Strategy for Allosteric Analysis Based on Protein-Patterned Stationary Phase in Microfluidic Chip

Abstract

An effective method is presented for the on-chip analysis of chiral interactions with a successful depression of nonspecific adsorption. The alumina gel-derived protein network on poly(methyl methacrylate) (PMMA) microchannel was explored to form a protein-stationary phase and then used to carry out electrophoresis for fast enantioseparation coupled with electrochemical detection. On the basis of the chemical modification of a synthesized copolymer containing silane-functionalized scaffold, alumina sol-gel could react readily with the silane groups and form steady microstructure on the chip surface achieving the encapsulation of functional biomolecules. Compared with the native PMMA microchannels, the modified surfaces exhibited much better wettability, more stable and enhanced electroosmotic mobility, and less nonspecific adsorption. The water contact angle and EOF of alumina-gel-derived PMMA substrate were 22 degrees and 4.3 x 10(-4) cm(2) V(-1) s(-1), compared to those of 73 degrees and 1.9 x 10(-4) cm(2) V(-1) s(-1) from the untreated one, respectively. Bovine serum albumin, acting as a target protein, could be stably and homogeneously immobilized in the modified PMMA microchannel to fabricate a protein-stationary phase. Under a mild condition, D- and L-tryptophan were efficiently separated with a resolution of 1.57. The as-prepared microchip can perform chiral separations within short time, indicating that the general protocol has the potential to provide a platform for high throughput screening of enantiomer candidates such as those biochemical drugs with protein targets and the research of receptor interactions.

Enantiomeric separation by microchip electrophoresis using bovine serum albumin conjugated magnetic core‐shell Fe3O4@Au nanocomposites as stationary phase

“…The resolution factor (Rs) was about 1.67, which is much better than that of 1.12 on BSA-encapsulated tetramethoxysilane-based hydrogel matrixes [48] and 1.35 on single-walled carbon nanotubes conjugated BSA stationary phase [27]. It is worth mentioning that the peak area and peak height of D-tryptophan are larger than that of L-tryptophan though the enantiomer ratio of D-and L-tryptophan is 1:2 [17,27]. Although the reason is still not exactly understood up to now, we presume that the reason for the different signal between D-tryptophan and L-tryptophan might be attributed to two aspects: one is the adsorption and electrode fouling from the irreversible electrochemical oxidation of D-tryptophan, which lead to reduce the effective surface area of the working electrode and obstruct the following electron transfer of L-tryptophan [49], the other is the lower electroactivity of L-tryptophan than D-tryptophan.…”

Fabrication, modification, and application of poly(methyl methacrylate) microfluidic chips

“…Recently, an alumina gel-derived network of BSA in PMMA microchannel was investigated to form a proteinstationary phase and then used to carry out electrophoresis for fast enantioseparation of DL-tryptophan coupled with amperometric detection [85]. Weng et al [86] have established a novel method of chiral separation based on proteinstationary phase immobilized in a PMMA microfluidic chip.…”

A Smart Surface in a Microfluidic Chip for Controlled Protein Separation

“…The channel plate began as a 4 2.5 cm PMMA substrate and was hot-imprinted with a micromachined silicon template to introduce a microchannel with about 50 mm depth and 135 mm width to the middle of the substrate. [39,40] Then, a sputtering process was implemented to cover the microchannel with gold as the base of the surface modification, and also the gold-modified microchannel surface would act as the working electrode in the subsequent electrochemistry experiments. Before the sputtering process, a plastic mask was positioned on the channel plate to only expose the desired regions including the microchannel and a 0.5 2.5 cm region at one end of the channel plate (the shadow regions in Scheme 2).…”

“…[41] At last, the two components, cover sheet and channel plate, were thermally bound together. [39,40] Synthesis of the IC and modification of the microchannel surface: The IC between MHA and b-CD was synthesized by following the method described in the literature. [20,21] MHA (20 mg) was added to an aqueous solution of b-CD (315 mg) in purified water (20 mL) with a molar ratio of 1:4 (excess b-CD was used to ensure that the majority of MHA in solution was bound by CD molecules).…”