The invention of the polymerase chain reaction (PCR) has caused a revolution in molecular biology, giving access to a method of amplifying deoxyribonucleic acid (DNA) molecules across several orders of magnitude. Since the first application of PCR in a microfluidic device was developed in 1998, an increasing number of researchers have continued the development of microfluidic PCR systems. In this review, we introduce recent developments in microfluidic-based space and time domain devices as well as discuss various designs integrated with multiple functions for sample preparation and detection. The development of isothermal nucleic acid amplification and digital PCR microfluidic devices within the last five years is also highlighted. Furthermore, we introduce various commercial microfluidic PCR devices.

/pdf/polymerase-chain-reaction-in-microfluidic-devices-4t1da93u6g.pdf

Polymerase chain reaction in microfluidic devices

Microfluidic chip electrophoresis has been widely employed for separation of various biochemical species owing to its advantages of low sample consumption, low cost, fast analysis, high throughput, and integration capability. In this article, we reviewed the development of four different modes of microfluidics-based electrophoresis technologies including capillary electrophoresis, gel electrophoresis, dielectrophoresis, and field (electric) flow fractionation. Coupling detection schemes on microfluidic electrophoresis platform were also reviewed such as optical, electrochemical, and mass spectrometry method. We further discussed the innovative applications of microfluidic electrophoresis for biomacromolecules (nucleic acids and proteins), biochemical small molecules (amino acids, metabolites, ions, etc.), and bioparticles (cells and pathogens) analysis. The future direction of microfluidic chip electrophoresis was predicted.

Microfluidic chip electrophoresis for biochemical analysis.

Polymerase chain reaction (PCR) amplifies specific fragment of DNA molecules and has been extensively applied in fields of pathogens and gene mutation detection, food safety and clinical diagnosis which on the other hand, holds the drawbacks of large size instrument, high heat dissipation etc. It has been demonstrated that microfluidics technique coupling with PCR reaction exhibits characteristics of integration, automatization, miniaturization, and portability. Meanwhile, various designed fabrication of microchip could contribute to diverse applications. In this review, we summarized major works about a variety of microfluidic chips equipped with several kinds of PCR techniques (PCR, RT-PCR, mPCR, dPCR) and detection methods like fluorescence, electrochemistry, and electrophoresis detection. The development and application of PCR-based microfluidic chip in pathogen and gene mutation detection, diseases prevention and diagnosis, DNA hybridization and low-volume sample treatment were also discussed. • Various fabrications of microfluidic chips equipped with PCR techniques have been presented. • Miniaturized microfluidic PCR chip showed rapid analysis, high sensitivity, high-throughput. • Bacteria, virus, gene mutation and DNA methylation detection could be achieved. • Integration of nucleic acid extraction, amplification and detection would be future direction. 

Present status of microfluidic PCR chip in nucleic acid detection and future perspective

https://espace.library.uq.edu.au/view/UQ:c044b82/UQc044b82_OA.pdf

A decade of microchip electrophoresis for clinical diagnostics - A review of 2008-2017.

In response to the world's medical community's need for accurate and immediate infectious pathogen detection, many researchers have focused on adapting the standard molecular diagnostic method of polymerase chain reaction (PCR) for point-of-care (POC) applications. PCR technology is not without its shortcomings; current platforms can be bulky, slow, and power-intensive. Although there have been some advances in microfluidic PCR devices, a simple-to-operate and fabricate PCR device is still lacking. In the first part of this paper, we introduce a compact plasmonic PCR thermocycler in which fast DNA amplification is derived from efficient photothermal heating of a colloidal reaction mixture containing gold nanorods (AuNRs) using a small-scale vertical-cavity surface-emitting laser (VCSEL). Using this method, we demonstrate 30 cycle-assay time of sub-ten minutes for successful Chlamydia trachomatis DNA amplification in 20 μL total PCR sample volume. In the second part, we report an ultrasensitive real-time amplicon detection strategy which is based on cycle-by-cycle monitoring of 260 nm absorption of the PCR sample. This was accomplished by irradiating the PCR sample using a UV LED and collecting the transmitted optical power with a photodetector. The UV absorption dependency on the nucleotides’ structural degree of freedom gives rise to distinctive features in the shape of UV amplification curves for the determination of PCR results, thus circumventing the need for the complicated design of target-specific probes or intercalating fluorophores. This amplicon quantification method has a high detection sensitivity of one DNA copy. This is the first demonstration of a compact plasmonic thermocycler combined with a real-time fluorophore-free quantitative amplicon detection system. The small footprint of our PCR device stems from hardware miniaturization, while abundant sample volume facilitates highly sensitive detection and fluid handling required for in-field sample analysis, thereby making it an excellent candidate for POC molecular diagnostics.

Plasmonic and label-free real-time quantitative PCR for point-of-care diagnostics.

Quantitative PCR (qPCR) has been widely used for the detection and monitoring of a variety of infectious diseases. PCR and CE were integrated into a microfluidic chip that was designed to achieve rapid real-time amplicon sampling, separation, and quantitation without requiring various probes. A novel chip design allows the overlapped execution of PCR and CE, minimizing the time required for CE analysis after each PCR cycle. The performance of the on-chip qPCR method was demonstrated using a 45-minutes model assay protocol for the phiX174 bacteriophage, and the multiplexing capability of the method was demonstrated by adding a second target, E. coli genomic DNA, to the model assay. The results indicate good sensitivity, reproducibility, and linearity over the tested assay range, 50 to 2 × 10(4) copies/25 μL reaction. Based on this performance, the on-chip qPCR method should be applicable to a wide variety of infectious disease detection and monitoring assays with the addition of suitable sample preparation protocols.

On-chip quantitative PCR using integrated real-time detection by capillary electrophoresis.

To enable reuse of expensive microchips in a clinical analysis apparatus, while constantly and efficiently obtaining accurate analysis results. A clinical analysis apparatus employs microchips to analyze substances, which are contained in samples and are targets of measurement. The clinical analysis apparatus is equipped with: a stocking section; a dispensing mechanism, for dispensing reagents stocked in the stocking section and samples to the microchips; and a measuring section, for measuring the measurement target substance within the samples. The measuring section includes: a dispensing station, at which the microchips are provided; a detecting station, for detecting the measurement target substance; and a cleansing station, at which microchips are cleansed following detection of the measurement target substance. The dispensing station, the detecting station, and the cleansing station are provided at a predetermined pitch from upstream to downstream positions. The microchips are continuously rotated through the stations to perform measurement repeatedly.

Clinical analysis apparatus

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On-chip quantitative PCR using integrated real-time detection by capillary electrophoresis.

Clinical analysis apparatus