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.
Highlights
Enzymes are the cornerstone of modern biotechnology
ACP has been extensively used for sample preparation given its efficiency in lysing bacteria namely
We used recombinase polymerase amplification (RPA) to assess the activity of nitrocellulose-bound ACP both when undisturbed (Fig. 1C) and when agitated (Fig. 1D)
Summary
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. These diagnostic tests are capable of target detection with high sensitivity and s pecificity[5], using three steps: (i) sample preparation, (ii) DNA amplification, and (iii) DNA detection Their main disadvantage is the requirement of high-end equipment and highly trained personnel, for carrying out these steps, which limits their use in POC devices. FTATM paper, introduces amplification inhibitors and requires a series of washing steps which makes its integration into POC diagnostics difficult[11,13,14,15] This is an inherent limitation that occurs when utilizing chemicals for lysis, which denature proteins nonspecifically and cannot be deactivated. For integration into POC NAATs, this introduces complexity since the minimum temperature required for ACP deactivation is 80 °C20
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