High sensitivity PCR assay in plastic micro reactors

Small volume operation and rapid thermal cycling have been subjects of numerous reports in micro reactor chip development. Sensitivity aspects of the micro PCR reactor have not been studied in detail, however, despite the fact that detection of rare targets or trace genomic material from clinical and/or environmental samples has been a great challenge for microfluidic devices. In this study, a serpentine shaped thin (0.75 mm) polycarbonate plastic PCR micro reactor was designed, constructed, and tested for not only its rapid operation and efficiency, but also its detection sensitivity and specificity, in amplification of Escherichia coli (E. coli) K12-specific gene fragment. At a template concentration as low as 10 E. coli cells (equivalent to 50 fg genomic DNA), a K12-specific gene product (221 bp) was adequately amplified with a total of 30 cycles in 30 min. Sensitivity of the PCR micro reactor was demonstrated with its ability to amplify K12-specific gene from 10 cells in the presence of 2% blood. Specificity of the polycarbonate PCR micro reactor was also proven through multiplex PCR and/or amplification of different pathogen-specific genes. This is, to our knowledge, the first systematic study of assay sensitivity and specificity performed in plastic, disposable micro PCR devices.     

An extrusion fluidic driving method for continuous-flow polymerase chain reaction on a microfluidic chip

A novel extrusion driving protocol was developed based on micro-fabricated polydimethylsiloxane (PDMS) pneumatic valves. High efficiency liquid transfer was performed by using entirely overlapping control channels and fluid channels. A 0.5-s time is sufficient for the transfer of 9 μL sample solution between two chambers in the microchip with a nitrogen pressure of 70 kPa. The driving method was used in a microfluidic polymerase chain reaction (PCR) system, and rapid cycling of the PCR mixture in a closed loop was achieved. The amplification of DNA was demonstrated via both three-stage and two-stage PCR thermal cycling on the microchips resulting in significant reduction of the PCR time. The amplifications of 144-bp and 200-bp DNA fragments were achieved within 24 min using a three-stage protocol with 30 thermal cycles, and 130-bp DNA fragments within 12 min by using 20 thermal cycles in the two-stage system, compared to about 2 h in benchtop PCR with the same number of thermal cycles.     

Capillary electrophoresis as a tool for optimization of multiplex PCR reactions

Copying multiple regions of a DNA molecule is routinely performed today using the polymerase chain reaction (PCR) in a process commonly referred to as multiplex PCR. The development of a multiplex PCR reaction involves designing primer sets and examining various combinations of those primer sets and different reaction components and/or thermal cycling conditions. The process of optimizing a multiplex PCR reaction in order to obtain a well-balanced set of amplicons can be time-consuming and labor-intensive. The rapid separation and quantitation capabilities of capillary electrophoresis make it an efficient technique to help in the multiplex PCR optimization process.     

Capillary electrophoresis as a tool for optimization of multiplex PCR reactions

Copying multiple regions of a DNA molecule is routinely performed today using the polymerase chain reaction (PCR) in a process commonly referred to as multiplex PCR. The development of a multiplex PCR reaction involves designing primer sets and examining various combinations of those primer sets and different reaction components and/or thermal cycling conditions. The process of optimizing a multiplex PCR reaction in order to obtain a well-balanced set of amplicons can be time-consuming and labor-intensive. The rapid separation and quantitation capabilities of capillary electrophoresis make it an efficient technique to help in the multiplex PCR optimization process.     

A nanoliter rotary device for polymerase chain reaction

Polymerase chain reaction (PCR) has revolutionized a variety of assays in biotechnology. The ability to implement PCR in disposable and reliable microfluidic chips will facilitate its use in applications such as rapid medical diagnostics, food control testing, and biological weapons detection. We fabricated a microfluidic chip with integrated heaters and plumbing in which various forms of PCR have been successfully demonstrated. The device uses only 12 nL of sample, one of the smallest sample volumes demonstrated to date. Minimizing the sample volume allows low power consumption, reduced reagent costs, and ultimately more rapid thermal cycling.     

Practical integration of polymerase chain reaction amplification and electrophoretic analysis in microfluidic devices for genetic analysis

An integrated system of a silicon-based microfabricated polymerase chain reaction (microPCR) 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 degrees C using an improved novel "joint-heating" scheme. Thermal cycling was digitally controlled with a temperature accuracy of +/- 0.2 degrees 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 microPCR was achieved in microchip, and application of the integrated microPCR-microchip 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.     

Miniaturized nucleic acid amplification systems for rapid and point-of-care diagnostics: A review

Point-of-care (POC) genetic diagnostics critically depends on miniaturization and integration of sample processing, nucleic acid amplification, and detection systems. Polymerase chain reaction (PCR) assays have extensively applied for the diagnosis of genetic markers of disease. Microfluidic chips for microPCR with different materials and designs have been reported. Temperature cycling systems with varying thermal masses and conductivities, thermal cycling times, flow-rates, and cross-sectional areas, have also been developed to reduce the nucleic acid amplification time. Similarly, isothermal amplification techniques (e.g., loop-mediated isothermal amplification or LAMP), which are still are emerging, have a better potential as an alternative to PCR for POC diagnostics. Isothermal amplification techniques have: (i) moderate incubation temperature leading to simplified heating and low power consumption, (ii) yield high amount of amplification products, which can be detected either visually or by simple detectors, (iii) allow direct genetic amplification from bacterial cells due to the superior tolerance to substances that typically inhibit PCR, (iv) have high specificity, and sensitivity, and (v) result in rapid detection often within 10–20 min. The aim of this review is to provide a better understanding of the advantages and limitations of microPCR and microLAMP systems for rapid and POC diagnostics.     

Microfabricated PCR-electrochemical device for simultaneous DNA amplification and detection

Microfabricated silicon/glass-based devices with functionalities of simultaneous polymerase chain reaction (PCR) target amplification and sequence-specific electrochemical (EC) detection have been successfully developed. The microchip-based device has a reaction chamber (volume of 8 microl) formed in a silicon substrate sealed by bonding to a glass substrate. Electrode materials such as gold and indium tin oxide (ITO) were patterned on the glass substrate and served as EC detection platforms where DNA probes were immobilized. Platinum temperature sensors and heaters were patterned on top of the silicon substrate for real-time, precise and rapid thermal cycling of the reaction chamber as well as for efficient target amplification by PCR. DNA analyses in the integrated PCR-EC microchip start with the asymmetric PCR amplification to produce single-stranded target amplicons, followed by immediate sequence-specific recognition of the PCR product as they hybridize to the probe-modified electrode. Two electrochemistry-based detection techniques including metal complex intercalators and nanogold particles are employed in the microdevice to achieve a sensitive detection of target DNA analytes. With the integrated PCR-EC microdevice, the detection of trace amounts of target DNA (as few as several hundred copies) is demonstrated. The ability to perform DNA amplification and EC sequence-specific product detection simultaneously in a single reaction chamber is a great leap towards the realization of a truly portable and integrated DNA analysis system.     

Under-three minute PCR: probing the limits of fast amplification

Nucleic acid amplification is enormously useful to the biotechnology and clinical diagnostic communities; however, to date point-of-use PCR has been hindered by thermal cycling architectures and protocols that do not allow for near-instantaneous results. In this work we demonstrate PCR amplification of synthetic SARS respiratory pathogenic targets and bacterial genomic DNA in less than three minutes in a hardware configuration utilizing convenient sample loading and disposal. Instead of sample miniaturization techniques, near-instantaneous heating and cooling of 5 μL reaction volumes is enabled by convective heat transfer of a thermal fluid through porous media combined with an integrated electrical heater. This method of rapid heat transfer has enabled 30 cycles of PCR amplification to be completed in as little as two minutes and eighteen seconds. Surprisingly, multiple enzymes have been shown to work at these breakthrough speeds on our system. A tool for measuring enzyme kinetics now exists and can allow polymerase optimization through directed evolution studies. Pairing this instrument technology with modified polymerases should result in a new paradigm for high-throughput, ultra-fast PCR and will hopefully improve our ability to quickly respond to the next viral pandemic.     

Dynamic temperature measurement in microfluidic devices using thermochromic liquid crystals

Thermochromic liquid crystals (TLCs) are used to explore the temperature transients during thermal cycling for microchip-based polymerase chain reaction (PCR). By analyzing the reflected spectra of the TLCs over time, temperature vs. time trajectories were extracted and overshoots/undershoots were estimated. To our knowledge, this is the first report of TLC-based dynamic temperature measurements in a microfluidic device for all PCR temperature stages.     

Performing microchannel temperature cycling reactions using reciprocating reagent shuttling along a radial temperature gradient

This study develops a novel temperature cycling strategy for executing temperature cycling reactions in laser-etched poly(methylmethacrylate) (PMMA) microfluidic chips. The developed microfluidic chip is circular in shape and is clamped in contact with a circular ITO heater chip of an equivalent diameter. Both chips are fabricated using an economic and versatile laser scribing process. Using this arrangement, a self-sustained radial temperature gradient is generated within the microfluidic chip without the need to thermally isolate the different temperature zones. This study demonstrates the temperature cycling capabilities of the reported microfluidic device by a polymerase chain reaction (PCR) process using ribulose 1,5-bisphosphate carboxylase large subunit (rbcL) gene as a template. The temperature ramping rate of the sample inside the microchannel is determined from the spectral change of a thermochromic liquid crystal (TLC) solution pumped into the channel. The present results confirm that a rapid thermal cycling effect is achieved despite the low thermal conductivity of the PMMA substrate. Using IR thermometry, it is found that the radial temperature gradient of the chip is approximately 2 degrees C mm(-1). The simple system presented in this study has considerable potential for miniaturizing complex integrated reactions requiring different cycling parameters.     

热解吸-电喷雾离子源-三重四极杆质谱法快速筛查火锅底料和肉汤中非法添加罂粟壳

通过简单的金属探针直接接触火锅底料和肉汤表面采集待测物,经热解吸离子源进一步热解吸和电喷雾离子化,最终进入三重四极杆质谱检测器在多反应监测模式下进行定性分析,实现了火锅底料和肉汤中罂粟壳的现场实时快速检测。结果表明,设置热解吸温度为260℃,以0.1%甲酸水溶液(含10 mmol/L甲酸铵)-乙腈(1∶1, v/v)作为注射溶剂、注射泵流速为200μL/h时,仪器响应值最优,灵敏度最高;5种生物碱中罂粟碱、那可丁、蒂巴因在火锅底料和肉汤中的检出限均为2μg/kg,可待因、吗啡在火锅底料中的检出限为10μg/kg,在肉汤中的检出限为5μg/kg。该法与罂粟壳胶体金卡片快检试剂盒相比,灵敏度具有明显优势。应用该法对50批次市售火锅底料、肉汤等样品进行检测,发现1批次鸡汤含有那可丁、罂粟碱、蒂巴因和吗啡4种生物碱,与高效液相色谱-三重四极杆质谱法的检测结果一致。由此说明该方法具有无需样品制备和色谱分离的特点,是一种快速、绿色、环保的分析方法,能够满足对食品中罂粟壳的快速定性分析。     

A disposable microfluidic cassette for DNA amplification and detection

A pneumatically driven, disposable, microfluidic cassette comprised of a polymerase chain reaction (PCR) thermal cycler, an incubation chamber to label PCR amplicons with up-converting phosphor (UPT) reporter particles, conduits, temperature-activated, normally closed hydrogel valves, and a lateral flow strip, was constructed and tested. The hydrogel valves, which were opened and closed with the aid of electrically controlled thermoelectric units, provided a simple means to seal the PCR reactor and suppress bubble formation. The hydrogel-based flow control was electronically addressable, leakage-free, and biocompatible. To test the device, a solution laden with genomic DNA isolated from B. cereus was introduced into the microfluidic cassette and a specific 305 bp fragment was amplified. The PCR amplicons were labelled with the phosphor (UPT) reporter particles, applied to the lateral flow strip, bound to pre-immobilized ligands, and detected with an IR laser that scanned the lateral flow strip and excited the phosphor (UPT) particles that, in turn, emitted light in the visible spectrum. The UPT particles do not bleach, they provide a permanent record, and they readily facilitate the filtering of background noise. The cassette described herein will be used for rapid testing at the point of care.     

LiNi_(0.8-y)Ti_yCo_(0.2)O_2电极材料中钛离子掺杂作用机理的研究

应用恒流充放电、非现场X射线粉末衍射 (ex situXRD)、电化学交流阻抗 (EIS)、程序控温脱附 质谱联用(TPD MS)等实验方法研究LiNi0. 8-yTiyCo0. 2O2电极材料钛离子的掺杂作用机理.结果表明,掺钛后的电极材料于充放电过程中的结构相变和晶格的膨胀收缩受到抑制,在高电位下的界面反应活性减弱,从而减小了由结构变化和界面反应引起的容量损失;同时,钛的掺杂增强了电极材料在脱锂状态下的结构稳定性,抑制了电极材料和电解液的分解或氧化反应,以上两个方面分别改善并提高了电极材料的充放电循环性能及其热稳定性.     

The role of thermal analysis in optimization of the electrochromic effect of nickel oxide thin films, prepared by the sol–gel method: Part II

Thin films and the corresponding xerogels were prepared from nickel acetate precursor using the sol–gel dip-coating technique. The differences in thermal stability of the two forms of samples were studied by dynamic and isothermal thermogravimetry. For thin films, the onset decomposition temperature of acetate groups was 230 °C and for the xerogel 250 °C. During thermal decomposition, the formation of nanosized nickel oxide took place. Carbonate ions, which were formed during thermal decompostion of acetate groups, remained either free or bidentately coordinated to nickel. In situ monochromatic optical transmittance changes showed that an optical stability up to the 100th cycle was already achieved for films heated for 15 min at the isothermal temperature (thermal decompositon 25%). Comparison of the results obtained for nickel sulfate (Part I) and nickel acetate precursors shows that at least two parameters, the precursor used and the degree of thermal treatment, have considerable influence on the thermal stability of the thin film and also on its electrochromic response during the cycling process.     

The effect of tungsten coating on the properties of diamond thick films

According to the physical properties of pure tungsten and tantalum and their carbides, a layer of tungsten thin films coating three tantalum filaments at a current of 750 A was prepared by us for the first time and called tungsten coating. Diamond thick films were deposited in a hot filament chemical vapor deposition (HFCVD) reactor using tantalum filaments before and after tungsten coating, respectively. The success of tungsten coating is verified by scanning electron microscopy examination of these filaments. The remarkable discrepancies in thermal conductivity, colors of diamond thick films, and electron dispersion spectroscopy of the films substantiate the success of tungsten coating, too. The effects of tungsten coating on the quality of diamond films were finally evaluated by Raman spectra. The text was submitted by the authors in English.     

Rapid amplification of genetically modified organisms using a circular ferrofluid-driven PCR microchip

The use of genetically modified organisms (GMOs) as food and in food products is becoming more and more widespread. Polymerase chain reaction (PCR) technology is extensively used for the detection of GMOs in food products in order to verify compliance with labeling requirements. In this paper, we present a novel close-loop ferrofluid-driven PCR microchip for rapid amplification of GMOs. The microchip was fabricated in polymethyl methacrylate by CO₂ laser ablation and was integrated with three temperature zones. PCR solution was contained in a circular closed microchannel and was driven by magnetic force generated by an external magnet through a small oil-based ferrofluid plug. Successful amplification of genetically modified soya and maize were achieved in less than 13 min. This PCR microchip combines advantages of cycling flexibility and quick temperature transitions associated with two existing microchip PCR techniques, and it provides a cost saving and less time-consuming way to conduct preliminary screening of GMOs. Figure Schematic of the circular ferrofluid-driven PCR microchip     

Ultrafast rotary PCR system for multiple influenza viral RNA detection

We presented a novel platform for an ultrafast PCR system, called the Rotary PCR Genetic Analyzer, which incorporates a thermal block and resistive temperature detector (RTD) for thermal cycling control, a disposable PCR microchip, and a stepper motor. The influenza viral RNAs from H3N2, H5N1, and H1N1 were simultaneously identified with high sensitivity and speed.     

Bulk-micromachined submicroliter-volume PCR chip with very rapid thermal response and low power consumption

The current paper describes the design, fabrication, and testing of a micromachined submicroliter-volume polymerase chain reaction (PCR) chip with a fast thermal response and very low power consumption. The chip consists of a bulk-micromachined Si component and hot-embossed poly(methyl methacrylate)(PMMA) component. The Si component contains an integral microheater and temperature sensor on a thermally well-isolated membrane, while the PMMA component contains a submicroliter-volume PCR chamber, valves, and channels. The micro hot membrane under the submicroliter-volume chamber is a silicon oxide/silicon nitride/silicon oxide (O/N/O) diaphragm with a thickness of 1.9 microm, resulting in a very low thermal mass. In experiments, the proposed chip only required 45 mW to heat the reaction chamber to 92 degrees C, the denaturation temperature of DNA, plus the heating and cooling rates are about 80 degrees C s(-1) and 60 degrees C s(-1), respectively. We validated, from the fluorescence results from DNA stained with SYBR Green I, that the proposed chip amplified the DNA from vector clone, containing tumor suppressor gene BRCA 1 (127 base pairs at 11th exon), after 30 thermal cycles of 3 s, 5 s, and 5 s at 92 degrees C, 55 degrees C, and 72 degrees C, respectively, in a 200 nL-volume chamber. As for specificity of DNA products, owing to difficulty in analyzing the very small volume PCR results from the micro chip, we vicariously employed the larger volume PCR products after cycling with the same sustaining temperatures as with the micro chip but with much slower ramping rates (3.3 degrees C s(-1) when rising, 2.5 degrees C s(-1) when cooling) within circa 20 minutes on a commercial PCR machine and confirmed the specificity to BRCA 1 (127 base pairs) with agarose gel electrophoresis. Accordingly, the fabricated micro chip demonstrated a very low power consumption and rapid thermal response, both of which are crucial to the development of a fully integrated and battery-powered instrument for a lab-on-a-chip DNA analysis.     

A disposable laser print-cut-laminate polyester microchip for multiplexed PCR via infra-red-mediated thermal control

Infrared (IR)-mediated thermal cycling system, a method proven to be a effective for sub-μL scale polymerase chain reaction (PCR) on microchips, has been integrated with DNA extraction and separation on a glass microchip in a fully integrated micro Total Analysis System by Easley et al., in 2006. IR-PCR has been demonstrated on both glass and PMMA microdevices where the fabrication (bonding) is not trivial. Polyester-toner (PeT) microfluidic devices have significant potential as cost-effective, disposable microdevices as a result of the ease of fabrication (∼$0.25 USD and <10 min per device) and availability of commercial substrates. For the first time, we demonstrate here the thermal cycling in PeT microchips on the IR-PCR system. Undesirable IR absorption by the black-toner bonding layer was eliminated with a spatial filter in the form of an aluminum foil mask. The solution heating rate for a black PeT microchip using a tungsten lamp was 10.1 ± 0.7 °C s⁻¹ with a cooling rate of roughly −12 ± 0.9 °C s⁻¹ assisted by forced air cooling. Dynamic surface passivation strategies allowed the successful amplification of a 520 bp fragment of the λ-phage genome (in 11 min) and a 1500 bp region of Azospirillum brasilense. Using a centrosymmetric chamber configuration in a multichamber PeT microchip, homogenous temperature distribution over all chambers was achieved with inter-chamber temperature differences at annealing, extension and denaturing steps of less than ±2 °C. The effectiveness of the multichamber system was demonstrated with the simultaneous amplification of a 390 bp amplicon of human β-globin gene in five PeT PCR microchambers. The relative PCR amplification efficiency with a human β-globin DNA fragment ranged from 70% to 90%, in comparison to conventional thermal cyclers, with an inter-chamber standard deviation of ∼10%. Development of PeT microchips for IR-PCR has the potential to provide rapid, low-volume amplification while also integrating PCR with extraction upstream and separation/detection downstream.