A new microchannel capillary flow assay (MCFA) platform with lyophilized chemiluminescence reagents for a smartphone-based POCT detecting malaria

The study addresses the need for low-cost, low-power, user-friendly point-of-care testing (POCT) systems capable of sensitive, quantitative detection of disease biomarkers in resource-limited settings. Despite advances in microfluidic lab-on-a-chip technologies, many immunoassays still require multiple liquid handling steps and bulky instrumentation, limiting deployment in the field. Smartphones offer integrated processing, display, connectivity, storage, and power, making them attractive as analyzers; however, built-in cameras are insufficient for ultra-sensitive optical detection required in immunodiagnostics. The authors propose a smartphone-powered analyzer interfaced via USB On-The-Go (OTG) to a sensitive optical detector, paired with a disposable microchannel capillary flow assay (MCFA) chip that autonomously performs a chemiluminescence-based sandwich ELISA using lyophilized reagents. The research question is whether a functionally designed capillary microfluidic chip with on-chip dried reagents and passive flow control can reliably execute the ELISA sequence and achieve clinically relevant sensitivity for malaria biomarker PfHRP2 detection, while using only smartphone power and minimal user steps.

Prior work shows microfluidic assays offer advantages (small size, low sample volume, portability, rapid detection, high sensitivity) and are central to POCT systems. Smartphone-based POCT has grown due to widespread adoption; many systems rely on smartphone cameras and image processing, but cameras often lack the sensitivity needed for ultra-sensitive immunodiagnostics. External detectors interfaced via audio jacks have been explored, but audio ports supply low and variable voltages, requiring rectification/boosting and increasing system complexity. USB-OTG offers both sufficient power (approximately 4.4–5.25 V) and digital data communication, and has been used for quantitative biomolecular detection. LFIA strips are popular but often provide qualitative results and suffer from lower sensitivity. Microfluidic ELISAs can improve LOD via enzyme amplification but typically require multiple liquid handling steps, demanding trained users. Dry reagents on-chip simplify workflows, remove cold-chain requirements, extend shelf life, and enable automation; lyophilization is widely used for drying biological reagents, and devices like Biosite Triage demonstrate multiplexed quantitative detection. Many autonomous microfluidic assays with dry reagents have used fluorescence, which needs excitation sources and increases power/complexity. Chemiluminescence (e.g., HRP-based) offers higher sensitivity than colorimetric or fluorescence methods and simpler detection hardware, but dry-reagent chemiluminescence implementations are rare. The authors previously validated drying of chemiluminescent substrate, motivating the current chemiluminescent MCFA approach.

System overview: The platform comprises (1) a disposable microchannel capillary flow assay (MCFA) lab chip fabricated from cyclic olefin copolymer (COC) with on-chip lyophilized reagents, (2) a sensitive, smartphone-attachable optical detector interfaced via USB-OTG for power and data, and (3) a smartphone running a custom app for display, analysis, storage, and control. MCFA chip design and operation: The chip implements a chemiluminescence-based sandwich ELISA with passive capillary flow and no external pumps. It features a sample loading chamber (~30 µL), two parallel fluidic paths, lyophilization chambers with micropillars for uniform drying, delay valves, a capillary stop valve, spiral reaction chambers (test, positive control, negative control), optical trenches to prevent crosstalk, a meandering high-resistance channel, a hydrophobic vent, and a high-capillary-pressure capillary pump for wash/waste collection. Surfaces are rendered sufficiently hydrophilic to promote capillary flow. - Two-path sequencing: A hydrophobic patch in the sample loading chamber splits the sample into Path-1 (detection antibody) and Path-2 (substrate). Path-1 contains a lyophilized HRP-labeled detection antibody (DAb-HRP) chamber; Path-2 contains a lyophilized chemiluminescent substrate chamber. Narrow capillary channels connect the sample reservoir to the lyophilization chambers to minimize back-diffusion and unintended enzyme-substrate interactions. - Flow control: Micropillars in lyophilization chambers create multiple small menisci enabling uniform drying and effective reconstitution. Descending/ascending ramps around these chambers confine reagents during lyophilization and slow flow to improve rehydration. Serial delay valves (meniscus enlargement features) retard flow fronts, promote bubble-free merging into single channels, and enhance reagent dissolution. A meandering channel in Path-2 imposes higher flow resistance to delay substrate arrival, allowing the antigen–DAb-HRP complex from Path-1 to form and bind at the capture-antibody-coated reaction chambers first. A hydrophobic vent on Path-2, opened by the user, releases trapped air to permit substrate entry after binding is complete. - Reaction chambers: Three spiral chambers serve as test (quantifies target), positive control (validates HRP conjugate), and negative control (blank). Optical trenches between spirals reduce chemiluminescent optical crosstalk. A downstream micro-pillared reservoir and meandering channel further slow flow to increase antibody–antigen reaction time and improve sensitivity. The capillary pump provides strong capillary suction to pull fluids through and collect wastes. Assay workflow: The user loads sample (e.g., serum) into the sample chamber. Capillary action splits flow into both paths, reconstituting dried DAb-HRP and substrate. Due to designed resistances and delay features, the antigen–DAb-HRP complex reaches and binds at capture antibody sites in the reaction chambers before substrate arrival. After a prescribed delay, the user opens the hydrophobic vent to allow the reconstituted substrate to reach the reaction chambers, where it reacts with HRP to generate chemiluminescent signal correlating with target PfHRP2 concentration. Total assay time is approximately 20 minutes after sample loading. Capillary physics and stop-valve: Flow control is governed by surface wettability, channel geometry, and resulting capillary pressures. Hydrophilic channel walls (low contact angle) increase negative capillary pressure and drive flow; hydrophobic regions provide barriers. A capillary stop valve with abrupt channel enlargement (β ≈ 90°) provides a pressure barrier preventing backflow from Path-1 into the substrate path, enabling correct sequencing. Finite-element two-phase flow simulations (COMSOL, level-set method) with contact angle 12°, liquid-air surface tension ~73 mN/m, and varying channel widths (50–150 µm) confirmed the stop valve blocks backflow while allowing continued forward flow, with estimated pressure barriers on the order of hundreds of Pa (e.g., ~800 Pa for 50 µm width and β = 90°). Materials and reagents: The chip substrate is COC. On-chip lyophilized reagents include HRP-conjugated detection antibody and chemiluminescent substrate (validated previously to be amenable to drying). Capture antibodies are immobilized in the reaction chambers (test, controls). Samples consisted of PfHRP2 spiked in artificial serum. Smartphone optical detection: A custom, high-sensitivity optical detector (e.g., photodiode-based) attaches to the smartphone via USB-OTG for both power (approximately 4.4–5.25 V available) and digital data transfer. The smartphone application handles real-time signal acquisition, display, storage, and analysis. Chemiluminescence detection requires no excitation source, minimizing power draw relative to fluorescence-based systems. Performance evaluation: The platform was validated by performing a sandwich ELISA for Plasmodium falciparum histidine-rich protein 2 (PfHRP2). The assay achieved a smartphone-read limit of detection (LOD) of 8 ng/mL in artificial serum, sufficient to detect active malaria infection. Each test required only sample loading and a single vent-opening step and completed within ~20 minutes.

- Developed a novel microchannel capillary flow assay (MCFA) chip executing chemiluminescent sandwich ELISA with on-chip lyophilized reagents and fully passive capillary flow control. - Integrated a USB-OTG smartphone-based analyzer with a custom optical detector for power and data communication, enabling portable, low-power POCT. - Achieved a limit of detection (LOD) of 8 ng/mL for PfHRP2 in artificial serum using the smartphone analyzer, adequate for detecting active malaria infection. - Autonomous assay sequencing accomplished via dual-path design, delay valves, meandering channels, and a capillary stop valve; no external pumps or complex user steps required (only sample loading and vent opening). - Optical trenches and spiral reaction chamber design minimized optical crosstalk and enhanced signal quality; assay completed within approximately 20 minutes. - Chip architecture includes internal quality controls (positive and negative control chambers) for run validation.

The findings demonstrate that carefully engineered capillary microfluidics with on-chip lyophilized reagents can autonomously execute a sensitive chemiluminescent sandwich ELISA without external pumping or complex user interaction. By leveraging USB-OTG, the smartphone provides both sufficient power and a digital communication link to a sensitive optical detector, overcoming limitations of smartphone cameras and audio-jack-powered devices. The achieved LOD of 8 ng/mL for PfHRP2 indicates performance suitable for clinically relevant malaria detection in resource-limited settings. The dual-path architecture, passive delay features, and stop-valve effectively maintain the correct ELISA sequence, while optical trenches reduce crosstalk, improving measurement fidelity. The platform’s modularity and reliance on general immunoassay principles suggest it can be adapted to other biomarkers, broadening its impact on POCT for infectious diseases.

This work introduces a new MCFA platform that combines capillary-driven microfluidics, on-chip lyophilized chemiluminescent reagents, and a USB-OTG smartphone-based optical detection system to deliver low-cost, portable, and user-friendly POCT. The system autonomously performs a chemiluminescent sandwich ELISA and achieves an 8 ng/mL LOD for PfHRP2 within about 20 minutes, validating its suitability for malaria diagnostics. The chip’s design elements (dual paths, delay valves, stop valve, optical trenches) ensure proper assay sequencing and signal integrity. Given its simple operation and reliance on broadly applicable immunochemistry, the platform can be customized to detect a variety of biomarkers, enabling hand-held POCT with networking capability. Future work could expand biomarker panels, evaluate performance with clinical samples, further ruggedize and mass-produce the chip, and refine the optical detector for enhanced sensitivity and manufacturability.