Low-concentration trypsin detection from a metal-enhanced fluorescence (MEF) platform: Towards the development of ultra-sensitive and rapid detection of proteolytic enzymes.

Proteolytic enzymes, which serve to degrade proteins to their amino acid building blocks, provide a distinct challenge for both diagnostics and biological research fields. Due to their ubiquitous presence in a wide variety of organisms and their involvement in disease, proteases have been identified as biomarkers for various conditions. Additionally, low-levels of proteases may interfere with biological investigation, as contamination with these enzymes can physically alter the protein of interest to researchers, resulting in protein concentration loss or subtler polypeptide clipping that leads to a loss of functionality. Low levels of proteolytic degradation also reduce the shelf-life of commercially important proteins. Many detection platforms have been developed to achieve low-concentration or low-activity detection of proteases, yet many suffer from limitations in analysis time, label stability, and ultimately sensitivity. Herein we demonstrate the potential utility of fluorescein derivatives as fluorescent labels in a new, turn-off enzymatic assay based on the principles of metal-enhanced fluorescence (MEF). For fluorescein sodium salt alone on nano-slivered 96-well plates, or Quanta Plates™, we report up to 11,000x enhancement for fluorophores within the effective coupling or enhancement volume region, defined as ~100 nm from the silver surface. We also report a 9% coefficient of variation, and detection on the picomolar concentration scale. Further, we demonstrate the use of fluorescein isothiocyanate-labeled YebF protein as a coating layer for a MEF-based, Quanta Plate™ enzymatic activity assay using trypsin as the model enzyme. From this MEF assay we achieve a detection limit of ~1.89 ng of enzyme (2.8 mBAEE activity units) which corresponds to a minimum fluorescence signal decrease of 10%. The relative success of this MEF assay sets the foundation for further development and the tuning of MEF platforms for proteolytic enzyme sensing not just for trypsin, but other proteases as well. In addition, we discuss the future development of ultra-fast detection of proteases via microwave-accelerated MEF (MAMEF) detection technologies.