Microfluidic Polymerase Chain Reaction Research Articles

Sensitive, microliter PCR with consensus degenerate primers for Epstein Barr virus amplification

Sensitive identification of the etiology of viral diseases is key to implementing appropriate prevention and treatment. The gold standard for virus identification is the polymerase chain reaction (PCR), a technique that allows for highly specific and sensitive detection of pathogens by exponentially amplifying a specific region of DNA from as little as a single copy through thermocycling a biochemical cocktail. Today, molecular biology laboratories use commercial instruments that operate in 0.5-2 h/analysis using reaction volumes of 5-50 μL contained within polymer tubes or chambers. Towards reducing this volume and maintaining performance, we present a semi-quantitative, systematic experimental study of how PCR yield is affected by tube/chamber substrate, surface-area-to-volume ratio (SA:V), and passivation methods. We perform PCR experiments using traditional PCR tubes as well as using disposable polymer microchips with 1 μL reaction volumes thermocycled using water baths. We report the first oil encapsulation microfluidic PCR method without fluid flow and its application to the first microfluidic amplification of Epstein Barr virus using consensus degenerate primers, a powerful and broad PCR method to screen for both known and novel members of a viral family. The limit of detection is measured as 140 starting copies of DNA from a starting concentration of 3 × 10(5) copies/mL, regarded as an accepted sensitivity threshold for diagnostic purposes, and reaction specificity was improved as compared to conventional methods. Also notable, these experiments were conducted with conventional reagent concentrations, rather than commonly spiked enzyme and/or template mixtures. This experimental study of the effects of substrate, SA:V, and passivation, together with sensitive and specific microfluidic PCR with consensus degenerate primers represent advances towards lower cost and higher throughput pathogen screening.

Applications of EWOD Systems for DNA Reaction and Analysis

A digital microfluidic system based on electrowetting-on-dielectric (EWOD) technology has attracted considerable attention because of its promising potential in biological, biochemical and biomedical applications. The digital microfluidic (EWOD) system enables a discrete droplet to be manipulated through reconfig-urable paths or channels defined by electrode actuation. Minute sample and reagent volume, precise droplet control, low energy consumption, low cost, rapid analysis, portablility for point-of-care diagnosis, flexibility for integrating with detector, multiple-step and simultaneous reaction processes are all advantages of EWOD system. Thus, it has become a promising and popular tool for bio-applications such as cell assays, proteomics, enzyme assays, immunoassays, DNA extraction, DNA repair and DNA amplification. In DNA-based applications, ordinarily, the DNA replication (e.g., DNA amplification) is necessary. Among the DNA replication techniques, Polymerase Chain Reaction (PCR) because of its simplicity and cost-effectiveness is the most common laboratory technique. Therefore, the development of EWOD-based microfluidic PCR system combined with other novel techniques for DNA amplification or DNA-based assays has extensively been reported. In this paper, the recent developments and studies of EWOD microfluidic system for DNA-based applications published in the last five years have been reviewed.

Bead-based polymerase chain reaction on a microchip.

We present a bead-based approach to microfluidic polymerase chain reaction (PCR), enabling fluorescent detection and sample conditioning in a single microchamber. Bead-based PCR, while not extensively investigated in microchip format, has been used in a variety of bioanalytical applications in recent years. We leverage the ability of bead-based PCR to accumulate fluorescent labels following DNA amplification to explore a novel DNA detection scheme on a microchip. The microchip uses an integrated microheater and temperature sensor for rapid control of thermal cycling temperatures, while the sample is held in a microchamber fabricated from (poly)dimethylsiloxane and coated with Parylene. The effects of key bead-based PCR parameters, including annealing temperature and concentration of microbeads in the reaction mixture, are studied to achieve optimized device sensitivity and detection time. The device is capable of detecting a synthetically prepared section of the Bordetella pertussis genome in as few as 10 temperature cycles with times as short as 15 min. We then demonstrate the use of the procedure in an integrated device; capturing, amplifying, detecting, and purifying template DNA in a single microfluidic chamber. These results show that this method is an effective method of DNA detection which is easily integrated in a microfluidic device to perform additional steps such as sample pre-conditioning.

Inhibition of on-chip PCR using PDMS–glass hybrid microfluidic chips

In the course of developing a microfluidic analytical platform incorporating the polymerase chain reaction (PCR) and subsequent capillary electrophoresis (CE) analysis for a variety of bio-assays, we examined PCR inhibition through surface interactions with the chip materials. Our devices perform PCR in a three-layer chip, a glass–poly(dimethylsiloxane)–glass sandwich in which the poly(dimethylsiloxane) (PDMS, a silicone rubber) layer is used for pneumatic membrane pumping and valving of the PCR reagents. Initial on-chip PCR–CE tests of BK virus replicated in multiple uncoated chips showed variable results, usually yielding no detectable product at the target sample concentrations used. Subsequent “chip-flush” experiments, where water or reagents were flushed through a chip and subsequently incorporated in off-chip PCR, highlighted bovine serum albumin (BSA) amongst other pre-treatments, chip materials and PCR recipes as being effective in mitigating inhibition. When the BSA channel pre-coating was applied to on-chip PCR–CE experiments, a substantial improvement (10× to 40×) in signal-to-noise (S/N) of the CE product peak was conferred, and was shown with high confidence despite high S/N variability. This is the first study to quantitatively examine BSA’s ability to reduce inhibition of PCR performed on PDMS chips, and one of very few microfluidic PCR inhibition studies of any kind to use a large number of microfluidic chips (~400). The simplicity and effectiveness of our BSA coating suggest that passivating materials applied to microfluidic device channel networks may provide a viable pathway for development of bio-compatible devices with reduced complexity and cost.

Evaluation of a digital microfluidic real-time PCR platform to detect DNA of Candida albicans in blood

Species of Candida frequently cause life-threatening infections in neonates, transplant and intensive care unit (ICU) patients, and others with compromised host defenses. The successful management of systemic candidiasis depends upon early, rapid diagnosis. Blood cultures are the standard diagnostic method, but identification requires days and less than half of the patients are positive. These limitations may be eliminated by using real-time polymerase chain reaction (PCR) to detect Candida DNA in the blood specimens of patients at risk. Here, we optimized a PCR protocol to detect 5-10 yeasts in low volumes of simulated and clinical specimens. We also used a mouse model of systemic candidiasis and determined that candidemia is optimally detectable during the first few days after infection. However, PCR tests are often costly, labor-intensive, and inconvenient for routine use. To address these obstacles, we evaluated the innovative microfluidic real-time PCR platform (Advanced Liquid Logic, Inc.), which has the potential for full automation and rapid turnaround. Eleven and nine of 16 specimens from individual patients with culture-proven candidemia tested positive for C. albicans DNA by conventional and microfluidic real-time PCR, respectively, for a combined sensitivity of 94%. The microfluidic platform offers a significant technical advance in the detection of microbial DNA in clinical specimens.

Plug-and-play, infrared, laser-mediated PCR in a microfluidic chip

Microfluidic polymerase chain reaction (PCR) systems have set milestones for small volume (100 nL-5 μL), amplification speed (100-400 s), and on-chip integration of upstream and downstream sample handling including purification and electrophoretic separation functionality. In practice, the microfluidic chips in these systems require either insertion of thermocouples or calibration prior to every amplification. These factors can offset the speed advantages of microfluidic PCR and have likely hindered commercialization. We present an infrared, laser-mediated, PCR system that features a single calibration, accurate and repeatable precision alignment, and systematic thermal modeling and management for reproducible, open-loop control of PCR in 1 μL chambers of a polymer microfluidic chip. Total cycle time is less than 12 min: 1 min to fill and seal, 10 min to amplify, and 1 min to recover the sample. We describe the design, basis for its operation, and the precision engineering in the system and microfluidic chip. From a single calibration, we demonstrate PCR amplification of a 500 bp amplicon from λ-phage DNA in multiple consecutive trials on the same instrument as well as multiple identical instruments. This simple, relatively low-cost plug-and-play design is thus accessible to persons who may not be skilled in assembly and engineering.

A Design of DSPIC Based Microfluidic PCR Chip Temperature Controlling System

In this paper a dsPIC microcontroller based temperature control system is developed for polymerase chain reaction (PCR) on-chip. PCR is one of the most important techniques in molecular biology and temperature control is the key technique of PCR. Yet, most of the works are based on PC, through an RS 232 interface to set and control the temperature. Here we design a temperature control system based on DSPIC Microcontroller. It can be much portable and cheaper than PC based systems. The system configuration mainly consists of a high precision, stable performance sensor PT1000, a signal amplification circuit, a PID algorithm. And then, we use MAX1978 to improve the accuracy of temperature control. After that we choose thermoelectric cooler (TEC) modules as actuator to improve system heating and cooling rate. It is believe that the portable PCR temperature control system will play an important role in the development of the Microfluidic PCR.

Microfluidic platform versus conventional real-time polymerase chain reaction for the detection of Mycoplasma pneumoniae in respiratory specimens

Rapid, accurate diagnosis of community-acquired pneumonia (CAP) due to Mycoplasma pneumoniae is compromised by low sensitivity of culture and serology. Polymerase chain reaction (PCR) has emerged as a sensitive method to detect M. pneumoniae DNA in clinical specimens. However, conventional real-time PCR is not cost-effective for routine or outpatient implementation. Here, we evaluate a novel microfluidic real-time PCR platform (Advanced Liquid Logic, Research Triangle Park, NC) that is rapid, portable, and fully automated. We enrolled patients with CAP and extracted DNA from nasopharyngeal wash (NPW) specimens using a biotinylated capture probe and streptavidin-coupled magnetic beads. Each extract was tested for M. pneumoniae-specific DNA by real-time PCR on both conventional and microfluidic platforms using Taqman probe and primers. Three of 59 (5.0%) NPWs were positive, and agreement between the methods was 98%. The microfluidic platform was equally sensitive but 3 times faster and offers an inexpensive and convenient diagnostic test for microbial DNA.

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.

An optimal design method for preventing air bubbles in high-temperature microfluidic devices

DNA analysis with the polymerase chain reaction (PCR) has become a routine part of medical diagnostics, environmental inspections, food evaluations, and biological studies. Furthermore, the development of a microscale PCR chip is an essential component of studies aimed at integrating PCR into a micro total analysis system (mu-TAS). However, the occurrence of air bubbles in microchannels complicates this process. In this study, we investigated a new technique based on the fluid dynamics of laminar flow that utilizes a small amount of mineral oil at the beginning of sample injection to prevent air bubbles from occurring in microchannels. We also further optimized the pressure, the length of the pressurizing channel and the volume of oil, thus making our microfluidic device more useful for high-temperature PCR. Additionally, quantitative continuous-flow PCR was performed using the optimized PCR chip in order to detect genetically modified (GM) maize. DNA was extracted from GM maize, MON 810, and non-GM maize at several concentrations from 0% (w/v) to 100% (w/v). The DNA amplification signals were then analyzed on the PCR chip using a laser-based system. The signal from our microfluidic PCR chip was found to increase in direct proportion to the initial GM maize concentration.

Microfluidic digital PCR enables rapid prenatal diagnosis of fetal aneuploidy

The purpose of this study was to demonstrate that digital polymerase chain reaction (PCR) enables rapid, allele independent molecular detection of fetal aneuploidy. Twenty-four amniocentesis and 16 chorionic villus samples were used for microfluidic digital PCR analysis. Three thousand and sixty PCR reactions were performed for each of the target chromosomes (X, Y, 13, 18, and 21), and the number of single molecule amplifications was compared to a reference. The difference between target and reference chromosome counts was used to determine the ploidy of each of the target chromosomes. Digital PCR accurately identified all cases of fetal trisomy (3 cases of trisomy 21, 3 cases of trisomy 18, and 2 cases of triosmy 13) in the 40 specimens analyzed. The remaining specimens were determined to have normal ploidy for the chromosomes tested. Microfluidic digital PCR allows detection of fetal chromosomal aneuploidy utilizing uncultured amniocytes and chorionic villus tissue in less than 6 hours.

Parallel DNA amplification by convective polymerase chain reaction with various annealing temperatures on a thermal gradient device

We present a thermal gradient convective polymerase chain reaction (PCR) for parallel DNA amplification with different annealing temperatures. The thermal gradient for microfluidic gradient PCR is produced by an innovative fin design whose formation principle is given. Without the need for a pump, the buoyancy forces continuously circulate reagents in a closed loop through different thermal zones, which brings self-actuated convective-flow PCR. In our prototype, we measured a temperature difference of about 45 °C along the gradient direction on the copper flake (45 × 40 × 4 mm). When the temperature of the hot zone is 90–97 °C and the temperature of the cold zone is 60–70 °C, the convection triggered two-temperature amplification of 112-bp fragment of Escherichia coli DNA. The time for amplification is less than 45 min. Interestingly, parallel DNA amplification with different annealing temperatures ranging from 60 to 70 °C was performed by this method. The PCR thermocycler demonstrated herein can be further scaled down and the loop length can be further reduced, and therefore the PCR times can be further reduced. These devices are suited as a platform for a new generation of low-power, portable DNA analysis systems.

Rapid detection of genetically modified organisms on a continuous-flow polymerase chain reaction microfluidics

The ability to perform DNA amplification on a microfluidic device is very appealing. In this study, a compact continuous-flow polymerase chain reaction (PCR) microfluidics was developed for rapid analysis of genetically modified organisms (GMOs) in genetically modified soybeans. The device consists of three pieces of copper and a transparent polytetrafluoroethylene capillary tube embedded in the spiral channel fabricated on the copper. On this device, the P35S and Tnos sequences were successfully amplified within 9 min, and the limit of detection of the DNA sample was estimated to be 0.005 ng μl −1. Furthermore, a duplex continuous-flow PCR was also reported for the detection of the P35S and Tnos sequences in GMOs simultaneously. This method was coupled with the intercalating dye SYBR Green I and the melting curve analysis of the amplified products. Using this method, temperature differences were identified by the specific melting temperature values of two sequences, and the limit of detection of the DNA sample was assessed to be 0.01 ng μl −1. Therefore, our results demonstrated that the continuous-flow PCR assay could discriminate the GMOs in a cost-saving and less time-consuming way.

An Innovative Rapid Thermal Cycling Device for Polymerase Chain Reaction

Polymerase chain reaction (PCR) is the most commonly used molecular biology technique to amplify nucleic acid (DNA and RNA) in vitro. This technique is highly temperature sensitive and thermal management has an important role in PCR operation in reaching the required temperature set points at each step of the process (denaturing, annealing and elongation). In this work, an innovative microfluidic PCR thermal cycling device is designed to increase the heating∕cooling thermal cycling speed while maintaining a uniform temperature distribution throughout the substrate containing the aqueous nucleic acid sample. The device design is incorporating the jet impingement and micro-channel thermal management technologies utilizing a properly arranged configuration filled with a porous medium. Porous Inserts are attractive choices in heat transfer augmentation. They provide a very large surface area for a given volume which is a key parameter in heat transfer processes. Various effective parameters that are relevant in optimizing this flexible thermal cycler are investigated such as thermal cycler configuration, thickness of inlet and exit fluid channels, fluid flow rate and velocity, the porous matrix material and properties, and utilization of thermal grease. An optimized case is established based on the effects of the cited parameters on the temperature ramp, temperature distribution and the required power for circulating the fluid in the thermal cycler. The results indicate that the heating∕cooling temperature ramp (temperature change per heating∕cooling cycling time) of the proposed device is considerably higher (150.82◻C∕s) than those in literature. In addition, the proposed PCR offers a very uniform temperature in the substrate while utilizing a low power.

Rapid microfluidic thermal cycler for polymerase chain reaction nucleic acid amplification

Polymerase chain reaction (PCR) is widely used in biochemical analysis to amplify DNA and RNA in vitro. The PCR process is highly temperature sensitive, and thermal management has an important role in PCR operation in reaching the required temperature set points at each step of the process. The goal of this research is to achieve a thermal technique to rapidly increase the heating/cooling thermal cycling speed while maintaining a uniform temperature distribution throughout the substrate containing the aqueous nucleic acid sample. In this work, an innovative microfluidic PCR thermal cycler, which utilizes a properly arranged configuration filled with a porous medium, is investigated. Various effective parameters that are relevant in optimizing this flexible heat exchanger are investigated such as heat exchanger geometry, flow rate, conductive plate, the porous matrix material, and utilization of thermal grease. An optimized case is established based on the effects of the cited parameters on the temperature distribution and the required power for circulating the fluid in the heat exchanger. The results indicate that the heating/cooling temperature ramp of the proposed PCR heat exchanger is considerably higher (150.82 °C/s) than those in the literature. In addition, the proposed PCR offers a very uniform temperature in the substrate while utilizing a low power.

Compatibility of Segmenting Fluids in Continuous-Flow Microfluidic PCR

Continuous flow offers notable advantages over batch processing for analytical applications like gene expression profiling of biological material, which demands very high processing. The technology of choice for future genetic analyzers will most likely use the polymerase chain reaction (PCR); therefore, high-throughput, high-speed PCR devices have raised enormous interest. Continuous-flow, biphasic PCR can meet these requirements but segmenting∕carrier fluids chemically compatible with the PCR are needed. The present paper compares several fluids in terms of compatibility with PCR and fluidic dynamics in a continuous, two-phase flow microfluidic device, and PCR efficiency was assessed quantitatively. The results represent the first step toward rational fluid design for biphasic continuous PCR.

Continuous‐Flow PCR Microfluidics for Rapid DNA Amplification Using Thin Film Heater with Low Thermal Mass

We have described a compact capillary‐based continuous‐flow polymerase chain reaction (PCR) microfluidics device, which uses flexible thin film heaters with low thermal mass to construct three isothermal zones. Due to the decreased thermal mass of flexible thin film heater, the low power supply and rapid thermal response was obtained. The energy consumption of a 33‐cycle continuous‐flow PCR was less than 0.0088 kW h, which is much lower than that of the metal block or liquid bath based capillary continuous‐flow PCR microfluidics. Special attention was also paid on the surface passivation of the capillary inner surface based on the competing bovine serum albumin (BSA), and the results showed the effect of dynamic passivation was superior to that of static passivation. With the help of the dynamic passivation, the 249 bp human β‐actin gene fragment was amplified in 15 min, which is several times faster than that of the conventional PCR machine. In addition, the effect of initial DNA template concentrations on continuous‐flow PCR was also investigated. The concentration limit of DNA template reached 0.18 ng µl−1, which can satisfy the requirements from different application fields.

A Prototype Continuous Flow Polymerase Chain Reaction LTCC Device

There is a growing need for remote biological sensing in both laboratory and harsh field environments. Sensing and detection of biological entities such as anthrax, Ebola and other micro-organisms of interest involves sampling of the environment, amplification, analysis and identification of the target DNA. A key component of such a sensor is a low cost, portable, reusable, continuous flow polymerase chain reaction (PCR) thermal cycler. Fabrication with low temperature co-fired ceramics (LTCC) can provide a reusable low cost device capable of operating in a wide range of environments The design and manufacture of a prototype continuous flow micro-fluidic PCR device using low temperature co-fired ceramic is presented. Initial modeling of flow characteristics and heat transfer was carried out in SolidWorks™. The prototype device employs resistance heaters below the channels, buried and surface thermocouples for temperature monitoring, and air gaps for thermal isolation.

Continuous‐flow Polymerase Chain Reaction Microfluidics by Using Spiral Capillary Channel Embedded on Copper

This paper presents a novel method for performing polymerase chain reaction (PCR) amplification by using spiral channel fabricated on copper where a transparent polytetrafluoroethylene (PTFE) capillary tube was embedded. The channel with 25 PCR cycles was gradually developed in a spiral manner from inner to outer. The durations of PCR mixture at the denaturation, annealing and extension zones were gradually lengthened at a given flow rate, which may benefit continuous‐flow PCR amplification as the synthesis ability of the Taq polymerase enzyme usually weakens with PCR time. Successful continuous‐flow amplification of DNA fragments has been demonstrated. The PCR products of 249, 500 and 982 bp fragments could be obviously observed when the flow rates of PCR mixture were 7.5, 7.5 and 3.0 mm s−1, respectively, and the required amplification times were about 25, 25, and 62 min, respectively. Besides, the successful segmented‐flow PCR of three samples (249, 500 and 982 bp) has also been reported, which demonstrates the present continuous‐flow PCR microfluidics can be developed for high‐throughput genetic analysis.

Continuous-flow polymerase chain reaction microfluidics based on polytetrafluoethylene capillary

The polytetrafluoroethylene (PTFE) capillary-based continuous-flow polymerase chain reaction (PCR) microfluidics were constructed by the flexible thin film heaters to form three constant temperature zones necessary for a PCR amplification, in which the PCR mixture could flow through the capillary mcirochannel continuously, thus leading to the successful completion of PCR exponential amplification. The effects of various concentrations of bovine serum albumin (BSA) in a 25-μl PCR mixture and flow rates of PCR mixture in the capillary microchannel, on continuous-flow PCR amplification, had been discussed in detail, where a PCR amplification of 249-bp human β-actin gene fragments was successfully performed within 15 and even 10 minutes, which is several times faster than the reaction time required (1–2 hours) with the conventional PCR machine.