SPEED: an integrated, smartphone-operated, handheld digital PCR Device for point-of-care testing

PCR enables selective amplification of DNA sequences and underpins major advances in genetics, diagnostics, and forensics. Digital PCR (dPCR), a first-generation quantitative technique that emerged with microfluidics, partitions a reaction into thousands to millions of wells or droplets such that each partition contains zero or one target molecule, enabling absolute quantification via Poisson statistics and detection of rare variants. Commercial cdPCR and ddPCR systems are typically bulky, complex, and costly due to discrete sample loading, thermal cycling, and detection modules, limiting their utility for point-of-care testing (POCT). Miniaturization using thermoelectric elements, smartphone-based optics, and remote control has shown promise. This work introduces and details SPEED, an integrated, handheld, smartphone-operated dPCR platform designed to overcome these barriers by combining compact thermal control, smartphone imaging/control, and chip-based partitioning on Si.

The paper highlights limitations of existing commercial dPCR platforms—bulkiness, labor-intensive workflows, and high costs of instruments and consumables—which hinder POCT adoption. Prior miniaturized systems commonly use thermoelectric (Peltier) elements for rapid thermal cycling and smartphone-based optical detection for compactness and affordability. Several smartphone-based dPCR devices have been reported and serve as the foundation for further miniaturization. The work positions SPEED within this trajectory, aiming for a fully integrated, portable solution with improved usability and cost profile.

System architecture: SPEED integrates fluorescence imaging, temperature control, and smartphone-based user interface in a 100 × 200 × 35 mm³ unit. A Huawei P40 smartphone provides control and imaging; results are processed externally and relayed back to the phone. Chip design and partitioning: Microwell-based cdPCR chips on Si were designed with partition diameters of 50, 20, 10, and 5 µm yielding approximately 26,448; 139,986; 475,272; and 1,656,000 partitions, respectively. Partitions are organized into six blocks to simplify identification and image processing. Chip size is 9 × 9 mm² to fit within a 14 × 14 mm² high-power thermoelectric generator footprint. Chip fabrication: Starting from a 100 mm Si wafer, a ~3.8 µm positive photoresist was spin-coated (1800 RPM), pre-baked at 110 °C for 80 s, UV-exposed (through Cr mask on soda lime) for 10.5 s at ~9.3 mJ·cm⁻², post-baked at 120 °C for 180 s, and developed (TMAH-based) for 27 s. Deep reactive ion etching formed well depths and diameters of 30, 20, 10, and 5 µm as designed. Residual photoresist was removed by O2 plasma; wafers were diced into 54 chips with a diamond blade; protective photoresist was removed with acetone followed by isopropanol, DI water rinse, and N2 drying. Surface treatment and cover: O2 plasma treatment (120 s) reduced Si surface contact angle from 95.3° to 35.5° to aid filling. A protective cover was fabricated from a 12 × 12 mm² coverslip coated with ~50 µm PDMS (spin-coated at 1500 RPM, cured on a sacrificial photoresist layer on Si) and ~2 µm Parylene-C (after bonding PDMS to glass and releasing with acetone). This cover mitigates evaporation and cross-contamination. Optical module: Four 470 nm LEDs provide excitation; filters and mirrors manage excitation and emission paths. The smartphone camera captures fluorescence images post-PCR. Image processing and analysis: Software performs illumination nonuniformity correction, conversion to monochrome, partition identification, skew correction, and image mask generation. Poisson statistics are applied to estimate target copy numbers from positive/negative partition counts. Temperature control hardware: A high-power-density TEC (~17 W·cm⁻² max dissipated power density) is soldered onto a copper heat-spreading structure. An Au-plated brass holder accommodates the chip, with an adjacent cavity for a Pt100 RTD. TEC surfaces are coated with In/Sn alloy (~120 °C melting point) to protect internal components during soldering. Power and control electronics: The TEC is driven by an H-bridge delivering current pulses at ~100 kHz up to ~5 A, filtered by LC circuitry to yield effective DC drive and minimize noise. Temperature transitions are controlled via PWM modulation of current direction and duty. A PID closed-loop controller uses Pt100 RTD feedback to track PCR temperature profiles. Software: Three software components include firmware on the system CPU; PC/Android user interface for protocol control and monitoring; and remote software for data processing and result return. A preheating/cooling PCR protocol UI is used to shorten cycle times. Validation: Chip/coverslip validated by filling with ~1.5 mM fluorescein (~3 µL), sealing with mineral oil (~10 µL), and imaging the same block before and after 40-cycle PCR protocol. Minimal fluorescence evaporation was observed, supporting cover performance. The SPEED device was further validated on a range of DNA targets to demonstrate applicability across fields.

- Integration and form factor: Complete, autonomous unit measuring 100 × 200 × 35 mm³; smartphone-operated (Huawei P40), 12 V DC powered. - Partition design: Chips with 50, 20, 10, and 5 µm wells provide ~26,448; 139,986; 475,272; and 1,656,000 partitions, enabling flexibility in sensitivity and dynamic range; partitions grouped into six blocks for robust imaging/processing. - Error considerations: Relative error improves markedly with increasing total partition number (N), consistent with Poisson statistics; microwell partitions selected to reduce partition uncertainty compared to droplets. - Surface engineering: O2 plasma treatment (120 s) reduced Si contact angle from 95.3° to 35.5°, facilitating consistent filling. - Cover design: PDMS (~50 µm) + Parylene-C (~2 µm) coated coverslip enabled reliable sealing; minimal fluorescence evaporation after 40-cycle protocol demonstrated cover performance. - Thermal system: TEC with ~17 W·cm⁻² maximum dissipated power density; H-bridge drive at ~100 kHz up to ~5 A; LC filtering minimized noise; PID control with Pt100 RTD for accurate temperature tracking. - Optics: Four 470 nm LEDs for excitation; filters/mirrors manage emission; smartphone imaging. - Workflow and software: Smartphone UI with preheating/cooling protocol to shorten cycle time; external data processing returns results to the device UI. - Validation: Device performance verified using various DNA targets, indicating broad applicability to bioanalytical and diagnostic contexts.

The SPEED system addresses the core challenge of translating dPCR to POCT by tightly integrating thermal cycling, fluorescence imaging, and smartphone-based control in a compact device. Using microwell partitions on Si reduces partition uncertainty, improves thermal uniformity, and supports mass production, thereby enhancing measurement precision and scalability. Increasing the number of partitions (up to ~1.66 million) lowers Poisson-driven relative error, directly improving quantification accuracy for low-abundance targets. The optical module with 470 nm LEDs and tailored filtering, along with image correction algorithms (illumination normalization, skew correction, and partition masking), supports robust fluorescence readout on a smartphone platform. The high-power-density TEC and PID control enable rapid, accurate temperature cycling compatible with PCR requirements. The validated cover system (PDMS/Parylene-C) ensures minimal evaporation and cross-contamination across cycles. Collectively, these design choices demonstrate that a handheld, smartphone-operated dPCR platform can achieve reliable performance suitable for diverse applications, advancing the feasibility of POCT deployment.

This work details the design, fabrication, and verification of SPEED, an integrated, handheld, smartphone-operated dPCR platform. Key contributions include: a Si-based microwell chip with configurable partition sizes and counts; a compact optical system using 470 nm LEDs and smartphone imaging; robust image-processing algorithms; and a high-power-density TEC with PID control for rapid thermal cycling. Surface engineering and a PDMS/Parylene-C cover ensured reliable chip loading and minimal evaporation. Validation with fluorescent filling and tests on various DNA targets demonstrate the system’s potential for broad bioanalytical and diagnostic use, particularly in POCT. Future work could focus on full on-device (smartphone) analysis to eliminate external processing, expanded clinical validation across more targets and sample types, integration of automated sample loading, and multiplexed assays to further enhance utility and throughput.