Polymerase Chain Reaction Microchip Research Articles

Polymerase Chain Reaction Microchip and PID Controller Based Thermal Cycler Design

Numerous specific procedures are needed for nucleic acid detection using traditional approaches. Because of the time and instrumentation requirements, poor detection limits, and other factors, they are behind in creating point-of-care (POC) testing. In addition, some of these traditional approaches can be extremely sensitive. Microfluidic technologies are being used to overcome these challenges by enabling the efficient and automatic performance of all analytical stages, such as sample pre-treatment, reactions, separations, and diagnostics in microchannels on a small chip. The use of microfluidics technology in polymerase chain reaction (PCR) technology has resulted in many advantages. PCR microchips and portable thermal cyclers that can be used for POC testing have enabled faster amplification with accurate results. This paper proposes a low-cost, fast enough thermal cycler for POC testing that uses a polymer-glass microchip and an affordable Peltier element to quickly heat samples. A laser cutter was used to build the microchip needed to perform PCR, which was designed using a computer-aided design (CAD) software. The heating and cooling elements employed in the creation of the thermal cycler were Peltier elements, heat sink, centrifugal fan, and CPU fan. A microcontroller and H-Bridge modules were used to control the temperature with the help of proportional-integral-derivative (PID) controller algorithm. After that, the circuit components and PID control algorithm—which was created for temperature control—were combined to create the PCR cycle. The analysis focuses on the heating and cooling performances of suggested thermal cycler.

A Thermocycler Using a Chip Resistor Heater and a Glass Microchip for a Portable and Rapid Microchip-Based PCR Device.

This study proposes a rapid and inexpensive thermocycler that enables rapid heating of samples using a thin glass chip and a cheap chip resistor to overcome the on-site diagnostic limitations of polymerase chain reaction (PCR). Microchip PCR devices have emerged to miniaturize conventional PCR systems and reduce operation time and cost. In general, PCR microchips require a thin-film heater fabricated through a semiconductor process, which is a complicated process, resulting in high costs. Therefore, this investigation substituted a general chip resistor for a thin-film heater. The proposed thermocycler consists of a compact glass microchip of 12.5 mm × 12.5 mm × 2 mm that could hold a 2 μL PCR sample and a surface-mounted chip resistor of 6432 size (6.4 mm × 3.2 mm). Improving heat transfer from the chip resistor heater to the PCR reaction chamber in the microchip was accomplished via the design and fabrication of a three-dimensional chip structure using selective laser-induced etching, a rapid prototyping technique that allowed to be embedded. The fabricated PCR microchip was combined with a thermistor temperature sensor, a blower fan, and a microcontroller. The assembled thermocycler could heat the sample at a maximum rate of 28.8 °C/s per second. When compared with a commercially available PCR apparatus running the same PCR protocol, the total PCR operating time with a DNA sample was reduced by about 20%.

Simulation and Optimization of a Single Heater Convective PCR Chip and Its Controller for Fast Salmonella Enteritidis Detection

The design and optimization of a Rayleigh- Bénard convection based polymerase chain reaction (PCR) microchip is presented. This PCR microchip simplifies the fabrication, implementation and control of microfluidic PCR while enhancing the detection of the bacterial pathogen Salmonella Enteritidis. The device's dimensions were optimized using multiphysics simulation then it was fabricated using high precision milling of Poly(methyl methacrylate) (PMMA) substrate. The device incorporated a single heater at the bottom end of its cylindrical chamber and two sensors at both ends. The heater was controlled using a fuzzy proportional, integral, derivative (PID) controller to maintain denaturation and annealing temperatures necessary for efficient PCR to take place. Salmonella Enteritidis DNA was successfully detected using the device while reducing the required assay time by 66%.

Development of a real-time microchip PCR system for portable plant disease diagnosis.

Rapid and accurate detection of plant pathogens in the field is crucial to prevent the proliferation of infected crops. Polymerase chain reaction (PCR) process is the most reliable and accepted method for plant pathogen diagnosis, however current conventional PCR machines are not portable and require additional post-processing steps to detect the amplified DNA (amplicon) of pathogens. Real-time PCR can directly quantify the amplicon during the DNA amplification without the need for post processing, thus more suitable for field operations, however still takes time and require large instruments that are costly and not portable. Microchip PCR systems have emerged in the past decade to miniaturize conventional PCR systems and to reduce operation time and cost. Real-time microchip PCR systems have also emerged, but unfortunately all reported portable real-time microchip PCR systems require various auxiliary instruments. Here we present a stand-alone real-time microchip PCR system composed of a PCR reaction chamber microchip with integrated thin-film heater, a compact fluorescence detector to detect amplified DNA, a microcontroller to control the entire thermocycling operation with data acquisition capability, and a battery. The entire system is 25×16×8 cm3 in size and 843 g in weight. The disposable microchip requires only 8-µl sample volume and a single PCR run consumes 110 mAh of power. A DNA extraction protocol, notably without the use of liquid nitrogen, chemicals, and other large lab equipment, was developed for field operations. The developed real-time microchip PCR system and the DNA extraction protocol were used to successfully detect six different fungal and bacterial plant pathogens with 100% success rate to a detection limit of 5 ng/8 µl sample.

Ferrofluid plug as valve and actuator for whole-cell PCR on chip

Ferrofluid plug was first used as valve and actuator to circumvent the evaporation of aqueous fluid in LOC. Moreover, this dynamic plug catered for aqueous fluid expansion during its heating in the microchannel. Whole-cell polymerase chain reaction (PCR) protocol was successfully implemented in a magnetically actuated closed-loop PCR microchip system. Here, the ferrofluid plug was used to reduce the evaporation of the PCR sample in the thermal lysis step. A whole-cell PCR model was used to demonstrate this concept. The DNA plasmid, pUC19, was transformed into DH5α competent cells. Primer pairs were designed to amplify region of interest in pUC19. The transformants were cultured and selected. Whole-cell PCR was performed on intact bacteria to screen for the presence of pUC19, containing the region of interests. Serial dilutions demonstrated the limit of detection for this system to be four bacterial cells. PCR amplifications were verified with regular thermocyclers and results were comparable.

An integrated PCR microfluidic chip incorporating aseptic electrochemical cell lysis and capillary electrophoresis amperometric DNA detection for rapid and quantitative genetic analysis

A fully integrated microchip for performing cell lysis, polymerase chain reaction (PCR) and quantitative analysis of DNA amplicons in a single step is described herein. The chip was built on glass substrate using an indium-tin-oxide (ITO) microheater and PDMS engraved microchannels, which integrated an electrochemical cell lysis zone, a continuous flow PCR module and capillary electrophoresis amperometric detection (CE-AD) system. The total length of the microchannel was 4625 mm for performing 25 cycles of flow-through PCR and was laid on a handheld form factor of 96 × 96 mm(2) area. The key to the fabrication of such a device lies in the use of a single medium to carry out different kinds of biochemical reactions and hence, a reagentless electrochemical cell lysis protocol was integrated on the microchip which was capable of lysing most cell types, including difficult to lyse gram positive bacteria. The lysate contained genomic DNA from a sample which was proven to be suitable for PCR reactions. Genetic analysis was successfully performed on the microchip with purified lambda phage genomic DNA and various cell types, including non-tumorigenic MCF-10A and tumorigenic MCF-7 human cell lines, gram negative bacteria Escherichia coli O157:H7, and gram positive bacteria Bacillus subtilis, at an optimized flow rate of 5 μl min(-1). For the detection of amplicon DNA, a CE-AD system was used, with semisolid alkaline agarose within the capillary microchannel to minimize interference from cell debris and for efficient resolution of DNA fragments. High signal to noise ratio during amperometric detection and the use of online FFT filtering protocol enhanced the limit of detection of DNA amplicons. Therefore, with a combination of portability, cost-effectiveness and performance, the proposed integrated PCR microchip can be used for one step genetic analysis of most of the cell types and will enable more accessible healthcare.

Development of PCR Microchip for Early Cancer Risk Prediction

An integrated continuous-flow microfluidic chip was fabricated on glass substrate with polydimethylsiloxane (PDMS)-based microchannels, cell lysis and Polymerase chain reaction (PCR) modules on the same chip. While gold-microelectrode was used for electrochemical cell lysis, indium-tin-oxide (ITO) microheater was used for thermal cycling during PCR reaction. The fabricated device was used for PCR amplification of pancreatic cancer DNA marker (SMAD4) from non-tumorigenic MCF10a human cell lines. The PCR product (193 bp) was verified for MCF10a cells by agarose gel electrophoresis after 20 cycles of reaction on the microchip, whereas no product was detected in case of tumorigenic MCF7 cells. The total time required for the entire reaction was less than 45 min. Therefore, the proposed microchip can be helpful in predicting the risk of metastatic cancer by analysis of genetic tumor markers from human samples and can also be used for other genetic analysis involving PCR reaction.

Integrated DNA purification, PCR, sample cleanup, and capillary electrophoresis microchip for forensic human identification

A fully integrated microdevice and process for forensic short tandem repeat (STR) analysis has been developed that includes sequence-specific DNA template purification, polymerase chain reaction (PCR), post-PCR cleanup and inline injection, and capillary electrophoresis (CE). Fragmented genomic DNA is hybridized with biotin-labeled capture oligos and pumped through a fluidized bed of magnetically immobilized streptavidin-coated beads in microchannels where the target DNA is bound to the beads. The bead-DNA conjugates are then transferred into a 250 nL PCR reactor for autosomal STR amplification using one biotin and one fluorescence-labeled primer. The resulting biotin-labeled PCR products are electrophoretically injected through a streptavidin-modified capture gel where they are captured to form a concentrated and purified injection plug. The thermally released sample plug is injected into a 14 cm long CE column for fragment separation and detection. The DNA template capture efficiency provided by the on-chip sequence-specific template purification is determined to be 5.4% using K562 standard DNA. This system can produce full 9-plex STR profiles from 2.5 ng input standard DNA and obtain STR profiles from oral swabs in about 3 hours. This fully integrated microsystem with sample-in-answer-out capability is a significant advance in the development of rapid, sensitive, and reliable micro-total analysis systems for on-site human identification.

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.

Design and numerical analysis of a joule-heating-induced continuous-flow polymerase chain reaction microchip

We reports a detailed study on a novel design of a Joule-heating-induced polymerase chain reaction (PCR) microchip directly heated by applying electric current to the ends of the geometric varied microchannel, which is simulated together with the factors acting on the temperature distribution. Treating the continuous flowing fluid in the channel as the resistance, different channel shapes would lead to the different resistances, which generates various heat when the fixed current is applied to the ends of channel. Based on the Ohm's law and the Joule-Thomson effect, three required sequential temperature zones are obtained. The temperature cycling is achieved and the factors that contribute to the temperature distribution are simulated and analyzed, such as the environment temperature, the heat transfer coefficient, the current density, the fluid velocity, and the channel shape. The design of the Joule-heating-induced PCR microchip sheds light on the future novel application of continuous-flow PCR. By applying certain conditions, thermal cycles that meet PCR requirements can be achieved.

High-Throughput Polymerase Chain Reaction in Parallel Circular Loops Using Magnetic Actuation

We report here a novel multichannel closed-loop magnetically actuated microchip for high-throughput polymerase chain reaction (PCR). This is achieved by designing a series of concentric circular channels on one microchip and exploiting a magnetic force to drive DNA samples flowing continuously through the closed loops. The magnetic force arises from an external permanent magnet through ferrofluid plugs inside the microchannels. The magnet enables simultaneous actuation of DNA samples in all the channels. As the samples go around the loops, they pass through three preset temperature zones. Parameters of PCR, such as incubation time, temperatures, and number of cycles, can be fully controlled and adjusted. High reproducibility was achieved for different channels in the same run and for the same channels in consecutive runs. Genetically modified organisms (GMOs) were amplified simultaneously using the developed device. This simple, reliable, and high-throughput PCR microchip would find wide applications in forensic, clinical, and biological fields.

Continuous flow polymerase chain reaction using a hybrid PMMA-PC microchip with improved heat tolerance

Recently, polymeric materials have been explored as more versatile alternatives for the fabrication of polymerase chain reaction (PCR) microchips. Poly(methyl methacrylate) (PMMA) is a popular substrate material due to its high mechanical stability, good chemical properties and most importantly, its suitability for cheap and simple CO2 laser ablation. However, it has a low glass transition temperature (Tg) of 105 °C, which is just above the denaturation temperature for PCR, thus the bond integrity is compromised. Polycarbonate (PC) is preferred as a substrate for PCR microchip as it has a higher Tg of 150 °C; but since its thermal properties are not suitable for CO2 laser light, the more expensive excimer laser has to be employed. Here we report a novel hybrid PMMA-PC microchip by bonding a PC cover plate with a PMMA substrate containing microchannel which is fabricated by CO2 laser ablation. This hybrid microchip has improved heat tolerance, such that the bonding integrity is sustained at the denaturation temperature. DNA amplification is found to be more efficiently performed in a PMMA-PC microchip than in a PMMA-PMMA microchip.

Novel fluorescence detection technique for non-contact temperature sensing in microchip PCR

DNA amplification using Polymerase Chain Reaction (PCR) in a small volume is used in Lab-on-a-chip systems involving DNA manipulation. For few microliters of volume of liquid, it becomes difficult to measure and monitor the thermal profile accurately and reproducibly, which is an essential requirement for successful amplification. Conventional temperature sensors are either not biocompatible or too large and hence positioned away from the liquid leading to calibration errors. In this work we present a fluorescence based detection technique that is completely biocompatible and measures directly the liquid temperature. PCR is demonstrated in a 3 μL silicon-glass microfabricated device using non-contact induction heating whose temperature is controlled using fluorescence feedback from SYBR green I dye molecules intercalated within sensor DNA. The performance is compared with temperature feedback using a thermocouple sensor. Melting curve followed by gel electrophoresis is used to confirm product specificity after the PCR cycles.

A circular ferrofluid driven microchip for rapid polymerase chain reaction

In the past few years, much attention has been paid to the development of miniaturized polymerase chain reaction (PCR) devices. After a continuous flow (CF) PCR chip was introduced, several CFPCR systems employing various pumping mechanisms were reported. However, the use of pumps increases cost and imposes a high requirement on microchip bonding integrity due to the application of high pressure. Other significant limitations of CFPCR devices include the large footprint of the microchip and the fixed cycle number which is dictated by the channel layout. In this paper, we present a novel circular close-loop ferrofluid driven microchip for rapid PCR. A small ferrofluid plug, containing sub-domain magnetic particles in a liquid carrier, is driven by an external magnet along the circular microchannel, which in turn propels the PCR mixture through three temperature zones. Amplification of a 500 bp lambda DNA fragment has been demonstrated on the polymethyl methacrylate (PMMA) PCR microchip fabricated by CO(2) laser ablation and bonded by a low pressure, high temperature technique. Successful PCR was achieved in less than 4 min. Effects of cycle number and cycle time on PCR products were investigated. Using a magnet as the actuator eliminates the need for expensive pumps and provides advantages of low cost, small power consumption, low requirement on bonding strength and flexible number of PCR cycles. Furthermore, the microchip has a much simpler design and smaller footprint compared to the rectangular serpentine CFPCR devices. To demonstrate its application in forensics, a 16-loci short tandem repeat (STR) sample was successfully amplified using the PCR microchip.

360 NFkB expression and disease outcome in prostate cancer

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 Int Ed 1999; 38 (8):1055–1058; Krishnan, M., Namasivayam, V., Lin, R., Pal, R., Burns, M.A. Microfabricated reaction and separation systems. Curr Opin Biotechnol 2001; 12:92–98; Schneegaβ, I., Köhler, J.M. Flow-through polymerase chain reactions in chip themocyclers. Rev Mol Biotechnol 2001; 82:101–121; deMello, A.J. DNA amplification: does ‘small’ really mean ‘efficient’? Lab Chip 2001; 1: 24N–29N; Mariella, Jr. R. MEMS for bio-assays. Biomed Microdevices 2002; 4 (2):77–87; deMello AJ. Microfluidics: DNA amplification moves on. Nature 2003; 422:28–29; Kricka, L.J., Wilding, P. Microchip PCR. Anal BioAnal Chem 2003; 377:820–825]. In this review, we survey the advances of the above aspects among the PCR microfluidic devices in detail. Finally, we also illuminate the potential and practical applications of PCR microfluidics to some fields such as microbial detection and disease diagnosis, based on the DNA/RNA templates used in PCR microfluidics. It is noted, especially, that this review is to help a novice in the field of on-chip PCR amplification to more easily find the original papers, because this review covers almost all of the papers related to on-chip PCR microfluidics.

Microchip PCR.

Miniaturization of genetic tests has become an important goal. This review surveys the current progress towards the miniaturization of tests based on the polymerase chain reaction (PCR). It examines the different types of PCR microchip designs, fabrication methods,and the components of a microchip PCR device. It also discusses the problems attributable to surface chemistry of microchip components (inhibition of PCR), and the static and dynamic surface passivation strategies developed for the solution of these difficulties