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.

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Polymerase chain reaction in microfluidic devices

A film-based integrated chip for gene amplification and electrochemical detection of pathogens causing foodborne illnesses.

Multiplexed digital polymerase chain reaction as a powerful diagnostic tool.

We developed a thermal digital microfluidic (T-DMF) device enabling ultrafast DNA melting curve analysis (MCA). Within 7 seconds, the T-DMF device succeeded in differentiating a melting point difference down to 1.6 °C with a variation of 0.3 °C in a tiny droplet sample (1.2 μL), which was 300 times faster and with 20 times less sample spending than the standard MCA (35 minutes, 25 μL) run in a commercial qPCR machine. Such a performance makes it possible for a rapid discrimination of single-nucleotide mutation relevant to prompt clinical decision-making. Also, aided by electronic intelligent control, the T-DMF device facilitates sample handling and pipelining in an automatic serial manner. An optimized oval-shaped thermal electrode is introduced to achieve high thermal uniformity. A device-sealing technique averts sample contamination and permits uninterrupted chemical/biological reactions. Simple fabrication using a single chromium layer fulfills both the thermal and typical transport electrode requirements. Capable of thermally modulating DNA samples with ultrafast MCA, this T-DMF device has the potential for a wide variety of life science analyses, especially for disease diagnosis and prognosis.

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Sub-7-second genotyping of single-nucleotide polymorphism by high-resolution melting curve analysis on a thermal digital microfluidic device

Hepatic rupture hemorrhage syndrome in chickens caused by a novel genotype avian hepatitis E virus.

Since its invention in 1985 the polymerase chain reaction (PCR) has become a well-established method for amplification and detection of segments of double-stranded DNA. Incorporation of fluorogenic probe or DNA intercalating dyes (such as SYBR Green) into the PCR mixture allowed real-time reaction monitoring and extraction of quantitative information (qPCR). Probes with different excitation spectra enable multiplex qPCR of several DNA segments using multi-channel optical detection systems. Here we show multiplex qPCR using an economical EvaGreen-based system with single optical channel detection. Previously reported non quantitative multiplex real-time PCR techniques based on intercalating dyes were conducted once the PCR is completed by performing melting curve analysis (MCA). The technique presented in this paper is both qualitative and quantitative as it provides information about the presence of multiple DNA strands as well as the number of starting copies in the tested sample. Besides important internal control, multiplex qPCR also allows detecting concentrations of more than one DNA strand within the same sample. Detection of the avian influenza virus H7N9 by PCR is a well established method. Multiplex qPCR greatly enhances its specificity as it is capable of distinguishing both haemagglutinin (HA) and neuraminidase (NA) genes as well as their ratio.

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Single Fluorescence Channel-based Multiplex Detection of Avian Influenza Virus by Quantitative PCR with Intercalating Dye

Cell adhesion and detachment are crucial components in cancer spreading, often leading to recurrence and patient death [1]. Probing the mechanical behavior at the whole cell level while the cell is undergoing spreading and detachment during would enhance our understanding on cancer metastasis. However, these processes are not well understood in a quantitative sense, especially for the cancer cells [2]. In this article, we propose a biohybrid micro-device for the investigation of cellular attachment and detachment dynamics. This device comprises of silicon nanowires as electromechanical strain sensors, embedded in a suspended doubly-clamped silicon dioxide (SiO2) microbridge (Fig. 1A & Fig. 2A) for breast cancer (MCF-7) cells seeding and attachment (Fig. 1B).

Detachment Dynamics of Cancer Cells

This paper reports the development of a silicon-based microfluidic device that enables ultra fast melting curve analysis (MCA) for genomic studies (DNA) and drug discovery (proteins). The system relies on the flow of nanoliter-sized discrete phase samples through a buried silicon channel. The samples are subjected to a temperature gradient generated by two external heaters along the channel. The channel also simultaneously serves as a light guide to illuminate the droplets and guide the emitted fluorescence to the detector outside the channel. The system provides a simple melting analysis of fluorescent-labeled molecular complexes, with high multiplexing capability and throughput. The chip-based solution is attractive due to nanolitre sized samples handling, resulting in fast heat transfer thus achieving ultra fast MCA for genomic studies and drug discoveries .

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High throughput melting curve analysis in monolithic silicon-based microfluidic device

System and method for for superheating and/or supercooling of liquids and use of the system and/or method, wherein the liquid is within a capillary tube, wherein there is at least one heating and/or cooling means for heating the liquid above boiling point of the liquid at ambient pressure or cooling the liquid below freezing point of the liquid at ambient pressure, wherein the at least one heating and/or cooling means is in thermal contact with the capillary tube in an area, in which there is liquid inside the capillary tube, and wherein the capillary tube is scratch-free at its inner surface.

System and method for superheating and/or supercooling of liquids and use of the system and/or method

The present invention is directed at a system for carrying out a thermal melt analysis of an organic compound, wherein the system comprises: a microchannel having a first opening and a second opening at opposite ends of the microchannel, wherein the temperature at the opposite ends of the microchannel is different thereby creating a temperature gradient between the opposite ends of the microchannel; a light source emitting light which is coupled into the microchannel through the first opening; a light detector arranged to detect a light signal exiting the microchannel through the second opening; and wherein the first opening of the microchannel is in fluid communication with at least one fluid source providing at least one organic compound. The present invention is also directed to a method of using this device.

Pavel Neuzil

Papers

Single Fluorescence Channel-based Multiplex Detection of Avian Influenza Virus by Quantitative PCR with Intercalating Dye

Detachment Dynamics of Cancer Cells

High throughput melting curve analysis in monolithic silicon-based microfluidic device

System and method for superheating and/or supercooling of liquids and use of the system and/or method

Method and system for thermal melt analysis