Immobilized G protein-coupled receptor is a versatile tool to study ligand-receptor interactions. In this work, we synthesized the immobilized alpha 1A adrenergic receptor (α 1A -AR), a GPCR subtype mediating smooth muscle contraction, through a site-selective covalent method that relies on the reaction between haloalkane dehalogenase tagged α 1A -AR and macroporous silica gel coated with 6-chlorohexanoic acid. To investigate thermodynamic and extra-thermodynamic parameters for ligand binding, we utilized the covalently immobilized receptor as stationary phase to perform frontal analysis and injection-amount dependent analysis as well as compared with the random immobilization method. Terazosin gave the association constant of 1.48 × 10 5 M −1 to α 1A -AR, indicating that the oriented immobilization of α 1A -AR enhances the ligand-binding activity by one order of magnitude in comparison with the random immobilization method (7.9 × 10 4 M −1 ). The binding of phentolamine and tamsulosin to the receptor was accompanied by a large absolute heat capacity (Δ C p ) of 1.28 ± 0.23 kJ mol −1 , demonstrating that the binding enthalpy and entropy appear to compensate for one another. These results indicated that the covalent immobilization of the receptor onto solid support has a profound impact on the ligand-binding activity of the receptor and the determination of ligand-receptor binding parameters. The receptor immobilized through the site-selective method will act as a benchmark for chromatographic determination of binding parameters in ligand-receptor interactions and can be used as an effective approach for rapid analysis of drug-protein interactions with high accuracy.
In this work, a three dimensional (3D) graphene-nitrogen doped carbon nanotubes (G-NCNTs) network was successfully fabricated on the surface of a glassy carbon (GC) electrode using the pulse potential method (PPM) in a graphene oxide-nitrogen doped carbon nanotubes (GO-NCNTs) dispersion. The morphological and characteristics of GO-NCNTs and G-NCNTs nanocomposites were investigated by atomic force microscopy (AFM), scanning electron microscopy (SEM), UV-vis spectroscopy, Raman spectroscopy, and electrochemical experiments. The 3DG-NCNTs network was applied as a new voltammetric material for the fabrication of an electrochemical platform for determination of urapidil. Systematic electrochemical tests demonstrate that the 3DG-NCNTs network modified GC electrode can effectively increase the response to the oxidation of urapidil. Under the optimum conditions, the electrochemical response was linear with urapidil concentrations in the range of 1.0 × 10−8~2.0 × 10−6mol·L−1, while a low detection limit of 5.0 × 10−9mol·L−1was obtained for urapidil. Moreover, the proposed sensing platform exhibited good results for sensitivity, reproducibility, selectivity, and stability, which makes it very suitable for use as an ideal inexpensive and rapid analytical method applicable for complex drug matrices.Keywords:3D graphene;graphene-nitrogen doped carbon nanotubes;pulse potentiostatic method;electrochemical sensor;urapidil
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