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.

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Fig: Schematic representation of the concepts. (A) Free ACP in solution inhibits RPA possibly by digesting the enzymes necessary for amplification; (B) drying ACP on nitrocellulose for immobilization. (C) Undisturbed ACP immobilized on nitrocellulose does not immediately inhibit RPA. (D) Agitation probably increases the rate at which RPA reagents come in contact with active ACP on the surface of nitrocellulose

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.

 

Chondrogiannis, G., Khaliliazar, S., Toldrà, A. et al. Nitrocellulose-bound achromopeptidase for point-of-care nucleic acid tests. Sci Rep11, 6140 (2021). https://doi.org/10.1038/s41598-021-85481-2

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