Application Of Microchip Electrophoresis In Biochemical Analysis

Currently,microchip electrophoresis?MCE?have been developed as powerful analytical techniques for analyzing the field of bio-analysis owing to their lower sample consumption and better separation efficiency,and proven versatility and potentiality.It is noteworthy that the wide application of MCE is relatively excellent in amino acids,antibiotics,peptides,drugs,nucleic acids,proteins and bacteria.Compared with traditional separation techniques such as HPLC and GC,MCE has gradually become one of the important technologies in biological sample research.The use of a microfluidic system includes sample processing,enrichment and separation.It can solve problems in many fields.Currently,MCE can study in many fields,such as the enrichment and detection of neurotransmitters in practical sample,the detection of pathogenic bacteria in food,the separation of nucleic acid,neurotransmitters in practical sample enrichment and separation of detection,detection of pathogenic bacteria in food,the separation of nucleic acid,the application of aptamer,as well as the separation of chiral compounds,MCE shows strong detection function in these research fields.In view of the strong advantages and research background of many fields in MCE,the electrophoresis analysis of small molecule and bacteria and the study of online enrichment technology were studied in this paper.The sensitivity of analysis was greatly improved by the combination of the modification of microchannel and online enrichment.The details are listed below:Part 1.A sensitive assay based on specific aptamer binding for the detection of Salmonella enterica serovar Typhimurium in milk samples by microchip electrophoresisThe detection of Salmonella enterica serovar Typhimurium?S.Typhimurium?is very important for the prevention of food poisoning and other infectious diseases.A simple and rapid strategy to test for bacteria based on specific aptamers by microchip electrophoresis?MCE?with laser-induced fluorescence?LIF?is described in this paper.The specificity of the reaction between specific aptamers and S.Typhimurium was explored in detail.The optimization of parameters such as fluorescent dye concentration,Mg²⁺concentration,incubation time,and pH of incubation solution was investigated.Under optimal conditions,rapid separation of the bacteria-aptamer complex and pure aptamers was achieved within 135 s using MCE-LIF with a relatively short detection length,with a limit of detection?S/N=3?of 3.37×10² CFU mL^-1 and a recovery rate of 95.8%.Finally,the proposed strategy was successfully applied to detect bacteria artificially added to milk.The results indicated that the established strategy could be used to analyze bacteria in food.Part 2.Simultaneous detection of Escherichia coli,Staphylococcus aureus,and Salmonella enterica serovar Typhimurium in food samples by microchip electrophoresis in combination with PCRA rapid and sensitive PCR based strategy in combination with microchip electrophoresis?MCE?was employed to simultaneously detect three foodborne pathogenic bacteria.Three pairs of primers were specially designed for the amplification of target genes from Escherichia coli?E.coli?,Staphylococcus aureus?S.aureus?and Salmonella enterica serovar Typhimurium?S.Typhimurium?.The PCR products along with standard DNA fragments were simultaneously employed to optimize the separation conditions in MCE.Under optimal conditions,detectable separation of the PCR products(0.15-0.56 ng?L^-1)from the three foodborne pathogenic bacteria was achieved within 135s?S/N=3?.The limits of detection of the three bacteria were concluded to be as low as 45 CFU mL^-1 for E.coli,62 CFU mL^-1for S.aureus and 42 CFU m L^-1 for S.Typhimurium,respectively.The RSD of migration time was in the range of 0.5-0.8%.We conclude that MCE along with PCR holds real potential for rapid analysis and detection of nucleic acids from routine foodborne pathogenic bacteria.Part 3.Selective fluorescence labeling and sensitive determination of Staphylococcusaureusbymicrochipelectrophoresiswitha multiple-concentration approachIn this paper we explore the use of fluorescently labelled vancomycin to specifically bind and detect S.aureus.In view of the specificity of vancomycin towards bacterial cell surfaces,we utilized Cy5 to label vancomycin?Cy5-Van?for the identification of S.aureus.Our experiments were designed to examine in greater detail the specificity of the reaction between Cy5-Van and S.aureus.Detection parameters such as the derivatization conditions,concentrations of buffer,pH value,response performance of Cy5-Van to bacterial surface,injection time and reversed-polarity time have been investigated and optimized.To develop a simple and quick assay for the detection of S.aureus at low concentrations,we propose to use the Cy5-Van for labeling the S.aureus coupled with an on-line multiple-concentration on microchip electrophoresis.Under the optimized conditions,the detection of S.aureus was achieved within 150 s with limits of detection?S/N=3?of 981 CFU m L^-1,and enhancement factors of 350-fold was obtained for S.aureus as compared to using the no concentration step.Part 4.Sensitive determination of neurotransmitters in urine by microchip electrophoresiswithmultiple-concentrationapproachescombining field-amplified and reversed-field stackingMicrochip electrophoresis?MCE?is particularly attractive as it provides high sensitivity and selectivity,short analysis time and low sample consumption.An on-line preconcentration strategy combining field-amplified stacking?FASS?and reversed-field stacking?RFS?was developed for efficient and sensitive analysis of neurotransmitters in real urine samples by MCE with laser induced fluorescence?LIF?detection.In this study,the multiple-preconcentration strategy greatly improves the sensitivity enhancement and surpasses other conventional analytical methods for neurotransmitters detection.Under optimal conditions,the separation of three neurotransmitters?dopamine,norepinephrine and serotonin?,was achieved within3 min with limits of detection?S/N=3?of 1.69,2.35,and 2.73 nM,respectively.The detection sensitivities were improved by 201-,182-,and 292-fold enhancement,for the three neurotransmitters respectively.Other evaluation parameters such as linear correlation coefficients were considered as satisfactory.A real urine sample was analyzed with recoveries of 101.8-106.4%.The proposed FASS-RFS-MCE method was characterized in terms of precision,linearity,accuracy and successfully applied for rapid and sensitive determination of three neurotransmitters in human urine.Part 5.Sensitive analysis of glutathione in bacteria and HaCaT cells by polydopamine/gold nanoparticle-coated microchip electrophoresis via on-line pre-concentration of field-amplified sample stackingIn this paper,polydopamine/gold nanoparticles?PDA/Au NPs?were used to construct a functional film on a glass microfluidic channel surface in microchip electrophoresis?MCE?for the separation of reduced glutathione?GSH?and oxidized glutathione?GSSH?.The formation of the PDA/Au NPs was characterized by scanning electron microscopy?SEM?,transmission electron microscope?TEM?,UV-vis spectra and ATR-FT-IR.An on-line pre-concentration strategy involving field-amplified sample stacking?FASS?was used to determine the sensitivity of the assay for measuring GSH and GSSH in bacteria(Escherichia coli,Staphylococcus aureus and Salmonella enterica serovar Typhimurium and HaCaT cells samples by MCE with laser induced fluorescence?LIF?detection.The influences of the separation voltage,the concentration of the running buffer and the p H value of running buffer,were carefully investigated.Using this studied method,GSH and GSSH could be simultaneously pre-concentrated and separated within 70 s.The limits of detection of GSH and GSSH were as low as 0.81 and 0.91 nM respectively?S/N=3?,which corresponded to approximately 180-to 301-fold improvements in peak height.Moreover,this method was successfully applied to the analysis of bacteria?E.coli,S.aureus and S.Typhimurium?and HaCaT cell samples with a satisfactory recovery rate.Part 6.Enantiomeric separation of tryptophan by open-tubular microchip electrophoresis using polydopamine/gold nanoparticles conjugated DNA as stationary phaseWe reported the use of polydopamine/Au nanoparticles/DNA?PDA/Au NPs/DNA?as a stationary phase for the separation of tryptophan enantiomers by microchip electrophoresis?MCE?.The stationary phase was formed by sequentially immobilizing PDA/Au NPs and DNA to the glass microfluidic channel surface using layer-by-layer assembly.The formation of PDA/Au NPs as a functional film involved the use of HAuCl₄ as an oxidant to trigger DA self-polymerization.DA was then used as a reductant to reduce HAuCl₄ to Au NPs.All These Au NPs were simultaneously deposited on the microchip surface by the adhesive PDA,and the thiolated DNA was strongly adsorbed onto the glass microfluidic channel surface through covalent gold-sulfur bonding.The functional film of the PDA/Au NPs/DNA was characterized by scanning electron microscopy,UV-vis spectra and ATR-FT-IR.The influence of separation voltage and the concentration and p H value of the running buffer were found to significantly influence separation.Successful baseline separation of tryptophan enantiomers was achieved within 65 s utilizing an effective separation length of 33 mm,and coupling with laser-induced fluorescence detection resulted in a resolution factor of 2.95.All these evaluation parameters,including stability and reproducibility,were considered satisfactory.The method outlined here,using the PDA/Au NPs/DNA-coated microfluidic channel as a versatile platform,may offer a processing strategy to prepare a functional surface on microfluidic chips.