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. 

Wearable sensors are a fast growing and exciting research area, the success of smart watches are a great example of the utility and demand for wearable sensing systems. The current state of the art routinely uses expensive and bulky equipment designed for long term use. There is a need for cheap and disposable wearable sensors to make single use measurements, primarily in the area of biomarker detection. Herein we report the ability to make cheap (0.22 USD/sensor), disposable, wearable sensors by stitching conductive gold coated threads into fabrics. These threads are easily functionalised with thiolate self-assembled monolayers which can be designed for the detection of a broad range of different biomarkers. This all textile sensing platform is ideally suited to be scaled up and has the added advantage of being stretchable with insignificant effect on the electrochemistry of the devices. As a proof of principle, the devices have been functionalised with a continuous glucose sensing system which was able to detect glucose in human sweat across the clinically relevant range (0.1-0.6 mM). The sensors have a sensitivity of 126 +/- 14 nA/mM of glucose and a limit of detection of 301 +/- 2 nM. This makes them ideally suited for biomarker detection in point-of-care sensing applications.

Nucleic acid amplification tests (NAATs) are very sensitive and specific methods, but they mainly rely on centralized laboratories and therefore are not suitable for point-of-care testing. Here, we present a 3D microfluidic paper-based electrochemical NAAT. These devices use off-the-shelf gold plasma-coated threads to integrate electroanalytical readouts using ex situ self-assembled monolayer formation on the threads prior to assembling into the paper device. They further include a sandwich hybridization assay with sample incubation, rinsing, and detection steps all integrated using movable stacks of filter papers to allow time-sequenced reactions. The devices use glass fiber substrates for storing recombinase polymerase amplification reagents and conducting the isothermal amplification. We used the paper-based device for the detection of the toxic microalgae Ostreopsis cf. ovata. The NAAT, completed in 95 min, attained a limit of detection of 0.06 pM target synthetic DNA and was able to detect 1 ng/mu L O. cf. ovata genomic DNA with negligible cross-reactivity from a closely related microalgae species. We think that the integration of thread electrodes within paper-based devices paves the way for digital one-time use NAATs and numerous other advanced electroanalytical paper- or textile-based devices.

Enzymes are the cornerstone of modern biotechnology. Achromopeptidase (ACP) is a well-known enzyme that hydrolyzes a number of proteins, notably proteins on the surface of Gram-positive bacteria. It is therefore used for sample preparation in nucleic acid tests. However, ACP inhibits DNA amplification which makes its integration difficult. Heat is commonly used to inactivate ACP, but it can be challenging to integrate heating into point-of-care devices. Here, we use recombinase polymerase amplification (RPA) together with ACP, and show that when ACP is immobilized on nitrocellulose paper, it retains its enzymatic function and can easily and rapidly be activated using agitation. The nitrocellulose-bound ACP does, however, not leak into the solution, preventing the need for deactivation through heat or by other means. Nitrocellulose-bound ACP thus opens new possibilities for paper-based Point-of-Care (POC) devices.

Rapid and accurate near-patient diagnostic tests outside well-equipped laboratories are essential in the fight against outbreaks of infectious diseases, especially when these can turn into pandemics. As severe acute respiratory syndrome CoV 2 (SARS-CoV-2) is the latest but probably not the last pandemic of the 21st century. Nucleic acid amplification tests (NAATs) identify pathogens at the molecular level by targeting specific gene sequences. NAATs are currently the gold standard of molecular diagnostics, given their reliability, sensitivity, and specificity. In addition, NAATs can provide quantitative results with a short turnaround time compared to conventional immunoassays or culturing methods. However, most NAATs necessitate centralized laboratories and trained health professionals and, to a large extend, fail to be point-of-care(POC).The biosensing field was inspired by the micro electronics revolution in the1980s, which led to the emergence of the micro-total analysis systems (μTAS)concept. μTAS was envisioned to miniaturized laboratory-based tests in single microfluidic devices. The combination of POC NAATs with μTAS can offer rapid, sensitive, and specific diagnostic tools of great importance in tackling diseases.In this thesis, we have utilized paper and textile materials as a platform for developing μTAS. These materials possess many features necessary for advanced μTAS, such as the ability to transport liquids, store reagents and embed electronic functions, making them ideal for integrating affordable, portable, and easy to manufacture μTAS for NAATs.We have specially developed NAATs with paper-based and thread-based electrochemical readout to provide quantitative responses with high sensitivity, specificity, and the possibility to connect to portable digital electronics. This work paves the way for robust sample-to-answer digital POC NAATs.

Snabba och noggranna diagnostiska tester som utförs nära patienten utanför välutrustade laboratorier är det mest avgörande sättet att ta itu med smittsamma sjukdomar, som i värsta fall kan förvandlas till en pandemi.Severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) är den senaste men förmodligen inte den sista pandemin under 2000-talet.Nukleinsyraamplifieringstest (NAAT) identifierar patogener på en molekylär nivå genom att rikta in på specifika gensekvenser. NAAT är för tillfället den gyllene standarden för molekylär diagnostisk teknik med tanke på desstillförlitlighet, känslighet, och specificitet Utöver detta ger NAAT kvantitativa resultat med en kort behandlingstid i motsats till konventionella immunanalyser eller odlingsmetoder. De flesta NAAT-metoder kräver dock centraliserade laboratorier och utbildad personal och är därmed inte anpassade för självtest nära patienten.Biosensorsfältet var inspirerat av mikroelektronikrevolutionen på 1980-talet, vilket ledde till uppkomsten av konceptet mikrototalanalyssystem(μTAS). Dessa system har som syfte att miniatyrisera laboratoriebaserade tester genom att utföra alla steg i enstaka mikrofluidanordningar.Kombineringen av patientnära NAAT-tester med μTAS kan därför erbjuda snabba, känsliga och specifika diagnostikverktyg och därmed ha stor påverkan för att förhindra överföring av infektionssjukdomar. I den här avhandlingen har vi använt papper och textila material som en platform utveckling av μTAS.Dessa material har många egenskaper som är nödvändiga för μTAS såsom förmågan att transportera vätskor, lagra reagenser och att integrera elektroniska funktioner, vilket gör dem ideala för att integrera prisvärda, bärbara och lättillverkade μTAS för NAAT-tester. Vi har speciellt utvecklat NAAT-tekniker med papper- och trådbaserad elektrokemisk avläsning som ger kvantitativa svar med hög känslighet,specificitet och möjlighet att ansluta till bärbara elektroniska enheter. Detta arbete banar vägen för robusta, digitala och patientnära prov-till-svar-tester som baseras på NAAT-teknik.

Abstract: One-dimensional substrates such as textile fibers and threads offer an excellent opportunity to realize sensors, actuators, energy harvesters/storage, microfluidics, and advanced therapies. A new generation of wearable devices made from smart threads offer ultimate flexibility and seamless integration with the human body and the garments that adorn them. This article reviews the state of the art in thread-based wearable devices for monitoring human activity and performance, diagnoses and manages medical conditions, and provides new and improved human–machine interfaces. In the area of new and improved human–machine interfaces, it discusses novel computing platforms enabled using thread-based electronics and batteries/capacitors. For physical activity monitoring, a review of wearable devices using strain sensing threads is provided. Thread-based devices that can monitor health from biological fluids such as total analysis systems, wearable sweat sensing patches, and smart sutures/smart bandages are also included. The article concludes with an outlook on how fibers and threads are expected to impact and revolutionize the next generation of wearable devices. Knowledge gaps and emerging opportunities are presented. 

Fiber-based biosensors enable a new approach in analytical diagnosticdevices. The majority of textile-based biosensors, however, rely oncolorimetric detection. Here a woven biosensor that integrates microfluidicsstructures in combination with an electroanalytical readout based on athiol-self-assembled monolayer (SAM) for Nucleic Acid Amplification Testing,NAATs is shown. Two types of fiber-based electrodes are systematicallycharacterized: pure gold microwires (bond wire) and off-the-shelf plasmagold-coated polyester multifilament threads to evaluate their potential to formSAMs on their surface and their electrochemical performance in woven textile.A woven electrochemical DNA (E-DNA) sensor using a SAM-based stem-loopprobe-modified gold microwire is fabricated. These sensors can specificallydetect unpurified, isothermally amplified genomic DNA of Staphylococcusepidermidis (10 copies/μL) by recombinase polymerase amplification (RPA).This work demonstrates that textile-based biosensors have the potential forintegrating and being employed as automated, sample-to-answer analyticaldevices for point-of-care (POC) diagnostics.

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.