Effective Construction of a High-Capacity Boronic Acid Layer on a Quartz Crystal Microbalance Chip for High-Density Antibody Immobilization

Boronic acids (BAs) provide strong potential in orientation immobilization of antibody and the modification method is crucial for efficiency optimization. A highly effective method has been developed for rapid antibody immobilization on gold electrodes through the electrodeposition of a BA–containing linker in this study. Aniline-based BA forms a condense layer while antibody could automatically immobilize on the surface of the electrode. Compare to traditional self-assembled monolayer method, the electrodeposition process dramatically reduces the modification time from days to seconds. It also enhances the immobilized efficiency from 95 to 408 (ng/cm2) with a strong preference being exhibited for shorter aniline-based linkers.

Compound (1-2-h)) can be collected. Yield 90%.  aqueous NaOH (1M,40 mL), the mixture is cooled down to 0°C and stirred for is added dropwise within 10 minutes and the reaction is warm up to room temperature stirred for another 12 hours.    (1-3-b). Yield 90%.
10% wt of Pd/C is gently added into the reaction at room temperature and stirred for a while. Ammonium formate (8 eq) is suspended 2 mL of water and mixed with 18 mL methanol. The mixture is injected into the reaction and raised temperature up to 40 °C for 8 hours. After the verification by TLC and ninhydrin stain, the mixture is cooled down to room temperature and filtered by celite in order to remove Pd/C. Redundant methanol is evaporated under reduced pressure and pure product (1-3-g) is obtained. Yield 75%.
EDC (1.1eq) and Hydroxybenzotriazole (HOBt, 1.5eq) are added into reactant subsequently under dry N2 atmosphere stirred for 2 hours. Dissolved in DMF (10mL), advenced product (1-3-b) is mixed in the reaction with triethylamine (1 eq) as base. After stirred for 12 hours, the reaction can be confirmed by TLC in ninhydrin stain. The resulting mixture is added 1mL into 50 mL centrifuge tube and fulfilled by 0.1M HClaq and centrifugared for 3 times each 20 minutes.
The solid suspension is dissolved in DCM and Methanol solvent mixture.
Column chromatography (DCM /Methanol, 10:1, v/v) gives the pure product can be collected. Yield 90%. Compound (1-4-b) is added to a solution of 100 mL EA with NaI (0.01eq) stirred vigorously for 5 minutes. The mixture is continues stirred for another 30 minutes after adding 1M H2O2. Confirming the reaction, mixture is extracted with EA, H2O and brine. The combined organic layer is dried over anhydrous MgSO4 and filtered, and solvents are evaporated under reduced pressure.
Product (1-4-c) is added into anhydrous dimethylforamide (10 mL) stirred for 10 minutes. Commercial 3-aminophenylboronic acid (1 eq) is sebsequently added into the mixture and stirred for another 10 minutes. Coupling reagent,  Compound (1-5-b) is added to a solution of 100 mL EA with NaI (0.01eq) stirred vigorously for 5 minutes. The mixture is continues stirred for another 30 minutes after adding 1M H2O2. Confirming the reaction, mixture is extracted with EA, H2O and brine. The combined organic layer is dried over anhydrous MgSO4 and filtered, and solvents are evaporated under reduced pressure.
Column chromatography with EA and hexane gives the pure product (1-5-c).