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.

A paper microfluidic device capable of conducting enzyme-linked assays is presented: a microfluidic enzyme-linked paper analytical device (μEL-PAD). The system exploits a wash-free sandwich coupling to form beads/analyte/enzyme complexes, which are subsequently added to the vertical flow device composed of wax-printed paper, waxed nitrocellulose membrane and absorbent/barrier layers. The nitrocellulose retains the bead complexes without disrupting the flow, enabling for an efficient washing step. The entrapped complexes then interact with the chromogenic substrate stored on the detection paper, generating a color change on it, quantified with an open-source smartphone software. This is a universal paper-based technology suitable for high-sensitivity quantification of many analytes, such as proteins or nucleic acids, with different enzyme-linked formats. Here, the potential of the μEL-PAD is demonstrated to detect DNA from Staphylococcus epidermidis. After generation of isothermally amplified genomic DNA from bacteria, Biotin/FITC-labeled products were analyzed with the μEL-PAD, exploiting streptavidin-coated beads and antiFITC-horseradish peroxidase. The μEL-PAD achieved a limit of detection (LOD) and quantification <10 genome copies/μL, these being at least 70- and 1000-fold lower, respectively, than a traditional lateral flow assay (LFA) exploiting immobilized streptavidin and antiFITC-gold nanoparticles. It is envisaged that the device will be a good option for low-cost, simple, quantitative, and sensitive paper-based point-of-care testing.

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. 

The easy and rapid spread of bacterial contamination and the risk it poses to human health makes evident the need for analytical methods alternative to conventional time-consuming laboratory-based techniques for bacterial detection. To tackle this demand, biosensors based on isothermal DNA amplification methods have emerged, which avoid the need for thermal cycling, thus facilitating their integration into small and low-cost devices for in situ monitoring. This review focuses on the breakthroughs made on biosensors based on isothermal amplification methods for the detection of bacteria in the field of food safety and environmental monitoring. Optical and electrochemical biosensors based on loop mediated isothermal amplification (LAMP), rolling circle amplification (RCA), recombinase polymerase amplification (RPA), helicase dependent amplification (HDA), strand displacement amplification (SDA), and isothermal strand displacement polymerisation (ISDPR) are described, and an overview of their current advantages and limitations is provided. Although further efforts are required to harness the potential of these emerging analytical techniques, the coalescence of the different isothermal amplification techniques with the wide variety of biosensing detection strategies provides multiple possibilities for the efficient detection of bacteria far beyond the laboratory bench.

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.

Harmful algal blooms (HABs) represent a growing threat to aquatic ecosystems and humans. Effective HAB management and mitigation efforts strongly rely on the availability of timely and in-situ tools for the detection of microalgae. In this sense, nudeic acid-based (molecular) methods are being considered for the unequivocal identification of miaoalgae as an attractive alternative to the currently used time-consuming and laboratory-based light microscopy techniques. This review provides an overview of the progress made on new molecular biotechnological tools for microalgal detection, particularly focusing on those that combine a nudeic acid (DNA or RNA) amplification step with detection. Different types of amplification processes (thermal and isothermal) and detection formats (e.g. microarrays, biosensors, lateral flows) are presented, and a comprehensive overview of their advantages and limitations is provided Although isothermal techniques are an attractive alternative to thermal amplification to reach in-situ analysis, further development is still required. Finally, current challenges, critical steps and future directions of the whole analysis process ( from sample procurement to in-situ implementation) are described.