Polymerase chain reaction (PCR) is a revolutionary technology in genetics, diagnostics, and forensics, enabling selective amplification of DNA sequences. Digital PCR (dPCR), a first-generation PCR technique, compartmentalizes the PCR master mix into numerous partitions (wells or emulsion droplets), leading to either one or zero target molecules per partition. This digitization allows for absolute quantification of target DNA copy numbers (cn) using Poisson statistics, overcoming limitations of standard PCR and qPCR. dPCR enables absolute quantification without standard curves, detects rare targets, and quantifies fractions of multiple targets, offering valuable insights in medical and biological sciences. Current commercial dPCR devices are bulky, expensive, labor-intensive, and have costly consumables, hindering point-of-care testing (POCT). Miniaturization efforts focus on chip structure improvements, often using thermoelectric elements (TECs) for heating and cooling. Smartphone-based optical detection and control systems further miniaturize these devices, improving affordability and robustness. Several smartphone-based dPCR devices have been developed.

The introduction section implicitly reviews the existing literature on dPCR technology, highlighting the limitations of commercial systems and the ongoing efforts to miniaturize the technology. It mentions the use of thermoelectric elements and smartphone integration as key advancements in this area, but does not explicitly cite specific publications.

The SPEED device integrates a Huawei P40 smartphone, a fluorescence imaging system (LED illumination), and a temperature control system based on a high-power density Peltier element. The smartphone's internal app controls the PCR protocol and TEC, capturing images after PCR completion. Data processing occurs externally, with results downloaded to the smartphone. The dPCR chip fabrication uses a silicon substrate for its thermal conductivity and mass production benefits. A photoresist is spin-coated on a silicon wafer, exposed to UV light, and developed. Deep reactive etching creates partitions of 50 μm, 20 μm, 10 μm, and 5 μm diameters, divided into six blocks for image processing. Oxygen plasma treatment makes the surface hydrophilic for easier loading of the PCR master mix. A protective cover is fabricated using a PDMS-Parylene-C-coated coverslip. The performance of the coverslip is validated using fluorescein solution. The temperature control system uses a high-power density TEC soldered onto a copper system for efficient heat dissipation. An Au-plated brass holder holds the dPCR chip, and the TEC surfaces are coated with In/Sn alloy. The TEC is driven by an H-bridge, with current pulses filtered to minimize noise. A PID closed feedback loop, using a Pt100 RTD, precisely controls the temperature according to the PCR protocol.

The relative error in dPCR results depends on the number of partitions (N) and is described by the equation: Error = max (|1 - e^{-λe^{N/λ}}|), where λ is the average copy number per partition. The error decreases significantly with increasing N. The design uses microwells instead of droplets due to lower partition uncertainty. Chips with various partition diameters (50 μm, 20 μm, 10 μm, and 5 μm) and total partition counts (26,448, 139,986, 475,272, and 1,656,000, respectively) were fabricated. The chip size (9 × 9) mm² is optimized to fit within the TEC dimensions (14 × 14) mm². Partitions were divided into six blocks to simplify image processing. The fabrication process involves spin-coating photoresist, UV exposure, development, deep reactive ion etching, and oxygen plasma treatment. The oxygen plasma treatment reduces the contact angle, improving hydrophilicity. A protective PDMS-Parylene-C coverslip prevents evaporation and cross-contamination. The validation using fluorescein solution showed minimal fluorescence evaporation after thermal cycling. The temperature control system employs a high-power density TEC with efficient heat dissipation. The PID closed-loop control system accurately maintains the desired temperature profile during PCR.

The SPEED device successfully integrates several components into a compact, smartphone-operated dPCR system for POCT. The use of silicon as a substrate, a high-power density TEC, and a well-designed optical and image processing system allows for rapid and accurate DNA quantification. The modular design allows for flexibility in the choice of partition size, catering to different sensitivity requirements. The results demonstrate the feasibility of using such miniaturized systems for rapid and affordable diagnostic applications. The integration with a smartphone simplifies operation and data analysis.

The SPEED device represents a significant advancement in point-of-care dPCR technology. Its compact design, smartphone integration, and accurate temperature control make it a promising tool for various applications. Future work could focus on further miniaturization, integration of sample preparation steps, and exploring the device's capabilities in real-world clinical settings.

The study does not explicitly mention limitations. However, potential limitations could include the need for external data processing, the impact of smartphone variations on device performance, and the long-term stability and reproducibility of the chip fabrication process.