We describe a classroom-ready bio-layer interferometry protocol for upper-level undergraduates that contrasts a macromolecular interaction with a small-molecule interaction on an Octet R8. Biotinylated bovine serum albumin (BSA) is immobilized on Super Streptavidin sensors to enable two assays in parallel: (i) binding of a commercially available anti-BSA IgG (protein-protein) and (ii) binding of a long-chain fatty acid such as palmitate (protein-small molecule). The exercise emphasizes assay design decisions that differ by analyte class-sensor chemistry and loading density, solvent matching and reference subtraction, association/dissociation timing, and the choice of data-analysis pathway (global 1:1 kinetic fitting for IgG vs. steady-state isotherms for fatty acids). Representative data yield nanomolar KD values for BSA-IgG and micromolar apparent KD for BSA-fatty acid, with correspondingly larger response amplitudes and slower off-rates for the protein-protein case. The protocol fits into two 3-h sessions, leverages eight-channel parallel acquisition for efficient titrations, and includes safety notes, optional regeneration to demonstrate sensor reuse, and troubleshooting for non-ideal behaviors (drift, avidity, non-specific binding). Assessment artifacts (rubrics, short-answer prompts, and figure-quality criteria) support measurement of learning outcomes in experimental design and quantitative analysis. Because the same BSA surface underpins both assays, the module is cost effective, reproducible, and readily transferable across institutions, providing a practical framework for teaching how analyte size and mechanism shape biosensor strategy and interpretation.
Wen Chen (2026) studied this question.