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The feasibility of immobilized protein-based biodetection relies critically on the activity of the immobilized proteins as well as the biocompatibility of the protein surface. Although many protein immobilization strategies have been developed with satisfied detection readout signals. Non-specific interactions caused by the protein-coating surface are still of great concern since they often interfere with or affect the reliability of detection. Herein, we developed a highly efficient G protein-coupled receptor (GPCR) immobilization method by the combination of polyethylene glycol (PEG) with a self-labeling enzyme-catalyzed reaction. The immobilization relies on the covalent interaction between the fusion tag of a target GPCR (kinase domain of epidermal growth factor receptor, EGFR) and its covalent inhibitor ibrutinib, which is modified on PEGylated silica gels. Two types of GPCRs, N-methyl-D-aspartate 2 A receptor (NMDAR 2A) and endothelin A receptor (ET A R), were used as examples to realize protein immobilization. The GPCR modified gels and the affinity columns packed with them have been extensively characterized, in terms of non-specific adsorptions, retention factor (k ′), half peak width (W 1/2), tailing factor (T f), theoretical plates (N), and association and dissociation constants of the ligands with the receptors. The immobilized GPCRs with reduced non-specific interactions and enhanced fouling resistance, salt tolerance, and chromatographic performance were clearly observed. We believe it is the first work to introduce PEGylation in GPCR immobilization and provide comprehensive proof-of-concept studies to illustrate the improved antifouling property, salt tolerance, and chromatographic performance. This method could be generally applicable in other immobilized protein-based technology for reliable biodetection. [Display omitted] • We developed a highly efficient GPCR immobilization method based on PEGylation. • Two types of GPCRs, NMDAR 2A and ET A R were applied to analyze the improvement of PEGylation. • PEGylated GPCR columns exhibited improved fouling resistance, salt tolerance, and chromatographic performance. • PEGylation provided more reliable measurements for the receptor-ligand interaction analysis. [ABSTRACT FROM AUTHOR]

• Two receptors were co-immobilized in equimolar amounts by bioorthogonal reaction. • Equations for revealing drug-dual target binding on heterogeneous surface were derived. • The method is reliable, time- and ligand-efficient with improved throughput. Characterization of the drug-target interactions is pivotal throughout the whole procedure of drug development. Most of the current assays, particularly, chromatographic methods lack the capacity to reveal drug adsorption on the muti-target surface. To this end, we derived a reliable and workable mathematical equation for revealing drug bindings to dual targets on the heterogeneous surface starting from the mass balance equation. The derivatization relied on the correlation of drug injection amounts with their retention factors. Experimental validation was performed by determining the binding parameters of three canonical drugs on a heterogeneous surface, which was fabricated by fusing angiotensin receptor type I and type II receptors (AT 1 R and AT 2 R) at the terminuses of circularly permuted HaloTag (cpHaloTag) and immobilizing the whole fusion protein onto 6-bromohexanoic acid modified silica gel. We proved that immobilized AT 1 R-cpHalo-AT 2 R maintained the original ligand- and antibody-binding activities of the two receptors in three weeks. The association constants of valsartan, candesartan, and telmisartan to AT 1 R were (6.26±0.14) × 105, (9.66±0.71) × 105, and (3.17±0.03) × 105 L/mol. In the same column, their association constants to AT 2 R were (1.25±0.04) × 104, (2.30±0.08) × 104, and (8.51±0.06) × 103 L/mol. The patterns of the association constants to AT 1 R/AT 2 R (candesartan>valsartan>telmisartan) were in good line with the data by performing nonlinear chromatography on control columns containing immobilized AT 1 R or AT 2 R alone. This provided proof of the fact that the derivatization allowed the determination of drug bindings on the heterogeneous surface with the utilization of a single series of injections and linear regression. We reasoned that is simple enough to model the bindings of drug adsorption on commercially available adsorbents in fundamental or industrial fields, thus having the potential to become a universal method for analyzing the bindings of a drug to the heterogeneous surface containing multiple targets. [ABSTRACT FROM AUTHOR]