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.     

An electronic DNA microarray technique for detection and differentiation of viable Campylobacter species

An electronic oligonucleotide microarray technique was developed for detection and differentiation of the viable Campylobacter species, C. jejuni, C. coli, and C. lari. This development consisted of four major components: identification of single nucleotide polymorphisms (SNPs) within the hsp60 gene as species markers, design of fluorescently labelled SNP-based reporters, development of an electronic microarray detection, and application of the integrated technique to analysis of Campylobacter species in food samples. A unique capability of this technique is the specific detection of viable cells and not dead ones. This is achieved by using mRNA of the 60 kDa heat-shock protein as the viability marker. The identification of two unique SNPs closely located at positions 291 and 294 of the hsp60 gene enabled the differentiation of the three Campylobacter species. This technique was able to detect as few as two viable Campylobacter cells. The analysis of 19 blind Campylobacter samples showed 100% agreement with their identities obtained using pulsed-field gel electrophoresis. The analysis of six chicken samples revealed the presence of C. coli in one of the samples.     

A universal multiplex PCR strategy for 100-plex amplification using a hydrophobically patterned microarray

Both basic research and clinical medicine have urgent demands for highly efficient strategies to simultaneously identify many different DNA sequences within a single tube. Effective and simultaneous amplification of multiple target sequences is a prerequisite for any successful multiple nucleic acid detection method. Multiplex PCR is one of the best choices for this purpose. However, due to the intrinsic interference and competition among primer pairs in the same tube, multiple rounds of highly empirical optimization procedures are usually required to establish a successful multiplex PCR reaction. To address this challenge, we report here a universal multiplex PCR strategy that is capable of over 100-plex amplification using a specially designed microarray in which hydrophilic microwells are patterned on a hydrophobic chip. On such an array, primer pairs tagged with a universal sequence are physically separated in individual hydrophilic microwells on an otherwise hydrophobic chip, enabling many unique PCR reactions to be proceeded simultaneously during the first step of the procedure. The PCR products are then isolated and further amplified from the universal sequences, producing a sufficient amount of material for analysis by conventional gel electrophoresis or DNA microarray technology. This strategy is abbreviated as "MPH&HPM" for "Multiplex PCR on a Hydrophobically and Hydrophilically Patterned Microarray". The feasibility of this method is first demonstrated by a multiplex PCR reaction for the simultaneous detection of eleven pneumonia-causing pathogens. Further, we demonstrate the power of this strategy with a highly successful 116-plex PCR reaction that required only little prior optimization. The effectiveness of the MPH&HPM strategy with clinical samples is then illustrated with the detection of deleted exons of the Duchenne Muscular Dystrophy (DMD) gene, the results are in excellent agreement with the clinical records. Because of its generality, simplicity, flexibility, specificity and capacity of more than 100-plex amplification, the MPH&HPM strategy should have broad applications in both laboratory research and clinical applications when multiplex nucleic acid analysis is required.     

Interlaboratory study of DNA extraction from multiple ground samples, multiplex real-time PCR, and multiplex qualitative PCR for individual kernel detection system of genetically modified maize

In many countries, the labeling of grains, feed, and foodstuff is mandatory if the genetically modified (GM) organism content exceeds a certain level of approved GM varieties. We previously developed an individual kernel detection system consisting of grinding individual kernels, DNA extraction from the individually ground kernels, GM detection using multiplex real-time PCR, and GM event detection using multiplex qualitative PCR to analyze the precise commingling level and varieties of GM maize in real sample grains. We performed the interlaboratory study of the DNA extraction with multiple ground samples, multiplex real-time PCR detection, and multiplex qualitative PCR detection to evaluate its applicability, practicality, and ruggedness for the individual kernel detection system of GM maize. DNA extraction with multiple ground samples, multiplex real-time PCR, and multiplex qualitative PCR were evaluated by five laboratories in Japan, and all results from these laboratories were consistent with the expected results in terms of the commingling level and event analysis. Thus, the DNA extraction with multiple ground samples, multiplex real-time PCR, and multiplex qualitative PCR for the individual kernel detection system is applicable and practicable in a laboratory to regulate the commingling level of GM maize grain for GM samples, including stacked GM maize.     

Fully integrated PCR-capillary electrophoresis microsystem for DNA analysis

A fully integrated genomic analysis microsystem including microfabricated heaters, temperature sensors, and PCR chambers directly connected to capillary electrophoretic separation channels has been constructed. Valves and hydrophobic vents provide controlled and sensorless sample positioning and immobilization into 200 nL PCR chambers. The use of microfabricated heating and temperature sensing elements improves the heating and cooling rates for the PCR reaction to 20 degree C s(-1). The amplified PCR product, labeled on-column with an intercalating fluorescent dye, is injected into the gel-filled capillary for electrophoretic analysis. Successful sex determination using a multiplex PCR reaction from human genomic DNA is demonstrated in less than 15 min. This device is an important step toward a microfabricated genomic microprocessor for use in forensics and point-of-care molecular medical diagnostics.     

Bead affinity chromatography in a temperature-controllable microsystem for biomarker detection

This paper describes a temperature-controllable bead affinity chromatography (BAC) in a microsystem for biomarker detection, and preparing samples for matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) analysis. Cancer marker proteins were captured in the microsystem by BAC with RNA aptamer-immobilized microbeads. The captured proteins were then denatured and released from the microbeads by controlling temperature. The microsystem consists of a microreactor for trapping microbeads and a temperature control unit for thermal treatment of the trapped beads. We used polymethylsilxoane or single crystalline silicon in fabricating two different types of reaction chamber to compare the differences in performance originated from the materials. Carcinoembryonic antigen was concentrated and purified from human serum using the microsystem and detected by MALDI-TOF MS to demonstrate the usefulness of the microsystem. The microsystem simplifies a sample preparation process required for protein analysis and cancer biomarker detection, which will accelerate the process of cancer research.     

DNA microarray: a high throughput approach for methylation detection

We described a DNA microarray-based method combined with bisulphite treatment of DNA and regular PCR to examine hyper-methylation in promoter 1A of APC gene. A set of oligonucleotide probes were designed and immobilized on the aldehyde-coated glass slides for detecting the methylation pattern of 15 selected CpG sites in the region. The methylation status of 30 colorectal tumor samples have been examined by both of methylation-specific PCR (MS-PCR) and the present microarray method. The methylation pattern of the 15 CpG sites for the samples have been obtained with the microarray. A total of 19 samples out of 30 were methylated by microarray, in which five samples cannot be detected by MS-PCR due to the methylated CpG patterns not accordant to the MS-PCR primers. The detecting ratio for methylation of APC gene of colorectal tumor samples increased from 46.7% with MS-PCR to 63.3% with the microarray, which successfully demonstrated that DNA microarray-based method not only can obtained the methylation patterns for the related genes, but also decrease the false-negative results of methylation status by the conventional MS-PCR for the investigated genes.     

A multiplex single nucleotide polymorphism genotyping method using ligase‐based mismatch discrimination and CE‐SSCP

Accuracy, simplicity, and cost‐effectiveness are the most important criteria for a genotyping method for SNPs compatible with clinical use. One method developed for SNP genotyping, ligase‐based discrimination, is considered the simplest for clinical diagnosis. However, multiplex assays using this method are limited by the detection method. Although CE has been introduced as an alternative to error prone microarray‐based detection, the design process and multiplex assay procedure are complicated because of the DNA size‐dependent separation principle. In this study, we developed a simple and accurate multiplex genotyping method using reaction condition‐optimized ligation and high‐resolution CE‐based SSCP. With this high‐resolution CE‐SSCP system, we are able to use similar‐sized probes, thereby eliminating the complex probe design step and simplifying the optimization process. We found that this method could accurately discriminate single‐base mismatches in SNPs of the tp53 gene, used as targets for multiplex detection.     

Development and application of a multiplex PCR system for drowning diagnosis

In recent years, the DNA detection of drowning-related diatoms, cyanobacteria, and aeromonas has gradually attracted interest from forensic scientists. In this study, we described the validation and application of a novel multiplex PCR system. This system integrated 12 fluorescently labelled primers designed to amplify specific genes of diatoms, cyanobacteria, and aeromonas. The specificity studies demonstrated that this multiplex PCR system could detect nine species of diatom, seven species of cyanobacteria, and five species of aeromonas, all of which were drowning-related and widely distributed in various water circumstance of southern China. The sensitivity studies indicated that the limit concentration of template DNA was 0.0125 ng. Besides, this multiplex PCR system had good performance in sizing precision and stability, but it is not suitable for degraded DNA samples. The application into forensic casework showed that all the tissue samples from ten nondrowning cases showed negative results, and the positive rates of lung, liver, kidney, and water samples from 30 drowning bodies were 100, 86.7, 90, and 100%, respectively. Combined with results of diatom tests of MD-VF-Auto SEM method, this multiplex PCR system could help rule out nondrowning bodies and provide extra evidences to support drowning diagnosis, especially for those cases with few diatoms observed. It is expected that this multiplex PCR system has great potential for forensic drowning diagnosis.     

DNA microarray fabricated on poly(acrylic acid) brushes-coated porous silicon by in situ rolling circle amplification

Microarrays hold considerable promise in large-scale biology on account of their analytical, massive and parallel nature. In a step toward further enabling such a capability, we describe the application of rolling circle amplification (RCA) for a sensitive and multiplex detection of nucleic acid targets on oligonucleotide-conjugated polymer brushes covalently grown from porous silicon. Both RCA and polymer brushes have been taken to increase the loading quantity of target molecules and thus improve the detection sensitivity without loss of multiplexing. Besides, polymer brushes were employed to protect porous silicon and to provide biologically simulated environments, making the attached biomolecules maintain bioactivity. This approach can reach a detection limit of 0.1 nM target analytes and three orders of magnitude dynamic range of 0.1-100 nM, with a fluorescence scanner. A two-colour DNA microarray was achieved via RCA of two kinds of circular DNA targets on one chip simultaneously. The porous silicon chip-based RCA technique is promising for the multiplex detection of deoxynucleic acids on microarrays.     

Development of an integrated microsystem for injection, transport and manipulation of encoded microbeads

An integrated microsystem for injection, transport and manipulation of encoded microbeads on a single microchip is presented. The device also incorporates a customized reaction chamber to process individual, optically encoded microbeads. This research illustrates how microfabrication technologies enable convenient integration of multiple capabilities of microbeads, controlled microfluidic injection, integration of heater elements and temperature sensors and detection of microbeads in a single microfluidic chip. A practical application for the integrated microsystem is confirmed by the ability to select a specific DNA sequence of interest from a 4 x 4 cDNA library. This application emphasizes the advantages of component integration for rapid bio-assay development in a complete microsystem.     

Microstructural reactors: Concept, development and application

In the recent years owing to growing necessity of raw material and energy economy and solution of ecological problems, a significant interest by the chemistry thechnologists is paid to the microsystem technology. One of directions in the development of microsystem technology is creation and application of microstructural reactors (microreactors). Currently world market proposes many samples of microstructural installations and even full-value chemical units for, in part, of organic synthesis. In Russia the microsystem technology is included to the list of priority directions of development of science and technology in 2001–2010 years.     

Programmed co-assembly of DNA-peptide hybrid microdroplets by phase separation

Biopolymers, including DNA and peptides have been used as excellent self-assembling building blocks for programmable single-component or hybrid materials, due to their controlled molecular interactions.However, combining two assembling principles of DNA-based programmability and peptide-based specific molecular interactions for hybrid structures to microscale has not yet been achieved. In this study,we describe a hybrid microsystem that emerges from the co-assembly of DNA origami structure and s...     

Imaging surface plasmon resonance for multiplex microassay sensing of mycotoxins

A prototype imaging surface plasmon resonance-based multiplex microimmunoassay for mycotoxins is described. A microarray of mycotoxin–protein conjugates was fabricated using a continuous flow microspotter device. A competitive inhibition immunoassay format was developed for the simultaneous detection of deoxynivalenol (DON) and zearalenone (ZEN), using a single sensor chip. Initial in-house validation showed limits of detection of 21 and 17 ng/mL for DON and 16 and 10 ng/mL for ZEN in extracts, which corresponds to 84 and 68 μg/kg for DON and 64 and 40 μg/kg for ZEN in maize and wheat samples, respectively. Finally, the results were critically compared with data obtained from liquid chromatography-mass spectrometry confirmatory analysis method and found to be in good agreement. The described multiplex immunoassay for the rapid screening of several mycotoxins meets European Union regulatory limits and represents a robust platform for mycotoxin analysis in food and feed samples.     

A microarray to measure repair of damaged plasmids by cell lysates

DNA repair mechanisms constitute major defences against agents that cause cancer, degenerative disease and aging. Different repair systems cooperate to maintain the integrity of genetic information. Investigations of DNA repair involvement in human pathology require an efficient tool that takes into account the variety and complexity of repair systems. We have developed a highly sensitive damaged plasmid microarray to quantify cell lysate excision/synthesis (ES) capacities using small amounts of proteins. This microsystem is based on efficient immobilization and conservation on hydrogel coated glass slides of plasmid DNA damaged with a panel of genotoxic agents. Fluorescent signals are generated from incorporation of labelled dNTPs by DNA excision-repair synthesis mechanisms at plasmid sites. Highly precise DNA repair phenotypes i.e. simultaneous quantitative measures of ES capacities toward seven lesions repaired by distinct repair pathways, are obtained. Applied to the characterization of xeroderma pigmentosum (XP) cells at basal level and in response to a low dose of UVB irradiation, the assay showed the multifunctional role of different XP proteins in cell protection against all types of damage. On the other hand, measurement of the ES of peripheral blood mononuclear cells from six donors revealed significant diversity between individuals. Our results illustrate the power of such a parallelized approach with high potential for several applications including the discovery of new cancer biomarkers and the screening of chemical agents modulating DNA repair systems.     

Multiplex detection platform for tumor markers and glucose in serum based on a microfluidic microparticle array

We present a multiplex detection platform based on a microfluidic microparticle array to detect proteins and glucose in serum simultaneously. Multiplex detection of proteins and glucose was performed using biofunctionalized microparticles arrayed on gel-based microstructures integrated in microfluidics. The microparticles immobilized on these microstructures showed high stability under microfluidic flow conditions. With arrays of antibody-coated microbeads, microfluidic quantitative immunoassays for two protein tumor markers, human chorionic gonadotropin (hCG) and prostate specific antigen (PSA) were performed in serum samples with detection limits bellow the cut-off values for cancer diagnosis. Parallel to the immunoassays, quantitative enzymatic assays for glucose in the physiological concentration range were performed. Multiplex detection was achieved by using a spatially encoded microarray. By patterning antibody-coated microbeads and enzyme-containing microparticles on a novel mixed structure array, we successfully demonstrated simultaneous immunoassays (binding based assay) for proteins and an enzymatic assay (reaction kinetic based assay) for glucose. Our microparticle arrays could be potentially used for the detection of multiple categories of biomolecules (proteins, small metabolites and DNA) for clinical diagnostics and other biological applications.     

Ultrafast diagnosis of the genetic-related disorders using the combined technologies of multiplex PCR and multichannel microchip electrophoresis.

For the diagnosis of unexplained male infertility a multiplex PCR for 6 markers, which are well-known as candidate genes for studying male infertility and located on the human Y-chromosome, has been designed. The multiplex PCR products have been separated on a 12 channel microchip electrophoresis system, which can analyze different samples simultaneously. By combining the technologies of multiplex PCR with multichannel microchip electrophoresis, the number of the DNA markers that can be screened simultaneously is increased to be 72 marker (12 x 6) in a single run while the electrophoresis analysis time is reduced to be only 180 s.     

Construction of two fluorescence‐labeled non‐combined DNA index system miniSTR multiplex systems to analyze degraded DNA samples in the Chinese Han Population

MiniSTR loci have been demonstrated to be an effective approach in recovering genetic information from degraded specimens, because of the reduced PCR amplicon sizes which improved the PCR efficiency. Eight non‐combined DNA index system miniSTR loci suitable for the Chinese Han Population were analyzed in 300 unrelated Chinese Han individuals using two novel five fluorescence‐labeled miniSTR multiplex systems(multiplex I: D10S1248, D2S441, D1S1677 and D9S2157; multiplex II: D9S1122, D10S1435, D12ATA63, D2S1776 and Amelogenin). The allele frequency distribution and forensic parameters in the Chinese Han Population were reported in this article. The Exact Test demonstrated that all loci surveyed here were found to be no deviation from Hardy–Weinberg equilibrium. The accumulated power of discrimination and power of exclusion for the eight loci were 0.999999992 and 0.98, respectively. The highly degraded DNA from artificially degraded samples and the degraded forensic case work samples was assessed with the two miniSTR multiplex systems, and the results showed that the systems were quite effective.     

Rapid methods for detection and enumeration of Campylobacter spp. in foods

Campylobacter spp. are the most commonly reported bacterial cause of acute diarrheal disease in humans throughout the world. Traditional cultural methods for the detection and quantitation of Campylobacterspp. are slow and tedious; therefore, specific, sensitive, and rapid methods for campylobacters are needed to collect sufficient data for risk assessment and food safety policy development. We developed several rapid methods based on polymerase chain reaction (PCR), DNA hybridization, hydrophobic grid membrane filters (HGMFs), and enzyme immunoassays (EIAs). A PCR assay targeting C. jejuni, combined with a simple sample preparation procedure, detects as few as 0.3 most probable number (MPN)/mL C. jejuni in naturally contaminated chicken rinses after 20-24 h enrichment. An HGMF-EIA method using a commercial polyclonal antibody for Campylobacter detects and enumerates thermophilic Campylobacter spp. from spiked chicken rinse and milk, and naturally contaminated chicken rinses. A C. jejuni-specific probe in an HGMF-DNA hybridization protocol specifically detects and quantitates C. jejuni in food samples. A dot-blot EIA combined with an MPN procedure quantitates thermophilic campylobacters from samples that might be difficult to filter through HGMFs.     

p16基因甲基化的芯片定量检测

p16基因的失活与多种肿瘤相关,但p16基因缺失率较低,突变更为罕见,p16基因启动子区CpG岛甲基化与其蛋白表达密切相关．DNA甲基化已成为目前研究的热点,现有的技术包括：Southernblot法、限制性内切酶-PCR法、DNA测序法、甲基化特异性PCR(MSP)、