Microfabricated Porous Membrane Structure for Sample Concentration and Electrophoretic Analysis (original) (raw)
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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.
A microfabricated CE chip for DNA pre-concentration and separation utilizing a normally closed valve
ELECTROPHORESIS, 2009
A simple, sequential DNA pre-concentration and separation method by using a micro-CE chip integrated with a normally closed valve, which is activated by pneumatic suction, has been developed. The CE chip is fabricated using PDMS. A surface treatment technique for coating a polymer bilayer with an anionic charge is applied to modify the surface of the microchannel. A normally closed valve with anionic surface charges forms a nanoscale channel that only allows the passage of electric current but traps the DNA samples so that they are pre-concentrated. After the pre-concentration step, a pneumatic suction force is applied to the normally closed valve. This presses down the valve membrane, which reconnects the channels. The DNA samples are then moved into a separation channel for further separation and analysis. Successful DNA pre-concentration and separation has been achieved. Fluorescent intensity at the pre-concentration area is increased by approximately 3570 times within 1.9 min of operation. The signals from the separation of fX174 DNA/HaeIII markers are enhanced approximately 41 times after 100 s of pre-concentration time, as compared with the results using a traditional cross-shaped micro-CE chip. These results clearly demonstrate that successful DNA pre-concentration for signal enhancement and separation analysis can be performed by using this new micro-CE chip.
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.
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. |
Integrated Cell Isolation and Polymerase Chain Reaction Analysis Using Silicon Microfilter Chambers
Analytical Biochemistry, 1998
White blood cells are isolated from whole blood in silicon-glass 4.5-l microchips containing a series of 3.5-m feature-sized 'weir-type' filters, formed by an etched silicon dam spanning the flow chamber. Genomic DNA targets, e.g., dystrophin gene, can be directly amplified using the polymerase chain reaction (PCR) from the white cells isolated on the filters. This dual function microchip provides a means to simplify nucleic acid analyses by integrating in a single device two key steps in the analytical procedure, namely, cell isolation and PCR.
DNA Purification on Microfluidic Devices with a Focus on Large Volume, Forensic Biological Samples
The development and application of microfluidic sample preparation methods to a wide range of sample types, including large-volume forensic samples undergoing genetic analysis, has the potential to greatly benefit the forensic and clinical communities. The design, development and optimization of a microfluidic solid phase extraction method applicable to the purification of DNA from forensic biological samples (vrSPE) obtained in large volumes is demonstrated. Illustration of the range of samples this method can handle, the successful application to environmentally-degraded DNA samples, mitochondrial DNA from blood and blood stains, and genomic DNA from bone are also shown. Integration of vrSPE with a secondary, orthogonal, purification method is demonstrated to be advantageous for the removal of the PCR inhibitors (e.g., indigo dye), and to outperform low volume microfluidic SPE (μSPE) and vrSPE in the removal of that inhibitor. The development of a microfluidic, forensic genetic analysis system, both in a modular and integrated form, is detailed. The modular system described, used three microfluidic devices for SPE, polymerase chain reaction (PCR), and microchip electrophoresis (ME), while the integrated system used a single device for both SPE and PCR. Each method was used for the successful, forensic STR analysis of buccal swab lysate. Finally, an exploratory excursion into the development of a PMMA (Plexiglas) microfluidic device for DNA purification is shown, with a focus on device bonding and surface modification. This work provides the next step towards developing a single-use, genetic analysis microdevice.
Design and fabrication of an integrated microsystem for microcapillary electrophoresis
Journal of Micromechanics and Microengineering, 2003
A capillary electrophoresis microsystem integrated with feed-through platinum electrodes was designed and fabricated for the separation of DNA fragments. A novel glass-to-silicon bonding technology, which allows anodic bonding of a glass wafer to a silicon wafer coated with a thick dielectric film by the inclusion of a thin intermediate amorphous silicon layer, was developed and employed to construct the microsystem. Despite the existence of a thick insulating material and non-uniform topography, robust devices without fluid leakage were obtained. Electrophoretic manipulation and separation of DNA fragments after capillary pre-treatment have been demonstrated and several operational considerations are discussed. The system performance suggests that silicon-based microsystems can be advantageous and practical for the fabrication of integrated microcapillary electrophoresis devices.
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.
DNA analysis on microfabricated electrophoretic devices with bubble cells
ELECTROPHORESIS, 2002
Microfluidic devices with bubble cells have been fabricated on poly(methyl methacrylate) (PMMA) plates and have been employed for the analysis of DNA using polyethylene oxide (PEO) solutions. First, the separation channel was fabricated using a wireimprinting method. Then, wires with greater sizes or a razor blade glued in a polycarbonate plate was used to fabricate bubble cells, with sizes of 190-650 mm. The improvements in resolution and sensitivity have been achieved for large DNA (. 603 base pair, bp) using such devices, which depend on the geometry of the bubble cell. The main contributor for optimal resolution is mainly due to DNA migration at lower electric field strengths inside the bubble cell. On the other hand, slight losses of resolution for small DNA fragments have been found mainly due to diffusion, supported by the loss of resolution when separating two small solutes. With a bubble cell of 75 mm (width)6500 mm (depth), the sensitivity improvement up to 17-fold has been achieved for the 271 bp fragment in the separation of FX-174/HaeIII DNA restriction fragments. We have also found that a microfluidic device with a bubble cell of 360 mm6360 mm is appropriate for DNA analysis. Such a device has been used for separating DNA ranging from 8 to 2176 bp and polymerase chain reaction (PCR) products amplified after 30 cycles, with rapidity and improvements in the sensitivity as well as resolution.