Rapid detection of high-risk HPV16 and HPV18 based on microchip electrophoresis

Researches on detection of human papillomavirus(HPV)high-risk samples were carried out by polymerase chain reaction(PCR)coupled with microchip electrophoresis(MCE).Herein,we introduced a simple,rapid,automated method for detecting high-risk samples HPV16 and HPV18.In this research,general primers were initially selected to obtain sufficient detectable yield by PCR to verify feasibility of MCM method for HPV detection,then type-specific primers were further used to evaluate the specificity of MCE method.The results indicated MCE method was capable of specifically detecting high-risk HPV16 and HPV18,and also enabled simultaneous detection of multiplex samples.This MCE method described here has been successfully applied to HPV detection and displayed excellent reliability demonstrating by sequencing results.The inherent capability of MCE facilitated HPV detection conducted in a small chip with automated,high throughput,massive parallelized analysis.We envision that MCE method will definitely pave a way for clinical diagnosis,and even on-site screening of cervical cancer.

Coupling microchip electrophoresis with MALDI-TOF-MS based on a freezing technique

A freezing technique protocol was proposed for coupling microchip electrophoresis with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry（MALD1-TOF-MS）.The microfluidic flow was frozen immediately after electrophoresis on microfluidic chip and the separated analyte molecules were kept in their zone pattern in the electrophoresis.Then,the frozen-chip was lyophilized and sent into TOF-MS instrument as a MALDI target,and the analyte molecules in the microfluidic channels were subjected to analysis by mass spectrometry.This approach could eliminate sample cross-contamination, providing a new interface for microchip electrophoresis and MALDI-MS.

Open channel-based microchip electrophoresis interfaced with mass spectrometry via electrostatic spray ionization

The coupling between open channel-based microchip electrophoresis and mass spectrometry via electrostatic spray ionization is proposed for in situ detection of fractionated analytes. Electrophoretic separation is performed in an open channel fabricated in a plastic substrate. The solvent of background electrolyte is evaporated from the open channel because of Joule heating during electrophoresis, leaving the dried electrophoretic bands to be directly analyzed by mass spectrometry via scanning electrostatic spray ionization. Proof-of-concept results are obtained with fluorescent dyes and antibiotics as the test samples, demonstrating an efficient on-chip detection platform based on the electrophoresis and electrostatic spray ionization mass spectrometry.

Selective fluorescence labeling and sensitive determination of Staphylococcus aureus by microchip electrophoresis with a multiple-concentration approach

The detection of Staphylococcus aureus （S. aureus） is very important as it is responsible for bacterial infectious diseases and food poisoning. In this paper we explored the application 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 （CyS-Van） for the identification of S. aureus. Our experiments were designed to examine in greater detail the specificity of the reaction between CyS- Van and S. aureus. Detection parameters such as the derivatization conditions, concentrations of buffer, pH value, response performance of CyS-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 5. aureus coupled with an on-line multiple-concentration in microcbip electrophoresis. Under the optimized conditions, the detection orS. aureus was achieved within 150 s with limit of detection （S/N = 3） of 981 CFU/mL, and 350- fold enhancement was obtained for S. aureus as compared to using the no concentration step. It is self- evident that this approach has great potential in the future for the analysis of S. aureus.

Analysis of arecoline in Semen Arecae decoction pieces by microchip capillary electrophoresis with contactless conductivity detection

A new method for the determination of arecoline in Semen Arecae decoction pieces by microchip capillary electrophoresis with contactless conductivity detection （MCE-CCD） was proposed. The effects of various electrophoretic operating parameters on the analysis of arecoline were studied. Under the optimal conditions, arecoline was rapidly separated and detected in 1 rain with good linearity over the concentration range of 20 1500 uM （r2=0.9991） and the detection limit of 5 uM （S/N=3）. The method was used for the analysis of arecoline satisfactorily with a recovery of 96.8 -104%.

Polydimethylsiloxane Microchip Capillary Electrophoresis for Determination of Cholesterol

Microchip capillary electrophoresis （MCE） has significant impact on diagnostic testing. One of the most importance in clinical analysis test is the determination of cholesterol level in blood, because its increase is associated with coronary heart disease which is a major cause of death world over. Polydimethylsiloxane （PDMS） was material used to make a MCE for determination of cholesterol. Hydrogen peroxide was generated from the oxidation of cholesterol with cholesterol oxidase （ChOx） and was detected electrochemically at a downstream gold （Au） wire electrode by amperometry. Various parameters, such as the detection potential, concentration of running buffer, pH of running buffer, separation voltage, injection time, and concentration of ChOx, were studied. The migration time of cholesterol is less than 100s and the calibration curve is linear from 50 mg.dL-1 to 250 mg-dL-1 with the coefficient of determination （R2） of 0.9955. Therefore, this proposed assay is very rapid and sensitive for the detection of cholesterol.

Rapid Determination of Free Amino Acids in Milk by Microchip Electrophoresis Coupled with Laser-induced Fluorescence Detection

A simple and sensitive method for determination of free amino acids in milk by microchip electrophoresis （MCE） coupled with laser-induced fluorescence （LIF） detection was developed. Seven kinds of standard amino ac- ids were derivated with sulfoindocyanine succinimidyl ester （Cy5） and then perfectly measured by MCE-LIF within 150 s. The parameters of MCE separation were carefully investigated to obtain the optimal conditions： 100 mmolo L^-1 sodium borate solution （pH 10.0） as running buffer solution, 0.8 kV as injection voltage, 2.2 kV as separation voltage etc. The linear range of the detection of amino acids was from 0.01 μmol·L^-1 to 1.0 μmol·L^-1 and the detection limit was as low as about 1.0 μmol·L^-1. This MCE-LIF method was applied to the measurements of free amino acids in actual milk samples and satisfactory experimental results were achieved.

Identification and Enumeration Method of Both Eukaryotic and Prokaryotic Microorganisms in Food Sample

The method to analyze both eukaryotic and prokaryotic microorganisms without preliminary microbial information of sample seemed to be useful not only for research and investigation of microorganisms but also for industry using microorganisms. In the present manuscript, preparation of a new DNA primers, new reference database for 18S rDNA for our newly developed method [1]- [3], and analyses of eukaryotic and prokaryotic microorganisms in fermentation products were presented. In komekouji, Aspergillus spp., was enumerated to be 46.5 × 106 MPN g-1, and Penicillium spp., was enumerated to be 1.5 × 106 MPN g-1. In dry yeast, Saccharomyces group, were enumerated to be 8600 × 106 MPN g-1. In komekouji-miso, no eukaryotic microorganism was detected, while the other Bacillus spp., was numerically dominant (21.5 × 106 MPN g-1) as prokaryotic microorganisms, followed by B. subtilis group (4.65 × 106 MPN g-1), and the other Firmicutes (3.7 × 106 MPN g-1). The komekouji-miso included lower number of Actinobacteria (0.15 × 106 MPN g-1), Burkhokderia sp. (1.5 × 106 MPN g-1), and the other α,β,γ-proteobacteria (0.12 × 106 MPN g-1). In sake-kasu, both prokaryote and eukaryote were not detected by the method. Present results indicated that using both universal primers for eukaryotic and prokaryotic microorganisms, each groups of prokaryotic and eukaryotic microorganisms were enumerated without any preliminary information nor setting up standard curve, which were required for real time PCR.

A Simple Evaluation System for Microbial Property in Soil and Manure

Analyses of microbial properties in soil and manure had always included the problem that there was no available standard method to evaluate microbial property. The one of the major problems was the vast diversity and the enormous population of soil microorganisms [1], the other was an existence of numerically dominant unculturable microorganisms which comprise 99% of soil habitat [2]. We evaluated whether our newly developed method, by which taxonomies and their number of each bacterial groups were estimated, could be used as evaluation method of microbial properties of soils and manures. In the forest soil, β-Proteobacteria, which included Burkholderia sp., Ralstonia sp., and Alcaligenes sp., was numerically dominant bacteria (3.64 × 106 MPN g-1 dry soil), followed by γ-Proteobacteria (1.32 × 106 MPN), δ-Proteobacteria (0.006 × 106 MPN), and the other gram negative bacteria (0.006 × 106 MPN). In the commercial manure, Actinobacteria, which included Streptoverticillium salmonis, Mycrococcus sp., Streptomyces bikiniensis, and Microbacterium ulmi, was numerically dominant bacterial group (30.8 × 106 MPN), followed by α-Proteobacteria (26.0 × 106 MPN), β-Proteobacteria (17.1 × 106 MPN), δ-Proteobacteria (11.2 × 106 MPN), the other Firmicutes (1.71 × 106 MPN), γ-Proteobacteria (0.5 × 106 MPN), and the other gram negative bacteria (0.05 × 106 MPN). In the upland field, the other Firmicutes, which included Paenibacillus sp., was numerically dominant bacteria (4.41 × 106 MPN), followed by Actinobacteria (2.14 × 106 MPN), Bacillus sp. (2.14 × 106 MPN), and γ-Proteobacteria (0.35 × 106 MPN). Although the precision of the affiliations became lower because of higher diversity of samples and the number of some Antinobacteria and Firmicutes might be underestimated by the used PCR condition, the method was found suitable as a candidate of a new evaluation system of soil and manure.

A New Evaluation Method for Antibiotic-Resistant Bacterial Groups in Environment

In the present manuscript it was presented whether spreading of antibiotic resistant bacterial groups in environment could be monitored by our newly developed method by enumerating antibiotic resistant bacterial groups in various biological wastes and composts. Although the numbers were not so high, diverse kinds of colistin resistant bacteria (25 mg·L-1) were included in row cattle feces (1.78 × 104 MPN g-1) and cattle feces manure (>3.84 × 104 MPN g-1). Compost originated from leftover food (>44.8 × 104 MPN g-1) and shochu lee (>320 × 104 MPN g-1) included higher numbers of chlortetracycline resistant Pseudomonas sp., (25 mg·L-1), and row cattle feces included higher numbers of chlortetracycline resistant Enterobacteriacea (15.7 × 104 MPN g-1), which mostly consisted from Pantoea sp. or Xenorhobdus doucetiae. Numbers of multi drug resistant bacteria, resistant to 25 mg·L-1 of ciprofloxacin, streptomycin, chloramphenicol, and ampicillin, were the highest in row cattle feces (>143.6 × 104 MPN g-1), followed by cattle feces manure (4.19 × 104 MPN g-1), and shochu lee (0.36 × 104 MPN g-1), which included diverse kinds of bacterial group. The present results indicated that higher numbers of multi drug resistant bacteria were typically found in row cattle feces, and the method was found suitable to enumerate and identify them. These results suggested that the method might become their environmental risk evaluation method.

High-Speed Analyzing PCR Products of M.tuberculosis Genome Stained by Ethidium Bromide on Microchip Gel Electrophoresis

The technique of microchip gel electrophoresis (MCGE) was used to analyze the polymerase chain reaction (PCR) products of M. tuberculosis Genome stained by ethidium bromide. The electrophoretic process was completed within 3-4 min and the results show that the technique of microchip electrophoresis is a high speed and high sensitivity analyzing method.

Dual-calibration coefficient:a more accurate protocol for simultaneous determination of superoxide and hydrogen peroxide in human Hep G2 cell extracts

We present a more accurate method for the quantification of superoxide anion （02-） and hydrogen peroxide （H202） simulta- neously in human HepG2 cell extracts. After the xanthine/xanthine oxidase system was added into cell extract which was devoid of O2＊- and H202, steady-state and in-situ produced O2＊- and H202 by xanthine/xanthine oxidase system was labeled by fluorescent probes and subsequently separated by microchip electrophoresis. Based on this method, two differential equations with the calibration coefficients were established for O2 and H202, respectively. Using the established dual-calibration coefficients, we obtained the calibrated concentrations of 02＊ and H202 that produced in human HepG2 cells, which were lower （0.66±0.03 and 0.82±0.04 lamol/L for 02＊-and H202, respectively） than that （0.85±0.03 and 0.96±0.03 gmol/L for O. and H202, respectively） obtained from statutory working curve. The proposed dual-calibration coefficient protocol takes into account both the complex matrix effect of the biological system and real time decaying of O2 ＊- and H202, providing a method with higher accuracy.