Fungal pathogenic nucleic acid detection achieved with a microfluidic microarray device

Detection of polymerase chain reaction (PCR) products obtained from cultured greenhouse fungal pathogens, Botrytis cinerea and Didymella bryoniae has been achieved using a previously developed microfluidic microarray assembly (MMA) device. The flexible probe construction and rapid DNA detection resulted from the use of centrifugal pumping in the steps of probe introduction and sample delivery, respectively. The line arrays of the oligonucleotide probes were “printed” on a CD-like glass chip using a polydimethylsiloxane (PDMS) polymer plate with radial microfluidic channels, and the sample hybridizations were conducted within the spiral channels on the second plate. The experimental conditions of probe immobilization and sample hybridization were optimized, and both complementary oligonucleotides and PCR products were tested. We were able to achieve adequate fluorescent signals with a sample load as small as 0.5 nM (1 μL) for oligonucleotide samples; for PCR products, we achieved detection at the level of 3 ng.     

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.     

Extraction,amplification and detection of DNA in microfluidic chip-based assays

This review covers three aspects of PCR-based microfluidic chip assays: sample preparation, target amplification, and product detection. We also discuss the challenges related to the miniaturization and integration of each assay and make a comparison between conventional and microfluidic schemes. In order to accomplish these essential assays without human intervention between individual steps, the micro-components for fluid manipulation become critical. We therefore summarize and discuss components such as microvalves (for fluid regulation), pumps (for fluid driving) and mixers (for blending fluids). By combining the above assays and microcomponents, DNA testing of multi-step bio-reactions in microfluidic chips may be achieved with minimal external control. The combination of assay schemes with the use of micro-components also leads to rapid methods for DNA testing via multi-step bioreactions. Contains 259 references. Figure

A graphical presentation of main PCR assays: DNA extraction from raw sample, target amplification by PCR and final product detection in conventional bench-top lab and miniaturized microfluidic chip.     

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.     

World-to-chip microfluidic interface with built-in valves for multichamber chip-based PCR assays

We report a practical world-to-chip microfluidic interfacing method with built-in valves suitable for microscale multichamber chip-based assays. One of the primary challenges associated with the successful commercialization of fully integrated microfluidic systems has been the lack of reliable world-to-chip microfluidic interconnections. After sample loading and sealing, leakage tests were conducted at 100 degrees C for 30 min and no detectable leakage flows were found during the test for 100 microchambers. To demonstrate the utility of our world-to-chip microfluidic interface, we designed a microscale PCR chip with four chambers and performed PCR assays. The PCR results yielded a 100% success rate with no contamination or leakage failures. In conclusion, we have introduced a simple and inexpensive microfluidic interfacing system for both sample loading and sealing with no dead volume, no leakage flow and biochemical compatibility.     

DNA purification from crude samples for human identification using gradient elution isotachophoresis

Gradient elution isotachophoresis (GEITP) was demonstrated for DNA purification, concentration, and quantification from crude samples, represented here by soiled buccal swabs, with minimal sample preparation prior to human identification using STR analysis. During GEITP, an electric field applied across leading and trailing electrolyte solutions resulted in isotachophoretic focusing of DNA at the interface between these solutions, while a pressure‐driven counterflow controlled the movement of the interface from the sample reservoir into a microfluidic capillary. This counterflow also prevented particulates from fouling or clogging the capillary and reduced or eliminated contamination of the delivered DNA by PCR inhibitors. On‐line DNA quantification using laser‐induced fluorescence compared favorably with quantitative PCR measurements and potentially eliminates the need for quantitative PCR prior to STR analysis. GEITP promises to address the need for a rapid and robust method to deliver DNA from crude samples to aid the forensic community in human identification.     

Recent progress of microfluidic sample preparation techniques

Sample preparation turns out to be one of the important procedures in complex sample analysis by affecting the accuracy, selectivity, and sensitivity of analytical results. However, the majority of the conventional sample preparation techniques still suffer from time-consuming and labor-intensive operations. These shortcomings can be addressed by reforming the sample preparation process in a microfluidic manner. Inheriting the advantages of rapid, high efficiency, low consumption, and easy integration, microfluidic sample preparation techniques receive increasing attention, including microfluidic phases separation, microfluidic field-assisted extraction, microfluidic membrane separation, and microfluidic chemical conversion. This review overviews the progress of microfluidic sample preparation techniques in the last 3 years based on more than 100 references, we highlight the implementation of typical sample preparation methods in the formats of microfluidics. Furthermore, the challenges and outlooks of the application of microfluidic sample preparation techniques are discussed.     

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.     

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.     

Detection of single-base mutations using 1-D microfluidic beads array

The application of a 1-D microfluidic beads array that is composed of individually addressable functionalized SiO2 beads has been demonstrated for detection of single-base mutations based on "sandwich" hybridization assay without additional sample labeling and PCR amplification. We concentrated on detection of mutations in the human p53 tumor suppressor gene with more than 50% mutation frequency in the known human cancers. Using a microinjection system, functionalized beads could be selectively and linearly arrayed in a single microfluidic channel comprising many periodic chambers. This 1-D microfluidic beads array was sufficiently sensitive to identify single-nucleotide mutations in 40 pM quantities of DNA targets and could discriminate the mutated alleles in an excess of nonmutated alleles at a level of one mutant in 100 wild-type sequences. The surface of beads was regenerated and rehybridized up to six times without obvious loss of signal. The entire reaction process was done at room temperature within minutes, and only 2-10 microL sample solution was needed to complete the whole detection process. The p53 genotypes of A549, CNE2, and SKBr-3 cell lines were also correctly evaluated by using mRNA extracts as target without need for sample labeling and amplification. Thus, this platform enabled rapid and exact discrimination of gene mutations with the advantages of reusability, simple handling of liquid, low cost, and little reagent consumption.     

Single-molecule emulsion PCR in microfluidic droplets

The application of microfluidic droplet PCR for single-molecule amplification and analysis has recently been extensively studied. Microfluidic droplet technology has the advantages of compartmentalizing reactions into discrete volumes, performing highly parallel reactions in monodisperse droplets, reducing cross-contamination between droplets, eliminating PCR bias and nonspecific amplification, as well as enabling fast amplification with rapid thermocycling. Here, we have reviewed the important technical breakthroughs of microfluidic droplet PCR in the past five years and their applications to single-molecule amplification and analysis, such as high-throughput screening, next generation DNA sequencing, and quantitative detection of rare mutations. Although the utilization of microfluidic droplet single-molecule PCR is still in the early stages, its great potential has already been demonstrated and will provide novel solutions to today's biomedical engineering challenges in single-molecule amplification and analysis.     

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

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

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.     

Analytical detection techniques for droplet microfluidics—A review

In the last decade, droplet-based microfluidics has undergone rapid progress in the fields of single-cell analysis, digital PCR, protein crystallization and high throughput screening. It has been proved to be a promising platform for performing chemical and biological experiments with ultra-small volumes (picoliter to nanoliter) and ultra-high throughput. The ability to analyze the content in droplet qualitatively and quantitatively is playing an increasing role in the development and application of droplet-based microfluidic systems. In this review, we summarized the analytical detection techniques used in droplet systems and discussed the advantage and disadvantage of each technique through its application. The analytical techniques mentioned in this paper include bright-field microscopy, fluorescence microscopy, laser induced fluorescence, Raman spectroscopy, electrochemistry, capillary electrophoresis, mass spectrometry, nuclear magnetic resonance spectroscopy, absorption detection, chemiluminescence, and sample pretreatment techniques. The importance of analytical detection techniques in enabling new applications is highlighted. We also discuss the future development direction of analytical detection techniques for droplet-based microfluidic systems.     

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.     

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.     

Preconcentration and separation of double-stranded DNA fragments by electrophoresis in plastic microfluidic devices

We have evaluated double-stranded DNA separations in microfluidic devices which were designed to couple a sample preconcentration step based on isotachophoresis (ITP) with a zone electrophoretic (ZE) separation step as a method to increase the concentration limit of detection in microfluidic devices. Developed at ACLARA BioSciences, these LabCard trade mark devices are plastic 32 channel chips, designed with a long sample injection channel segment to increase the sample loading. These chips were designed to allow stacking of the sample into a narrow band using discontinuous ITP buffers, and subsequent separation in the ZE mode in sieving polymer solutions. Compared to chip ZE, the sensitivity was increased by 40-fold and we showed baseline resolution of all fragments in the PhiX174/HaeIII DNA digest. The total analysis time was 3 min/sample, or less than 100 min per LabCard device. The resolution for multiplexed PCR samples was the same as obtained in chip ZE. The limit of detection was 9 fg/microL of DNA in 0.1xpolymerase chain reaction (PCR) buffers using confocal fluorescence detection following 488 nm laser excitation with thiazole orange as the fluorescent intercalating dye.     

On-line cell lysis and DNA extraction on a microfluidic biochip fabricated by microelectromechanical system technology

Integrating cell lysis and DNA purification process into a micrototal analytical system (microTAS) is one critical step for the analysis of nucleic acids. On-chip cell lysis based on a chemical method is realized by sufficient blend of blood sample and the lyzing reagent. In this paper two mixing models, T-type mixing model and sandwich-type mixing model, are proposed and simulation of those models is conducted. Result of simulation shows that the sandwich-type mixing model with coiled channel performs best and this model is further used to construct the microfluidic biochip for on-line cell lysis and DNA extraction. The result of simulation is further verified by experiments. It asserts that more than 80% mixing of blood sample and lyzing reagent which guarantees that completed cell lysis can be achieved near the inlet location when the cell/buffer velocity ratio is less than 1:5. After cell lysis, DNA extraction by means of a solid-phase method is implemented by using porous silicon matrix which is integrated in the biochip. During continuous flow process in the microchip, rapid cell lysis and PCR-amplifiable genomic DNA purification can be achieved within 20 min. The potential of this microfluidic biochip is illustrated by pretreating a whole blood sample, which shows the possibility of integration of sample preparation, PCR, and separation on a single device to work as portable point-of-care medical diagnostic system.