DNA analysis on microfabricated electrophoretic devices with bubble cells (original) (raw)
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Electrophoresis, 2003
We have evaluated double-stranded DNA separations in microfluidic devices which were designed to couple a sample preconcentration step based on isotachophoresis (ITP) with a zone electrophoretic (ZE) separation step as a method to increase the concentration limit of detection in microfluidic devices. Developed at ACLARA BioSciences, these LabCard™ devices are plastic 32 channel chips, designed with a long sample injection channel segment to increase the sample loading. These chips were designed to allow stacking of the sample into a narrow band using discontinuous ITP buffers, and subsequent separation in the ZE mode in sieving polymer solutions. Compared to chip ZE, the sensitivity was increased by 40-fold and we showed baseline resolution of all fragments in the ΦX174/HaeIII DNA digest. The total analysis time was 3 min/sample, or less than 100 min per LabCard device. The resolution for multiplexed PCR samples was the same as obtained in chip ZE. The limit of detection was 9 fg/μL of DNA in 0.1×polymerase chain reaction (PCR) buffers using confocal fluorescence detection following 488 nm laser excitation with thiazole orange as the fluorescent intercalating dye.
Analytical Chemistry, 2002
An isothermal signal amplification technique for specific DNA sequences, known as cycling probe technology (CPT), was performed within a microfluidic chip. The presence of DNA from methicillin-resistant Staphylococcus aureus was determined by signal amplification of a specific DNA sequence. The microfluidic device consisted of four channels intersecting to mix the sample and reagents within 55 s, as they were directed toward the reactor coil by electrokinetic pumping. The 160-nL CPT reactor occupied ∼220 mm 2 . Gel-free capillary electrophoresis separation of the biotin-and fluorescein-labeled probe from the probe fragments was performed on-chip following the on-chip reaction. An off-chip CPT reaction, with on-chip separation gave a detection limit of 2 fM (0.03 amol) target DNA and an amplification factor of 85 000. Calibration curves, linear at <5% probe fragmentation, obeyed a power law relationship with an argument of 0.5 [target] at higher target DNA concentrations for both on-chip and off-chip CPT reaction and analysis. An amplification factor of 42 000 at 250 fM target (25 000 target molecules) was observed on-chip, but the reaction was ∼4 times less sensitive than off-chip under the conditions used. Relative SD values for on-chip CPT were 0.8% for the peak migration times, 9% for the area of intact probe peak, and 8% for the fragment/probe peak area ratio Routine use of gene sequences for sample analysis requires the development of new methods for performing analytical assays, in terms of the reagents and reactions used, as well as the technology for performing those assays. 1 Microfluidic devices offer an attractive approach to miniaturizing the amounts of reagent required and automating or speeding up the analyses. 2-8 The focus to date has been on performing DNA separations within capillary gel electrophoresis (CGE) microchips for sequencing or sizing 9-12 and on integrating the polymerase chain reaction 13-21 (PCR). PCR is a common DNA sample preparation step, in which the target † University of Alberta. ‡ Both first and second authors have contributed equally. § Defence Research Establishment Suffield. |
Electrophoresis, 2003
An integrated system of a silicon-based microfabricated polymerase chain reaction (μPCR) chamber and microfabricated electrophoretic glass chips have been developed. The PCR chamber was made of silicon and had aluminum heaters and temperature sensors integrated on the glass anodically bonded cover. Temperature uniformity in the reaction chamber was ±0.3°C using an improved novel “joint-heating” scheme. Thermal cycling was digitally controlled with a temperature accuracy of ± 0.2°C. Small operating volumes together with high thermal conductivity of silicon made the device well suited to rapid cycling; 16 s/cycle were demonstrated. For analysis of the PCR products, the chamber output was transferred to the glass microchip by pressure. Analysis time of PCR amplified genomic DNA was obtained in the microchip in less than 180 s. The analysis procedure employed was reproducible, simple and practical by using viscous sieving solutions of hydroxypropylmethylcellulose and dynamically coated microchip channels with poly(vinylpyrrolidone). DNA fragments that differ in size by 18 base pairs (bp) were resolved. Analysis of genomic male and female amplified DNA by μPCR was achieved in microchip, and application of the integrated μPCR-μchip for the identification of bird sex was tested. Genomic DNA samples from several bird species such as pigeon and chicken were analyzed. Hence, the system could be used as well to determine the sex of avian species.
Journal of Chromatography A, 2005
This paper deals with dynamic coating of the microchannels fabricated on poly(methyl methacrylate) (PMMA) chips and DNA separation by microchip electrophoresis (MCE). After testing a number of polymers, including 2-hydroxyethyl cellulose, hydroxypropylmethyl cellulose, different sizes of poly(ethylene oxide) (PEO), and poly(vinyl pyrrolidone) (PVP), we found that coating of the PMMA microchannels with PEO(M r = 6.0 × 10 5 g/mol) on the first layer is essential to minimize the interaction of DNA with PMMA surface. To achieve high efficiency, multilayer coating of PMMA chips with PEO, PVP, and PEO containing gold nanoparticles [PEO(GNP)] is important. A 2-(PEO-PVP)-PEO(GNP) PMMA chip, which was repeatedly coated with 1.0% PEO and 5.0% PVP twice, and then coated with 0.75% PEO(GNP) each for 30 min, provided a high efficiency (up to 1.7 × 10 6 plates/m) for the separation of DNA markers V (pBR 322/HaeIII digest) and VI (pBR 328/BglI digest and pBR 328/HinfI digest) when using 0.75% PEO(GNP). With such a high efficiency, we demonstrated the separation of hsp65 gene fragments of Mycobacterium HaeIII digests by MCE within 90 s. The advantages of this approach to DNA analysis include ease of filling the microchannel with 0.75% PEO(GNP), rapidity, and reproducibility.
Disposable polyester–toner electrophoresis microchips for DNA analysis
The Analyst, 2012
Microchip electrophoresis has become a powerful tool for DNA separation, offering all of the advantages typically associated with miniaturized techniques: high speed, high resolution, ease of automation, and great versatility for both routine and research applications. Various substrate materials have been used to produce microchips for DNA separations, including conventional (glass, silicon, and quartz) and alternative (polymers) platforms. In this study, we perform DNA separation in a simple and low-cost polyester-toner (PeT)-based electrophoresis microchip. PeT devices were fabricated by a directprinting process using a 600 dpi-resolution laser printer. DNA separations were performed on PeT chip with channels filled with polymer solutions (0.5% m/v hydroxyethylcellulose or hydroxypropylcellulose) at electric fields ranging from 100 to 300 V cm À1 . Separation of DNA fragments between 100 and 1000 bp, with good correlation of the size of DNA fragments and mobility, was achieved in this system. Although the mobility increased with increasing electric field, separations showed the same profile regardless of the electric field. The system provided good separation efficiency (215 000 plates per m for the 500 bp fragment) and the separation was completed in 4 min for 1000 bp fragment ladder. The cost of a given chip is approximately $0.15 and it takes less than 10 minutes to prepare a single device.
Analytical Chemistry, 1996
Microfabricated silicon PCR reactors and glass capillary electrophoresis (CE) chips have been successfully coupled to form an integrated DNA analysis system. This construct combines the rapid thermal cycling capabilities of microfabricated PCR devices (10°C/s heating, 2.5°C/s cooling) with the high-speed (<120 s) DNA separations provided by microfabricated CE chips. The PCR chamber and the CE chip were directly linked through a photolithographically fabricated channel filled with hydroxyethylcellulose sieving matrix. Electrophoretic injection directly from the PCR chamber through the cross injection channel was used as an "electrophoretic valve" to couple the PCR and CE devices on-chip. To demonstrate the functionality of this system, a 15 min PCR amplification of a-globin target cloned in M13 was immediately followed by high-speed CE chip separation in under 120 s, providing a rapid PCR-CE analysis in under 20 min. A rapid assay for genomic Salmonella DNA was performed in under 45 min, demonstrating that challenging amplifications of diagnostically interesting targets can also be performed. Real-time monitoring of PCR target amplification in these integrated PCR-CE devices is also feasible. Amplification of the-globin target as a function of cycle number was directly monitored for two different reactions starting with 4 × 10 7 and 4 × 10 5 copies of DNA template. This work establishes the feasibility of performing high-speed DNA analyses in microfabricated integrated fluidic systems.
Philosophical Transactions of the Royal Society of London. Series A: Mathematical, Physical and Engineering Sciences, 2004
Many routine genomic-analysis assays rely on gel electrophoresis to perform sizeselective fractionation of DNA fragments in the size range below 1 kb in length. Over the past decade, impressive progress has been made towards the development of microfabricated electrophoresis systems to conduct these assays in a microfluidic lab-on-a-chip format. Since these devices are inexpensive, require only nanolitre sample volumes, and do not rely on the availability of a pre-existing laboratory infrastructure, they are readily deployable in remote field locations for use in a variety of medical and biosensing applications. The design and construction of microfabricated electrophoresis devices poses a variety of challenges, including the need to achieve high-resolution separations over distances of a few centimetres or less, and the need to easily interface with additional microfluidic components to produce self-contained integrated DNA-analysis systems. In this paper, we review recent efforts to develop devices to satisfy these requirements and live up to the promise of fulfilling the growing need for inexpensive portable genomic-analysis equipment.
Capillary electrophoresis microchip for direct amperometric detection of DNA fragments
ELECTROPHORESIS, 2011
Detection and quantitation of nucleic acids have gained much importance in the last couple of decades, especially in the post-human genome project era. Such processes are tedious, time consuming and require expensive reagents and equipment. Therefore, in the present study, we demonstrated a simple process for the separation and analysis of small DNA fragments using capillary electrophoretic amperometric detection on an inexpensive disposable glass microchip. The device used polydimethylsiloxane engraved microchannel and Au/Ti in-channel microelectrodes for sample detection. The DNA fragments were separated under low electric field (20 V/cm) for improved detection sensitivity and to retain the biomolecules in their native conformation. With a low sample requirement (as low as 1 mL) and high reproducibility, the proposed microchip device was successful in resolution and detection of DNA fragments of various lengths.
PCR microfluidic devices for DNA amplification
Biotechnology Advances, 2006
The miniaturization of biological and chemical analytical devices by micro-electro-mechanical-systems (MEMS) technology has posed a vital influence on such fields as medical diagnostics, microbial detection and other bio-analysis. Among many miniaturized analytical devices, the polymerase chain reaction (PCR) microchip/microdevices are studied extensively, and thus great progress has been made on aspects of on-chip micromachining (fabrication, bonding and sealing), choice of substrate materials, surface chemistry and architecture of reaction vessel, handling of necessary sample fluid, controlling of three or two-step temperature thermocycling, detection of amplified nucleic acid products, integration with other analytical functional units such as sample preparation, capillary electrophoresis (CE), DNA microarray hybridization, etc. However, little has been done on the review of above-mentioned facets of the PCR microchips/microdevices including the two formats of flow-through and stationary chamber in spite of several earlier reviews [Zorbas, H. Miniature continuous-flow polymerase chain reaction: a breakthrough? Angew Chem