A versatile polydopamine platform for facile preparation of protein stationary phase for chip-based open tubular capillary electrochromatography enantioseparation

• Chip-based OT-CEC with PDA/BSA conjugates as stationary phase was fabricated.
• PDA coating served as a versatile platform for protein stationary phase preparation.
• Good biocompatibility of PDA coatings well maintained the biological activity of BSA.
• This protocol simplified protein stationary phase fabrication of OT-CEC microdevice.

A novel, simple, and economical method for the preparation of chiral stationary phases for chip-based enantioselective open tubular capillary electrochromatography (OT-CEC) using polydopamine (PDA) coating as an adhesive layer was reported for the first time. After the poly(dimethylsiloxane) (PDMS) microfluidic chip was filled with dopamine (DA) solution, PDA film was gradually formed and deposited on the inner wall of microchannel as permanent coating via the oxidation of DA by the oxygen dissolved in the solution. Due to possessing plentiful catechol and amine functional groups, PDA coating can serve as a versatile multifunctional platform for further secondary reactions, leading to tailoring of the coatings for protein bioconjugation by the thiols and amines via Michael addition or Schiff base reactions. Bovine serum albumin (BSA), acting as a target protein, was then stably and homogeneously immobilized in the PDA-coated PDMS microchannel to fabricate a novel protein stationary phase. Compared with the native PDMS microchannels, the modified surfaces exhibited much better wettability, more stable and enhanced electroosmotic mobility, and less nonspecific adsorption. The water contact angle and electroosmotic flow of PDA/BSA-coated PDMS substrate were measured to be 44° and 2.83 × 10−4 cm2 V−1 s−1, compared to those of 112° and 2.10 × 10−4 cm2 V−1 s−1 from the untreated one, respectively. Under a mild condition, d- and l-tryptophan were efficiently separated with a resolution of 1.68 within 130 s utilizing a separation length of 37 mm coupled with in-column amperometric detection on the PDA/BSA-coated PDMS microchips. This present versatile platform, facile conjugation of biomolecules onto microchip surfaces via mussel adhesive protein inspired coatings, may offer new processing strategies to prepare a biomimetic surface design on microfluidic chips, which is promising in high-throughput and complex biological analysis.