Micro- and Nanotechnologies
SLAS2018 Innovation Award Finalist: Microfluidic Siphoning Array (MSA) – A Novel Scalable Digital PCR Integrated Platform
Tuesday, February 6
4:30 PM - 5:00 PM
Digital PCR offers compelling advantages over the current gold standard qPCR with the ability to: 1) detect rare events (high sensitivity); 2) be less prone to inhibition (high specificity); 3) quantify nucleic acids in an absolute manner without a standard-curve (high precision). However, widespread adoption has not occurred despite the proven advantages of dPCR over qPCR as existing dPCR platforms are capital intensive, cost prohibitive, have workflows with many steps and are not easily accessible to automation.
We have developed the patented Microfluidic Siphoning Array (MSA) Technology where bulk qPCR reagents can be partitioned into “lollipops”, a novel injection molded microfluidic device coupled with a semi-permeable thin film. The key advantages of the MSA include (a) Open source chemistry, which allows direct assay translation from qPCR to dPCR without tailored reagent formulations. (b) Experiment-to-experiment reproducibility with high fidelity microstructures with fixed number and known volume partitions insensitive to liquid handling errors. (c) Low cost due to a highly scalable manufacturing process (d) No cross talk with physically isolated partitions. The prototype device utilizes a standard format, making it compatible with automated liquid handlers. There are 8 units per prototype device, and 5,000 partitions per unit (1 μL total assay volume), with the model to create “application-specific” dPCR consumables balancing between throughput and sensitivity. To enable walkaway dPCR workflow with the MSA device, an instrument integrating pneumatic control, thermal cycling, and optical imaging was developed. An Image J pipeline was used to subtract background, extract fluorescent intensities from each lollipop, and converting them into scattered plot before applying Poisson statistics for absolute quantification. An HIV viral load assay and a Copy Number Variation (CNV) analysis assay were demonstrated with the prototype platform and achieved equal performance to the current market leader. An additional benefit of this platform is that real-time imaging of each partition during the thermal cycling process can be monitored, minimizing false positives, and enabling digital high-resolution melt analysis (dHRMA) to further improve multiplexing capability.
In summary, the advantages enabled by this platform include lowering both the workflow and cost barriers of digital PCR without compromising the performance in precision, and sensitivity. The real-time imaging capability allows background subtraction to minimize false positives, as well as digital melt analysis to improve multiplexity. We envision a scalable and automatable digital PCR platform which can easily be integrated to research laboratories, and extend to the clinic.