Affinity-Driven Two-Step Immobilization of Endogenous Natural GABAA Receptor to Quantify Drug–Protein Analysis with Enhanced Sensitivity and Accuracy

Immobilized proteins have been conceived as promising for developing assays, including biosensors and chromatographic methods. Conventional efforts are geared toward the attachment of purified or recombinant protein, while lacking strategies for capturing endogenous protein in live cells. Herein, we developed a rapid, two-step method for the target- and site-selective immobilization of endogenous natural γ-amino butyric acid receptors (GABA_AR) directly from live hippocampal neuronal cells (HT22 cells). The method employed a ligand-tethered reagent as a recognition motif and a derivative of acyl-imidazole as an alkyne donor, which selectively introduces an alkyne click handle into specific sites of GABA_AR. Subsequent click reaction between the alkyne-protein and azide-modified dye or support surface enables visualization of the receptor in live cells, as well as fabrication of GABA_AR-immobilized surface plasmon resonance (SPR) sensor chips or affinity chromatography stationary phases. Compared to a typical affinity-driven one-step capture method, this approach significantly shortens the immobilization time from 6 h to 15 min while preserving receptor functionality. The immobilized endogenous GABA_AR was successfully applied in two analytical platforms: as an SPR sensor chip for enhancing sensitivity in revealing drug-receptor interactions, and as a stationary phase in affinity chromatography, where it markedly improved resolution, selectivity, and accuracy in analyzing drug-receptor interactions. Such unique features indicate that the two-step strategy may be a versatile tool for modifying endogenous natural proteins in live cells while minimizing the influence on their functions. It has great potential to facilitate the construction of affinity analytical assays using immobilized endogenous proteins.