Microchip Electrophoresis System Research Articles

Detection of miRNA in Cell Cultures by Using Microchip Electrophoresis with a Fluorescence-Labeled Riboprobe

The analysis of a microRNA (miRNA), miR-222 isolated from the PC12 cell line, was performed by use of the ribonuclease (RNase) protection assay, cyanine 5 (Cy5)-labeled miR-222 riboprobe, and a Hitachi SV1210 microchip electrophoresis system, which can be used to evaluate the integrity of total RNA. The fluorescence intensity corresponding to the protected RNA fragment increased in a dose-dependent manner with respect to the complementary-strand RNA. More highly sensitive detection of miRNA by microchip electrophoresis than by conventional method using fluorescence-labeled riboprobe could be obtained in 180 s. An obvious increase in miR-222 expression induced by nerve growth factor in PC12 cells could be observed. These results clearly indicate the potential of microchip electrophoresis for the analysis of miRNA using RNase protection assay with a fluorescence-labeled riboprobe.

Fast screening of rice knockout mutants by multi-channel microchip electrophoresis

A multi-channel microchip electrophoresis (MC-ME) system with a laser-induced fluorescence detector was developed for the fast simultaneous detection of rice knockout mutants in genetically modified (GM) rice. In addition, three parallel separation channels were fabricated on a glass microchip to investigate the possibility of high-throughput screening of amplified-polymerase chain reaction products representing wild-type rice and mutants. The MC-ME system was developed to simultaneously record data on all channels using specifically designed electrodes for an even distribution of electric fields, an expanded laser beam for excitation, a 10× objective lens to capture emissions, and a charge coupled device camera for detection. Under a programmed electric field strength and a sieving gel matrix of 0.7% poly(ethylene oxide) (Mr=8,000,000), T-DNA-inserted rice mutants, two standard wild-type rice lines, and six rice knockout mutants were analyzed within 4min using three parallel channels on the microchip. Compared to conventional microchip electrophoresis, the MC-ME method is a valid and practical way to effectively analyze multiple samples in parallel for the identification of GM rice without any loss of resolving power or reproducibility. The MC-ME method was more than 15 times faster than traditional slab gel electrophoresis and proved to be a powerful tool for high-throughput screening of GM rice with high sensitivity, efficiency, and reproducibility.

Microchip electrophoresis coupled with on‐line magnetic separation and chemiluminescence detection for multiplexed immunoassay

A facile and universal strategy for multiplexed immunoassay is proposed. The strategy is based on microchip electrophoresis (MCE) coupled with on-line magnetic separation and chemiluminescence (CL) detection. The system consisted of a microchip, an electromagnet, and a photomultiplier. The realization of multiplexed immunoassay protocol involves sampling magnetic nanoparticles (MNPs) labeled antibodies, N-(4-aminobutyl)-N-ethyl-isoluminol (ABEI) labeled antigens and free antigens in the precolumn reactor, on-line immunoreaction, capturing the MNPs-immunocomplexes, and the separation of unconjugated ABEI-labeled antigens. After on-line magnetic separation, the free ABEI-labeled antigens were transported into the separation channel, and mixed with hydrogen peroxide (H(2) O(2) ) in the presence of horseradish peroxidase in the postcolumn reactor, and producing CL emission. Using this arrangement, multiple analytes could be measured simultaneously by performing the technical operations for a single assay. As a proof-of-concept, the multiplexed immunoassay was evaluated for the simultaneous determination of five model analytes (i.e. hydrocortisone, corticosterone, digoxin, testosterone, and estriol). The results exhibited excellent precision and sensitivity, the relative standard deviations for nine times detection were lower than 4.7% for all the five components, and the detection limits of five analytes were in the range of 3.6-4.9 nM. The MCE system was validated using two human serum-based control samples containing five analytes.

Influence of Dna–Dye Complex Stability on Separation Resolution in Microchip Electrophoresis

Different markers have been used to label DNA for sample detection in gel electrophoresis. Intercalating dyes, (e.g., YOYO) have been widely used to label DNA for sample detection, because they do not require the use of radioisotopes, covalent attachment or enzyme reactions. The labeling of DNA fragments can be achieved by simply mixing solutions of the intercalating dye and DNA sample. However, the separation quality of DNA labeled with intercalating dyes is greatly influenced by the buffer used, which affects the DNA-dye complex stability. In this study, we investigated the effects of DNA-dye complex stability on separation resolution of dsDNA migrating in a photopolymerized polyacrylamide gel by measuring mobility and dispersion coefficients on a microfluidic chip and comparing predicted separation resolution under different dye and buffer conditions. We found that a buffer containing tetrapentylammonium (NPe(4)(+)) yielded better separation resolution than the frequently used TBE buffer on our microchip electrophoresis system.

High‐voltage power supplies to capillary and microchip electrophoresis

Over the past years, the development of capillary electrophoresis (CE) and microchip electrophoresis (ME) systems has grown due to instrumental simplicity and wide application. In both CE and ME, the application of a high voltage (HV) is a crucial step in the electrokinetic (EK) injection and separation processes. Particularly on ME devices, EK injection is often performed with three different modes: gated, pinched, and unpinched. In all these cases, different potential values may be applied to one or multiple channels to control the injection of small sample volumes as well as the separation process. For this reason, the construction of reliable HV power supplies (HVPS) is required. This review covers the advances of the development of commercial and laboratory-built HVPS for CE and ME. Moreover, it intends to be a guide for new developers of electrophoresis instrumentation.

Aberrant DNA methylation of tumor‐related genes in oral rinse

The early detection of oral squamous cell carcinoma (OSCC) is important, and a screening test with high sensitivity and specificity is urgently needed. Therefore, in this study, the authors investigated the methylation status of tumor-related genes with the objective of establishing a noninvasive method for the detection of OSCC. Oral rinse samples were obtained from 34 patients with OSCC and from 24 healthy individuals (controls). The methylation status of 13 genes was determined by using methylation-specific polymerase chain reaction analysis and was quantified using a microchip electrophoresis system. Promoter methylation in each participant was screened by receiver operating characteristic analysis, and the utility of each gene's methylation status, alone and in combination with other genes, was evaluated as a tool for oral cancer detection. Eight of the 13 genes had significantly higher levels of DNA methylation in samples from patients with OSCC than in controls. The genes E-cadherin (ECAD), transmembrane protein with epidermal growth factor-like and 2 follistatin-like domains 2 (TMEFF2), retinoic acid receptor beta (RARβ), and O-6 methylguanine DNA methyltransferase (MGMT) had high sensitivity (>75%) and specificity for the detection of oral cancer. OSCC was detected with 100% sensitivity and 87.5% specificity using a combination of ECAD, TMEFF2, RARβ, and MGMT and with 97.1% sensitivity and 91.7% specificity using a combination of ECAD, TMEFF2, and MGMT. The aberrant methylation of a combination of marker genes present in oral rinse samples was used to detect OSCC with >90% sensitivity and specificity. The detection of methylated marker genes from oral rinse samples has great potential for the noninvasive detection of OSCC.

Pigment epithelial cells isolated from human peripheral iridectomies have limited properties of retinal stem cells

The identification of cells with properties of retinal progenitor cells (RPCs) in the adult human ciliary margin (CM) prompted a number of studies of their proliferative and differentiation potential. One of the remaining challenges is to find a feasible method of isolating RPCs from the patient's eye. In the human CM, only the iris pigment epithelium (IPE) is easily obtained by a minimally invasive procedure. In the light of recent studies questioning the existence of RPCs in the adult mammalian CM, we wanted to assess the potential of the adult human IPE as source of RPCs. The IPE were isolated from peripheral iridectomies during glaucoma surgery, and IPE and ciliary body (CB) epithelium were also isolated from post-mortem tissue. Cells were cultivated in sphere-promoting conditions or as monolayers. Whole-tissue samples, undifferentiated and differentiated cells were studied by immunocytochemistry, RT-PCR and transmission electron microscopy. The adult human IPE, like the CB, expressed markers of RPCs such as Pax6, Sox2 and Nestin in vivo. Both sphere-promoting and monolayer cultures preserved this phenotype. However, both IPE/CB cultures expressed markers of differentiated epithelial cells such as Claudin, microphtalmia-associated transcription factor (MITF) and Cytokeratin-19. Ultrastructurally, IPE spheres displayed epithelial-like junctions and contained mature melanosomes. After induced differentiation, IPE-derived cells showed only partial neuronal differentiation expressing β-III-tubulin, Map-2 and Rhodopsin, whereas no mature glial markers were found. Proliferative cells with some properties of RPCs can be isolated from the adult human IPE by peripheral iridectomies. Yet, many cells retain properties of differentiated epithelial cells and lack central properties of somatic stem cells.

Evaluación del cobas 4800 CT/NG test para la detección de Chlamydia trachomatis

To evaluate the new automated system cobas 4800 CT/NG test for detection of Chlamydia trachomatis in urogenital specimens. We analyzed 696 specimens (488 swabs from urethral or cervical specimens, and 208 urines) to detect C. trachomatis. The results of the cobas 4800 CT/NG test (c4800) were compared to those obtained with Cobas AMPLICOR CT/NG test (CAM). Discordant results were analyzed with a conventional PCR assay and microchip electrophoresis system in agarose gel, MultiNA. We made two simultaneous analyses. In the first one, we compared the results obtained with swab specimens using the c4800 system and CAM. In this case, the sensitivity, the specificity, the positive and negative predictive values (PPV and NPV) were: 77.9%, 100%, 100% and 96% respectively. In the second one, we compared the results obtained for urine and its corresponding swab specimens on the c4800. The values obtained were: 100%, 98.9%, 92.9% and 100% respectively. The kappa values of these comparisons were: 0.857 for swab specimens on the c4800 and CAM, and 0.957 for urine versus swab specimens on the c4800. The results obtained with c4800 system were completely comparable with those obtained with CAM. We also noted an excellent correlation with these results when we compared swab specimens with their urine samples in the c4800 system. Therefore this sample type could be used routinely to diagnose infections in men and women.

One-way multiplexed immunoassay strategy for simultaneous determination of multi-analytes by microchip electrophoresis

An integrated microchip electrophoresis (MCE) system with online immunoreaction and laser induced fluorescence (LIF) detection has been developed for simultaneous determination of multi-analytes. In this system, the multiplexed immunoreactions between multiple antibody-immobilized glass beads with analytes and respective fluorescently labeled antigens were performed in a sample reservoir. After online incubation, the immunoreaction solution was injected into a one-way separation channel, and free fluorescently labeled antigens were separated and detected in the separation channel. With the help of glass beads, the immunocomplex can not move into the separation channel, which simplifies the separation of fluorescently labeled antigens. With the use of phenobarbital (PB), phenytoin (PHT), carbamazepine (CBZ) and theophylline (Th) as proof-of-principle analytes, the one-way multiplexed immunoassay could be completed within 20 min, resulting in a response curve over the range of 4.0-400 nM for each analyte. Detection limits (S/N = 3) for the drugs tested were in the range of 1.8 × 10(-9) to 2.5 × 10(-9) M. Compared with the conventional immunoassays, this assay is simple, rapid, sensitive and low cost, and provides an accurate procedure for a multiplex immunoassay. The present method has been applied for the simultaneous determination of PB, PHT, CBZ and Th in human serum, which showed a promise of automated clinical application.

Microchip electrophoresis system for the evaluation of RNA quality

RNAの分解は，臨床検体の遺伝子発現の分析を行うにあたって，重要な問題である．とくに，RNAの十分な品質は，定量PCRやマイクロアレイ解析のようなmRNA測定の精度を決定する重要な因子である．RNAサンプルのRNA収量と品質を評価し，マイクロチップ電気泳動システムによるRNA Integrity Number（RIN）を得た．冷凍保存したRNAと培養細胞から抽出したRNAの全サンプルは，9.8以上のRINを示し，アガロース電気泳動においても高分子量であった．また，RINは，凍結融解の繰り返しによる影響を受けなかった．したがって，これらの結果は，RINが保存中のRNAの品質を適切に評価しうる指標であることを示唆している．

M-marker 2009, a Marker set for Mapping Medaka Mutants Using PCR Length Polymorphisms with an Automated Microchip Gel Electrophoresis System

We have developed a highly effective marker set for assigning medaka mutants to specific linkage groups (LG) in bulk segregation analysis by PCR product length polymorphisms (PLPs). The marker set permits analysis using an automated microchip electrophoresis system as well as conventional agarose gel electrophoresis.

Multiplexed p53 Mutation Detection by Microchip Electro‐phoresis with Laser‐Induced Fluorescence Detector

AbstractA form of single‐strand DNA‐conformation polymorphism analysis (SSCP) employing nondenaturing slab gel electrophoresis is applicable to the genetic diagnosis of mutations at exons 7, 8 and 9 of the p53 gene. Recently, microchip electrophoresis (ME) systems have been used in SSCP analysis instead of conventional slab gel electrophoresis in terms of speed, sensitivity and automation. The aim of the present study was to investigate the application of SSCP and ME analysis as a rapid and effective method to the detection of mutations for exons 7, 8 and 9 of the p53 gene. It was found that using the electric field strength 260 V/cm and the sieving matrix of 4 mg/mL poly(ethylene oxide) was very useful to achieve better resolution and fast detection of mutations at exons 7, 8 and 9 of p53 gene. Under the optimized conditions, mutations at exons 7–9 of p53 gene were analyzed within 60 s and the relative standard deviation values of the migration times were less than 5.81% (n=5). The detection limit can be as low as 1 ng·L−1.

Validation of a microchip electrophoresis system as a DNA amplification control

As part of the normal procedure in a forensic DNA laboratory, a quality control step of the amplified DNA is often implemented to ensure the correct amplification of the sample before it is analysed in downstream applications. A validation study was undertaken to investigate a new microchip electrophoresis system (MultiNa, Shimadzu Corporation) claiming high resolution and sensitivity compared to routine polyacrylamide gel electrophoresis (PAGE). An array of STR multiplexes (AmpFISTR™ SGM+, GenePrint ® FFFL, PowerPlex™ 16, PowerPlex™ Y, an in-house Y-STR multiplex and AmpFISTR™ Profiler) was tested under both standard and low copy number PCR parameters to evaluate the accuracy, reproducibility and sensitivity of this technique. These tests showed that the microchip system did not have improved sensitivity compared to PAGE though had increased resolution and high reproducibility between samples.

Effects of heterogeneous electron‐transfer rate on the resolution of electrophoretic separations based on microfluidics with end‐column electrochemical detection

We demonstrate here that the electrode kinetics of an electrochemical detector contributes greatly to the resolution of the analyte bands in microchip electrophoresis systems with amperometric detection. The separation performance in terms of resolution and theoretical plate number can be improved and tailored by selecting or modifying the working electrode and/or by controlling the detection potential. Such improvements in the separation performance reflect the influence of the heterogeneous electron-transfer rate of electroactive analytes upon the post-channel band broadening, as illustrated for catechol and hydrazine compounds. The electrode kinetics thus has a profound effect not only on the sensitivity of electrochemical detectors but on the separation efficiency and the overall performance of microchip electrochemistry systems.

Continuous Operation of Microfabricated Electrophoresis Devices for 24 Hours and Application to Chemical Monitoring of Living Cells

Microchip electrophoresis is an emerging analytical technology with several useful attributes including rapid separation time, small sample requirements, and automation. In numerous potential applications, such as chemical monitoring or high-throughput screening, it may be desirable to use a system for many analyses without operator intervention; however, long-term operation of microchip electrophoresis systems has received little attention. We have developed a microchip electrophoresis system that can automatically inject samples at 6 s intervals for 24 h resulting in collection of 14,400 assays in one session. Continuous operation time of a prototype of the device was limited to 2 h due to degradation of reagents and electrophoresis buffers on the chip; however, modification so that all reagents were continuously perfused into reservoirs on the device ensured fresh reagents were always used for analysis and enabled extended operating sessions. The electrophoresis chip incorporated a cell perfusion chamber and reagent addition channels to allow chemical monitoring of fluid around cells cultured on the chip by serial electrophoretic immunoassays. The immunoassay had detection limits of 0.4 nM for insulin and generated approximately 4% relative standard deviation over an entire 24 h period with no evidence of signal drift. The combined system was used to monitor insulin secretion from single islets of Langerhans for 6-39 h. The monitoring experiments revealed that islets have secretion dynamics that include spontaneous oscillations after extended nonoscillating periods and possible ultradian rhythms.

Comparison of Four Carbon Fiber Electrodes in Microfluidic Chip Integrated With Electrochemical Detector

A microchip electrophoresis system with integrated amperometric detector was developed. Using 0.02 M Tris-HCl (pH = 8) as buffer, at the detection potential of 0.6 V ( vs. Ag/AgCl), dopamine (DA) and isoprenaline (IP) were detected by chip-CE with end-column amperometric detection using a carbon fiber microelectrode (CFME), a carbon fiber nanoelectrode (CFNE), a carbon fiber microelectrode modified by single-walled carbon nanotubes (SWNTs-CFME), and a carbon fiber microdisc electrode (CFMDE). The resolution of DA and IP are 0.64, 1.06, 0.61 and 1.22, and the sensitivity of DA ( S/ N = 3) are 1.7 × 10 −7, 5.9 × 10 −8, 2.3 × 10 −8, and 5.3 × 10 −7, respectively. As the CFNE had high resolution and sensitivity, the nanoelectrode-based microchip CE system was successfully applied to the determination of DA in cultured rat pheochromocytoma (PC12) cells, and the average content of DA in an individual PC12 cell was 0.57 ± 0.07 fmol ( n = 5), which was in good agreement with that reported in the literatures.

最新のキャピラリー電気泳動とチップ電気泳動 マイクロチップ電気泳動装置ＭＣＥ‐２０２“ＭｕｌｔｉＮＡ”の概要と応用例―ノロウイルス検出並びに食肉の品種鑑別―

This paper describes the concept of MCE-202 “MultiNA”, a new microchip electrophoresis system for DNA / RNA analysis. MCE-202 “MultiNA” realizes high-throughput and cost-effective analysis with originally developed reusable microchips and reagent kits. Application data for Norovirus detection and meat identification are also presented.

Pharmaceutical applications of isoelectric focusing on microchip with imaged UV detection

For the first time, the application of a commercial Shimadzu microchip electrophoresis system MCE-2010 equipped with an imaging UV detector for isoelectric focusing (IEF) of therapeutic proteins is reported. By proper adjustment of the pH gradient, samples with p I values ranging from 2.85 to 10.3 can be focused to the imaged part of the separation channel. Three therapeutic proteins (hirudin, erythropoietin, and bevacizumab) have been successfully focused on the microchip, and the results have been compared to conventional capillary IEF in terms of peak profile, p I values, and reproducibility.

Floating resistivity detector for microchip electrophoresis

A newly developed conductivity detector, the floating resistivity detector (FRD), for microchip electrophoresis was introduced in this work. The detector design permits decoupling of the detection circuit from the high separation voltage without compromising separation efficiency. This greatly simplifies the integration of microchip electrophoresis systems. Its method of detection relies on platinum electrodes being dipped in two buffer-filled branched detection probe reservoirs on the microchip device. In this way, analytes passing through the detection window will not pass through and subsequently adsorb onto the electrodes, alleviating problems of electrode fouling due to analyte contamination and surface reactions. A customized microchip design was proposed and optimized stepwise for the new FRD system. Each branched detection probe was determined to be 4.50 mm long with a 0.075 mm detection window gap between them. The distance between the detection window and buffer waste reservoir was determined to be 1.50 mm. The optimized microchip design was subsequently used in the analysis of four groups of analytes - inorganic cations, amino acids, aminoglycosides antibiotics, and biomarkers. Based on the preliminary results obtained, the detection limits were in the range of 0.4-0.7 mg/L for the inorganic cations and 1.5-15 mg/L for the amino compounds.

An optimized microchip electrophoresis system for mutation detection by tandem SSCP and heteroduplex analysis for p53 gene exons 5–9

With the complete sequencing of the human genome, there is a growing need for rapid, highly sensitive genetic mutation detection methods suitable for clinical implementation. DNA-based diagnostics such as single-strand conformational polymorphism (SSCP) and heteroduplex analysis (HA) are commonly used in research laboratories to screen for mutations, but the slab gel electrophoresis (SGE) format is ill-suited for routine clinical use. The translation of these assays from SGE to microfluidic chips offers significant speed, cost, and sensitivity advantages; however, numerous parameters must be optimized to provide highly sensitive mutation detection. Here we present a methodical study of system parameters including polymer matrix, wall coating, analysis temperature, and electric field strengths on the effectiveness of mutation detection by tandem SSCP/HA for DNA samples from exons 5-9 of the p53 gene. The effects of polymer matrix concentration and average molar mass were studied for linear polyacrylamide (LPA) solutions. We determined that a matrix of 8% w/v 600 kDa LPA provides the most reliable SSCP/HA mutation detection on chips. The inclusion of a small amount of the dynamic wall-coating polymer poly-N-hydroxyethylacrylamide in the matrix substantially improves the resolution of SSCP conformers and extends the coating lifetime. We investigated electrophoresis temperatures between 17 and 35 degrees C and found that the lowest temperature accessible on our chip electrophoresis system gives the best condition for high sensitivity of the tandem SSCP/HA method, especially for the SSCP conformers. Finally, the use of electrical fields between 350 and 450 V/cm provided rapid separations (<10 min) with well-resolved DNA peaks for both SSCP and HA.