| Microchip electrophoresis (MCE) is capable of conducting rapid analysis, reducing sample consumption, integrating and automating multiple analytical processes. It has been widely applied in some areas of research, such as biochemistry, medical laboratory science, pharmaceutical synthesis, drug screening, environmental monitoring and so on. However, the concentration limits of detection of MCE are often inadequate for the measurement of low concentration sample, because of the characteristically low injection sample volume, short optical path length and short separation distance. But enhancement of the path length is hardly applicable to MCE because it is difficult to change the depth of the microchannel at the detection point. One way of improving the sensitivity is to integrate an online sample stacking method.On the basis of the capillary electrophoresis detection of bacteria, some researchers have explored the possibility of analyzing bacteria using MCE. In this paper, MCE coupled with laser-induced fluorescence detection was applied to analysis the bacterial cells. In order to detect the low concentration sample, we do some further researches on improving detection sensitivity utilizing on-line concentration techniques. The details are listed below:Part1. Rapid determination of amino acids in milk by microchip electrophoresis coupled with laser-induced fluorescence detectionA simple and sensitive method for determination of amino acids in milk by microchip electrophoresis (MCE) coupled with laser-induced fluorescence (LIF) detection was developed. Seven kinds of standard amino acids were derivated with sulfoindocyanine succinimidyl ester (Cy5) and then perfectly measured by MCE-LIF within150s. The parameters of MCE separation were carefully investigated to obtain the optimal conditions:100mM sodium borate solution (pH10.0) as running buffer, 0.8kV as injection voltage,22kV as separation voltage etc. The linear range of the standard amino acid solutions was from0.01μM to1.0μM and the detection limit was as low as about1.0nM. This MCE-LIF method was applied to the measurements of amino acids in actual milk samples and satisfactory experimental results were achieved.Part2. Microchip electrophoresis of bacteria using lipid-based liquid crystalline nanoparticlesThe aggregation and adhesion of bacterial cells is a serious disadvantage for electrophoretic separations of bacteria. In this study, lipid-based liquid crystalline nanoparticles were used as a pseudostationary phase to minimise the bacterial aggregation and adsorption to the inner walls of microchannels. Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus and Lactobacillus rhamnosus were selected as analytes and were separated by microchip electrophoresis (MCE) with laser-induced fluorescence (LIF) detection using4.5mM Tris(hydroxymethyl) amminomethane (TRIS)-4.5mM boric acid-0.1mM ethylenediamine tetraacetate (EDTA)(TBE) containing poly(ethylene oxide)(PEO) and lipid-based nanoparticles as the running buffer. The mechanism of lipid-based nanoparticles affecting bacterial adhesion and aggregation was discussed and supported by zeta potential experiments. Under the optimal conditions, the three species of bacteria were identified with patterned peaks. This proposed MCE method using lipid-based nanoparticles as running buffer additives was also used to analyse a real yogurt sample, and valuable bacterial information was obtained by the electropherograms.Part3. Ultrasensitive detection of bacteria by microchip electrophoresis based on multiple concentration approaches combining chitosan sweeping, field-amplified sample stacking and reversed-field stacking. This paper describes an on-chip multiple concentration method combining chitosan (CS) sweeping, reversed-field stacking and field-amplified sample stacking for highly efficient detection of bacteria. Escherichia coli was selected as a model bacterium to investigate the efficiency of this multiple concentration method. CS was the most suitable sweeping agent for microchip electrophoresis (MCE), replacing the usually used cetyltrimethylammonium bromide (CTAB) for capillary electrophoresis (CE). The additive taurine had a synergistic effect by enhancing the interaction between CS and the surface of the bacteria, thus improving the analysis sensitivity. All steps of the concentration method and related mechanisms are described and discussed in detail. A concentration enhancement factor of approximately six thousand was obtained using this concentration method under optimal conditions as compared to using no concentration step, and the detection limit of E.coli was145CFU/mL. The multiple concentration methodology was also applied for the quantification of bacteria in surface water, and satisfactory results were achieved. The application of this methodology showed that the concentration enhancement of bacteria clearly conferred advantageous sensitivity, speed and sample volume compared to established methods.Part4. Study of on-line concentration of bacteria based on Cy5-Vancomycine responsing to bacteriaIn this paper we utilized Cy5to label vancomycin and to identify microorganism bacteria in biological sample coupled with on-line enrichment of the method reported in previous chapter. Compared with the method of nucleic acid labeling, this method avoid background nucleic interference from biological sample. This paper discussed the derivative conditions, the response performance of Cy5-Van to bacterial surface and some mechanism are discussed.Part5. On-line sanple pre-concentration for highly sensitivity detection of three antibiotics in microchip electrophoresis We reported a highly effective on-chip preconcentration method in a commercially available "sigle-cross"glass chip.by combining filed-amplified sample injection with reversed voltage control and micellar-electrokinetic chromatography for high sensitivive detection antibiotics. A low conductivity sample buffer plug was introduced into the separation channel. The sample anions become stacked at the interface between the low-and high-conductivity buffers due to the higher electric field strength in the low-conductivity buffer driving faster migration of the anions relative to the running buffer. PSS added into running buffer to improve the resolution of three antibiotics. Under the optimal conditions, the detection sensitivity for Gentamicin, Vancomycine, Kanamycin was improved by72,135and102fold, respectively, relative to without concentration mode. This proconcentration scheme will be of particular significance to the practical use of MCE in the emerging miniaturized analytical instrumentation. |
PDF Full Text Request