Analytical systems based on isothermal nucleic acid amplification tests (NAATs) paired with electroanalytical detection enable cost-effective, sensitive, and specific digital pathogen detection for various in situ applications such as point-of-care medical diagnostics, food safety monitoring, and environmental surveillance. Self-assembled monolayers (SAMs) on gold surfaces are reliable platforms for electroanalytical DNA biosensors. However, the lack of automation and scalability often limits traditional chip-based systems. To address these challenges, we propose a continuous thread-based device that enables multiple electrochemical readings on a functionalized working electrode Au thread with a single connection point. We demonstrate the possibility of rolling the thread on a spool, which enables easy manipulation in a roll-to-roll architecture for high-throughput applications. As a proof of concept, we have demonstrated the detection of recombinase polymerase amplification (RPA) isothermally amplified DNA from the two toxic microalgae species Ostreopsis cf. ovata and Ostreopsis cf. siamensis by performing a sandwich hybridization assay (SHA) with electrochemical readout.

Thread-based microfluidics, which rely on capillary forces in threads for liquid flow, are a promising alternative to conventional microfluidics, as they can be easily integrated into wearable textile-based biosensors. We present here advanced textile-based microfluidic devices fabricated by machine stitching, using only commercially available textiles. We stitch a polyester “Coolmax®” yarn with enhanced wicking abilities into both hydrophobic fabric and hydrophobically treated stretchable fabric, that serve as non-wicking substrates. In doing so we construct textile microfluidics capable of performing a wide variety of functions, including mixing and separation in 2D and 3D configurations. Furthermore, we integrate a stitched microfluidic device into a wearable T-shirt and show that this device can collect, transport, and detect sweat from the wearer’s skin. These can also be machine-washed, making them inherently reusable. Finally, we integrate electrochemical sensors into the textile-based microfluidic devices using stitched gold-coated yarns to detect analytes in the microfluidic yarns. Our stitched textile-based microfluidic devices hold promise for wearable diagnostic applications. This novel, bottom-up fabrication using machine stitching is scalable, reproducible, low-cost, and compatible with the existing textile manufacturing industry.

The realization of electrochemical nucleic acid amplification tests (NAATs) at the point of care (POC) is highly desirable, but it remains a challenge given their high cost and lack of true portability/miniaturization. Here we show that mass-produced, industrial standardized, printed circuit boards (PCBs) can be repurposed to act as near-zero cost electrodes for self-assembled monolayer-based DNA biosensing, and further integration with a custom-designed and low-cost portable potentiostat. To show the analytical capability of this system, we developed a NAAT using isothermal recombinase polymerase amplification, bypassing the need of thermal cyclers, followed by an electrochemical readout relying on a sandwich hybridization assay. We used our sensor and device for analytical detection of the toxic microalgae Ostreopsis cf. ovata as a proof of concept. This work shows the potential of PCBs and open-source electronics to be used as powerful POC DNA biosensors at a low-cost. 

Nucleic acid amplification tests (NAATs) are powerful medical diagnostic tools for point-of-care (POC) and other field applications. However, traditional methods like qquantitative PCR (qPCR) require complex, expensive equipment and trained operators, limiting their use to centralized labs. Isothermal alternatives, like Loop-mediated Isothermal Amplification (LAMP), are better adapted for POC devices. Lab-on-PCB systems have the potential to overcome the challenges faced by conventional microfabrication-based systems. This study presents a novel lab-on-PCB device for RNA amplification and electrochemical detection using reverse transcription LAMP (RT-LAMP) of SARS-CoV-2. The system consists of only two disposable PCB-based chips making it close to zero cost. One PCB is for heating and DNA amplification, while the other is for electrochemical detection using Cyclic Voltammetry with a redox-active intercalating probe. The PCB slides are connected to a compact electronic device (<10 USD) for controlling the heating and electroanalytical readout. Using this device, we achieved successful rapid (<1 hour) nucleic amplification and detection at a target concentration of 100 copies/reaction. This work represents a notable step toward developing integrated, portable NAAT devices for POC diagnostics.