食品中沙门菌和单增李斯特菌的多重PCR-芯片电泳快速检测

建立了食品中沙门菌和单增李斯特菌的多重PCR-芯片电泳快速检测方法。根据沙门菌和单增李斯特菌的特征基因合成2对特异性引物,优化聚合酶链反应(PCR)体系,采用芯片毛细管电泳快速检测食品中上述2种致病菌的多重PCR扩增产物。在优化的实验条件下,6 min内即可完成沙门菌和单增李斯特菌的同时检测;迁移时间的日内精密度为0.20%～1.7%,日间精密度3.7%～4.5%。     

Coupling chemiluminescence with capillary electrophoresis to analyze single human red blood cells

In this paper, we describe a new method for determination of hemoglobin of single red blood cells by coupling chemiluminescence with capillary electrophoresis (CL-CE). The chemiluminescent detection is based on the catalytic effects of hemoglobin on the luminol-hydrogen peroxide reaction. The conditions of chemiluminescent reaction and capillary electrophoresis were investigated. Hemoglobin in human blood samples was detected with the present method, the linear range from 1.7 μg mL⁻¹ to 6.8 μg mL⁻¹ was tested, and the correlation coefficient of 0.997 and low detection limit of 0.17 μg mL⁻¹ (approximately 2.2 pg, S/N = 3) were obtained. Cell injection procedure was improved, and the method was successfully used to determine hemoglobin of single red blood cells and the statistical result of the average content of hemoglobin in 26 human red blood cells was 23.6 pg. Compared to other current methods, CE with CL system is simple, sensitive and will become an attractive alternative method for single cell analysis.     

High-throughput gender determination using automated denaturant gel capillary electrophoresis

Sex determination of anonymous samples is a requirement before analysis of DNA variation on X or Y chromosomes. Based on this, we designed a method for screening samples on different DNA capillary sequencing instruments with a sensitivity that is able to quantify sex chromosome abnormalities. The two different amelogenin alleles sited on the X and Y chromosomes were polymerase chain reaction amplified with the same set of primers and separated by denaturant capillary electrophoresis (DCE). Sex chromosome ratios could be reproducibly determined with a relative standard deviation of 8.7%, which is sufficient to distinguish a normal XY karyotype from an XYY karyotype associated with Klinefelter syndrome. Reconstruction experiments demonstrated sensitivity down to a simulated Y:X allelic ratio of 1:127 in all three instruments, enabling the prediction of sex chromosomal aneuploidies. When tested on anonymous pooled and single samples, DCE gave a good prediction of the male to female ratio in pools of 1000 blood donors. In conclusion, DCE is a simple and robust method for sex determination that can be readily performed on commercially available CE systems.     

Determination of formaldehyde in single cell by capillary electrophoresis with LIF detection

As a small molecule gas, formaldehyde (FA) is the simplest carbonyl active material and plays an important role in the carbon cycle of metabolism. However, due to the volatile nature of the gas, it is difficult to accurately quantify its content, which limits the study of the mechanism of action in life activities. Thus, an efficient approach to quantitative detection of FA in cells especially in single cell is urgent needed. Nevertheless, no method for quantifying FA in single cell has been reported to date. In this work, a fluorescent probe N‐propyl‐4‐hydrazino‐naphthalimide (NPHNA), which has highly desirable attributes and has been applied to living biological samples, was chosen as labeling reagent to detect endogenous FA at single cell level. After optimization of separation conditions, fast baseline separation of the FA derivative N‐propyl‐4‐hydrazone‐naphthalimide product (NPHNA‐FA) and NPHNA was achieved in about 5 min by CE with LIF detection. The detection limit for FA was 5 amol (S/N ratio of 3). The developed method was validated by the measurements of intracellular levels of FA in single cell.     

Determination of glutathione in single HepG2 cells by capillary electrophoresis with reduced graphene oxide modified microelectrode

Determination of intracellular bioactive species will afford beneficial information related to cell metabolism, signal transduction, cell function, and disease treatment. In this study, the electrochemically reduced graphene oxide modified carbon fiber microdisk electrode (ER‐GOME) was used as a detector of CZE‐electrochemical detection and developed to detect glutathione (GSH). The electrocatalytic activity of the modified microelectrode was characterized by cyclic voltammetry. Under optimized experimental conditions, the concentration linear range of GSH was from 1 to 60 μM. When the S/N ratio was 3, the concentration detection limit was 1 μM. Compared with the unmodified carbon fiber microdisk electrode, the sensitivity was enhanced more than five times. With the use of this method, the average contents of GSH in single HepG2 cells were found to be 7.13 ± 1.11 fmol (n = 10). Compared with gold/mercury amalgam microelectrode, which was usually used in determining GSH, the electrochemically reduced graphene oxide modified carbon fiber microdisk electrode was friendly to environment for free mercury. Furthermore, there were several merits of the novel electrochemical detector coupled with CE, such as comparative repeatability, easy fabrication, and high sensitivity, hold great potential for the single‐cell assay.     

Simultaneous detection of single nucleotide polymorphisms and copy number variations in the CYP2D6 gene by multiplex polymerase chain reaction combined with capillary electrophoresis

CYP2D6 (cytochrome P450 2D6) is one of the most important enzymes involved in drug metabolism, and CYP2D6 gene variants may cause toxic effects of therapeutic drugs or treatment failure. In this research, a rapid and simple method for genotyping the most common mutant alleles in the Asian population (CYP2D6*1/*1, CYP2D6*1/*10, CYP2D6*10/*10, CYP2D6*1/*5, CYP2D6*5/*10, and CYP2D6*5/*5) was developed by allele-specific polymerase chain reaction (AS-PCR) combined with capillary electrophoresis (CE). We designed a second mismatch nucleotide next to the single nucleotide polymorphism (SNP) site in allele-specific primers to increase the difference in PCR amplification. Besides, we established simulation equations to predict the CYP2D6 genotypes by analyzing the DNA patterns in the CE chromatograms. The multiplex PCR combined with CE method was applied to test 50 patients, and all of the test results were compared with the DNA sequencing method, long-PCR method and real-time PCR method. The correlation of the analytical results between the proposed method and other methods were higher than 90%, and the proposed method is superior to other methods for being able to simultaneous detection of SNPs and copy number variations (CNV). Furthermore, we compared the plasma concentration of aripiprazole (a CYP2D6 substrate) and its major metabolites with the genotype of 25 patients. The results demonstrate the proposed genotyping method is effective for estimating the activity of the CYP2D6 enzyme and shows potential for application in personalized medicine. Similar approach can be applied to simultaneous detection of SNPs and CNVs of other genes.     

Ultrasensitive determination of cobalt in single hair by capillary electrophoresis using chemiluminescence detector

By use of the high separation capability of capillary electrophoresis (CE) and ultrasensitive chemiluminescence (CL) detection, a CE-CL method was proposed for the ultrasensitive determination of trace cobalt in a single hair, which is potentially useful in construction of the fingerprint of trace elements in the hair collected from crime scenes. In this work, the CE experimental conditions, CL experimental conditions and the digestion methods for the analysis of a single hair sample for cobalt were investigated in detail. The relative limit of detection (LOD, 3σ) was 0.01 ng/mL, and the absolute LOD was 2.4 × 10^− 16 g considering the sampling volume of 24 nL. Using a dry digestion method, the analytical results for certified reference hair samples by the proposed method were in good agreement with the certified values. Finally, this method was successfully used to detect trace cobalt in a single hair from three adults. It has potential applications in forensic analysis.     

Analysis of serotonin release from single neuron soma using capillary electrophoresis and laser-induced fluorescence with a pulsed deep-UV NeCu laser

The use of capillary electrophoresis (CE) with laser-induced fluorescence excited by ultraviolet (UV) lasers in the range 200–300 nm has been restricted by the available wavelengths and expense of UV lasers. The integration of a NeCu deep UV laser operating at 248.6 nm with a single channel CE system with post-column sheath flow detection allows detection limits for serotonin and tryptophan of 3.9×10^-8 M and 4.5×10^-8 M respectively. Single cell analysis of serotonergic metacerebral cells from the sea slug Aplysia californica yields a value of 800±85 fmol of serotonin in each cell soma. For the first time, serotonin is directly detected in electrically stimulated release from single metacerebral cell soma, with approximately 4% of the serotonin contained in the soma released from a semi-intact preparation with a 2 min electrical stimulation.     

High-throughput single nucleotide polymorphism typing by fluorescent single-strand conformation polymorphism analysis with capillary electrophoresis

In this study, we performed high-throughput and precise single nucleotide polymorphism (SNP) typing by fluorescent capillary electrophoresis single-strand conformation polymorphism (CE-SSCP) analysis. A system composed of a multicapillary DNA analyzer, a newly developed sieving matrix, four different colors of fluorescent labels, and a multiplex polymerase chain reaction (PCR) enabled low-cost and highly reliable SNP typing. Moreover, this system enabled the estimation of SNP allele frequencies using pooled DNA samples, which should be beneficial for large-scale association studies. Thus, fluorescent CE-SSCP analysis is a useful method for large-scale SNP typing.     

Determination of ascorbic acid in individual rat hepatocyte cells based on capillary electrophoresis with electrochemiluminescence detection

A novel method to detect ascorbic acid (AA) in individual rat hepatocyte cells was developed by combining CE with electrochemiluminescence (ECL) based on tris(2,2'-bipyridine) ruthenium(II) (Ru(bpy)(3)2+). A single cell, followed by 0.1% SDS as cell lysis solution, was injected into the inlet of the separation capillary by electromigration. After optimizing the analytical conditions, the RSDs of migration time and peak height were 0.38% and 2.6% for 1.0x10(-5) M AA (n=10), respectively. The linear range of AA was from 1.0x10(-8) to 5.0x10(-5) M with a correlation coefficient of 0.9979 and the LOD (S/N=3) was estimated to be 1.0x10(-8) M. This method has been successfully applied to determine AA in single rat hepatocytes and the amount of AA in seven rat hepatocytes ranged from 16 to 62 fmol. The above results demonstrated that CE coupled with ECL is convenient, sensitive, and will become an attractive alternative method for single-cell analysis.     

Separation and determination of enkephalin-related peptides using capillary electrophoresis

CZE with UV-absorption detection has been used for the separation and determination of enkephalin-related peptides. The experimental conditions, such as pH and concentration of running buffer, applied voltage, injection method, and time, were investigated in detail. Excellent separation efficiency could be obtained for ten enkephalin-related peptides with a 50 microm (ID) x 58 cm capillary using sodium dihydrogen phosphate as the running buffer (pH 3.11) when 20 kV of applied voltage was used. The concentration detection limits were found to be in the range of 0.31-1.94 microg/mL (defined as S/N = 3). The proposed method has been applied to analyze the spiked cerebrospinal fluid (CSF) sample, and the results showed that CZE is a powerful technique for separation and detection of the above biological peptides.     

Measurement of alkaline phosphatase isoenzymes in individual mouse bone marrow fibroblast cells based on capillary electrophoresis with on-capillary enzyme-catalyzed reaction and electrochemical detection

A novel method for the determination of alkaline phosphatase (ALP) isoenzymes in individual fibroblast cells of mouse bone marrow was developed by combining capillary electrophoresis with an on-capillary enzyme-catalyzed reaction and electrochemical detection. In this method, a single an cell, followed by 5.0 x 10(-2) mol/L Na2B4O7- 3.0 x 10(-2) mol/L NaCl (pH 9.8) as cell lysis solution, was injected into the inlet of the separation capillary by electromigration. The cell was lysed by applying a voltage of 2 kV. The ALP isoenzymes in the cell were preseparated at 20 kV for 1 min, and then allowed to react for 30 min with disodium phenyl phosphate as enzyme substrate in the running buffer. ALP converted disodium phenyl phosphate into its product, phenol, at a relatively high reaction rate without consumption, with resultant amplification of the signal on prolonged reaction time, producing an adequate amount of product for final detection. A mass detection limit as low as 3.5 x 10(-21) mol/L (corresponding to 1.5 nU) was achieved. Finally, the two zones of products generated by ALP isoenzymes were detected at the outlet of the capillary by using the end-capillary amperometric detection at a carbon fiber microdisk bundle electrode with a constant potential.     

Stuffer‐free multiplex ligation‐dependent probe amplification based on conformation‐sensitive capillary electrophoresis: A novel technology for robust multiplex determination of copy number variation

Developing diagnostic tools based on the application of known disease/phenotype‐associated copy number variations (CNVs) requires the capacity to measure CNVs in a multiplex format with sufficient reliability and methodological simplicity. In this study, we developed a reliable and user‐friendly multiplex CNV detection method, termed stuffer‐free MLPA‐CE‐SSCP, that combines a variation of multiplex ligation‐dependent probe amplification (MLPA) with CE‐SSCP. In this variation, MLPA probes were designed without the conventionally required stuffer sequences. To separate the similar‐sized stuffer‐free MLPA products, we adopted CE‐SSCP rather than length‐dependent conventional CE analysis. An examination of the genomic DNA from five cell lines known to vary in X‐chromosome copy number (1–5) revealed that copy number determinations using stuffer‐free MLPA‐CE‐SSCP were more accurate than those of conventional MLPA, and the CV of the measured copy numbers was significantly lower. Applying our system to measure the CNVs on autosomes between two HapMap individuals, we found that all peaks for CNV targets showed the expected copy number changes. Taken together, our results indicate that this new strategy can overcome the limitations of conventional MLPA, which are mainly related to long probe length and difficulties of probe preparation.     

In‐line coupled single drop liquid–liquid–liquid microextraction with capillary electrophoresis for determining fluoroquinolones in water samples

A simple in‐line single drop liquid–liquid–liquid microextraction (SD‐LLLME) coupled with CE for the determination of two fluoroquinolones was developed. The method is capable to quantify trace amount of analytes in water samples and to improve the sensitivity of CE detection. For the SD‐LLLME, a thin layer of organic phase was used to separate a drop of 0.1 M NaOH hanging at the inlet of the capillary from the aqueous donor phase. By this way, the analytes were extracted to the acceptor phase through the organic layer based on their acidic/basic dissociation equilibrium. The drop was immersed into the organic phase during 10 min for extraction and then it is directly injected into the capillary for the analysis. Parameters such as type and volume of organic solvent phase, aqueous donor, and acceptor phases and extraction time and temperature were optimized. The enrichment factor was calculated, resulting 40‐fold for enrofloxacin (ENR) and sixfold for ciprofloxacin (CIP). The linear range were 20–400 μg/L for ENR and 60–400 μg/L for CIP. The detection limits were 10.1 μg/L and 55.3 μg/L for ENR and CIP, respectively, and a good reproducibility was obtained (4.4% for ENR and 5.6% for CIP). Two real water samples were analysed applying the new method and the obtained results presented satisfactory recovery percentages (90–100.3%).     

Quantitative-competitive polymerase chain reaction coupled with slab gel and capillary electrophoresis for the detection of roundup ready soybean and maize

The aim of the present study was to develop a quantitative-competitive PCR (QC-PCR) method to detect DNA from transgenic herbicide-resistant (roundup ready, RR) soybean and maize. Since no QC-PCR system for the quantification of RR maize had been published at the time of writing, a specific competitor DNA for transgenic event was developed. For the QC-PCR of RR-soybean, a commercially available competitor was employed. These internal standards were calibrated by coamplifying with mixtures containing RR-soybean and maize DNAs. The calibrated QC-PCR systems were applied to certified RR-soybean and maize flour mixtures in order to demonstrate their suitability not only for the quantification of the glyphosate resistance traits in DNA matrices, but also in practically relevant samples. In addition, a special focus of the present work was to compare the detection of QC-PCR products by slab gel and CGE with UV detection. CGE permitted the precise detection of transgenic events also below the equivalence points; while in slab gel electrophoresis, due to the low sensitivity the quantification of genetically modified DNA was allowed only at the equivalence point.     

A covalent capillary coating of diazoresin and polyglycerol dendrimer for protein analysis using capillary electrophoresis

Overcoming proteins adsorption on the inner surface of capillary has attracted increasing attention recently. By using the unique photochemistry reaction of diazoresin (DR), a new covalent capillary coating was prepared on the fused‐silica capillary through layer‐by‐layer self‐assembly of DR with polyglycerol (PG) dendrimer. The separation performance of covalently DR/PG‐dendrimer coated capillary noticeably exceeded the bare capillary and the noncovalently linked DR/PG‐dendrimer capillary. A baseline separation of lysozyme, myoglobin, bovine serum albumin, and ribonuclease A was achieved using CE within 20 min. Besides, the covalently linked DR/PG‐dendrimer coating has the remarkable stability and reproducibility. Especially, compared with the traditional method which use highly toxic and moisture‐sensitive silane coupling agent, this method seems to be a simple and environmental friendly way to prepare the covalently coated capillaries for CE.     

Telomerase activity measurement in magnetically captured epithelial cells: comparison of slab-gel and capillary electrophoresis

We have compared telomerase activity measurements by slab-gel and capillary electrophoresis in cultured cells (A549 and H125 human cancer cell lines) and in cells isolated from clinical peripheral blood specimens epithelial cells of patients with lung and esophageal cancer. Telomerase activity was determined using the telomerase repeat amplification protocol (TRAP) assay with phosphoimager scanning of slab-gels and by laser-induced fluorescence capillary electrophoresis (LIF-CE). Experiments using A549 and H125 cells were performed to determine the reproducibility of each method and to identify the contribution of each stage of the TRAP/polymerase chain reaction (PCR) assay to the variability. In these experiments, it was found that more than half of the overall variability (coefficient of variation, CV = 35%) of the slab-gel method and almost all of the overall variability (CV = 20%) of the CE method was due to the PCR stage of the TRAP assay. In the clinical samples, classification as positive or negative was by visual inspection of the slab-gel and CE electropherograms for the presence of the characteristic 6 base-pair TRAP ladder and by GeneScan analysis of the CE. We examined several criteria including the use of 3, 4, or 5 TRAP bands as the definition of a positive test. Using the slab-gel method, the 5-band criterion gave 40% sensitivity with 100% specificity (no false positives in inactive controls). The CE method yielded a comparable 38% sensitivity and 100% specificity using this criterion. These data indicate that detection of telomerase activity in epithelial cells isolated from peripheral blood has a useful level of sensitivity and specificity and may be useful in the detection and monitoring of aerodigestive cancers. However, analysis by slab-gel is cumbersome and the precision is poor (inter-replicate CV = 20%) compared to LIF-CE (CV = 5%). A high-throughput CE-LIF detection platform will be indispensable for validation studies of telomerase activity measurements.     

Selective preconcentration of amino acids and peptides using single drop microextraction in-line coupled with capillary electrophoresis

Single drop microextraction (SDME) can be in-line coupled with capillary electrophoresis by attaching a drop to the tip of a capillary. With a 2-layer drop comprised of an aqueous basic acceptor phase covered with a thin organic layer, acidic analytes in an aqueous acidic donor phase can be extracted into the organic layer and then back-extracted into the acceptor phase. However, preconcentration of amino acids and peptides by SDME is difficult since their zwitterionic properties prevent them from being partitioned in the middle organic phase. When amino acids were derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), amino acids without a charged side chain were converted to carboxylic acids. In the acidic donor phase, those NBD-amino acids were predominantly neutral and they were successfully concentrated into the basic acceptor phase. In the meantime, amino acids with a charged side chain after NBD-F derivatization were not concentrated via SDME. With this selective SDME, we were able to extract acidic and neutral amino acids obtaining several hundred-fold enrichments within 5 min at 25 °C, while leaving basic amino acids—Arg, Lys, and His—in the acidic donor phase. Furthermore, detection sensitivity was enhanced by employing laser-induced fluorescence detection. We then applied this technique to the selective concentration of peptides.     

Genotyping of two single nucleotide polymorphisms in 5,10-methylenetetrahydrofolate reductase by multiplex polymerase chain reaction and capillary electrophoresis

Two single nucleotide polymorphisms (SNPs) of 5,10-methylenetetrahydrofolate reductase (MTHFR) gene, A1298C and C677T, were widely considered to be related with various neoplasia disorders. We established a simple and effective capillary electrophoresis (CE) method for detection of two SNPs in MTHFR gene simultaneously. DNA samples were amplified by multiplex PCR with universal fluorescence-labeled primer and analyzed by single-strand conformation polymorphism (SSCP)-CE method. The CE method was performed using 1.5% hydroxyethyl cellulose in 1× TBE buffer containing 1 M urea. The PCR products after SSCP procedure were electrokinetically injected at −10 kV, 30 s. Separation voltage was −6 kV and the temperature was set at 20 °C. The optimal SSCP-CE method was applied to detect two polymorphisms in MTHFR gene of acute lymphoblastic leukemia (ALL) and attention-deficit/hyperactivity disorder (ADHD) patients. Genotyping results were evaluated in terms of relationships between outcomes for ADHD patients after ALL chemotherapy and ALL disease. The SSCP-CE method and multiplex PCR with universal fluorescence primer were used as the fast technique for screening two SNPs in MTHFR gene, A1298C and C677T. The genotyping data were coincident with DNA sequencing. This SSCP-CE method was found feasible for detecting mutation of MTHFR gene in populations.