微流控芯片电泳激光诱导荧光检测p53外显子上的突变点

利用单链构象多态性(SSCP),建立微流控芯片电泳(ME)联合激光诱导荧光检测(LIF)技术,检测人类p53基因点突变的方法。设计不同碱基长度的p53单链序列,针对易突变的外显子7,8,9进行SSCP分析,分离野生与突变的单链DNA序列;研究了筛分介质聚乙烯基氧化物(PEO)的浓度,场强对芯片电泳行为的影响。在PEO质量分数为0.5%,分离场强为260V/cm时,100 s之内就可以实现样品p53外显子7,8,9的野生型与突变型碱基的分离检测。     

Rapid mutation detection in complex genes by heteroduplex analysis with capillary array electrophoresis

Mutational analysis of large multiexon genes without prevalent mutations is a laborious undertaking that requires the use of a high-throughput scanning technique. The Human Genome Project has enabled the development of powerful techniques for mutation detection in large multiexon genes. We have transferred heteroduplex analysis (HA) by conformation-sensitive gel electrophoresis of the two major breast cancer (BC) predisposing genes, BRCA1 and BRCA2, to a multicapillary DNA sequencer in order to increase the throughput of this technique. This new method that we have called heteroduplex analysis by capillary array electrophoresis (HA-CAE) is based on the use of multiplex-polymerase chain reaction (PCR), different fluorescent labels and HA in a 16-capillary DNA sequencer. To date, a total of 114 different DNA sequence variants (19 insertions/deletions and 95 single-nucleotide substitutions - SNS) of BRCA1 and BRCA2 from 431 unrelated BC families have been successfully detected by HA-CAE. In addition, we have optimized the multiplex-PCR conditions for the colorectal cancer genes MLH1 and MSH2 in order to analyze them by HA-CAE. Both genes have been amplified in 13 multiplex groups, which contain the 35 exons, and their corresponding flanking intronic sequences. MLH1 and MSH2 have been analyzed in nine hereditary nonpolyposis colorectal cancer patients, and we have found six different DNA changes: one complex deletion/insertion mutation in MLH1 exon 19 and another five SNS. Only the complex mutation and one SNS may be classified as cancer-prone mutations. Our experience has revealed that HA-CAE is a simple, fast, reproducible and sensitive method to scan the sequences of complex genes.     

Electrochemiluminescence detection system for microchip capillary electrophoresis and its application to pharmaceutical analysis

We describe an efficient and easily fabricated electrochemiluminescence detection system for microchip capillary electrophoresis. A 300-μm-diameter platinum disc working electrode was embedded in a titanium tube which provides an adequate holding for working electrode and acts as counter electrode. We also have designed a simplified detection cell with a guide channel for the electrode. The integrated working-counter electrode can be easily aligned to the outlet of the separation channel through the guide channel. The functionality of the system was demonstrated by separation and detection of proline and tripropylamine. The response to proline is linear in the range from 5 μM to 5,000 μM, and the detection limit is 1.0 μM (S/N?=?3). The system was further applied to the determination of chlorpromazine hydrochloride in pharmaceutical formulations. The system is believed to have potential applications in pharmaceutical analysis.

A parylene-based dual channel micro-electrophoresis system for rapid mutation detection via heteroduplex analysis

A new dual channel micro-electrophoresis system for rapid mutation detection based on heteroduplex analysis was designed and implemented. Mutation detection was successfully achieved in a total separation length of 250 microm in less than 3 min for a 590 bp DNA sample harboring a 3 bp mutation causing an amino acid change. Parylene-C was used as the structural material for fabricating the micro-channels as it provides conformal deposition, transparency, biocompatibility, and low background fluorescence without any surface treatment. A new dual channel architecture was derived from the traditional cross-channel layout by forming two identical channels with independent sample loading and waste reservoirs. The control of injected sample volume was accomplished by a new u-turn injection technique with pull-back method. The use of heteroduplex analysis as a mutation detection method on a cross-linked polyacrylamide medium provided accurate mutation detection in an extremely short length and time. The presence of two channels on the microchip offers the opportunity of comparing the sample to be tested with a desired control sample rapidly, which is very critical for the accuracy and reliability of the mutation analyses, especially for clinical and research purposes.     

Integration of combined heteroduplex/restriction fragment length polymorphism analysis on an electrophoresis microchip for the detection of hereditary haemochromatosis

This work describes an integrated method of enzymatic digestion, heteroduplex analysis (HA) and electrophoretic sizing on a microfluidic chip. HA techniques based on microchip electrophoresis are capable of the high sensitivity detection of subtle mutations such as single nucleotide polymorphisms (SNPs) but are not readily able to detect homozygous mutant genotypes. Such homozygous conditions are commonly encountered with the gene implicated in hereditary haemochromatosis, HFE. We employed the restriction fragment length polymorphism (RFLP) method of mutation detection to complement the HA method in a rapid novel on-chip procedure that separated digested PCR fragments to reliably determine the presence or absence of the most important mutations associated with haemochromatosis. This method was able to distinguish the homozygous mutant, heterozygous and homozygous wildtype genotypes. The mutations investigated here (C282Y, H63D and S65C) are often the mutation targets used in the genetic testing for haemochromatosis. This method provides the extremely specific digestion methods needed for the analysis of the known and relatively common mutations that have a significant probability of occurring in a homozygous form. However, the high sensitivity of the HA method is useful in detecting other mutations of lesser likelihood which, by virtue of their rarity, are likely to be present only in a heterozygous form. Although the conventional methods of analysing these mutations require as much as a day to perform, this microchip method, even without robotics or multiplexed operation, can be performed in about 10 min per sample.     

Rapid analysis of amino acids in Japanese green tea by microchip electrophoresis using plastic microchip and fluorescence detection

Microchip electrophoresis for the short-time analysis of amino acids in Japanese green tea was developed. The amino acids in Japanese green tea were derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F). The derivatives were filtered and directly analyzed by electrophoresis on a plastic microchip with a 31-mm long separation channel with fluorescence detection. Amino acid analysis of Japanese green tea was improved by removing polyphenols using a polyvinylpolypyrrolidone pretreatment. Elution profiles of NBD-amino acids were examined under different running buffer conditions, and the sodium dodecyl sulphate in the running buffer exhibited a dramatically high-separation efficiency of amino acids by inhibiting their adsorption on the channel walls. Under the optimized conditions (5 mM phosphate buffer (pH 5.5) containing 0.05 mM sodium dodecylsulfate as running buffer), the main amino acids contained in Japanese green tea were well separated within 2 min, and theanine (1475 mg/100 g tea leaf), Arg (408 mg/100 g tea leaf) and Gln (217 mg/100 g tea leaf) were detected in Japanese green tea.     

新型芯片电泳柱端安培检测系统的构建与评价

自行设计开发了一套便于与电泳芯片集成的一体式柱端安培检测池系统．该系统由整块透明有机玻璃精密加工而成,包括电泳芯片支架和安培检测池两部分,芯片可通过芯片插槽和不锈钢夹具固定在芯片支架上,各种检测用电极可直接通过螺母固定在安培检测池中．以100μmol／L的DA为模式分析物,分别采用直径为100、300和500μm的铂金圆盘电极与表观直径为240μm的碳纤维电极作为工作电极均在该装置上实现了良好组装和高灵敏检测．采用碳纤维工作电极对该系统的检测参数进行了优化．测试结果表明该系统在电化学清洗程序下连续六次测定100μmol／L多巴胺的峰电流相对标准偏差为3．2％,保留时间相对标准偏差为0．5％,DA的检测限为0．4μmol／L（按照S／N=3计）．该系统体积小巧,测试稳定,检测灵敏度较高,工作电极更换方便,适合作为芯片电泳柱端安培检测通用平台．     

An integrated method for mutation detection using on-chip sample preparation, single-stranded conformation polymorphism, and heteroduplex analysis

This work integrates rapid techniques for mutation detection by producing single-stranded DNA and (renatured) double-stranded DNA on-chip, labeling these with fluorescent DNA stains and then performing two complementary methods of mutation detection-single stranded conformation polymorphism (SSCP) analysis and heteroduplex analysis (HA). This involves the denaturation of double-stranded polymerase chain reaction (PCR) product into single-stranded DNA, the mutation analysis of the single-stranded DNA by SSCP and the rehybridized double-stranded DNA by HA. These steps were performed entirely on-chip within several minutes of operation. The combination of these two mutation detection methods on-chip provides a highly sensitive method of mutation detection for either genotyping or screening. Many mutation analysis methods rely upon fluorescently labeled samples from a PCR with fluorescently labeled primers. By labeling on-chip we not only attain improved signal strength, but the method is considerably more versatile. Although we used PCR products in this work, this method could be used to analyze DNA from any source. We believe that this combination of several procedures on a single chip represents a significant step in the development of higher levels of integration upon microfluidic devices.     

Single-strand conformation polymorphism analysis to detect the p53 mutation in colon tumor samples by capillary electrophoresis

The polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) technique is developed for the detection of point mutations in DNA samples, and is very useful in the research of tumors. The traditional SSCP was carried out with slab gel electrophoresis (SGE), but this is time-consuming and labor-intensive, particularly for clinical diagnoses. We have developed a capillary electrophoresis (CE) method for SSCP detection with a linear polyacrylamide gel solution as the sieving matrix. Twenty colon tumor samples were detected with SSCP-CE and the point mutation in exon 7 of the p53 gene was found in six of the samples. Based on the sequencing results, the accuracy of SSCP-CE was better than that of SSCP-SGE. We hope this rapid and convenient method could be applied in the clinical diagnosis of tumors soon.     

Effect of polymer matrix and glycerol on rapid single-strand conformation polymorphism analysis by capillary and microchip electrophoresis for detection of mutations in K-ras gene

We present the rapid single-strand conformation polymorphism (SSCP) analysis by capillary and microchip electrophoresis to detect the mutations in K-ras gene. Parameters that might affect the analysis of mutation in K-ras gene, such as the polymer and the additive in the sieving matrix, have been studied systematically. Under the optimal conditions, the analysis of seven mutants of K-ras gene could be finished within 10 min by capillary electrophoresis (CE). Furthermore, with the wild-type gene as the inner standard, the analysis accuracy of mutations could be improved. In addition, by studying the properties of polymer solutions, the matrix suitable for microchip electrophoresis was found, and the detection of mutations in K-ras gene could be further shortened to 1 min.     

Efficient mutation detection in MEN1 gene using a combination of single-strand conformation polymorphism (MDGA) and heteroduplex analysis

For facilitated genotypic analysis of multiple endocrine neoplasia type 1 (MEN1), a familial syndrome associated with tumors of the parathyroid and neuroendocrine tissues, we developed two screening methods, heteroduplex mutation assay (HMA) and mutation detection gel analysis (MDGA), both based on electrophoretic discrimination of polymerase chain reaction (PCR) products, to detect the mutations. Forty-three genomic DNA samples were used for the evaluation of these techniques. The whole coding region of MEN1 was PCR-amplified with fluorescent primers and then denatured/renatured before electrophoresis on an automated sequencer. 100% of the mutations were detected, subsequently confirmed and identified by sequencing. "Negative" samples were used to evaluate the specificity and reproducibility of the two techniques. The combination of the two methods allows high throughput cost-effective mutation screening which is less laborious than systematic sequencing of the whole coding region of MEN1. Together, these methods provide an efficient screen for MEN1 mutations.     

Single-strand conformation polymorphism for p53 mutation by a combination of neutral pH buffer and temperature gradient in capillary electrophoresis

A large number of point mutations in the p53 gene have been detected by capillary zone electrophoresis via single-strand conformation polymorphism (SSCP) analysis. A much improved detection sensitivity was obtained via the following modifications in running conditions: use of low-viscosity 3% hydroxyethylcellulose (HEC), a neutral pH (pH 6.8) buffer, in which the standard Tris moiety was substituted with a 2-(N-morpholino)ethanesulfonic acid (MES)/Tris mixture, use of SYBR Green II for improved fluorescent signal at the lower pH adopted; and, finally, the use of a temperature gradient in the 15-25 degrees C interval, for favoring the conformational transitions in the mutated samples. The typical temperature gradient activated had a slope of 2 degrees C/min and were induced externally. A total of 24 samples from affected patients, both in the homo- and heterozygous state, were analyzed. All the mutations could be detected by this improved protocol, raising the sensitivity from the standard ca. 80% of conventional SSCP to essentially 100% with the present methodology. All the mutations were confirmed by sequence analysis of the affected samples.     

An ultrasensitive colorimeter assay strategy for p53 mutation assisted by nicking endonuclease signal amplification

A novel catalytic colorimetric assay assisted by nicking endonuclease signal amplification (NESA) was developed. With the signal amplification, the detection limit of the p53 target gene can be as low as 1 pM, which is nearly 5 orders of magnitude lower than that of other previously reported colorimetric DNA detection strategies based on catalytic DNAzyme.     

A sensitive and rapid immunoassay for quantification of testosterone by microchip electrophoresis with enhanced chemiluminescence detection

A sensitive and rapid approach to perform testosterone (T) competitive immunoassay by microchip electrophoresis (MCE) with chemiluminescence (CL) detection was developed. The assay is based on the competitive immunoreactions between T and N-(4-aminobutyl)-N-ethylisoluminol-labeled T (ABEI-T) with a limited amount of antibody (Ab), and the rapid electrophoretic separation of an equilibrated mixture of ABEI-T-Ab complex and free ABEI-T, followed by CL detection using horseradish peroxidase-catalyzed ABEI-H(2)O(2) system. Free ABEI-T and the ABEI-T-Ab complex are well separated within 30 s under the assay conditions. The developed method could be used to determine T with good precision and a detection limit lower than 1.0 nM. This method was applied for the quantification of T in human serum. The results demonstrated that the current MCE-CL-based competitive immunoassay maybe served as an alternative tool for clinical analysis.     

Optimum sample medium for single-nucleotide polymorphism and mutation detection by capillary electrophoresis

Capillary electrophoresis (CE) is a versatile analytical platform widely used for nucleic acids analysis. Its applications in research and diagnostics include scanning and screening for mutations and polymorphisms by such reliable methods as single-strand conformation polymorphism (SSCP), heteroduplex analysis (HA), and combined SSCP/HA. This study, aimed at the further development of these methods, is focused on detailed sample-media characteristics. Factors affecting single-strand conformer stability and DNA intake efficiency were analyzed. The sample media optimal for efficient mutation or SNP detection were determined, and complex SSCP-CE patterns arising from unpurified PCR products were explained. It turns out, that the nondenaturing aqueous media assure both efficient DNA intake, and single-strand conformers stability required for the SSCP and combined SSCP/HA. The results of this study are applicable to all these areas of biomedical research, in which capillary electrophoresis is used for the characterization of nucleic acids.     

Latex bead immobilisation in PDMS matrix for the detection of p53 gene point mutation and anti-HIV-1 capsid protein antibodies

Two diagnostic chemiluminescent biochips were developed for either the detection of p53 gene point mutation or the serological detection of anti-HIV-1 p24 capsid protein. Both biochips were composed of 24 microarrays of latex beads spots (4×4) (150 m in diameter, 800 m spacing) entrapped in a poly(dimethylsiloxane) elastomer (PDMS). The latex beads, bearing oligonucleotide sequences or capsid protein, were spotted with a conventional piezoelectric spotter and subsequently transferred at the PDMS interface. The electron microscopy observation of the biochips showed how homogeneous and well distributed the spots could be. Point mutation detection on the codon 273 of the p53 gene was performed on the basis of the melting temperature difference between the perfect match sequence and the one base pair mismatch sequence. The hybridisation of a 20-mer oligonucleotide form the codon 273 including a one base pair mutation in its sequence on a biochip arrayed with non-muted and the muted complementary sequences, enabled a clear discrimination at 56°C between muted and wild sequences. Moreover, the quantitative measurement of the amount of muted sequence in a sample was possible in the range 0.4–4 pmol. Serological measurement of anti-HIV-1 p24 capsid protein on the biochip, prepared with 1-m-diameter latex beads, enabled the detection of monoclonal antibodies in the range 1.55–775 ng mL^–1. Such a range could be lowered to 0.775 ng mL^–1 when using 50-nm-diameter beads, which generated a higher specific surface. The validation of the biochip for the detection of anti-HIV-1 capsid protein antibodies was performed in human sera from seropositive and seronegative patients. The positivity of the sera was easily discriminated at serum dilutions below 1:1,000.     

An ellipsoidal mirror for detection of laser-induced fluorescence in capillary electrophoresis system: applications for labelled antibody analysis

An LIF detector was integrated into a CE system which uses a ball lens to focus the laser beam on the CE capillary. The detector employs an ellipsoid that is glued on the capillary window, to permit the collection of the fluorescence in the capillary. This 'trapped' fluorescence stays in the capillary because the angle of the silica/air interface is greater than the critical angle. The performance of this new detector setup is found to be identical to the collinear setup using the same ball lens. An application to the analysis of FITC-labeled IgG was optimized using a 14 cm effective length capillary. The LOD of an FITC-labeled IgG2 at an excitation wavelength of 488 nm was 150 pg/mL, which was 10 times better than the LOD recorded with slab gel silver staining. Using a tetramethylrhodamine (TAMRA)-labeled IgG2 and a 532 nm excitation wavelength the LOD is 50 pg/mL. The electropherograms of four different commercial FITC conjugates of IgG were studied. The presence of aggregates was observed in two samples while close kinetics of reduction was observed between free aggregates and high aggregates concentration samples. The integrated LIF detector provides an extremely powerful and convenient tool for antibody analysis and should be useful for therapeutic MAb control in pharmaceutical facilities.     

A modified electrode for the electrochemical detection of biogenic amines and their amino acid precursors separated by microchip capillary electrophoresis

The use of a mixed-valent ruthenium oxide/hexacyanoruthenate polymeric film electrochemically deposited onto glassy carbon electrodes is proposed here for the detection of biogenic amines and their amino acid precursors, following their separation by microchip capillary electrophoresis. The ability of this ruthenium coating to electrocatalyze the oxidation of aliphatic and heterocyclic amines, as well as their amino acid precursors, was checked by using ethanolamine, tryptamine and tryptophane as prototype compounds and adopting a 25 mM sulphuric acid as the electrolyte in the detection cell, where a constant potential of 1.05 V versus Ag/AgCl, 3 M KCl was applied to the modified working electrode. Optimization of parameters affecting both detection and separation steps led to satisfactory separations when performed by using a 20 mM phosphate running buffer (pH 2.5) and applying a high voltage of 2.5 kV both in the separation and in the electrokinetic injection (duration 4 s). The recorded peaks were characterized by good repeatability (RSD ≤ 3.6%), high sensitivity and a wide linear range. Detection limits of 23 μM (1.4 mg/L), 27 μM (4.3 mg/L) and 34 μM (6.8 mg/L) were inferred for ethanolamine, tryptamine and tryptophane, respectively. The approach proposed here was also applied for the analysis of some double malt dark beers spiked with a controlled amount of the analytes considered.     

Homocyclic o-dicarboxaldehydes: Derivatization reagents for sensitive analysis of amino acids and related compounds by capillary and microchip electrophoresis

Amino acids are essential compounds for living organisms, and their determination in biological fluids is crucial for the clinical analysis and diagnosis of many diseases. However, the detection of most amino acids is hindered by the lack of a strong chromophore/fluorophore or electrochemically active group in their chemical structures. The highly sensitive determination of amino acids often requires derivatization. Capillary electrophoresis is a separation technique with excellent characteristics for the analysis of amino acids in biological fluids. Moreover, it offers the possibility of precapillary, on-capillary, or postcapillary derivatization. Each derivatization approach has specific demands in terms of the chemistry involved in the derivatization, which is discussed in this review. The family of homocyclic o-dicarboxaldehyde compounds, namely o-phthalaldehyde, naphthalene-2,3-dicarboxaldehyde, and anthracene-2,3-dicarboxaldehyde, are powerful derivatization reagents for the determination of amino acids and related compounds. In the presence of suitable nucleophiles they react with the primary amino group to form both fluorescent and electroactive derivatives. Moreover, the reaction rate enables all of the derivatization approaches mentioned above. This review focuses on articles that deal with using these reagents for the derivatization of amino acids and related compounds for ultraviolet-visible spectrometry, fluorescence, or electrochemical detection. Applications in capillary and microchip electrophoresis are summarized and discussed.     

An image analysis suite for spot detection and spot matching in two-dimensional electrophoresis gels

We propose a suite of novel algorithms for image analysis of protein expression images obtained from 2-D electrophoresis. These algorithms are a segmentation algorithm for protein spot identification, and an algorithm for matching protein spots from two corresponding images for differential expression study. The proposed segmentation algorithm employs the watershed transformation, k-means analysis, and distance transform to locate the centroids and to extract the regions of the proteins spots. The proposed spot matching algorithm is an integration of the hierarchical-based and optimization-based methods. The hierarchical method is first used to find corresponding pairs of protein spots satisfying the local cross-correlation and overlapping constraints. The matching energy function based on local structure similarity, image similarity, and spatial constraints is then formulated and optimized. Our new algorithm suite has been extensively tested on synthetic and actual 2-D gel images from various biological experiments, and in quantitative comparisons with ImageMaster2D Platinum the proposed algorithms exhibit better spot detection and spot matching.