集成核酸提取的实时荧光PCR微全分析系统

集成核酸提取的实时荧光PCR微全分析系统将核酸提取、PCR扩增与实时荧光检测进行整合,在同一块微流控芯片上实现了核酸分析过程的全自动和全封闭,具有试剂用量少、分析速度快、操作简便等优点。本研究采用微机械加工技术制作集成核酸提取微流控芯片的阳极模,使用组合模具法和注塑法制作具有3D通道的PDMS基片,与玻璃基底通过等离子体键合封装成集成核酸提取芯片。构建了由微流体速度可调节(0~10 mL/min)的驱动控制装置、温控精度可达0.1℃的TEC温控平台、CCD检测功能模块等组成的微全分析系统。以人类血液裂解液为样品,采用硅胶膜进行芯片上核酸提取。系统根据设置好的时序自动执行,以2 mL/min的流体驱动速度完成20μL裂解液上样、清洗;以1 mL/min的流体驱动速度完成DNA洗脱,抽取PCR试剂与之混合注入到反应腔。提取的基因组DNA以链上内参基因GAPDH为检测对象,并以传统手工提取为对照,在该系统平台上进行PCR扩增和熔解曲线分析实验。片上PCR扩增结果显示,扩增曲线明显,Ct值分别为25.3和26.9。扩增产物进行熔解曲线分析得到的熔解温度一致,均为89.9℃。结果表明,此系统能够自动化、全封闭的在微流控芯片上完成核酸提取、PCR扩增与实时定量分析。     

Sequential microfluidic droplet processing for rapid DNA extraction

This work describes a novel droplet-based microfluidic device, which enables sequential droplet processing for rapid DNA extraction. The microdevice consists of a droplet generation unit, two reagent addition units and three droplet splitting units. The loading/washing/elution steps required for DNA extraction were carried out by sequential microfluidic droplet processing. The movement of superparamagnetic beads, which were used as extraction supports, was controlled with magnetic field. The microdevice could generate about 100 droplets per min, and it took about 1 min for each droplet to perform the whole extraction process. The extraction efficiency was measured to be 46% for λ-DNA, and the extracted DNA could be used in subsequent genetic analysis such as PCR, demonstrating the potential of the device for fast DNA extraction.     

Disposable on‐chip microfluidic system for buccal cell lysis,DNA purification,and polymerase chain reaction

This paper reports the development of a disposable, integrated biochip for DNA sample preparation and PCR. The hybrid biochip (25 × 45 mm) is composed of a disposable PDMS layer with a microchannel chamber and reusable glass substrate integrated with a microheater and thermal microsensor. Lysis, purification, and PCR can be performed sequentially on this microfluidic device. Cell lysis is achieved by heat and purification is performed by mechanical filtration. Passive check valves are integrated to enable sample preparation and PCR in a fixed sequence. Reactor temperature is needed to lysis and PCR reaction is controlled within ±1°C by PID controller of LabVIEW software. Buccal epithelial cell lysis, DNA purification, and SY158 gene PCR amplification were successfully performed on this novel chip. Our experiments confirm that the entire process, except the off‐chip gel electrophoresis, requires only approximately 1 h for completion. This disposable microfluidic chip for sample preparation and PCR can be easily united with other technologies to realize a fully integrated DNA chip.     

Quantitative and qualitative analysis of a microfluidic DNA extraction system using a nanoporous AlO(x) membrane

A nanoporous aluminium oxide membrane was integrated into a microfluidic system designed to extract hgDNA (human genomic DNA) from lysed whole blood. The effectiveness of this extraction system was determined by passing known concentrations of purified hgDNA through nanoporous membranes with varying pore sizes and measuring the amount of hgDNA deposited on the membrane while also varying salt concentration in the solution. DNA extraction efficiency increased as the salt concentration increased and nanopore size decreased. Based on these results, hgDNA was extracted from whole blood while varying salt concentration, nanopore size and elution buffer to find the conditions that yield the maximum concentration of hgDNA. The optimal conditions were found to be using a low-salt lysis solution, 100 nm pores, and a cationic elution buffer. Under these conditions the combination of flow and ionic disruption were sufficient to elute the hgDNA from the membrane. The extracted hgDNA sample was analysed and evaluated using PCR (polymerase chain reaction) to determine whether the eluted sample contained PCR inhibition factors. Eluted samples from the microfluidic system were amplified without any inhibition effects. PCR using extracted samples was demonstrated for several genes of interest. This microfluidic DNA extraction system based on embedded membranes will reduce the time, space and reagents needed for DNA analysis in microfluidic systems and will prove valuable for sample preparation in lab-on-a-chip applications.     

Platinum nanoparticle-facilitated reflective surfaces for non-contact temperature control in microfluidic devices for PCR amplification

The polymerase chain reaction (PCR) is critical for amplification of target sequences of DNA or RNA that have clinical, biological or forensic relevance. While extrinsic Fabry-Perot interferometry (EFPI) has been shown to be adequate for non-contact temperature sensing, the difficulty in defining a reflective surface that is semi-reflective, non-reactive for PCR compatibility and adherent for thermal bonding has limited its exploitation. Through the incorporation of a reflective surface fabricated using a thermally driven self-assembly of a platinum nanoparticle monolayer on the surface of the microfluidic chamber, an enhanced EFPI signal results, allowing for non-contact microfluidic temperature control instrumentation that uses infrared-mediated heating, convective forced-air cooling, and interferometic temperature sensing. The interferometer is originally calibrated with a miniature copper-constantan thermocouple in the PCR chamber resulting in temperature sensitivities of -22.0 to -32.8 nm·°C(-1), depending on the chamber depth. This universal calibration enables accurate temperature control in any device with arbitrary dimensions, thereby allowing versatility in various applications. Uniquely, this non-contact temperature control for PCR thermocycling is applied to the amplification of STR loci for human genetic profiling, where nine STR loci are successfully amplified for human identification using the EFPI-based non-contact thermocycling.     

一种可绝对定量核酸的数字PCR微流控芯片

构建了一种新型的可进行核酸单分子扩增和核酸绝对定量的数字聚合酶链式反应(数字PCR)微流控芯片. 应用多层软光刻技术, 以聚二甲基硅氧烷(PDMS)作为芯片材料, 盖玻片作为基底制作了具有3层结构以及微阀控制功能的微流控芯片. 芯片的大小与载玻片相当, 可同时检测4个样品, 每个样品通入芯片后平均分配到640个反应小室, 每个小室的体积为6 nL. 以从肺癌细胞A549中提取的18sRNA为样品检测了该芯片的可行性. 将样品稀释数倍后通入芯片, 核酸分子随机分布在640个小室中并扩增. 核酸分子在芯片中的分布符合泊松分布原理, 当样品中待测核酸分子平均拷贝数低于0.5个/小室时, 则每个反应小室包含0个或1个分子. 经过PCR扩增后, 有模板分子的小室检测结果为阳性反应, 而无模板分子的小室为阴性反应, 最后通过计数阳性反应室的个数, 可绝对定量原始待测样品中的目标DNA分子拷贝数. 实验结果表明, 该数字 PCR芯片可实现DNA单分子反应和核酸绝对定量, 具有成本低、 灵敏度高、 节省时间和试剂以及操作简单等优点, 为数字PCR方法在普通实验室的应用提供了一种新途径, 可用于癌症及感染性疾病的早期诊断、 单细胞分析、 产前诊断以及各种细菌病毒的核酸检验等研究.     

A surface topography assisted droplet manipulation platform for biomarker detection and pathogen identification

This paper reports a droplet microfluidic, sample-to-answer platform for the detection of disease biomarkers and infectious pathogens using crude biosamples. The platform exploited the dual functionality of silica superparamagnetic particles (SSP) for solid phase extraction of DNA and magnetic actuation. This enabled the integration of sample preparation and genetic analysis within discrete droplets, including the steps of cell lysis, DNA binding, washing, elution, amplification and detection. The microfluidic device was self contained, with all reagents stored in droplets, thereby eliminating the need for fluidic coupling to external reagent reservoirs. The device incorporated unique surface topographic features to assist droplet manipulation. Pairs of micro-elevations were created to form slits that facilitated efficient splitting of SSP from droplets. In addition, a compact sample handling stage, which integrated the magnet manipulator, the droplet microfluidic device and a Peltier thermal cycler, was built for convenient droplet manipulation and real-time detection. The feasibility of the platform was demonstrated by analysing ovarian cancer biomarker Rsf-1 and detecting Escherichia coli with real time polymerase chain reaction and real time helicase dependent amplification.     

DNA purification using dynamic solid-phase extraction on a rotationally-driven polyethylene-terephthalate microdevice

We report the development of a disposable polyester toner centrifugal device for semi-automated, dynamic solid phase DNA extraction (dSPE) from whole blood samples. The integration of a novel adhesive and hydrophobic valving with a simple and low cost microfabrication method allowed for sequential addition of reagents without the need for external equipment for fluid flow control. The spin-dSPE method yielded an average extraction efficiency of ∼45% from 0.6 μL of whole blood. The device performed single sample extractions or accommodate up to four samples for simultaneous DNA extraction, with PCR-readiness DNA confirmed by effective amplification of a β-globin gene. The purity of the DNA was challenged by a multiplex amplification with 16 targeted amplification sites. Successful multiplexed amplification could routinely be obtained using the purified DNA collected post an on-chip extraction, with the results comparable to those obtained with commercial DNA extraction methods. This proof-of-principle work represents a significant step towards a fully-automated low cost DNA extraction device.     

Integrated microelectronic device for label-free nucleic acid amplification and detection

We present an integrated microelectronic device for amplification and label-free detection of nucleic acids. Amplification by polymerase chain reaction (PCR) is achieved with on-chip metal resistive heaters, temperature sensors, and microfluidic valves. We demonstrate a rapid thermocycling with rates of up to 50 degrees C s(-1) and a PCR product yield equivalent to that of a bench-top system. Amplicons within the PCR product are detected by their intrinsic charge with a silicon field-effect sensor. Similar to existing optical approaches with intercalators such as SYBR Green, our sensing approach can directly detect standard double-stranded PCR product, while in contrast, our sensor does not require labeling reagents. By combining amplification and detection on the same device, we show that the presence or absence of a particular DNA sequence can be determined by converting the analog surface potential output of the field-effect sensor to a simple digital true/false readout.     

One-Step Nucleic Acid Purification and Noise-Resistant Polymerase Chain Reaction by Electrokinetic Concentration for Ultralow-Abundance Nucleic Acid Detection

Nucleic acid amplification tests (NAATs)integrated on a chip hold great promise for point-of-care diagnostics. Currently, nucleic acid (NA) purification remains time-consuming and labor-intensive, and it takes extensive efforts to optimize the amplification chemistry. Using selective electrokinetic concentration, we report one-step, liquid-phase NA purification that is simpler and faster than conventional solid-phase extraction. By further re-concentrating NAs and performing polymerase chain reaction (PCR) in a microfluidic chamber, our platform suppresses non-specific amplification caused by non-optimal PCR designs. We achieved the detection of 5 copies of M. tuberculosis genomic DNA (equaling 0.3 cell) in real biofluids using both optimized and non-optimal PCR designs, which is 10- and 1000-fold fewer than those of the standard bench-top method, respectively. By simplifying the workflow and shortening the development cycle of NAATs, our platform may find use in point-of-care diagnosis.     

Microfluidic DNA amplification—A review

The application of microfluidic devices for DNA amplification has recently been extensively studied. Here, we review the important development of microfluidic polymerase chain reaction (PCR) devices and discuss the underlying physical principles for the optimal design and operation of the device. In particular, we focus on continuous-flow microfluidic PCR on-chip, which can be readily implemented as an integrated function of a micro-total-analysis system. To overcome sample carryover contamination and surface adsorption associated with microfluidic PCR, microdroplet technology has recently been utilized to perform PCR in droplets, which can eliminate the synthesis of short chimeric products, shorten thermal-cycling time, and offers great potential for single DNA molecule and single-cell amplification. The work on chip-based PCR in droplets is highlighted.     

Genotyping from saliva with a one-step microdevice

This paper presents a disposable microfluidic device for on-chip lysing, PCR, and analysis in one continuous-flow process. Male-female sex determination was performed with human saliva in less than 20 min from spit to finish, and requiring only seconds of manual sample handling. This genetic analysis was based on the amplification and detection of the DYZ1 repeat region unique to the Y-chromosome. The flow-through microfluidic chip consisted of a single serpentine channel designed to guide samples through 42 heating and cooling cycles. Cycling was performed by matching the local channel geometry to a steady-state temperature gradient established across the microfluidic chip. 38 channel segments were designed for rapid low volume PCR, and four were optimized for spatial DNA melting analysis. Fluorescence detection was used to monitor the amplification and to capture the melting signature of the amplicon was performed with a basic 8-bit CCD camera. The microfluidic device itself was fabricated from microscope slides and a double-sided tape. The simplicity of the system and its robust performance combine in an elegant solution for lab-on-a-chip genetic analysis.     

Developments toward a complete micro-total analysis system for Duchenne muscular dystrophy diagnosis

The diagnosis of Duchenne muscular dystrophy (DMD) has historically utilized either PCR or requires Southern blot analysis, a southern blot analysis, however, is not amenable to incorporation in a microdevice format. A PCR amplification-based method has been developed, and we have previously coupled this amplification with microchip separation of the PCR fragments for DMD diagnosis. Diagnoses of affected patients were performed by comparing exon concentrations to those of control samples amplified at the same time. To accurately identify mutations in patient samples, this work established normal ranges for the concentration of each amplified exon fragment using control samples amplified over successive days. Our studies show that the number of cycles used in the amplification process affects this range. Affected patient samples were analyzed using these normal ranges and the mutations detected by Southern blot analysis were also diagnosed using the microchip separation method.

Employing the microchip separation method decreases the time required for the analysis, but the time required for DNA purification and PCR amplification must also be decreased for faster total analysis of patient samples. Development of microchip methods for these processing steps is one approach for reducing the individual times, while also providing the possibility of integrating these steps in a single device. Here we report on the microchip extraction of genomic DNA from whole blood using a novel sol–gel matrix that is easily formed in microdevices. IR-mediated PCR amplification of a β-globin fragment from genomic DNA followed by electrophoretic analysis on a single integrated microdevice is presented for the first time. Work towards the development of a micro-total analysis device for DMD diagnosis, through integration of all processing steps on a single device, is also discussed.     

Solid phase DNA extraction on PDMS and direct amplification

In this paper we report an innovative use of Poly(DiMethyl)Siloxane (PDMS) to design a microfluidic device that combines, for the first time, in one single reaction chamber, DNA purification from a complex biological sample (blood) without elution and PCR without surface passivation agents. This result is achieved by exploiting the spontaneous chemical structure and nanomorphology of the material after casting. The observed surface organization leads to spontaneous DNA adsorption. This property allows on-chip complete protocols of purification of complex biological samples to be performed directly, starting from cells lysis. Amplification by PCR is performed directly on the adsorbed DNA, avoiding the elution process that is normally required by DNA purification protocols. The use of one single microfluidic volume for both DNA purification and amplification dramatically simplifies the structure of microfluidic devices for DNA preparation. X-Ray Photoelectron Spectroscopy (XPS) was used to analyze the surface chemical composition. Atomic Force Microscopy (AFM) and Field Emission Scanning Electron Microscopy (FESEM) were employed to assess the morphological nanostructure of the PDMS-chips. A confocal fluorescence analysis was utilized to check DNA distribution inside the chip.     

1-Million droplet array with wide-field fluorescence imaging for digital PCR

Digital droplet reactors are useful as chemical and biological containers to discretize reagents into picolitre or nanolitre volumes for analysis of single cells, organisms, or molecules. However, most DNA based assays require processing of samples on the order of tens of microlitres and contain as few as one to as many as millions of fragments to be detected. Presented in this work is a droplet microfluidic platform and fluorescence imaging setup designed to better meet the needs of the high-throughput and high-dynamic-range by integrating multiple high-throughput droplet processing schemes on the chip. The design is capable of generating over 1-million, monodisperse, 50 picolitre droplets in 2-7 minutes that then self-assemble into high density 3-dimensional sphere packing configurations in a large viewing chamber for visualization and analysis. This device then undergoes on-chip polymerase chain reaction (PCR) amplification and fluorescence detection to digitally quantify the sample's nucleic acid contents. Wide-field fluorescence images are captured using a low cost 21-megapixel digital camera and macro-lens with an 8-12 cm(2) field-of-view at 1× to 0.85× magnification, respectively. We demonstrate both end-point and real-time imaging ability to perform on-chip quantitative digital PCR analysis of the entire droplet array. Compared to previous work, this highly integrated design yields a 100-fold increase in the number of on-chip digitized reactors with simultaneous fluorescence imaging for digital PCR based assays.     

Circumventing air bubbles in microfluidic systems and quantitative continuous-flow PCR applications

Polymerase chain reaction (PCR) is an essential part of research based on genomics or cell analysis. The development of a microfluidic device that would be suitable for high-temperature-based reactions therefore becomes an important contribution towards the integration of micro-total analysis systems (μTAS). However, problems associated with the generation of air bubbles in the microchannels before the introduction of the assay liquid, which we call the “initial start-up” in this study, made the flow irregular and unstable. In this report, we have tried to address these problems by adapting a novel liquid-flow method for high-temperature-based reactions. A PDMS-based microfluidic device was fabricated by soft-lithography techniques and placed on a cartridge heater. The generation of the air bubbles was prevented by introducing the fluorinated oil, an inert and highly viscous liquid, as the cap just before the introduction of the sample solutions into the microchannels. The technique was applied for continuous-flow PCR, which could perform PCR on-chip in a microfluidic system. For the evaluation of practical accuracy, plasmid DNA that serves as a reference molecule for the quantification of genetically modified (GM) maize was used as the template DNA for continuous-flow PCR. After PCR, the products were collected in a vial and analyzed by gel electrophoresis to confirm the accuracy of the results. Additionally, quantitative continuous-flow PCR was performed using TaqMan technology on our PCR device. A laser detection system was also used for the quantitative PCR method. We observed a linear relationship between the threshold cycle (Ct) and the initial DNA concentration. These results showed that it would be possible to quantify the initial copies of the template DNA on our microfluidic device. Accurate quantitative DNA analysis in microfluidic systems is required for the integration of PCR with μTAS, thus we anticipate that our device would have promising potential for applications in a wide range of research.     

Functional integration of DNA purification and concentration into a real time micro-PCR chip

Microfluidic devices for on-chip amplification of DNA from various biological and environmental samples have gained extensive attention over the past decades with many applications including molecular diagnostics of disease, food safety and biological warfare testing. But the integration of sample preparation functions into the chip remains a major hurdle for practical application of the chip-based diagnostic system. We present a PCR-based molecular diagnostic device comprised of a microfabricated chip and a centrifugal force assisted liquid handling tube (CLHT) that is designed to carry out concentration and purification of DNA and subsequent amplification of the target gene in a single chip. The reaction chamber of the chip contains an array of pillar structures to increase the surface area for capturing DNA from a raw sample of macro volume in the presence of kosmotropic agents. The CLHT was designed to provide an effective interface between sample preparation and the microfluidic PCR chip. We have characterized the effect of various fluidic parameters including DNA capture, amplification efficiency and centrifugal pressure generated upon varying sample volume. We also evaluated the performance of this system for quantitative detection of E. coli O157:H7. From the samples containing 10(1) to 10(4) cells per mL, the C(T) value linearly increased from 25.1 to 34.8 with an R(2) value greater than 0.98. With the effectiveness and simplicity of operation, this system will provide an effective interface between macro and micro systems and bridge chip-based molecular diagnosis with practical applications.     

One‐Step Nucleic Acid Purification and Noise‐Resistant Polymerase Chain Reaction by Electrokinetic Concentration for Ultralow‐Abundance Nucleic Acid Detection

Nucleic acid amplification tests (NAATs)integrated on a chip hold great promise for point‐of‐care diagnostics. Currently, nucleic acid (NA) purification remains time‐consuming and labor‐intensive, and it takes extensive efforts to optimize the amplification chemistry. Using selective electrokinetic concentration, we report one‐step, liquid‐phase NA purification that is simpler and faster than conventional solid‐phase extraction. By further re‐concentrating NAs and performing polymerase chain reaction (PCR) in a microfluidic chamber, our platform suppresses non‐specific amplification caused by non‐optimal PCR designs. We achieved the detection of 5 copies of M. tuberculosis genomic DNA (equaling 0.3 cell) in real biofluids using both optimized and non‐optimal PCR designs, which is 10‐ and 1000‐fold fewer than those of the standard bench‐top method, respectively. By simplifying the workflow and shortening the development cycle of NAATs, our platform may find use in point‐of‐care diagnosis.