Molecular Imprinting Microfluidics For Chiral Separation

Enantioseparation is of paramount importance since chiral compounds exhibit different properties in biochemical systems. Exploring new enantioseparation techniques with low cost and high efficiency is urgent, and provides huge challenge and opportunity to the researchers and market. Recently, the development of the microfluidic enantioseparation provides feasibility for the miniaturized enantioseparation with short analysis time and low consumption of reagent and sample, showing a promising avenue for high-through enantioscreening of chiral compound. Some microfluidic enantioseparation methods by using enzyme, chromatography and electrophoresis have been developed and showed fast analysis speed. However, molecular imprinting technique (MIT), a well-known means for the preparation of biomimetic recognition matrices using molecularly imprinted polymer (MIP) with good binding affinity, stability and specificity toward the target molecules, has not been used to combine with microfluidic technique yet, although one of the widest applications of MIP as chiral stationary phases for enantioseparation has been developed by coupling with high-performance liquid chromatography and capillary electrochromatography with the predetermined elution order. This dissertation first introduced MIT into microfluidic analysis for the enantioseparation of chiral compounds, and four methods were proposed for microfluidic enantioseparation:1. Enantioseparation and amperometric detection of chiral compounds by in situ molecular imprinting on the microchannel wallThe MIT was first introduced into the microchannel of a microfluidic device (MD) to form in situ the imprinted polymer for fast enantioseparation of chiral compounds. The MIP was in situ chemically polymerized on the microchannel wall using acrylamide as the functional monomer and ethylene glycol dimethacrylate (EDMA) as the cross-linker, and characterized by scanning electron microscopy, atomic force microscopy, and infrared spectroscopy. Under the optimized conditions, such as optimal preparation of MIP, composition and pH of mobile phase, and separation voltage, the model enantiomers, tert-butoxycarbonyl-D-tryptophan (Boc-D-Trp) and Boc-L-Trp, could be baseline separated within 75 s. The linear ranges for amperometric detection of the enantiomers using carbon fiber microdisk electrode at +1.2 V (vs Ag/AgCl) were from 75 to 4000?M and 400 to 4000?M with the detection limits of 20 and 140?M, respectively. The MIP-microchip electrochromatography provided a powerful protocol for separation and detection of Boc-Trp enantiomers within a short analytical time. The molecular imprinting on microchannel wall opens a promising avenue for fast enantioscreening of chiral compounds.2. Molecularly imprinted magnetic nanoparticles as tunable stationary phase located in microfluidic channel for enantioseparationA MD integrated with molecularly imprinted magnetic nanoparticles (MIP-MNPs) as stationary phase was designed for rapid enantioseparation by capillary electrochromatography. The nanoparticles were synthesized by the co-polymerization of methacrylic acid and EDMA on 3-(methacryloyloxy)propyltrimethoxysilane-functionalized MNPs (25-nm diameter) in the presence of template molecule, and characterized with infrared spectroscopy, thermal gravimetric analysis, and transmission electron microscope. MIP-MNPs (200-nm diameter) could be localized as stationary phase in the microchannel of MD with the tunable packing length by the help of an external magnetic field. Using S-ofloxacin as the template molecule, the preparation of MIP-MNPs, the composition and pH of mobile phase, and the separation voltage were optimized to obtain baseline separation of ofloxacin enantiomers within 195 s. The analytical performance could be conveniently improved by varying the packing length of nanoparticles zone, showing an advantage over the conventional packed capillary electrochromatography. The linear ranges for amperometric detection of the enantiomers using carbon fiber microdisk electrode at +1.0V (vs Ag/AgCl) were from 1.0 to 500?M and 5.0 to 500?M with the detection limits of 0.4 and 2.0?M, respectively. The magnetically tunable MIP-MNPs-MD could be expanded to localize more than one kind of template-imprinted magnetic nanoparticles for realizing simultaneous analysis of different kinds of chiral compounds.3. Simultaneous multiple enantioseparation with one-pot imprinted microfluidic channel by microchip capillary electrochromatographyA multi-template imprinted microchannel was prepared by a one-pot in situ imprinting process. The imprinted microchannel led to a novel chip-based strategy for simultaneous multiple enantioseparation. The one-pot imprinting process formed a multi-template imprinted porous thin layer (about 2?m) on the inner wall of capillary, which was characterized by scanning electron microscopy, infrared spectroscopy, and solid-state UV-vis spectroscopy. By fixing the imprinted capillary to a support substrate composed of poly(dimethylsiloxane) on a glass slide, a multi-analyte microchip was thus conveniently constructed. Using L-tyrosine (L-Tyr) and L-Trp as the template molecules, two pairs of enantiomers were simultaneously baseline separated in a 6-cm separation channel within 120 s under the optimized preparation and electrochromatographic conditions. The separation showed excellent efficiency. The linear ranges for amperometric detection of four analytes using carbon fiber microdisk electrode at+1.2 V (vs Ag/AgCl) were from 20 to 500?M for racemic Tyr and Trp. This multi-template imprinting strategy could be expanded for simultaneous separation and detection of more pairs of enantiomers within a short analytical time. It would open a promising avenue for high-throughput screening of chiral compounds.4. Superporous monolithic imprinted microfluidic channel for chip-based enantioseparationA method for chip-based enantioseparation by superporous monolithic imprinted microfluidic channel was successfully developed. The superporous monolith anchored onto the surface of the microchannel wall was prepared by in situ chemically copolymerized, and characterized by scanning electron microscopy, infrared spectroscopy, and solid-state UV-vis spectroscopy. The monolithic network with high porosity gave a large surface area, good permeability, low mass-transfer resistance, leading to a high efficiency. A portable microchip was conveniently constructed by integrating the imprinted capillary with 5-cm length as the separation channel and a carbon fiber microdisk working electrode for amperometric detection. Using L-Tyr as the template molecule, Tyr enantiomers were baseline separated within 55 s under the optimized preparation and separation conditions. The linear ranges for on-line amperometric detection of the enantiomers were from 20 to 2400?M for racemic Tyr. Compared with the surface imprinting methods, the superporous monolithic imprinting strategy exhibited a higher efficiency of enantioseparation and column capacity. It opens a promising avenue for high-throughput screening of chiral compounds.