The cost, speed, and accuracy of diagnosing infectious diseases are crucial in maintaining global health. The recent COVID-19 pandemic specifically emphasized the importance of timely, accurate, and early detection in preventing widespread transmission. Polymerase chain reaction (PCR) has been the standard for diagnosing infectious diseases for the past few decades, particularly during the pandemic. PCR-based detection was adopted as the definitive diagnosis method because of its high sensitivity, accuracy, and rapid results compared to other techniques.
Following the COVID-19 pandemic, the development of point-of-care (PoC) type low-cost and rapid PCR devices has been in high demand, driven by improvements in power consumption, portability, and sample preparation methods. Photothermal PCR is one of the typical developments that promises a fast, sensitive, and reliable PoC detection of infection. However, photothermal PCR has been limited in academic research with a single or a few concurrent quantitative (qPCRs) only and has not been explored beyond PoC applications, despite its low-power and energy-efficient thermal cycling method, because of the following major limitations in the photothermal qPCRs:
- Use of open reaction chambers, often wells requiring mineral oils, resulting in inconvenient handling of PCR reagent samples and discarding the used reaction chambers
- High cost related to photothermal reaction chamber fabrication, as they require costly novel metals such as gold nano layer deposition or their nanoparticles.
- Scaling up the number of qPCR is limited because of a bottleneck in the simultaneous measurement of fluorescence intensity and temperature.
Thus, we developed a disposable photothermal PCR chip which is affordable because of fabrication using high-throughput roll-to-roll (R2R) inline printing and imprinting method and utilizes carbon-black instead of gold in mini-wells for fast light-to-heat conversion-based thermal cycles. In addition, a qPCR device, idream-qPCR, features an innovative off-axis mirror-based fluorescence intensity measurement method to monitor multiplexed 16 qPCR reactions, up to three fluorescence channels and a long-wave infrared (LWIR) thermal sensor to monitor thermal cycles of 16 PCR reactions. This innovative design, along with multi-band optical filters, facilitated concurrent multi-fluorescence intensities and non-contact temperature measurement without any actuating components. The idream-qPCR, an innovative infectious disease diagnostic device, leverages rapid photothermal conversion to complete RT-qPCR assay within 17 min, which includes 5 min of RT, 25 s of initial denaturation, and 40 thermal cycles in a multi-target chip. idream-qPCR is suitable for remote and low-budget medical facilities because of its low cost, power efficiency, compatibility for cloud connectivity, and requiring minimal infrastructure.
Three innovations in idream-qPCR
Conventional fluorescence intensity measurement systems in qPCR devices require measurement using a sensor from a vertical direction and excite the fluorophore from a side with the help of a dichroic mirror, installed above the qPCR reagent chamber. Additionally, in photothermal qPCR, the heating light is sourced from the bottom in most cases. This scenario is a bottleneck for scaling up the number of simultaneous photothermal qPCRs, as they require precise temperature control in all reaction chambers. The temperature in each reaction chamber is highly dependent on the parameters, such as the efficiencies of the heating LEDs and homogeneity in the photothermal surface thickness. Thus, measuring the temperature of all reactions using a long-wave infrared (LWIR) thermal sensor can resolve the issue, but requires a vertical field-of-view. In the idream-qPCR device, the innovative setup using the off-axis mirror-based three-channel fluorescence intensity measurement system clears the vertical space required for non-contact temperature monitoring, allowing concurrent real-time temperature and fluorescence intensity measurement of all reaction chambers, shown in Figure 1. Additionally, using a multi-band filter, the implementation expanded to actuation-less multiple-excitation fluorescence intensity measurement.
We designed a mini-well reaction chamber having a special neck-tie shape as shown in Figure 2 below, which eliminated the contamination during sample loading and reduced bubble accumulation during thermal cyclers, reducing errors in fluorescence intensity measurement. Additionally, we used transparent adhesive film to laminate the graphite-PDMS chip instead of mineral oils to cover the PCR reagent, which enhanced sample handling before and after PCR, allowing contamination-free disposal. This PCR chip was fabricated using R2R gravure printing of carbon black film and imprinting graphite mixed PDMS mini-wells in a 4x4 array format at the rate of 0.46 m2 per min, enabling low-cost photothermal heating.
We validated idream-qPCR by amplifying and detecting SARS-CoV-2 N1 72 bp, RdRP 100 bp, and E 113 bp genes using FAM, TAMRA, and CY5 fluorescent dyes, respectively, showcasing the device's multi-target and multiplexing capabilities. The device demonstrated an impressive PCR efficiency of 102.5% and a limit-of-detection (LoD) equivalent to 0.85 copies/µL (or 4.7 copies/reaction), which was comparable to a conventional qPCR device.
Inside idream-qPCR
The photothermal cycler used 90 W for rapid thermal cycling of 40 cycles under 13 min using sixteen 940 nm LEDs with a high temperature accuracy of ±1 °C. The temperature was continuously measured vertically using a thermal camera, providing real-time feedback of all 16 mini-wells for 16 PID controllers controlling 16 mini-wells individually, implemented as shown in the system block diagram in Figure 4. The idream-qPCR device is compact and portable, measuring 80 × 190 × 270 mm³ and weighing just 2.9 kg. It was built from aluminum 6061 with a black anodized surface, minimizing undesired reflections during fluorescence intensity measurements, enhancing durability, and isolating electromagnetic interference.
idream-qPCR was developed to extend the application of rapid and energy-efficient photothermal qPCR devices beyond PoC and implement them into laboratory environments. Its low cost and quick turnaround time make it ideal for use in remote or small medical clinics, where a medium-throughput RT-qPCR test is required because of factors such as high costs, low operational speed, the requirement of trained personnel, and the inability to use POC devices by the elderly population.
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