Point-of-care testing (POCT) is crucial for diagnosing diseases in resource-limited settings. While microfluidic-based immunodiagnostics offer advantages like reduced sample volume and portability, the need for inexpensive, low-power, and user-friendly systems remains. This paper addresses this need by developing a smartphone-based POCT analyzer using a novel MCFA platform. Smartphones are ubiquitous, offering features like a display, data processing capabilities, storage, and a power source. However, smartphone cameras lack the sensitivity required for ultra-high sensitive optical detection needed in immunodiagnostics. Previous attempts using smartphone audio jacks for power and communication have limitations due to low voltage. This research utilizes the smartphone's USB On-The-Go (OTG) port, which provides both power and data communication. Lateral flow immunoassays (LFIAs), while popular, suffer from inconsistencies in sensitivity and qualitative readouts. Microchannel-based immunoassays with enzyme-based signal amplification offer improved LOD. Utilizing dry reagents (lyophilized) simplifies the assay, reduces user intervention, eliminates the need for cold chain storage, and extends shelf life. While fluorescence-based detection is common in autonomous microfluidic assays with dry reagents, it requires an excitation source, increasing cost and complexity. Chemiluminescence offers superior sensitivity with a simpler detection protocol. This research leverages previously validated methods for drying chemiluminescent substrates to create a microfluidic platform for chemiluminescence-based sandwich ELISA. The MCFA chip design employs passive flow control, eliminating external pumps. The system is validated by detecting the malaria biomarker Plasmodium falciparum histidine rich protein 2 (PfHRP2), which is vital for detecting P. falciparum malaria and known for higher sensitivity compared to other diagnostic tests.

The introduction section extensively reviews existing literature on POCT systems, microfluidic assays, smartphone-based diagnostics, and the challenges associated with each. It highlights the advantages and disadvantages of various approaches, such as fluorescence-based detection versus chemiluminescence, liquid reagent handling versus lyophilized reagents, and the use of different smartphone ports for power and communication. The review establishes the context for the proposed MCFA platform and its advantages.

The paper details the design and fabrication of the MCFA lab-on-a-chip device. The chip uses passive flow control based on capillary action, leveraging the hydrophilicity of the microchannels to guide reagent flow. The design incorporates features such as a sample loading chamber, separate channels for the detection antibody and chemiluminescent substrate (both lyophilized), delay valves to control reagent delivery timing, a stop valve to prevent backflow, multiple spiral reaction chambers, and a capillary pump for waste removal. Equations are provided to describe capillary flow (Q=ΔP/Rf and Pcap) and demonstrate how channel geometry and surface properties affect flow rate and pressure. The design minimizes optical crosstalk using trenches between reaction chambers. Micropillar structures in lyophilization chambers ensure uniform drying. The stop-valve design and function are explained, including an equation (ΔP equation) for pressure barrier generation and simulation results from COMSOL Multiphysics showing its effectiveness. The paper describes the smartphone-based optical detector, its connection to the smartphone via USB-OTG for power and data transfer, and the software used for data acquisition and analysis. The experimental procedure for the sandwich ELISA using PfHRP2 as the target biomarker is outlined, including the sample preparation, the assay steps performed on the MCFA chip, and the chemiluminescence detection and analysis. The dimensions and volumes of various chip components are summarized in a table.

The MCFA platform, coupled with the smartphone-based detector, successfully detected PfHRP2 in artificial serum with a limit of detection (LOD) of 8 ng/mL. This LOD is sufficiently sensitive to detect active malarial infection. The entire assay process, including sample loading and signal detection, is completed within 20 minutes. The system demonstrates the successful integration of a passive microfluidic device with a smartphone platform for point-of-care diagnostics. The use of lyophilized reagents eliminates the need for liquid handling and cold chain storage, enhancing the practicality and affordability of the system. The system's portability and ease of use make it ideal for resource-limited settings. The results from COMSOL simulations validated the stop-valve design, confirming its ability to prevent backflow and ensure proper reagent sequencing. The design of the microchannels, with varying capillary pressures, facilitated the sequential flow of reagents and accurate performance of the sandwich ELISA. The presence of positive and negative controls on the chip allowed for quality control and validation of each assay. The use of optical trenches effectively reduced crosstalk during chemiluminescence detection.

The findings demonstrate the feasibility of a low-cost, portable, and user-friendly POCT system for malaria diagnosis. The high sensitivity achieved with chemiluminescence detection, coupled with the passive flow control of the MCFA chip, results in a robust and reliable assay. The successful integration of the MCFA chip with a smartphone analyzer highlights the potential of using readily available technology for point-of-care diagnostics in resource-limited areas. The use of lyophilized reagents addresses many practical challenges associated with reagent storage and handling. Future research could focus on adapting this platform for detecting other infectious diseases, integrating more sophisticated data analysis algorithms on the smartphone, and conducting field trials in resource-limited settings.

This research successfully developed a novel microchannel capillary flow assay (MCFA) platform coupled with a smartphone-based analyzer for sensitive and rapid malaria detection. The use of lyophilized chemiluminescence reagents and passive flow control enhances the system's portability, user-friendliness, and cost-effectiveness. Future work will concentrate on expanding the platform’s applications to other diseases.

The study was conducted using artificial serum, which may not perfectly mimic the characteristics of real patient samples. Further validation with clinical samples is needed. The current design is optimized for PfHRP2; adaptation to other biomarkers might require microfluidic channel optimization. The long-term stability of lyophilized reagents within the chip needs to be further evaluated.