Study Of Norepinephrine-Functionalized PDMS Microchip And Application In The Separation Of Biological Sample

Microchip capillary electrophoresis system was developed in the early 1990 s, using the micro-machining technology, on the glass, quartz and polymer materials, the micro channel can be produced for electrophoretic separation by electric field force as the driving force and ions or molecules differences in electromigration behavior or distribution. Thus the sample injection, reaction, separation and detection processes were completed on the size of the square centimeter chip. Because of its advantages such as short analysis time, high-throughput, low sample and reagent consumption, easy miniaturization and integration etc., it has become an important research direction in the field of analytical chemistry. However, these defects include the extreme hydrophobicity of PDMS microchip and low density of charge on the surface of PDMS microchip, the unstable electroosmotic flow?EOF? and easy adsorption of analytes onto the microchip channel surface, etc. which greatly hinder its application scope in biological sample analysis and detection field. So it has become an urgent problem to look for new coating materials and prepare tne novel stationary phase for separation and analysis of biological samples in the PDMS microchannel.Commonly known as a neurotransmitter, norepinephrine?NE?, a derivative of dopamine, is a small-molecule mimic of the adhesive proteins. Under alkaline conditions, it could be self-polymerization to form chemically active adherent films on virtually any material surfaces. Using the self-polymerization of norepinephrine?NE? and its favorable film-forming property, a simple and green preparation approach was developed to modify a poly?dimethylsiloxane??PDMS? channel. After the PNE-coated PDMS surface, it not only greatly improved the hydrophilicity, but also obtained stable electroosmotic flow, which opens up a promising avenue for high-throughput screening of chiral compounds.In this paper, the modified PDMS microchannel with PNE was applied to the separation and analysis of biological samples, which were as follows.1. In this paper, using the self-polymerization of norepinephrine?NE? and its favorable film-forming property, a simple and green preparation approach was developed to modify a poly?dimethylsiloxane??PDMS? channel for enantioseparation of chiral compounds. After the PDMS microchip was filled with NE solution, poly?norepinephrine??PNE? film was gradually formed and deposited on the inner wall of microchannel as permanent coating via the oxidation of NE by the oxygen dissolved in the solution. Due to possessing plentiful catechol and amine functional groups, the PNE-coated PDMS surface exhibited much better wettability, more stable and suppressed electroosmotic mobility, and less nonspecific adsorption. The water contact angle and electroosmotic flow of PNE-coated PDMS substrate were measured to be 13° and 1.68×10-4 cm2 V-1 s-1, compared to those of 108° and 2.24×10-4 cm2 V-1 s-1 from the untreated one, respectively. Different kinds of chiral compounds, such as amino acid enantiomer, drug enantiomer, and peptide enantiomer were efficiently separated utilizing a separation length of 37 mm coupled with in-column amperometric detection on the PNE-coated PDMS microchips. This facile mussel-inspired PNE-based microchip system exhibited strong recognition ability, high-performance, admirable reproducibility and stability, which may have potential use in the complex biological analysis.2. A newly designed molecularly imprinted polymer?MIP? material was developed and successfully used as recognition element for enantioselective recognition by microchip electrophoresis. In this work, molecularly imprinted polymers were facilely prepared employing Fe₃O₄ nanoparticles?NPs? as the supporting substrate and norepinephrine as the functional monomer in the presence of template molecule in a weak alkaline solution. After extracting the embedded template molecules, the produced imprinted Fe₃O₄@polynorepinephrine?MIP-Fe₃O₄@PNE? NPs have cavities complementary to three dimensional shape of template molecules favoring high binding capacity and magnetism property for easy manipulation. The MIP-Fe₃O₄@PNE NPs prepared with L-tryptophan, L-valine, L-threonine, Gly-L-Phe, S-?-?-ofloxacin or S-?-?-binaphthol as template molecules were packed in the polydimethylsiloxane microchannel via magnetic field as novel stationary phase to successful enantioseparation of corresponding target analysts. The MIP-Fe₃O₄@PNE NPs-based microchip electrophoresis system exhibited strong recognition ability, excellent high-performance, admirable reproducibility and stability, which provided a powerful protocol for separation enantiomers within a short analytical time and opened up an avenue for multiplex chiral compound assay in various systems.3. In a weak alkaline solution, the thin adherent poly?norepinephrine??PNE? film was spontaneously formed on the surface of Fe₃O₄ nanoparticles?NPs? to produce the Fe₃O₄@PNE NPs through the self-polymerization of norepinephrine and employing Fe₃O₄ NPs as the supporting substrate. Due to the good magnetic properties, Fe₃O₄@PNE NPs can be easily fixed in the PDMS channels under the external magnetic field; on the other hand, the catechol hydroxyl groups in the PNE can chelate Ti⁴⁺ to form Fe₃O₄@PNE-Ti⁴⁺composite in the PDMS channels. When peptides are phosphorylated by PKA in the presence of ATP, the Fe₃O₄@PNE-Ti⁴⁺ coated in the PDMS channels can specifically coordinate to the phosphorylated sites of phosphopeptides via multicoordinative interactions, but the non-phosphorylated peptides can't bind with the Ti⁴⁺. Thus phosphorylated peptides and non-phosphorylated peptides can be separated successfully, and fast and sensitive detection of PKA can be attained.