Spinning Disk Platform for Microfluidic Digital Polymerase Chain Reaction
ABSTRACT An inexpensive plastic disk disposable was designed for digital polymerase chain reaction (PCR) applications with a microfluidic architecture that passively compartmentalizes a sample into 1000 nanoliter-sized wells by centrifugation. Well volumes of 33 nL were attained with a 16% volume coefficient of variation (CV). A rapid air thermocycler with aggregate real-time fluorescence detection was used, achieving PCR cycle times of 33 s and 94% PCR efficiency, with a melting curve to validate product specificity. A CCD camera acquired a fluorescent image of the disk following PCR, and the well intensity frequency distribution and Poisson distribution statistics were used to count the positive wells on the disk to determine the number of template molecules amplified. A 300 bp plasmid DNA product was amplified within the disk and analyzed in 50 min with 58-1000 wells containing plasmid template. Target concentrations measured by the spinning disk platform were 3 times less than that predicted by absorbance measurements. The spinning disk platform reduces disposable cost, instrument complexity, and thermocycling time compared to other current digital PCR platforms.
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- "We used capillary PCR and a PCR melting approach for preliminary characterization of extraction output and establishment of RT-PCR protocols. The RT-PCR disks were loaded first by pipetting 10 µL each of PCR mix and mineral oil (M5904, Sigma-Aldrich Corporation), dyed with Oil Red O (Matheson Coleman & Bell, Gardena, CA), into the individual loading reservoirs of the disk and the disk was then spun at 4000 rpm for 5 min to move the fluid from the middle to the outside of the disk . Disk images before and after the PCR runs were taken and samples were centrifuged into collecting vials and subjected to melting or gel electrophoresis as required. "
ABSTRACT: This work reports design and characterization results for an integrated microfluidics-based biosensor being developed for field-based Foot-and-Mouth-Disease Virus (FMDV) detection. This paper outlines the specific challenges towards integration of such a device and describes coating materials and characterization of the different modules using appropriate viral analogues. The modules include: a sample preparation module for viral RNA extraction using a disposable silica filter and an on-chip RT PCR module for detection with high sensitivity and specificity. INTRODUCTION FMDV is highly contagious and prevalent in the most economically important animals worldwide . FMDV exists in seven different serotypes, the early identification of which is important in understanding the disease and its potential spread in the population. Our laboratory is developing a field-based analysis tool for rapid FMDV serotype identification from clinical samples that range from vesicular fluid to feet or nose epithelium to whole blood. The overall goal of this project is to create an integrated system with sample in-answer out capability. The requirement of distinguishing different serotypes poses a significant challenge towards automation and total integration due to the inherent complexity and potential contamination issues. This necessitates an amplification approach that is capable of rapidly processing multiple samples at once without any cross-talk. For this purpose our initial work has used an automated microfluidic extraction unit (Figure 1) with a spinning disk platform (Figure 2) for RT-PCR that will allow us to analyze the different FMDV serotypes with high sensitivity in less than three hours with our current analysis protocol. The current version of the RT-PCR disk contains 5 separate channels with individual loading chambers for different samples. Figure 1 is a photograph of the microfluidic extraction system in its current format. The top portion consists of a PDMS microfluidic platform complete with on-chip valves (31 pneumatic valves), reservoir pumps (9 chambers that also work as pumps), and a disposable extraction filter. The system is controlled by a LabView program that operates a sequence of solenoid valves to run the RNA extraction protocol, which uses the same chemical sequence as the Qiagen RNeasy Mini spin kit. FABRICATION Extraction chamber module: The microfluidic chip for flow control with on-chip microvalves was fabricated using three-layers: a fluid channel layer, a membrane, and a valve control channel layer. A thin membrane layer serves as the valve and pump actuator separating the fluid channel layer and a pneumatic valve control channel layer . The fluid and control microchannel layers are molded in PDMS using xurographically-patterned tapes as the mold. An off-the-shelf thin silicone sheet was used as the membrane layer [Bisco HT 6135, Rogers Corp, CT]. The layers were bonded together by activating the surfaces with a corona discharge treatment. As the final step, access holes for valves, inlets and outlets were cored into the molded PDMS to allow fluidic connections to the fluid and control channels. The on-chip pneumatic valves are actuated by applying vacuum to the control channel layer. Spinning disk RT-PCR module: The disk for RT-PCR uses centrifugal force to move fluid into 5 short microfluidic channels with individual inlets located at the center to run separate chemistries for different viral serotypes. The disk for RT-PCR is fabricated from thin film polycarbonates to create an inexpensive disposable, requiring only centrifugation for fluid control. The entire disk is 120 mm in diameter, the size of a CD, and is 375 m thick (see Figure 2). The channel layer disks were manufactured using the process of xurography [3, 4].
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ABSTRACT: We present a new method for aliquoting liquids on the centrifugal microfluidic platform. Aliquoting is an essential unit operation to perform multiple parallel assays (“geometric multiplexing”) from one individual sample, such as genotyping by real-time polymerase chain reactions (PCR), or homogeneous immunoassay panels. Our method is a two-stage process with an initial metering phase and a subsequent transport phase initiated by switching a centrifugo-pneumatic valve. The method enables aliquoting liquids into completely separated reaction cavities. It includes precise metering that is independent on the volume of pre-stored reagents in the receiving cavities. It further excludes any cross-contamination between the receiving cavities. We characterized the performance for prototypes fabricated by three different technologies: micro-milling, thermoforming of foils, and injection molding. An initial volume of ~90μl was split into 8 aliquots of 10μl volume each plus a waste reservoir on a thermoformed foil disk resulting in a coefficient of variation (CV) of the metered volumes of 3.6%. A similar volume of ~105μl was split into 16 aliquots of 6μl volume each on micro-milled and injection-molded disks and the corresponding CVs were 2.8 and 2.2%, respectively. Thus, the compatibility of the novel aliquoting structure to the aforementioned prototyping and production technologies is demonstrated. Additionally, the important question of achievable volume precision of the aliquoting structure with respect to the production tolerances inherent to each of these production technologies is addressed experimentally and theoretically. The new method is amenable to low cost mass production, since it does not require any post-replication surface modifications like hydrophobic patches. KeywordsLab-on-a-chip–Centrifugal microfluidics–Aliquoting–Multiplexing–PCR–PneumaticMicrofluidics and Nanofluidics 06/2011; 10(6):1279-1288. DOI:10.1007/s10404-010-0759-0
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ABSTRACT: This study presents an optical microfluidic platform and method for performing real-time polymerase chain reactions of MDA-MB-231 breast cancer cell DNA within droplet-in-oil micro-reactors. Illumination of the droplets using a low-power (20–40 mW) infrared (IR) laser at 1,460 nm provides a simple approach for droplet manipulation and rapid thermal cycling. The nanoliter droplet volumes allow for extremely fast amplification times, from cell lysis to assay completion in 15 min or less. Droplets containing lysis buffer and subsequently master mix solutions are optically positioned in mineral oil to coalesce with droplets containing live cells on a Petri dish surface for reverse-transcription polymerase chain reactions (RT-PCR). The optical PCR setup is also shown to amplify DNA in droplets containing single or multiple cells and distinguish between methylated and unmethylated BRCA1 promoters in microdroplets containing sample at the single-cell level. Melting curves generated using IR heating indicates a melting temperature of 86 °C for the 255-bp amplicon. The results are consistent with standard PCR and methylation-specific protocols performed in a commercial system. The simplicity of the droplet-in-oil Petri dish platform provides an easy and efficient tool for DNA analysis from live cells, and can be integrated with other microfluidic technologies for complex and large-scale assays.Microfluidics and Nanofluidics 12/2012; 13(6). DOI:10.1007/s10404-012-1016-5