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.

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.

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.

No abstract

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.