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

https://doi.org/10.1016/j.saa.2019.117739Get rights and content

Highlights

  • A new metal-enhanced fluorescence (MEF) based enzymatic activity assay is reported.

  • Optimization is done with silvered 96 well plates and fluorescein isothiocyanate.

  • An enhancement value of 11,000x is reported for fluorescein on silvered plates.

  • For the model enzyme trypsin, a detection limit of 2 ng is achieved for the assay.

Abstract

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.

Introduction

Proteolytic enzymes are ubiquitous across a broad range of biological organisms, playing critical roles in physiological and pathogenic cellular mechanisms involving protein degradation. These enzymes, which are essential in digestive processes, reduce proteins down to their amino acid components and therefore present a unique challenge to biological and chemical research [1,2]. In fact, proteolytic degradation is a leading cause of protein instability in preparations resulting in decreased shelf-life. In the case of quantitative detection of protein, even low concentrations of protease impurities could lead to protein degradation during analysis, thus skewing results in biological assays and preventing accurate quantitation. In addition, proteases are well-known biomarkers for diseases such as acute pancreatitis and cystic fibrosis [[2], [3], [4]] and are frequently involved in health conditions including cancer, emphysema, viral infections, and neurodegenerative diseases [1,5]. Due to these factors, detection of low levels of proteolytic activity is necessary for diagnostics, biologics manufacturing, and biological research.

Numerous methods have been employed for the detection of proteolytic enzymes, ranging from the commonly used enzyme-linked immunosorbent assay (ELISA) to techniques such as mass spectroscopy [1]. Included in this spectrum of detection methods are fluorescence-based assays [1,2,6,7], which in general are relatively quick and facile; however, these assays may still be limited by long incubation times, degradation of fluorophores during exposure and detection, and low signal to noise ratios due to biological autofluorescence from sample media. To address these limitations, we propose the incorporation of the Metal-Enhanced Fluorescence (MEF) technology into fluorescence-based enzymatic assays, using commercially available MEF platforms such as Quanta Plates™ (www.ursabioscience.com).

MEF has become an established biotechnology platform for sensing and diagnostics. In essence, MEF works by amplifying fluorescent signals in the near-field of metal nanoparticles thereby improving signal to noise ratios, which ultimately allows for more sensitive detection [8,9]. Although the mechanism of MEF is not entirely understood, in principle MEF arises from the coupling of fluorophore quanta to metal nanoparticle plasmons such that the coupled system may radiate as a single system. This coupled system yields enhancement from a combination of both absorption and emission amplification [8]. MEF has been demonstrated for many fluorophores—both small molecule [10] and nano-sized [11,12]—as well as with various plasmonic materials [[13], [14], [15]]. In addition to signal amplification, the coupled system frequently demonstrates much improved photophysical qualities such as fluorophore photostability [9,16]. These attributes present a strong basis for development of more sensitive and tunable assays.

Indeed, MEF-based assays are distinct from traditional fluorescence detection assays in that the fluorescent label is attached directly to the metal nanoparticle surface within an optimal near-field distance to the plasmonic substrate, rather than diffusing freely within the sample well [[17], [18], [19]]. Only fluorophores within this near-field range (also defined herein as the effective enhancement or coupling region volume), approximately 25 nm from the nanoparticle surface, [8] couple to localized surface plasmons and exhibit significant fluorescence enhancement relative to free-space fluorophores. Probe solutions are, therefore, used only to coat the nanoparticle surfaces, and solvents containing un-bound probe could then be potentially collected and re-used for future platform functionalization before the assay samples themselves are added. Comparatively, many commonly used fluorescent probes do not typically couple to the analysis platform surfaces such as plastic, requiring direct use of the probe in higher concentrations, thus leading to higher chemical requirements for a single assay as compared to a MEF-based design. The potential for MEF technology application in enzymatic assays is further reinforced by the development of microwave-accelerated MEF, or MAMEF, which permits the detection of very low concentrations or low activity agents in tandem with an ultra-fast assay design [17,[20], [21], [22], [23], [24]]. In these systems, low-power microwaves increase the rate of mass transportation and molecular diffusion at the metal nanoparticle surfaces, shortening lengthy incubation times required to generate low detection limits in otherwise identical fluorescence-based assays. Previous work demonstrated the utility of this platform, achieving picogram per milliliter detection limits of the anthrax protective antigen exotoxin in under an hour [17].

Herein we explore the foundational development and potential of a MEF-based sensing platform for the detection of proteases using trypsin as a model enzyme. Trypsin is a serine protease that hydrolyzes peptide and ester bonds of lysine and arginine residues. Using Quanta Plates™ we developed a turn-off fluorescent assay using fluorescein isothiocyanate (FITC)-labeled YebF protein for detection of trypsin activity. The data reported provides a foundation for the future tuning and development of even more sensitive proteolytic enzyme detection platforms based on the principles of metal-enhanced fluorescence.

Section snippets

Determination of absolute detection, enhancement factor, and coefficient of variation for fluorescein sodium salt in Quanta Plates™

Silvered 96-well plates, or Quanta Plates™, were purchased from URSA BioScience (www.ursabioscience.com) for use as the plasmonic platform in this study (Fig. 1). To determine the suitability of a fluorescein-derived probe as the assay fluorophore component, the detectable concentration range was determined for fluorescein sodium salt (Sigma Aldrich) in the Quanta Plates™. To determine this range, a 1 μM solution of fluorescein sodium salt was prepared in deionized water. Serial dilutions were

Analysis of fluorescein sodium salt in Quanta Plates™

Fluorescein and its derivatives are commonly used fluorescent labels that have been evaluated with plasmonic platforms for a metal-enhanced fluorescence effect [[26], [27], [28]]. To demonstrate that a fluorescein-based probe would be suitable for the Quanta Plate™ platform and for the model protease assay described, fluorescence from a 1 μM solution of fluorescein sodium salt was analyzed both on blank 96-well plates and within the Quanta Plate™ wells (Fig. 2a). From these spectra, a MEF value

Conclusions

Here we report the development of a turn-off, MEF-based assay for the detection of proteolytic enzymes using Quanta Plates™. On this plasmonic platform we report strong enhancement of the fluorescein sodium salt fluorophore emission by proximity to the silvered surfaces, with detectable fluorescence as low as picomolar concentrations. Additionally, we report a ~9% coefficient of variation for fluorescein sodium salt in the Quanta Plate™ wells, confirming the possible utility of this

Author contributions

All information reported was written by Rachael Knoblauch and edited by all additional authors. Experiments were designed by and executed under the supervision of Dr. Chris D. Geddes. Undergraduate researcher Eric Lucas conducted the implementation of experiments and collection of data. Additional data analysis was performed by Rachael Knoblauch. Labeling proteins were provided by Dr. Sheldon Broedel Jr. with the assistance of Mandie Combs-Bosse.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgement

This work was supported by the National Science Foundation Graduate Research Fellowship Program (2018262827), the HHS/NIH/National Institute of General Medical Sciences (NIGMS) through the Chemistry/Biology Interface Program at the University of Maryland Baltimore County (5T32GM066706), and the Institute of Fluorescence at the University of Maryland Baltimore County Internal Funding. The authors would like to thank Dr. Sheldon Broedel and Mandie Combs-Bosse for the collaboration on FITC-YebF

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