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

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.

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

“…Ongoing research should focus on three specific areas: (i) shortening of the incubation times of the sandwich hybridization assay as well as performing stability studies of the SAMs on PCBs to achieve ready-to-use platforms; 59 (ii) integration of RPA on chip by using heating, 60,61 by performing a solid-phase amplification, 62 and by using microfluidics on PCBs 60 to achieve a true lab-on-a-chip device; and (iii) inclusion of a sample preparation step on chip by exploiting magnetic beads-powered cell capture 63 and enzymatic cell lysis and DNA extraction steps using paper. 7 Our demonstration of SAM-based PCB sensors will likely be applicable to many other SAM-based sensors. 23,30,53,64 Our prototype clearly demonstrates substantial superiority in terms of cost and portability over existing alternatives.…”

“…4 The utilization of the PCR tests during this pandemic has also made it clearer that "one size does not fit all" when it regards to public health testing, and that point-of-care (POC) tests with much higher dissemination can provide valuable information and significantly contribute to public health. 5 The realization of a low-cost and automated POC NAAT is however challenging, as it requires many steps including: sample preparation with sample collection and DNA extraction, 6,7 DNA amplification, 8,9 and DNA detection. 10,11 Each of these steps including their subparts must be simplified and minimized for integration into POC devices.…”

“…The realization of a low-cost and automated POC NAAT is however challenging, as it requires many steps including: sample preparation with sample collection and DNA extraction, 6,7 DNA amplification, 8,9 and DNA detection. 10,11 Each of these steps including their subparts must be simplified and minimized for integration into POC devices.…”

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

“…Future work should include improvement in three specific areas: (i) Sample collection and preparation, for example, using paper-based purification as well as DNA extraction with cell lysis; (ii) Reduction of incubation times for the assay by shortening the time of the SHA assay and by fully integrating the RPA in the paper; and (iii) Integration with open-source electronics to enable electrochemical readout with simple potentiostats and to automate the liquid handling steps, e.g., using electrical valves. , …”

“…Nevertheless, our isothermal amplification and electroanalyt- ical detection embedded in paper microfluidics require less manual intervention and, more importantly, has the potential to be portable, facilitating testing in resource-limited areas, which is not possible with centralized PCR. Future work should include improvement in three specific areas: (i) Sample collection and preparation, for example, using paper-based purification as well as DNA extraction with cell lysis; 59 (ii) Reduction of incubation times for the assay by shortening the time of the SHA assay and by fully integrating the RPA in the paper; and (iii) Integration with open-source electronics to enable electrochemical readout with simple potentiostats 60 and to automate the liquid handling steps, e.g., using electrical valves. 61…”