Portable electroanalytical nucleic acid amplification tests using printed circuit boards and open-source electronics.

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.

Electroanalytical Paper-Based Nucleic Acid Amplification Biosensors with Integrated Thread Electrodes.

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/µL 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.

Toward Continuous Molecular Testing Using Gold-Coated Threads as Multi-Target Electrochemical Biosensors.

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.

Nitrocellulose-bound achromopeptidase for point-of-care nucleic acid tests.

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.

Woven Electroanalytical Biosensor for Nucleic Acid Amplification Tests.

Fiber-based biosensors enable a new approach in analytical diagnostic devices. The majority of textile-based biosensors, however, rely on colorimetric detection. Here a woven biosensor that integrates microfluidics structures in combination with an electroanalytical readout based on a thiol-self-assembled monolayer (SAM) for Nucleic Acid Amplification Testing, NAATs is shown. Two types of fiber-based electrodes are systematically characterized: pure gold microwires (bond wire) and off-the-shelf plasma gold-coated polyester multifilament threads to evaluate their potential to form SAMs on their surface and their electrochemical performance in woven textile. A woven electrochemical DNA (E-DNA) sensor using a SAM-based stem-loop probe-modified gold microwire is fabricated. These sensors can specifically detect unpurified, isothermally amplified genomic DNA of Staphylococcus epidermidis (10 copies/µL) by recombinase polymerase amplification (RPA). This work demonstrates that textile-based biosensors have the potential for integrating and being employed as automated, sample-to-answer analytical devices for point-of-care (POC) diagnostics.

A disposable, wearable, flexible, stitched textile electrochemical biosensing platform.

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.

Rapid prototyping of heterostructured organic microelectronics using wax printing, filtration, and transfer.

Conducting polymers are the natural choice for soft electronics. However, the main challenge is to pattern conducting polymers using a simple and rapid method to manufacture advanced devices. Filtration of conducting particle dispersions using a patterned membrane is a promising method. Here, we show the rapid prototyping of various micropatterned organic electronic heterostructures of PEDOT:PSS by inducing the formation of microscopic hydrogels, which are then filtered through membranes containing printed hydrophobic wax micropatterns. The hydrogels are retained on the un-patterned, hydrophilic regions, forming micropatterns, achieving a resolution reaching 100 µm. We further solve the problem of forming stacked devices by transferring the acidified PEDOT:PSS micropattern using the adhesive tape transfer method to form vertical heterostructures with other micropatterned electronic colloids such as CNTs, which are patterned using a similar technique. We demonstrate a number of different heterostructure devices including micro supercapacitors and organic electrochemical transistors and also demonstrate the use of acidified PEDOT:PSS microstructures in cell cultures to enable bioelectronics.

Electrochemical Detection of Genomic DNA Utilizing Recombinase Polymerase Amplification and Stem-Loop Probe.

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/µL Staphylococcus epidermidis DNA with a total time to result of 70-75 min.