基于热阻梯度加热系统的温度梯度芯片毛细管电泳用于DNA突变检测

建立了一种简单、可靠的空间温度梯度芯片毛细管电泳DNA突变分析系统, 制作了热阻呈梯度均匀变化的硅橡胶(PDMS)基片, 利用其热阻变化对热传导的影响, 在基片表面形成稳定的空间温度梯度. 通过改变PDMS基片的厚度差, 可得到范围不同的温度梯度, 且形成的温度梯度在6 h内保持稳定. 利用该温度梯度加热装置对玻璃微流控芯片进行加热, 在10 ℃温度梯度范围内对209 bp的DNA突变标准样品进行分离检测, 单次样品分析时间为8.3 min, 并成功用于3例大肠癌患者石蜡组织切片中K-ras基因突变的检测.     

Fluorescence-based single-strand conformation polymorphism analysis of mutations by capillary electrophoresis

Capillary electrophoresis in combination with fluorescence-based single-strand conformation polymorphism (SSCP) analysis was used to screen for known mutations as well as for unknown mutations. The mutations causing hemochromatosis and thrombogenetic diseases (factor V Leiden mutation and prothrombin mutation) are well defined. Familial hypercholesterolemia is caused by mutations in the low density lipoprotein (LDL) receptor gene. Because the mutations are heterogeneously localized in all 18 exons of the LDL receptor gene, effective screening procedures are necessary. The three well known mutations and 59 of 61 previously characterized mutations in the LDL receptor gene were detected by a distinct abnormal fragment pattern in capillary electrophoresis. The remaining two mutations in the LDL receptor gene showed only slight abnormalities under standard electrophoresis conditions (13 kV, 30 degrees C, 30 min). However, the abnormal pattern could be amplified by increasing the electrophoresis temperature. In all cases, heterozygous and homozygous mutations could clearly be differentiated from wild-type alleles. Because of the high efficiency of mutation detection, capillary electrophoresis in combination with fluorescence-based SSCP analysis would be attractive for the detection of well-defined mutations as well as for the screening of unknown mutations. The accuracy and the degree of automation make this technique well suited for routine genetic diagnosis.     

Integration of gene amplification and capillary gel electrophoresis on a polydimethylsiloxane-glass hybrid microchip

We report on the development of a hybrid polydimethylsiloxane (PDMS)-glass microchip for genetic analysis by functional integration of polymerase chain reaction (PCR) and capillary gel electrophoresis (CGE), and on related temperature control systems for PCR on a PDMS-glass hybrid microchip. The microchip was produced by molding PDMS against a microfabricated master with comparatively simple and inexpensive methods. PCR was successfully carried out on the PDMS-glass hybrid microchip with 500 bp target of lambdaDNA and the amplified gene was subsequently analyzed by CGE on the same PDMS-glass microchip. The chip could be considered as an inexpensive single-use apparatus compared to glass or silicon-made microchips for the same purpose.     

Polymer solutions and entropic-based systems for double-stranded DNA capillary electrophoresis and microchip electrophoresis

We give an overview of recent development of low-viscosity polymer solutions and entropic trapping networks for double-stranded DNA (dsDNA) separations by conventional capillary electrophoresis and microchip electrophoresis. Theoretical models for describing separation mechanisms, commonly used noncross-linked polymer solutions, thermoresponsive (viscosity-adjustable) polymer solutions, and novel entropic trapping networks are included. The thermoresponsive polymer solutions can be loaded at one temperature into microchannels at lower viscosities, and used in separation at another temperature at entanglement threshold concentrations and higher viscosities. The entropic-based separations use only arrays of regular obstacles acting as size-separations and do not need viscous polymer solutions. These progresses have potential in integration to automated capillary and microfluidic chip systems, enabling better reusability of separation microchannels, much shorter DNA separation times, and higher reproducibility due to less matrix degradation.     

Top-down analysis of basic proteins by microchip capillary electrophoresis mass spectrometry

A system of microchip capillary electrophoresis/electrospray ionization mass spectrometry (microchip-CE/ESI-MS) for rapid characterization of proteins has been developed. Capillary electrophoresis (CE) enables rapid analysis of a sample present in very small quantity, such as at femtomole levels, at high resolution. Faster CE/MS analysis is expected by downsizing the normal capillary to the microchip (microchip) capillary. Although rapidity and high resolution are advantages of CE separation, electroosmotic flow (EOF) instability caused by the interaction between proteins and the microchannel surface results in low reproducibility in the analysis of basic proteins under neutral pH conditions. By coating the microchannel surface with a basic polymer, polyE-323, basic proteins, which have pI values of over 7.5, could be separated and detected by microchip-CE/MS on quadrupole (Q) and time-of-flight (TOF) hybrid instruments. By increasing the cone and collision voltages during the analysis by microchip-CE/ESI-MS of a small protein, some product ions, which contain the sequence information, could also be obtained, i.e., 'top-down' analysis of the protein could be accomplished with this microchip-CE/MS system. To our knowledge, this is the first report of 'top-down' analysis of a protein by microchip-CE/MS. Since it requires a much shorter time and a smaller sample amount for analysis than the conventional liquid chromatography (LC)/ESI-MS method, microchip-CE/MS promises to be suitable for the high-throughput characterization of proteins.     

Capillary and microelectrophoretic separations of ligase detection reaction products produced from low-abundant point mutations in genomic DNA

Capillary gel electrophoresis (CGE) and polymer-based microelectrophoretic platforms were investigated to analyze low-abundant point mutations in certain gene fragments with high diagnostic value for colorectal cancers. The electrophoretic separations were carried out on single-stranded DNA (ssDNA) products generated from an allele-specific ligation assay (ligase detection reaction, LDR), which was used to screen for a single base mutation at codon 12 in the K-ras oncogene. The presence of the mutation generated a ssDNA fragment that was >40 base pairs (bp) in length, while the primers used for the ligation assay were <30 bp in length. Various separation matrices were investigated, with the success of the matrix assessed by its ability to resolve the ligation product from the large molar excess of unligated primers when the mutant allele was lower in copy number compared to the wild-type allele. Using CGE, LDR product models (44 and 51 bp) could be analyzed in a cross-linked polyacrylamide gel with a 1000-fold molar excess of LDR primers (25 bp) in approximately 45 min. However, when using linear polyacrylamide gels, these same fragments could not be detected due to significant electrokinetic biasing during injection. A poly(methylmethacrylate) (PMMA) microchip of 3.5 cm effective column length was used with a 4% linear polyacrylamide gel to analyze the products generated from an LDR. When the reaction contained a 100-fold molar excess of wild-type DNA compared to a G12.2D mutant allele, the 44 bp ligation product could be effectively resolved from unligated primers in under 120 s, nearly 17 times faster than the CGE format. In addition, sample cleanup was simplified using the microchip format by not requiring desalting of the LDR prior to loading.     

Fast electrophoretic analysis of individual mitochondria using microchip capillary electrophoresis with laser induced fluorescence detection

The analysis of mitochondria by capillary electrophoresis usually takes longer than 20 min per replicate which may compromise the quality of the mitochondria due to degradation. In addition, low sample consumption may be beneficial in the analysis of rare or difficult samples. In this report, we demonstrate the ability to analyze individual mitochondrial events in picoliter-volume samples (approximately 80 pL) taken from a bovine liver preparation using microchip capillary electrophoresis with laser-induced fluorescence detection (micro-chip CE-LIF). Using a commercial "double-T" glass microchip, the sample was electrokinetically loaded in the "double-T" intersection and then subjected to electrophoretic separation along the main separation channel. In order to decrease interactions of mitochondria with channel walls during the analysis, poly(vinyl alcohol) was used as a dynamic coating. This procedure eliminates the need for complicated covalent surface modifications within the channels that were previously used in capillary electrophoresis methods. For analysis, mitochondria, isolated from bovine liver tissue, were selectively labelled using 10-nonyl acridine orange (NAO). The results consist of electropherograms where each mitochondrial event is a narrow spike (240 +/- 44 ms). While the spike intensity is representative of its NAO content, its migration time is used to calculate and describe its electrophoretic mobility, which is a property still largely unexplored for intracellular organelles. The five-fold decrease in separation time (4 min for microchip versus 20 min for capillary electrophoresis) makes microchip electrophoretic separations of organelles a faster, sensitive, low-sample volume alternative for the characterization of individual organelle properties and for investigations of subcellular heterogeneity.     

Detection of K-ras exon 1 mutations by constant denaturant capillary electrophoresis

Among various mutation detection methods, constant denaturant capillary electrophoresis (CDCE) is one of the most common techniques for rapid identification of known or unknown mutations. In this report, a CDCE analysis method with homemade linear polyacrylamide (LPA) kit was developed on ABI 310 genetic analyzer, the effect and relationship of various denaturing factors in CDCE analysis were investigated and K-ras gene mutations of 31 coloerctal cancer patients were detected. Results indicate that, with the increase of chemical danaturant concentration, the optimum temperature was lowered, and when the concentration of urea (formamide) was higher than 7 M (40%), the homoduplex and heteroduplex of mutant samples were separated with difficulty. Detection results of K-ras gene in colorectal samples indicated that mutations were present in eight (26%) of 31 patients; most mutations were localized in codon 12, which is thought to be a critical step and plays an important role in human colorectal carcinogenesisas.     

Rapid analysis of atorvastatin calcium using capillary electrophoresis and microchip electrophoresis

In this work, a capillary electrophoretic method for the rapid quantitation of atorvastatin (AT) in a lipitor tablet was investigated and developed. Method development included studies of the effect of applied potential, buffer concentration, buffer pH, and hydrodynamic injection time on the electrophoretic separation. The method was validated with regard to linearity, precision, specificity, LOD, and LOQ. The optimum electrophoretic separation conditions were 25 mM sodium acetate buffer at pH 6, with a separation voltage of 25 kV using a 50 microm capillary of 33 cm total length. Sodium diclofenac was used as an internal standard. Analysis of AT in a commercial lipitor tablet by electrophoresis gave quite high efficiency, coupled with an analysis time of less than 1.2 min in comparison to LC. Once the separation was optimized on capillary, it was further miniaturized to a microchip platform, with linear imaging UV detection using microchip electrophoresis (MCE). Linear imaging UV detection allowed for real-time monitoring of the analyte movement on chip, so that the optimum separation time could be easily determined. This microchip electrophoretic method was compared to the CE method with regard to speed, efficiency, precision, and LOD. This work represents the most rapid and first reported analysis of AT using MCE.     

Rapid sizing of microsatellite alleles by gel electrophoresis on microfabricated channels: application to the D19S394 tetranucleotide repeat for cosegregation study of familial hypercholesterolemia.

This study evaluated the applicability of microchip electrophoresis to the sizing of microsatellites suitable to genetic, clinical and forensic applications. The evaluation was performed with the D19S394 tetranucleotide (AAAG) repeat characterized by a wide variation in the repeat number (1-17) and a short recombination distance from the low-density lipoprotein (LDL)-receptor gene that makes it suitable to cosegregation analysis of familial hypercholesterolemia (FH). The study was performed with 70 carriers of two LDL-receptor mutations common in northern Italy (i.e., the 4 bp insertion in exon 10 known as FH-Savona and the D200G missense mutation in the exon 4, known as FH-Padova 1) and 100 healthy controls. The polymerase chain reaction (PCR) amplification products prepared with a cosolvent PCR protocol and an antibody-protected polymerase were directly analyzed with an apparatus for high-voltage capillary electrophoresis on microchips and laser-induced fluorescence detection equipped with chips for the analysis of 25-500 bp dsDNA fragments. The test could not be extended to dinucleotide repeats due to the resolution characteristics of the available microchip. This novel approach was able to distinguish 17 microsatellite alleles varying from 0 to 17 repeats. Many of these alleles were quite rare, but the seven more abundant accounted for over the 70% of allele distribution in control population. The standard deviation in the sizing of the most abundant alleles ranged from +0.60 to +/- 0.75 bp. This indicated that the size attribution to a conventional allele using the +/- 1 bp range around it allowed a confidence limit above the 80 %. The sizing of D19S394 obtained this way allowed the cosegregation analysis with both the FH mutations tested. Therefore, this innovative approach to microsatellite sizing was much simpler, but equally effective as traditional capillary electrophoresis, at least with tetranucleotide repeats.     

Fast extraction, amplification and analysis of genes from human blood

In order to shorten the time spent on the sample preparation for gene analysis, a novel method was proposed through the combination of fast DNA extraction and purification by Generation capture disk, amplification by capillary polymerase chain reaction, and confirmation of amplification products by microchip electrophoresis. With this method, 3 microL blood was enough to obtain adequate target fragments in human genes. Under the optimal conditions in each step, the sample preparation for eight fragments in beta-globin gene and four fragments in ras gene could be finished within 20 min. Since all the experiments were performed on commercial instruments, this method showed a wide range of applicability. In addition, other advantages such as fast speed and low consumption of samples were demonstrated. All these merits proved that such a combination method was of great potential for the clinical diagnostics.     

Towards a portable microchip system with integrated thermal control and polymer waveguides for real-time PCR

A novel real-time PCR microchip platform with integrated thermal system and polymer waveguides has been developed. The integrated polymer optical system for real-time monitoring of PCR was fabricated in the same SU-8 layer as the PCR chamber, without additional masking steps. Two suitable DNA binding dyes, SYTOX Orange and TO-PRO-3, were selected and tested for the real-time PCR processes. As a model, cadF gene of Campylobacter jejuni has been amplified on the microchip. Using the integrated optical system of the real-time PCR microchip, the measured cycle threshold values of the real-time PCR performed with a dilution series of C. jejuni DNA template (2 to 200 pg/microL) could be quantitatively detected and compared with a conventional post-PCR analysis (DNA gel electrophoresis). The presented approach provided reliable real-time quantitative information of the PCR amplification of the targeted gene. With the integrated optical system, the reaction dynamics at any location inside the micro reaction chamber can easily be monitored.     

Plastic microchip electrophoresis for genetic screening: the analysis of polymerase chain reactions products of fragile X (CGG)n alleles

Clinical screening of abnormal chromosomes associated with fragile X syndrome (FXS) demands a high-throughput method including DNA sizing and detection of the amplified products. This study is to explore the use of polymer microchip electrophoresis for the analysis of polymerase chain reaction (PCR) products of fragile X (CGG)n alleles to facilitate a fast exclusion test of FXS. The sequences flanking the CGG-repeat of FMR1 gene was amplified by betaine-PCR and the amplified products were desalted and then analyzed by microchips which were fabricated on poly(methyl methacrylate) (PMMA) substrate. The PCR bands with more than six CGG-repeats in difference could be clearly distinguished in less than 3 min by microchip electrophoresis with a separation length of 6 cm. It was found that the signal was greatly enhanced with the use of both covalent (Cy5) and intercalating dye (TORRO-3), which has never been demonstrated before. We tested the method by reanalysis of twelve samples from males and six samples from females. For female samples with less than six repeat differences, Southern blotting method was performed to confirm or exclude the findings from microchips. It was found that the test results from all male and female samples show a 100% correlation between the microchip electrophoresis and the existing methods.     

Multiplexed p53 Mutation Detection by Microchip Electrophoresis with Laser-Induced Fluorescence Detector

A 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.     

Integrated circuit microchip system with multiplex capillary electrophoresis module for DNA analysis

In this paper, we describe the use of an integrated circuit (IC) microchip system as a detector in multiplex capillary electrophoresis (CE). This combination of multiplex capillary gel electrophoresis and the IC microchip technology represents a novel approach to DNA analysis on the microchip platform. Separation of DNA ladders using a multiplex CE microsystem of four capillaries was monitored simultaneously using the IC microchip system. The IC microchip-CE system has advantages such as low cost, rapid analysis, compactness, and multiplex capability, and has great potential as an alternative system to conventional capillary array gel electrophoresis systems based on charge-coupled device (CCD) detection.     

Native and sodium dodecyl sulfate-capillary gel electrophoresis of proteins on a single microchip

Simultaneous electrophoresis of both native and Sodium dodecyl sulfate (SDS) proteins was observed on a single microchip within 20 min. The capillary array prevented lateral diffusion of SDS components and avoided cross contamination of native protein samples. The planar sputtered electrode format provided a more uniform distribution of separation voltage into each of the 36 parallel microchannel capillaries than platinum wire electrodes commonly used in conventional electrophoresis. The customized geometry of the stacking capillary machined into the cover plate of the microchip facilitated reproducible sample injection without the requirement for stacking gel. Polyimide served as a mask and facilitated insulation of the anode and cathode to prevent electrode lift off and deterioration during continuous electrophoresis, even at a constant current of 8 mA. Improved protein separation was observed during capillary electrophoresis at lower currents. Ferguson plot analysis confirmed the electrophoretic mobility of native globular proteins in accordance with their charge and size. Corresponding Ferguson plot analysis of SDS-associated proteins on the same chip confirmed separation of marker proteins according to their molecular weight.     

微流体芯片电泳技术对人血清蛋白的快速分离

通过微流体芯片电泳技术分离人血清蛋白,探讨了常见十字形微流体芯片上样品的电动进样与分离过程,分析了在十字芯片上的进样时间和电压设置对后续样品检测和定量的影响。采用的缓冲体系为:100mmol/L H3BO3,50mmol/L NaCl,5%Dextran(以NaOH调至pH 8.3),该缓冲液能够有效分离人血清蛋白中的白蛋白(Albumin)和4种球蛋白(α1-,α2-,β-,和γ-globulin),并且给出了它们在该缓冲体系中的淌度估算范围为5.15×10-5～47.2×10-5 cm2/(V.s)。在芯片上2min之内可以完成进样和分离,相比于常用的毛细管区带电泳,提高了分析速度。     

High-sensitivity capillary gel electrophoretic analysis of DNA fragments on an electrophoresis microchip using electrokinetic injection with transient isotachophoretic preconcentration

The research adopted a single-channel microchip as the probe, and focused electrokinetic injection combined with transient isotachophoresis preconcentration technique on capillary electrophoresis microchip to improve the analytical sensitivity of DNA fragments. The channel length, channel width and channel depth of the used microchip were 40.5 mm, and 110 and 50 μm, respectively. The separation was detected by CCD (charge-coupled device) (effective LENGTH=25 mm, 260 nm). A 1/100 diluted sample (0.2 mg/l of each DNA fragment) of commercially available stepladder DNA sample could be baseline separated in 120 s with S/N=2–5. Compared with conventional chip gel electrophoresis, the proposed method is ideally suited to improve the sensitivity of DNA analysis by chip electrophoresis.     

High-speed electrophoretic analysis of 1-phenyl-3-methyl-5-pyrazolone derivatives of monosaccharides on a quartz microchip with whole-channel UV detection

1-Phenyl-3-methyl-5-pyrazolone (PMP) derivatives of monosaccharides were analyzed by electrophoresis on a quartz microchip with whole-channel UV detection. Rapid separation of PMP derivatives of aldopentoses was achieved by plain-zone electrophoresis in a neutral phosphate buffer with the height equivalent to a theoretical plate at the micrometer level. Zone electrophoresis as borate complexes was also successful for the separation of PMP derivatives of a few aldoses, which were separated within 1 min. Separation by microchip electrophoresis was compared to that by capillary electrophoresis, and the difference was discussed in terms of column efficiency and sample column capacity.     

Serial processing of biological reactions using flow-through microfluidic devices: coupled PCR/LDR for the detection of low-abundant DNA point mutations

We have fabricated a flow-through biochip consisting of passive elements for the analysis of single base mutations in genomic DNA using polycarbonate (PC) as the substrate. The biochip was configured to carry out two processing steps on the input sample, a primary polymerase chain reaction (PCR) followed by an allele-specific ligation detection reaction (LDR) for scoring the presence of low abundant point mutations in genomic DNA. The operation of the device was demonstrated by detecting single nucleotide polymorphisms in gene fragments (K-ras) that carry high diagnostic value for colorectal cancers. The effect of carryover from the primary PCR on the subsequent LDR was investigated in terms of LDR yield and fidelity. We found that a post-PCR treatment step prior to the LDR phase of the assay was not essential. As a consequence, a thermal cycling microchip was used for a sequential PCR/LDR in a simple continuous-flow format, in which the following three steps were carried out: (1) exponential amplification of the gene fragments from genomic DNA; (2) mixing of the resultant PCR product(s) with an LDR cocktail via a Y-shaped passive micromixer; and (3) ligation of two primers (discriminating primer that carried the complement base to the mutation locus being interrogated and a common primer) only when the particular mutation was present in the genomic DNA. We successfully demonstrated the ability to detect one mutant DNA in 1000 normal sequences with the integrated microfluidic system. The PCR/LDR assay using the microchip performed the entire assay at a relatively fast processing speed: 18.7 min for 30 rounds of PCR, 4.1 min for 13 rounds of LDR (total processing time = ca. 22.8 min) and could screen multiple mutations simultaneously in a multiplexed format. In addition, the low cost of the biochip due to the fact that it was fabricated from polymers using replication technologies and consisted of passive elements makes the platform amenable to clinical diagnostics, where one-time use devices are required to eliminate false positives resulting from carryover contamination.