An Allosteric Assay for Identifying Ligands Binding to β 2 Adrenergic Receptor by Surface-Enhanced Raman Scattering (SERS)-Active Nanoparticles

The development of conventional orthosteric drugs targeting G protein-coupled receptors (GPCRs) is often hindered by off-target effects and low subtype selectivity. Allosteric ligands enable precise control of receptor signaling through selective conformational changes, highlighting their critical importance as a promising therapeutic strategy and as a key frontier in modern drug discovery. To enhance GPCR ligand screening efficacy, surface-enhanced Raman spectroscopy (SERS), with sensitivity and narrow spectral peaks, was employed to outline a methodology based on allosteric aptamer binding and stabilizing conformations of the β 2 adrenergic receptor (β 2 AR) for identifying GPCR ligands by fabricating a sandwich-type SERS sensor. The method involved immobilization of β 2 AR on magnetic plasmonic nanoparticles by label-free click chemistry, probing and stabilization of the allosteric conformational changes by RNA aptamer, and amplification and detection of the signal by SERS-active nanoparticles. The success of the allosteric β 2 AR assay was confirmed through multiple analytical techniques, including SERS, SEM, UV–vis spectroscopy, and particle size analysis. This methodology demonstrates broad applicability, characterized by rapid response, high sensitivity, and excellent selectivity. BI167107 could be detected within a range of 10 –9 –10 –6 M, with a detection limit of 49 pM. Such a sandwich biosensor successfully identified the agonist, antagonist, and allosteric inhibitor of β 2 AR from the ligand complex in both aqueous solution and serum. This method is expected to become an alternative for high-throughput and multiplexed drug screening when it comes to a compound library.