Recent progress in microfluidics and optical systems has made enormous impact in the advancement of nucleic acid amplification and detection. However, commercial and currently reported microfluidic PCR devices have not yet found their utilization in point-of-care (POC) applications. This is due to long amplification time, high power requirement, and bulky size of commercial PCR machines or cost-inefficiency, complex fabrication and operation of microfluidic chips. In this work, we present a compact PCR device in which fast amplification is accomplished by photothermal heating of gold nanorods evenly dispersed in PCR reaction by a vertical-cavity surface-emitting laser (VCSEL). This thermocycler offers sub-ten-minute amplification time for 30 thermal cycles with high temperature stability and PCR products comparable to conventional bench-top machines. The proposed device is approximately 100mm×50mm×50mm in size, and its small footprint is obtained by hardware miniaturization. Retaining conventional sample volumes (20μL) makes our device more user-friendly in terms of sample loading and capable of more sensitive amplicon detection for on-site assays. Also, its cost-effectiveness due to disposable AuNRs and inexpensive light source outweigh surface plasmon heating methods utilizing embedded Au films with limited lifetimes and other previously presented plasmonic thermocyclers.
There is a growing focus to adapt Polymerase Chain Reaction (PCR) to point-of-care (POC) testing to provide for a low-cost, rapid and reliable diagnostic instrument. Many studies proposed the integration of microfluidics with fluorophore-assisted or electrochemical amplicon detection methods to introduce a real-time miniature device for POC applications. However, their practicality in POC testing is limited due to their complex microfabrication, high cost, and intrinsic challenges due to their intercalation and hybridization-based detection. In this paper, we present a purely optical methodology without the addition of non-PCR reagents (electroactive or fluorogenic DNA intercalators) to enhance the reliability in quantitative PCR measurement of DNA yield. The determination of PCR results and DNA amplicon quantification are realized by monitoring transmitted power of a 260nm LED in PCR reaction at every thermal cycle. The least-square fits to transmission data demonstrate distinctive features to classify positive vs. negative PCRs and to quantify amplified products. This real-time UV monitoring system was combined with a VCSEL-based plasmonic thermocycler to accomplish fast amplification and detection in a simple and small-scaled footprint applicable for POC diagnostics.
The high mortality rate in developing countries stemming from poverty and diseases, and the pressure on healthcare budgets in developed countries have evoked a major concern in healthcare delivery. The need for less costly and patientcentered healthcare delivery brings point-of-care (POC) testing to the fore. POC devices help to eliminate the overhead associated with centralized bench-top laboratory instruments. Although handheld devices such as glucose biosensor strip exist, POC devices for molecular techniques such as Polymerase Chain Reaction (PCR) are new and emerging. PCR makes it possible to replicate DNA and generate millions of copies from a single strand. This finds applications in the medical field to identify and detect infectious diseases. Conventional PCR equipment is expensive and requires a significant amount of personnel time and space to setup and run in the laboratory. We have recently demonstrated a rapid and low-cost PCR thermocycler based on laser heating of gold nanoparticles suspended in the PCR tube. A critical aspect of PCR systems is the need to detect amplified products in real time. Here we show that by measuring the optical absorption of the suspended gold nanoparticles at a single wavelength during thermocycling, it is possible to detect amplified PCR products in real time. We investigate several different signal processing approaches in order to determine the most sensitive monitoring technique. This method makes it possible to distinguish between negative and positive PCRs with starting copy numbers as low as 10,000 genome copies per microliter.