Polymerase Chain Reaction Chamber Research Articles

A 3D-Printed Microfluidic Device for qPCR Detection of Macrolide-Resistant Mutations of Mycoplasma pneumoniae.

Mycoplasma pneumonia (MP) is a common respiratory infection generally treated with macrolides, but resistance mutations against macrolides are often detected in mycoplasma pneumoniae in China. Rapid and accurate identification of mycoplasma pneumoniae and its mutant type is necessary for precise medication. This paper presents a 3D-printed microfluidic device to achieve this. By 3D printing, the stereoscopic structures such as microvalves, reservoirs, drainage tubes, and connectors were fabricated in one step. The device integrated commercial polymerase chain reaction (PCR) tubes as PCR chambers. The detection was a sample-to-answer procedure. First, the sample, a PCR mix, and mineral oil were respectively added to the reservoirs on the device. Next, the device automatically mixed the sample with the PCR mix and evenly dispensed the mixed solution and mineral oil into the PCR chambers, which were preloaded with the specified primers and probes. Subsequently, quantitative real-time PCR (qPCR) was carried out with the homemade instrument. Within 80 min, mycoplasma pneumoniae and its mutation type in the clinical samples were determined, which was verified by DNA sequencing. The easy-to-make and easy-to-use device provides a rapid and integrated detection approach for pathogens and antibiotic resistance mutations, which is urgently needed on the infection scene and in hospital emergency departments.

Liquid marbles as biochemical reactors for the polymerase chain reaction.

The polymerase chain reaction (PCR) is a popular and well-established DNA amplification technique. Technological and engineering advancements in the field of microfluidics have fuelled the progress of polymerase chain reaction (PCR) technology in the last three decades. Advances in microfluidics-based PCR technology have significantly reduced the sample volume and thermal cycling time. Further advances led to novel and accurate techniques such as the digital PCR. However, contamination of PCR samples, lack of reusability of the microfluidic PCR platforms, complexity in instrumentation and operation remain as some of the significant drawbacks of conventional microfluidic PCR platforms. Liquid marbles, the recently emerging microfluidic platform, could potentially resolve these drawbacks. This paper reports the first liquid marble based polymerase chain reaction. We demonstrated an experimental setup for the liquid-marble based PCR with a humidity-controlled chamber and an embedded thermal cycler. A concentrated salt solution was used to control the humidity of the PCR chamber which in turn reduces the evaporation rate of the liquid marble. The successful PCR of microbial source tracking markers for faecal contamination was achieved with the system, indicating potential application in water quality monitoring.

A Disposable and Multi-Chamber Film-Based PCR Chip for Detection of Foodborne Pathogen.

Since the increment of the threat to public health caused by foodborne pathogens, researches have been widely studied on developing the miniaturized detection system for the on-site pathogen detection. In the study, we focused on the development of portable, robust, and disposable film-based polymerase chain reaction (PCR) chip containing a multiplex chamber for simultaneous gene amplification. In order to simply fabricate and operate a film-based PCR chip, different kinds of PCR chambers were designed and fabricated using polyethylene terephthalate (PET) and polyvinyl chloride (PVC) adhesive film, in comparison with commercial PCR, which employs a stereotyped system at a bench-top scale. No reagent leakage was confirmed during the PCR thermal cycling using the film PCR chip, which indicates that the film PCR chip is structurally stable for rapid heat cycling for DNA amplification. Owing to use of the thin film to fabricate the PCR chip, we are able to realize fast thermal transfer from the heat block that leads to short PCR amplification time. Moreover, using the film PCR chip, we could even amplify the target pathogen with 10 CFU mL−1. The artificially infected milk with various concentration of Bacillus cereus was successfully amplified on a single film PCR chip. On the basis of the reliable results, the developed film PCR chip could be a useful tool as a POCT device to detect foodborne pathogens via genetic analysis.

A comprehensive methodology for design and development of an integrated microheater for on-chip DNA amplification

This paper presents the design and development of an optimized aluminum microheater integrated onto a biochip for the amplification of DNA using polymerase chain reaction (PCR). A coupled 3D finite element electro-thermal simulation has been used to aid in the design of the microheater and the PCR reactor. The microheater has a special shape, designed to provide a uniform temperature throughout the PCR chamber. The microreactor is fabricated at the center of a 20 mm × 20 mm silicon chip. It has a meandered shape, a volume of 1.89 µl and occupies a square area with a side of 3.8 mm. Microchannels to transport fluid in and out of the reactor are also provided. After heater design optimization, the simulated temperature of the fluid volume within the PCR chamber is very uniform (95% of the volume has a temperature within ±0.2 °C when the average temperature is 60 °C). This result is validated by DNA melting point experiments, showing a very similar uniformity. A PCR experiment, consisting of 50 cycles of amplification is conducted to demonstrate functionality of the system; amplifications is uniform across the reactor with variation of the threshold cycle within about 0.5 units.

A Built-In Temperature Sensor in an Integrated Microheater

Chip-based microheaters have been widely used in many applications, including gas sensors, flow meters, mass sensors, and polymerase chain reaction chambers, where accurate monitoring of temperature is critical. The temperature measurement is conventionally done with the aid of a separate sensor, which may add to the cost and inaccuracy. In this paper, a built-in temperature sensing method is provided for the microheaters. The resistor-based microheater relies on Joule heating mechanism and its resistance is dependent upon its own body temperature, implying that the microheater has an inherent temperature sensing mechanism. It is found that an intermittent temperature sampling in the middle of the heating cycle does not disturb the body temperature if the temperature sampling voltage and pulsewidth are sufficiently low and short, respectively. The built-in temperature sensing is attributed to the electrical time constant being few orders of magnitude smaller than the thermal time constant. The temperature estimation results using the built-in method show excellent agreement with the benchmark measurements from an infrared pyrometer.

An air-pressure-free elastomeric valve for integrated nucleic acid analysis by capillary electrophoresis

We present a new elastomeric valve for integrated nucleic acid analysis by capillary electrophoresis. The valve functions include metering to capture a designated volume of biological sample into a polymerase chain reaction (PCR) chamber, sealing to preserve the sample during PCR cycling, and transfer of the PCR-products and on-chip formamide post-processing for the analysis of DNA fragments by capillary gel electrophoresis. This new valve differs from prior art polydimethylsiloxane (PDMS) valves in that the valve is not actuated externally by air-pressure or vacuum so that it simplifies a DNA analysis system by eliminating the need for an air-pressure or vacuum source, and off-cartridge solenoid valves, control circuit boards and software. Instead, the new valve is actuated by a thermal cycling peltier assembly integrated within the hardware instrument that tightly comes in contact with a microfluidic cartridge for thermal activation during PCR, so that it spontaneously closes the valve without an additional actuator system. The valve has bumps in the designated locations so that it has a self-alignment that does not require precise alignment of a valve actuator. Moreover, the thickness of the new valve is around 600 μm with an additional bump height of 400 μm so that it is easy to handle and very feasible to fabricate by injection molding compared to other PDMS valves whose thicknesses are around 30–100 μm. The new valve provided over 95% of metering performance in filling the fixed volume of the PCR chamber, preserved over 97% of the sample volume during PCR, and showed very comparable capillary electrophoresis peak heights to the benchtop assay tube controls with very consistent transfer volume of the PCR-product and on-chip formamide. The new valve can perform a core function for integrated nucleic acid analysis by capillary electrophoresis.

Analysis of Thermal Performance in a Bidirectional Thermocycler by Including Thermal Contact Characteristics

This paper illustrates an application of a technique for predicting the thermal characteristics of a bidirectional thermocycling device for polymerase chain reaction (PCR). The micromilling chamber is oscillated by a servo motor and contacted with different isothermal heating blocks to successfully amplify the DNA templates. Because a comprehensive database of contact resistance factors does not exist, it causes researchers to not take thermal contact resistance into consideration at all. We are motivated to accurately determine the thermal characteristics of the reaction chamber with thermal contact effects existing between the heater surface and the chamber surface. Numerical results show that the thermal contact effects between the heating blocks and the reaction chamber dominate the temperature variations and the ramping rates inside the PCR chamber. However, the influences of various temperatures of the ambient conditions on the sample temperature during three PCR steps can be negligible. The experimental temperature profiles are compared well with the numerical simulations by considering the thermal contact conductance coefficient which is empirical by the experimental fitting. To take thermal contact conductance coefficients into consideration in the thermal simulation is recommended to predict a reasonable temperature profile of the reaction chamber during various thermal cycling processes. Finally, the PCR experiments present that Hygromycin B DNA templates are amplified successfully. Furthermore, our group is the first group to introduce the thermal contact effect into theoretical study that has been applied to the design of a PCR device, and to perform the PCR process in a bidirectional thermocycler.

An Integrated Nucleic Acid Extraction Microchip for Real-time PCR Micro Total Analysis

Abstract A real-time polymerase chain reaction (PCR) micro total analysis system (μ-TAS) was constructed by integrating nucleic acid extraction and PCR amplification with real-time fluorescent detection on a same microfluidic chip, allowing fully automated and on-chip analysis. This approach has the advantages such as low sample consumption, fast analysis and simple operation. Micromachining technology was used to fabricate the anodic molds of integrated nucleic acid extraction microfluidic chip. A polydimethylsiloxane (PDMS) substrate with 3D channels was manufactured by a combination of molds and an injection molding method. The glass substrate and the chip were bonded together using a plasma treatment. The μ-TAS included a microfluidic control device by which micro fluidic velocity (0–10 mL min −1 ) could be adjusted, a TEC platform with a precision of temperature control of 0.1 °C, and a CCD detection module. The DNA of human blood was extracted using a silica gel membrane method on the microfluidic chip. The DNA extraction and detection were preset in the μ-TAS. Human blood lysate (20 μL) was loaded into the extraction chamber and then washed at a speed of 2 mL min −1 . DNA and PCR reagents were mixed and then driven into the PCR chamber at a speed of 1 mL min −1 . The reference gene GAPDH in extracted genome DNA was amplified by PCR and verified by melting curve analysis. The results of nucleic acid extraction method on the chip were compared with those obtained using a standard manual centrifuge extraction method. The on-chip PCR amplifications gave obvious amplification curves, with C T values of 25.3 and 26.9 respectively. The melting temperature of all the amplification products was 89.9 °C. The results validated that the chip-based method and corresponding device could realize the extraction, amplification and detection of nucleic acid automatically.

An integrated, cellulose membrane-based PCR chamber

We report the design, fabrication, and testing of a microfluidic device for molecular diagnostics that sites lysis, solid-phase extraction of nucleic acids, polymerase chain reaction (PCR) amplification, and real-time fluorescence detection in a single chamber. This streamlined design considerably simplifies the fabrication and operation of chip-based nucleic acid assays for detection of pathogens and other disease markers. A single 25-μL PCR chamber in a plastic chip houses a cellulose-based filter membrane (Whatman FTA®) for extraction of nucleic acids from the sample. Nucleic acids captured on the filter serve as a template for in situ PCR amplification. The single-use (disposable) microfluidics chip mates with a portable instrument that provides double-sided heating of the PCR chamber for rapid thermal cycling, and incorporates, real time fluorescence detection using a low-cost, miniaturized (ESE GmbH) fluorescence reader. We optimized multiple-pass sample loading of the FTA® membrane, vacuum drying of membrane, and membrane wash steps to improve extraction efficiency in a microfluidics format. The chip was tested with samples of Bacillus cereus bacterial culture, and showed a limit of detection of ~103 target cells. The adaptation of this chip to practical microfluidics applications incorporating integrated pouches for storage and fluid actuation, and pre-loading of dry-stored PCR reagents is discussed, along with potential near-term applications.

A temperature control method for shortening thermal cycling time to achieve rapid polymerase chain reaction (PCR) in a disposable polymer microfluidic device

We present a temperature control method capable of effectively shortening the thermal cycling time of polymerase chain reaction (PCR) in a disposable polymer microfluidic device with an external heater and a temperature sensor. The method employs optimized temperature overshooting and undershooting steps to achieve a rapid ramping between the temperature steps for DNA denaturation, annealing and extension. The temperature dynamics within the microfluidic PCR chamber was characterized and the overshooting and undershooting parameters were optimized using the temperature-dependent fluorescence signal from Rhodamine B. The method was validated with the PCR amplification of mecA gene (162 bp) from methicillin-resistant Staphylococcus aureus bacterium (MRSA), where the time for 30 cycles was reduced from 50 min (without over- and undershooting) to 20 min.

Microfluidic Chip Fabrication and Method to Detect Influenza

Fast and effective diagnostics play an important role in controlling infectious disease by enabling effective patient management and treatment. Here, we present an integrated microfluidic thermoplastic chip with the ability to amplify influenza A virus in patient nasopharyngeal (NP) swabs and aspirates. Upon loading the patient sample, the microfluidic device sequentially carries out on-chip cell lysis, RNA purification and concentration steps within the solid phase extraction (SPE), reverse transcription (RT) and polymerase chain reaction (PCR) in RT-PCR chambers, respectively. End-point detection is performed using an off-chip Bioanalyzer (Agilent Technologies, Santa Clara, CA). For peripherals, we used a single syringe pump to drive reagent and samples, while two thin film heaters were used as the heat sources for the RT and PCR chambers. The chip is designed to be single layer and suitable for high throughput manufacturing to reduce the fabrication time and cost. The microfluidic chip provides a platform to analyze a wide variety of virus and bacteria, limited only by changes in reagent design needed to detect new pathogens of interest.

The Effect of Thermal Contact Resistance on Heat Management in a Shuttling PCR System

A novel shuttling polymerase chain reaction (PCR) system is assembled to make temperature uniform in the reaction chamber. The chamber is oscillated by a servo motor and contacted with three different isothermal zones to complete several thermal cycles. The home-made computer code is utilized to investigate the influences of operational parameters on the temperature inside the chamber. Numerical results show that the contact resistances between the heating blocks and the reaction chamber dominate the temperatures inside the PCR chamber. In this work a PCR system that is composed of the PID controller, the moving stage, three aluminum blocks for three different isothermal zones and a reaction chamber is also developed. Experimental results demonstrated that the stability of this shuttling PCR system is confirmed. And results show that DNA templates provided with the yT&A® cloning vector are amplified successfully in this PCR system.

Integrated slidable and valveless polymerase chain reaction–capillary electrophoresis microdevice for pathogen detection

An integrated slidable and valveless polymerase chain reaction (PCR) and capillary electrophoresis (CE) microdevice has been developed for pathogen detection on a portable genetic analysis microsystem. The proposed microdevice does not need any microvalve or external pneumatic actuators, thereby simplifying the chip fabrication and the chip operation process. The slidable PCR–CE chip consists of three glass layers: a channel wafer that includes a sample injection and CE microchannel, a Ti/Pt electrode-patterned resistance temperature detector (RTD) wafer, and a slidable plate in which a PCR chamber was fabricated. First, the slidable PCR plate was placed in the sample injection zone to load a PCR cocktail, and then moved to the PCR zone for thermal cycling. After PCR, the slidable PCR chamber was switched to the CE region for separation, the resultant amplicons were then analyzed by a miniaturized laser-induced fluorescence detector. Non-specific DNA adsorption was prevented by treating the glass surface with hydrophobic decyltrichlorosilane, and the leakage and evaporation of the PCR cocktail during the movement and PCR could be minimized by surrounding the slidable chamber with mineral oil. The target protein A gene (101 bp) of Staphylococcus aureus (S. aureus) was detected with a limit of detection (LOD) of 6 copies, even with a background of 1000-fold excess of Escherichia coli K12 (E. Coli K12) templates. This demonstrates a highly sensitive and selective genetic analysis was achieved on our slidable and valveless PCR–CE microdevice.

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.

Fast detection of genetic information by an optimized PCR in an interchangeable chip

In this paper, we report the construction of a polymerase chain reaction (PCR) device for fast amplification and detection of DNA. This device consists of an interchangeable PCR chamber, a temperature control component as well as an optical detection system. The DNA amplification happens on an interchangeable chip with the volumes as low as 1.25 μl, while the heating and cooling rate was as fast as 12.7°C/second ensuring that the total time needed of only 25 min to complete the 35 cycle PCR amplification. An optimized PCR with two-temperature approach for denaturing and annealing (Td and Ta) of DNA was also formulated with the PCR chip, with which the amplification of male-specific sex determining region Y (SRY) gene marker by utilizing raw saliva was successfully achieved and the genetic identification was in-situ detected right after PCR by the optical detection system.

A portable, integrated analyzer for microfluidic – based molecular analysis

A portable, fully automated analyzer that provides actuation and flow control to a disposable, self-contained, microfluidic cassette ("chip") for point-of-care, molecular testing is described. The analyzer provides mechanical actuation to compress pouches that pump liquids in the cassette, to open and close diaphragm valves for flow control, and to induce vibrations that enhance stirring. The analyzer also provides thermal actuation for the temperature cycling needed for polymerase chain reaction (PCR) amplification of nucleic acids and for various drying processes. To improve the temperature uniformity of the PCR chamber, the system utilizes a double-sided heating/cooling scheme with a custom feedforward, variable, structural proportional-integral-derivative (FVSPID) controller. The analyzer includes a programmable central processing unit that directs the sequence and timing of the various operations and that is interfaced with a computer. The disposable cassette receives a sample, and it carries out cell lysis, nucleic acid isolation, concentration, and purification, thermal cycling, and either real time or lateral flow (LF) based detection. The system's operation was demonstrated by processing saliva samples spiked with B. cereus cells. The amplicons were detected with a lateral flow assay using upconverting phosphor reporter particles. This system is particularly suited for use in regions lacking centralized laboratory facilities and skilled personnel.

Development of a real-world direct interface for integrated DNA extraction and amplification in a microfluidic device

Integrated DNA extraction and amplification have been carried out in a microfluidic device using electro-osmotic pumping (EOP) for fluidic control. All the necessary reagents for performing both DNA extraction and polymerase chain reaction (PCR) amplification were pre-loaded into the microfluidic device following encapsulation in agarose gel. Buccal cells were collected using OmniSwabs [Whatman™, UK] and manually added to a chaotropic binding/lysis solution pre-loaded into the microfluidic device. The released DNA was then adsorbed onto a silica monolith contained within the DNA extraction chamber and the microfluidic device sealed using polymer electrodes. The washing and elution steps for DNA extraction were carried out using EOP, resulting in transfer of the eluted DNA into the PCR chamber. Thermal cycling, achieved using a Peltier element, resulted in amplification of the Amelogenin locus as confirmed using conventional capillary gel electrophoresis. It was demonstrated that the PCR reagents could be stored in the microfluidic device for at least 8 weeks at 4 °C with no significant loss of activity. Such methodology lends itself to the production of 'ready-to-use' microfluidic devices containing all the necessary reagents for sample processing, with many obvious applications in forensics and clinical medicine.

A microfluidic device integrated with multichamber polymerase chain reaction and multichannel separation for genetic analysis

This work describes a microfluidic device integrated with multichamber polymerase chain reaction (PCR) and multichannel separation for parallel genetic analysis. The microdevice consists of three functional units: temperature control, multiple PCR (four chambers PCR), and multiple channel separation (four separation channels, each channel connected to a PCR chamber). Platinum (Pt)/titanium (Ti) microheater was used to ensure homogeneous temperature field, and Pt-chip sensor was used for temperature monitoring. The interface between chip-PCR and chip separation was simplified by connecting the PCR chamber with separation channel directly. After chip-PCR, PCR products were introduced into parallel separation channels for subsequent separation/detection by applying an electric field automatically. This microdevice was successfully applied for detection of pathogens including hepatitis B virus (HBV) and Mycobacterium tuberculosis (MTB), and genotyping of human leucocyte antigen (HLA)-B27 as well, demonstrating the feasibility of the integrated microdevice for parallel genetic analysis.

A large volume, portable, real-time PCR reactor

A point-of-care, diagnostic system incorporating a portable thermal cycler and a compact fluorescent detector for real-time, polymerase chain reaction (PCR) on disposable, plastic microfluidic reactors with relatively large reaction volume (ranging from 10 µL to 100 µL) is described. To maintain temperature uniformity and a relatively fast temperature ramping rate, the system utilizes double-sided heater that features a master, thermoelectric element and a thermal waveguide connected to a second thermoelectric element. The waveguide has an aperture for optical coupling between a miniature, fluorescent reader and the PCR reaction chamber. The temperature control is accomplished with a modified, feedforward, variable structural proportional-integral-derivative controller. The temperature of the liquid in the reaction chamber tracks the set-point temperature with an accuracy of ± 0.1 °C. The transition times from one temperature to another are minimized with controllable overshoots (< 2 °C) and undershoots (< 5 °C). The disposable, single-use PCR chip can be quickly inserted into a thermal cycler/reader unit for point-of-care diagnostics applications. The large reaction chamber allows convenient pre-storing of dried, paraffin-encapsulated PCR reagents (polymerase, primers, dNTPs, dyes, and buffers) in the PCR chamber. The reagents are reconstituted "just in time" by heating during the PCR process. The system was tested with viral and bacterial nucleic acid targets.

Nanostructured biochip for label-free and real-time optical detection of polymerase chain reaction

In this report, Au-coated nanostructured biochip with functionalized thiolated primers on its surface is developed for label-free and real-time optical detection of polymerase chain reaction (PCR). A PCR chamber of 150 μm in thickness containing Au-coated nanostructured substrate in the bottom layer was bordered with SU-8 100 walls. After immobilization of 5′-thiolated primers on the surface, simultaneous DNA amplification and detection were performed without any labeled molecules via the relative reflected intensity (RRI) of Au-coated nanostructured substrate. When human genomic DNA at several concentrations of 0.2, 0.5 and 1 ng μL −1 was included in the initial DNA samples, the increases in the RRI peak values were clearly observed with the increasing PCR cycle numbers. We found that the starting point of the optical signal, which was divergent from the background in our PCR biochip, was around 3–4 cycles, much lower than that of the fluorescent real-time PCR analysis (around 23–25 cycles). Our proposed PCR device using Au-coated nanostructured substrate holds noteworthy promise for rapid, label-free and real-time DNA detection for point-of-care testing (POCT) applications.