title: Nanopore-based electrical and label-free sensing of enzyme activity in blood serum.
abstract: A generic strategy to expand the analytical scope of electrical nanopore sensing is presented. We specifically and electrically detect the activity of a diagnostically relevant hydrolytic enzyme and remove the analytically harmful interference from the biochemically complex sample matrix of blood serum. Our strategy is demonstrated at the example of the renin protease which is involved in regulation of blood pressure. The analysis scheme exploits a new approach to reduce sample complexity while generating a specific read-out signal. Within a single spin-column (i), the protease cleaves a resin-tethered peptide substrate (ii) which is affinity-purified using the same multifunctional resin to remove interfering blood serum components, followed by (iii) detecting the peptide via electrical nanopore recordings. Our approach is beneficial in several ways. First, by eliminating serum components, we overcome limitations of nanopore sensing when challenging samples lead to membrane instability and a poor signal-to-noise ratio. Second, the label-free sensing avoids drawbacks of currently used radiolabel-immunoassays for renin. Finally, the strategy of simultaneous generation and purification of a signal peptide within a multifunctional resin can very likely be expanded to other hydrolytic enzymes dissolved in any analyte matrix and exploited for analytical read-out methods other than nanopore sensing.
authors: Department of Chemistry, Institute of Structural and Molecular Biology, University College London , London, England, United Kingdom.|School of Chemistry, University of Southampton , Southampton, England, United Kingdom.
authors: Mikiembo
authors: M
authors: Kukwikila
authors: Department of Chemistry, Institute of Structural and Molecular Biology, University College London , London, England, United Kingdom.
authors: Stefan
authors: S
authors: Howorka
mesh_terms: D000595:Amino Acid Sequence; D001774:Blood Chemical Analysis / Q000379:methods*; D004560:Electricity; D006801:Humans; D008969:Molecular Sequence Data; D058608:Nanopores; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D059748:Proteolysis; D012083:Renin / Q000097:blood* / Q000378:metabolism*; D059629:Signal-To-Noise Ratio
chemical_list: D010455:Peptides; D012083:Renin