Abstract

Abstract Introduction Bio-layer interferometry (BLI) has gained significant interest as a label-free technique for the detection and kinetic analysis of diverse biomolecules such as antibodies, proteins, and small molecules. The technology relies on the phase shift-wavelength correlation generated between interference patterns at the tip of the biosensor probe where molecules associate and dissociate. However, current biosensors face challenges regarding sensitivity with small molecules/peptides and compatibility with large biomolecules like nanomaterials. Traditional BLI often produces inverted signals when nanomaterials bind which hinders accurate kinetics analysis. Overcoming these limitations is crucial for expanding the range of applications and enhancing the performance of BLI-based detection systems. Significance In this study, we have developed an improved BLI sensor, Gator® SA XT, which features newly designed streptavidin-based surface capable of loading biotinylated ligands as small as 1.5 kDa. Compared to traditional BLI streptavidin probes, the SA XT probes exhibit a 3-5 times higher signal intensity. Moreover, the incorporation of a novel optical coating layer enables the detection of large biomolecules such as lipid nanoparticles without signal inversion. This advancement in biosensor technology facilitates the detection of ligands and their analytes at lower concentrations and expands the range of compatible analytes for BLI-based applications. Methods To enhance the sensitivity of the interference patterns, we utilized a proprietary optical coating layer with a refractive index significantly lower than that of proteins and other biomolecules. We assessed the sensitivity and sensing distance of the optical coating layer using a layer-by-layer model system. Binding cycles of biotinylated protein A and human IgG were repeated until the theoretical biolayer thickness reached approximately 700 nm. Results Comparative analysis of binding signals between the newly designed SA XT probes and traditional SA probes were conducted for various biomolecules. The SA XT probes demonstrated significantly higher binding signals for oligos (2.8-fold), peptides (3.0-fold), Protein A (4.1-fold), PDL1 (4.5-fold), and IgG (4.3-fold). Furthermore, the unique optical properties of the SA XT probes prevented signal inversion enabling the detection of biomolecules as large as 2 MDa. Using a layer-by-layer model system, the SA XT probes successfully detected a biolayer thickness of 700nm without signal inversion. Additionally, we demonstrated the detection of lipid nanoparticles and subsequent biomolecule bindings using the SA XT probes. Conclusions In conclusion, we have designed a novel biosensor for BLI that enables the detection of a wider range of biomolecules with high sensitivity. The SA XT probes, coupled with the proprietary optical coating layer, have overcome the limitations of traditional BLI probes and facilitated the generation of reliable and high-quality kinetics data for various applications. This advancement expands the analytical capabilities of researchers and opens new avenues for investigating biomolecular interactions.