Nucleic acid amplification tests (NAATs) are powerful medical diagnostic tools for point-of-care (POC) and other field applications. However, traditional methods like quantitative 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 nucleic acid amplification and electrochemical detection using reverse transcription LAMP (RT-LAMP) of SARS-CoV-2. The system consists of two disposable PCB-based chips making it close to zero cost. One PCB is for heating and nucleic acid amplification, while the other is for electrochemical detection using Cyclic Voltammetry (CV) with a redox-active intercalator. 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 (< 1.5 h) nucleic acid amplification and detection at a target concentration of 10 copies/reaction. This work represents a notable step toward developing integrated, portable NAAT devices for POC diagnostics.

Point-of-need devices perform analytical tests that help inform decisions where they are needed, away from modern lab infrastructure, be it in-field or in resource-poor settings. They have many applications, including veterinary medicine, agriculture, food safety, environmental monitoring, and forensics. In medical diagnostics, such devices are called point-of-care tests, and they could help combat societal challenges such as the spread of epidemic diseases and providing adequate healthcare in developing countries. Point-of-care devices could also be wearable to non-invasively monitor body fluids such as sweat or urine from the patient. Ideal point-of-care devices conform with the REASSURED criteria, that they should be Real-time connected, Easy to collect samples, Affordable, Sensitive, Specific, User-friendly, Rapid, robust, Equipment-free, environmentally friendly, and Deliverable to the end user.

We have here developed Point-of-need devices based on textiles and Printed Circuit Boards (PCBs); both well-established technologies that could offer low-cost mass production using existing industrial resources. Specifically, we have made electrochemical biosensors based on gold-coated yarn in a rolling architecture, as well as combined with wicking Coolmax® yarn acting as microfluidic channels in wearable systems, enabling advanced textile-based diagnostic devices suitable for automation or machine-stitching into fabrics. We also showed biosensors based on gold-coated PCBs that can connect to portable potentiostats for electrochemical detection and have integrated heating for isothermal nucleic acid amplification.

Fälttestsenheter utför analytiska tester som hjälper till att informera beslut där de behövs, separat från modern labbinfrastruktur, vare sig det är i fält eller i resurssvaga områden. De har många tillämpningar, inklusive veterinärmedicin, jordbruk, livsmedelssäkerhet, miljöövervakning och kriminalteknik. Inom medicinsk diagnostik kallas sådana apparater patientnära tester, och de kan hjälpa till att bekämpa samhällsutmaningar såsom spridning av epidemiska sjukdomar och tillhandahållande av adekvat sjukvård i utvecklingsländer. Patientnära testenheter kan också vara bärbara för att icke-invasivt övervaka kroppsvätskor såsom svett eller urin från patienten. Idealiska patientnära enheter korresponderar med REASSURED-kriterierna, att de ska vara realtidsanslutna, lätta att samla in prover, prisvärda, känsliga, specifika, användarvänliga, snabba, robusta, utrustningsfria, miljövänliga och leveransbara till slutanvändaren. Vi har här utvecklat fälttestenheter baserade på textilmaterial och mönsterkort; båda väletablerade teknologier som skulle kunna erbjuda massproduktion till låg kostnad med hjälp av befintliga industriella resurser. Specifikt har vi tillverkat elektrokemiska biosensorer baserade på guldbelagt garn i en rullande arkitektur, samt kombinerat med vätande Coolmax®-garn som fungerar som mikrofluidkanaler i bärbara system, vilket möjliggör avancerade textilbaserade diagnostiska enheter lämpliga för automatisering eller maskinsömnad i tyger. Vi visade också biosensorer baserade på guldbelagda PCB som kan ansluta till bärbara potentiostater för elektrokemisk detektering ha integrerad värmefunktion för isotermisk nukleinsyraamplifiering.

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.

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.

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 tests integrated into digital point-of-care (POC) diagnostic systems have great potential for the future of health care. However, current methods of DNA amplification and detection require bulky and expensive equipment, many steps, and long process times, which complicate their integration into POC devices. We have combined an isothermal DNA amplification method, recombinase polymerase amplification, with an electrochemical stem-loop (S-L) probe DNA detection technique. By combining these methods, we have created a system that is able to specifically amplify and detect as few as 10 copies/mu L Staphylococcus epidermidis DNA with a total time to result of 70-75 min.

Angiogenesis plays an important role in cancer and ophthalmic disorders such as age-related macular degeneration and diabetic retinopathy. The vascular endothelial growth factor (VEGF) family and corresponding receptors are regulators of angiogenesis and have been much investigated as therapeutic targets. The aim of this work was to generate antagonistic VEGFR2-specific affinity proteins having adjustable pharmacokinetic properties allowing for either therapy or molecular imaging. Two antagonistic Affibody molecules that were cross-reactive for human and murine VEGFR2 were selected by phage and bacterial display. Surprisingly, although both binders independently blocked VEGF-A binding, competition assays revealed interaction with non-overlapping epitopes on the receptor. Biparatopic molecules, comprising the two Affibody domains, were hence engineered to potentially increase affinity even further through avidity. Moreover, an albumin-binding domain was included for half-life extension in future in vivo experiments. The best-performing of the biparatopic constructs demonstrated up to 180-fold slower dissociation than the monomers. The new Affibody constructs were also able to specifically target VEGFR2 on human cells, while simultaneously binding to albumin, as well as inhibit VEGF-induced signaling. In summary, we have generated small antagonistic biparatopic Affibody molecules with high affinity for VEGFR2, which have potential for both future therapeutic and diagnostic purposes in angiogenesis-related diseases.

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.

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.