The human immunodeficiency virustype1(HIV- 1)protease is a retroviral aspartyl protease, and has been recognized as the essential element in maturation of the infectious virus.  Accordingly, HIV-1 protease has been an important target for drug therapy.  Here, we report a rapid, sensitive, and label-free nanopore sensing method for the detection of HIV-1 protease, in which an engineered alpha-hemolysin is utilized as the sensing element, while an unmodified peptide is used as the substrate.  Under a fixed potential bias applied across the nanopore membrane, the activity of the HIV-1 protease can be detected and quantified by monitoring the change in the ionic current passing through the nanopore, which is due to the cleavage of the peptide by the HIV-1 protease.  The method is sensitive (can detect HIV-1 protease at pico- to nanomolar levels) and selective (other proteases will not interfere with the detection).  The substrate-based nanopore sensing approach should find useful application in the development of nanopore sensors for other proteolyticenzymes.

Real-time label-free measurement of HIV-1 protease activity by nanopore analysis

248th National Meeting (2014)

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