The detection of clinically relevant viral pathogens is an essential task performed by medical microbiology laboratories, to help establish diagnosis, guide the subsequent treatment and contribute to public health surveillance, including monitoring of emerging agents. The introduction of molecular detection methods and especially nucleic acid amplification techniques, such as polymerase chain reaction (PCR) [1
], has revolutionized the capabilities of diagnostic virology laboratories in large part due to the increased sensitivity of detection and improved turnaround time. Since different viruses can have similar clinical presentations, patients typically have to be tested for the presence of several different viruses at the same time; this is leading increasingly to the setting up of testing panels for several viruses linked to a specific syndrome. Some typical examples include gastroenteritis virus panels, viral encephalitis panels and of course respiratory virus panels. In practice, “panels” can be performed in two different ways; several different assays on a single sample with multiple targets/markers by using spectral multiplexing or, by utilizing spatial multiplexing based parallel PCR assays where separate individual sample and target mixtures are prepared and amplified in parallel [3
PCR is used in the screening and detection of numerous infectious viral or bacterial species, by amplifying the target nucleic acids extracted from patient sample, over several orders of magnitude [1
]. Quantitative polymerase chain reaction (qPCR) furthermore facilitates real-time detection of target nucleic acid during the amplification process and allows for quantitation of the initial amount of template [4
]. PCR at microscale leads to a reduction in the reaction time, bio-sample/reagent volume [6
]. Following the early development of the conventional close channel microfluidics based PCR micro-devices [6
], handling the PCR sample volume in form of rapidly dispensed, discrete microliter or smaller droplets has become a preferred method of choice for PCR micro-devices [10
]. Most microfluidic PCR systems have focused primarily on either reducing the PCR reaction volume (down to few hundred nanoliter) [12
] or reducing the PCR reaction time (~10 min) [12
]. The requirement of high surfactant concentration in the continuous oil phase to stabilize the PCR droplets, lack of individual addressing of the multiple droplets and the excessive need for off-chip overhead (pumps, plumbing, valves) are just a few of the challenging issues driving the development of miniaturized PCR set-ups towards droplet microfluidic (DMF). Among the available DMF methods, electro-actuation of droplets on patterned substrates (Glass, Silicon, Polymer, etc.
) is the most effective means of precision dispensing and subsequently handling multitude of bio-sample and reagents using a miniaturized device. Electrowetting (EW) or, EW-on-dielectric (EWOD) [17
] has been successfully utilized to demonstrate PCR reactions at microscale [19
], however, the necessity of active electrode switching to facilitate droplet motion, in the digital microfluidic technique, results in a substantial electrical overhead, especially for the multiplexed, chip based bio-assay schemes. Recently, our work has demonstrated that a continuous droplet transport scheme, which enables droplet transport and thermal cycling without the requirement of active electrode switching [11
], can be an effective solution for a PCR micro-device with reduced electrical overhead requirement for a multiplexed diagnosis system. Here active droplet transport is facilitated by electrostatic or, droplet-dielectrophoresis (D-DEP) based electro-actuation technique, which utilizes herring-bone shaped electrode arrays to facilitate droplet transport and thermal cycling [11
In cases pertaining to the detection and quantification of RNA viruses, for example Influenza viruses, Hepatitis C virus, Measles virus, SARS-CoV and Ebola virus, detection by PCR requires transcribing the viral RNA extracted from virions through a reverse transcription reaction, to yield complementary DNA (cDNA) molecules. Apart from virology, other major applications of RT-PCR include analysis of gene expression from target cells and detection of certain genetic diseases. In quantitative analysis of RT-PCR (qRT-PCR), a reaction mixture containing both a reverse transcriptase enzyme and a thermo-stable DNA polymerase (TAQ) is used so that the two enzymatic reactions (reverse transcription and PCR amplification) can be performed serially through temperature control, as an integrated two-step process. On a miniaturized scale, RT-PCR reactions have been achieved by utilizing microfluidic methods for manipulating nucleic acid samples and PCR reagents including the use of continuous flow techniques [6
] and discrete droplet based microfluidics [10
]. The majority of microfluidic implementations for RT-PCR assays have been targeted to gene expression analysis, where a large amount of genomic molecules has helped towards lowering the reaction volumes to sub-microliters [12
]. However, in clinical diagnostic applications, for the detection of trace quantities of viral RNA in a matrix sample that often contains an abundance of genomic DNA from the human host, the emphasis is placed on the reliable, rapid detection [15
The evolution of PCR technologies over the last two decades suggests the need for further improvement towards the performance and reliability of PCR systems. The droplet digital PCR (ddPCR™, Bio-Rad, Hercules, CA, USA) system commercially available from Bio-Rad incorporates the close-channel microfluidic based droplet generation method to create a large library (~15,000) of sub-microliter droplets, which are dispensed from a large PCR sample/reagent mixture (~20 μL) [16
]. This approach allows for one step detection and quantification of extracted nucleic acid and it is an excellent example of PCR technology that illustrates the feasible integration of microfluidics into such commercial systems.
Reports of large PCR sample arrays of sub-microliter reactions have also been reported for high-throughput qRT-PCR, with as few as five copies of template RNA in each reaction [14
]. However, detection of a panel of infectious viruses (such as the respiratory virus panel) in human samples utilizing a qRT-PCR microchip remains to be realized. Examples of recent single RT-PCR applications using microfluidics suggest a detection time (reverse transcription and thermal cycling) of up to an hour, using as low as 2–5 μL PCR volume [14
]. We have previously designed a microchip utilizing electrostatic/droplet-DEP (D-DEP) electro-actuation method and integrated thermostatic zones (micro-heaters and resistive temperature sensors) to achieve single qRT-PCR amplification of in vitro
synthesized Influenza viral RNA [11
], with a detection threshold of less than 10 copies of template RNA in the PCR reaction volume. We have also investigated the scalability of PCR sample volume in our device application, over the range of 1–10 μL [11
], which is industrially accepted for viral detection in clinical samples. In this work, we have modified the previously designed continuous, D-DEP electrode architecture for the PCR thermal cycling to produce a spatially multiplexed PCR micro-device, suitable for carrying out several different qRT-PCR reactions in parallel (up to eight assays per chip) and with a built-in flexibility to accommodate different cycling parameters for each reaction. The performance of this micro-device is illustrated by the parallel execution of assays for the detection of Influenza A virus and Influenza B virus in different panels of clinical samples. The reported multiplexed qRT-PCR assays are a first demonstration of a D-DEP based DMF device for analysing multiple clinically relevant viral pathogens in panels of extracted patient samples.
This present investigation demonstrates and furthermore extends the applicability of the continuous D-DEP based droplet transport method for parallel, spatially multiplexed qRT-PCR reactions on a nano-textured DMF chip. The improved micro-electrode architecture accommodates up to eight parallel, qRT-PCR reactions. As a proof of principle, detection of Influenza A and B viruses from clinical samples was conducted using a blind panel. Influenza A and B were accurately identified and quantified using the standard quantification method, in the two micro-device based qRT-PCR assays. The outcomes of the repeated blind panel experiments confirm that the micro-device can successfully handle more than one nucleic acid samples and markers over an array of parallel, spatially multiplexed DMF micro-electrodes, to screen for a panel of viral/infectious diseases. The efficiency of chip based qRT-PCR assays were reasonably within the accepted industrial benchmark (PCR efficiency ~94%–97%) and the completion time for the sample loading/mixing, RT-reactions and up to 38 PCR thermal cycles for up to eight different PCR droplets was found to be ~35–40 min, again comparable to that of a commercial fast qRT-PCR equipment. The detection limit, as identified using the chip based standard quantification process, for the multiplexed qRT-PCR micro-device was found to be <10 copies of RNA templates/PCR reaction. The micro-device furthermore offers future integration of both spatial (parallel qPCR reactions with differed targets) and spectral (multiple target markers in same PCR assay) multiplexing to screen for a larger panel of infectious agents. As a next step in the development, our focus is to improve the up-stream sample handling to achieve serial dilution of RNA samples and facilitate on-chip mixing and preparation of the reagent mixture and dispensing of multitude of sample droplets to suitably address the multiplexed qRT-PCR tracks. In addition, we will focus on the development of a separate sample extraction and purification chip to separate, lyse and concentrate target DNA/RNA from clinical patient samples, in preparation for the qRT-PCR amplification and detection stage. These proposed developments will lead to a portable sample-to-detection microsystem, suitable for example for field analysis of human, live-stock and food borne pathogens.