Enzyme-Instructed Formation of β-Sheet-Rich Nanoplatelets for Label-Free Protease Sensing

Emma Olson, Justin S. Plaut, Sarah A. Barnhill, Sinan Sabuncu, Corey M. Dambacher, Sean D. Speese, Srivathsan V. Ranganathan, Bruce P. Branchaud, Adem Yildirim

Research output: Contribution to journalArticlepeer-review

Abstract

Dysregulated proteolytic activity has been observed in various human diseases, including cancer, neurodegenerative disorders, and cardiovascular diseases. Thus, there is an immense need to develop simple and sensitive methods to monitor specific protease activities in biological solutions for the detection and prognosis of these diseases. In this article, we describe a fluorogenic label-free protease detection method using a rationally designed β-sheet-rich nanoplatelet-forming peptide precursor and an intercalating dye, thioflavin T. Hydrolysis of the peptide by the target protease triggers the formation of β-sheet-rich self-assembled 3 nm-thick nanoplatelets. In situ intercalation of thioflavin T into these β-sheet domains results in a strong enhancement of the dye's fluorescence, allowing sensitive detection of protease activity with high signal-to-noise ratios (up to 45-fold). The proposed concept was demonstrated to detect the activity of legumain, a cysteine protease that was found to be over-expressed in several cancers, with a detection limit of ∼0.2 nM. In addition, assay conditions were optimized to detect legumain activity in human plasma. Importantly, both assay components can be commercially obtained, and no time-consuming conjugation reactions and purification steps are required. Thus, the method described here holds great potential in various protease-detection applications, with its simplicity and low cost.

Original languageEnglish (US)
Pages (from-to)7800-7810
Number of pages11
JournalACS Applied Nano Materials
Volume4
Issue number8
DOIs
StatePublished - Aug 27 2021

Keywords

  • label-free sensing
  • legumain
  • peptide self-assembly
  • protease detection
  • thioflavin T
  • β-sheet

ASJC Scopus subject areas

  • Materials Science(all)

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