Label–free, turn–on fluorescent sensor for trypsin activity assay and inhibitor screening

doi:10.1016/j.talanta.2016.09.011

Talanta

Volume 161, 1 December 2016, Pages 535-540

https://doi.org/10.1016/j.talanta.2016.09.011 Get rights and content

Highlights

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A simple, label–free and turn–on fluorescent sensor for trypsin was fabricated.
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Cytochrome c was selected as the natural substance of trypsin.
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Trypsin cleaved cytochrome c into heme–peptide fragment.
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Heme–peptide fragment catalyzed H₂O₂ oxidizing thiamine to fluorescent thiochrome.
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The method was applied to screen the inhibitor of trypsin.

Abstract

The development of new detection methods for proteases activity assay is important in clinical diagnostics and drug development. In this work, a simple, label–free, and turn–on fluorescent sensor was fabricated for trypsin, a protease produced in the pancreas. Cytochrome c, a natural substance of trypsin, could be selectively cleaved by trypsin into heme–peptide fragment. The produced heme–peptide fragment exhibited an intensive catalytic role on the H₂O₂–mediated the oxidation of thiamine to form strong fluorescent thiochrome. The fluorescence intensity was closely dependent on the amount of trypsin presented. The procedure allowed the measurement of trypsin over the range of 0.5–20.0 μg/mL with a detection limit of 0.125 μg/mL. The sensor showed better precision with a relative standard deviation of 1.6% for the measurement of 1.0 μg/mL trypsin solution (n=11). This sensing system was applied to screen the inhibitor of trypsin, the IC₅₀ values were calculated to be 12.71 ng/mL for the trypsin inhibitor from soybean and 2.0 μg/mL for benzamidine hydrochloride, respectively, demonstrating its potential application in drug development and related diseases treatment.

Graphical abstract

Introduction

Many efforts have been devoted to developing new detection methods and new detection techniques toward biological macromolecules, e.g. protein and nucleic acid, as the result of increasing attention paid to human health, and diagnosis and treatment of disease [1]. Protease, also known as proteolytic enzyme, exists widely in natural organisms and plays important roles in catalyzing the hydrolytic cleavage of specific peptide bonds in the target proteins into smaller fragments [2]. Proteases are involved in the control of a variety of physiological and pathological processes [3], [4]. On the other hand, proteases are also implicated in many physiological diseases. The abnormal level of protease would lead to a number of diseases such as inflammation, cancer, Alzheimer's [5], [6]. More and more studies have been paid to develop simple and sensitive methods toward the detection of protease activity and the related inhibitor screening [7], [8], [9], [10], [11], [12], which is significance to the disease diagnosis and treatment as well as to the drug research and development. Among the reported techniques, fluorescence–based assays are more attractive due to its high sensitivity, easy operation and rapid response. The probes of labeled specific peptide sequence were often designed and used as the enzyme substrates in most of fluorescent–based methods [10], [11], [12]. However, chemical modification of peptide substrate may depress the affinity of the probe to the target protease and thus reduce the detection sensitivity. In addition, the synthesis and modification of specific peptide substrate are more tedious and expensive. Hence, it is highly desirable to develop simple and sensitive fluorescent method for the assay of protease activity by employing a natural substrate in a label–free mode.

Trypsin is an important serine protease produced by the pancreas. It promotes the pancreatic proenzymes into the active forms and controls pancreatic exocrine function. A deficiency in the trypsin level can cause pancreatic diseases such as meconium ileus and hereditary pancreatitis [13]. The development of simple and convenient method for trypsin activity assay and its inhibitor screening has therapeutic implications for these pancreatic diseases. Cytochrome c is a highly water–soluble hemeprotein widely found in plants, animals, and unicellular organisms [14]. As an electron transfer protein, cytochrome c can act as an efficient fluorescence quencher through the electron transfer between the fluorophore and the heme moiety of cytochrome c [15]. Cytochrome c could be selectively cleaved by trypsin into smaller fragments on the C–terminal side of arginine and lysine residues [16]. The isoelectric point (pI) of the heme–peptide fragment formed (7.0) was significant different with that of cytochrome c (pI 10.2) [17]. This difference renders the scientists the opportunity to develop fluorescent method for trypsin detection by using cytochrome c as the natural substrate, in which cytochrome c acts as a fluorescent quencher [18], [19], [20], [21], [22]. In the presence of trypsin, cytochrome c could be degraded into the heme–peptide fragment in the form of Cys–Ala–Gln–Cys–His–Thr–Val–Glu–Lys–heme at pH 8.9 [23], [24]. Zhang et al. [25] and our previous work [26] indicated that the formed heme–peptide fragment had peroxidase–like activity and could catalyze the oxidation of organic molecule, such as luminol and 3,3′,5,5′–tetramethylbenzidine, in the presence of hydrogen peroxide (H₂O₂). These properties have been explored to develop chemiluminescent and colorimetric sensor for the detection of trypsin.

The main aim of the present work is to develop a fluorescent sensor for trypsin assay by using peroxidase–like activity of heme–peptide fragment. Scheme 1 depicts the schematic diagram of this strategy for fluorescent sensing of trypsin. Thiamine, a fluorescent substrate of peroxidase [27], was employed as the model substrate of heme–peptide fragment. As expected, the heme–peptide fragment exhibited an intensively catalytic role on the H₂O₂–mediated oxidation of thiamine to produce strong fluorescent thiochrome. This fluorescent sensor has several advantages. Firstly, cytochrome c was used as a natural enzyme substrate. Thus, no specific peptide chain is synthesized, making the operation more simple and convenient. Secondly, no label is required on the substrate, which would not only reduce the cost, but also improve the sensitivity. This sensing system has been successfully applied to trypsin inhibitor screening, demonstrating its potential application in drug development.

Section snippets

Apparatus

Fluorescence measurement was carried out on a F–2700 spectrofluorometer (Hitachi, Japan) with slit widths of both 5 nm for excitation and emission, respectively. The hydrolysis of cytochrome c by trypsin was incubated at 37 °C in the DF–101S constant temperature heating magnetic stirrer (Zhengzhou Kefeng Instrument Equipment Co. Ltd., China). The pH of the buffer solution was measured with a PB–10 pH meter (Sartorius Scientific Instrument Co. Ltd., Germany).

Chemicals

All chemicals were of analytical grade,

Proof–of–principle of assay strategy

As shown in Fig. 1, curve a, H₂O₂ hardly oxidized thiamine to produce fluorescent thiochrome in the absence of a catalyst and thus the fluorescence signal was very weak. Cytochrome c was previously reported to catalyze the oxidation of organic molecules in the presence of H₂O₂ [28], but it showed almost no catalytic effect on the oxidation of thiamine by H₂O₂ in this work. The fluorescence signal of thiamine–H₂O₂ system in the presence of cytochrome c showed no observable difference with that

Conclusion

In summary, we developed a label–free, turn–on fluorescent sensor for trypsin activity assay by using cytochrome c as the natural substrate. The hydrolysate of cytochrome c by trypsin, heme–peptide fragment, exhibited strong catalytic activity on the H₂O₂–mediated oxidation of thiamine to strong fluorescent thiochrome. The sensor had better selectivity for trypsin detection and allowed the quantitative determination of trypsin from 0.5 μg/mL to 20.0 μg/mL. Most importantly, this sensor has been

Acknowledgment

The work was supported by the Fundamental Research Funds for the Central Universities (Grant No.: GK201603029).

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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.

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Label–free, turn–on fluorescent sensor for trypsin activity assay and inhibitor screening

Highlights

Abstract

Graphical abstract

Introduction

Section snippets

Apparatus

Chemicals

Proof–of–principle of assay strategy

Conclusion

Acknowledgment

J. Biol. Chem.

Sens. Actuators B Chem.

Talanta

Biosens. Bioelectron.

Biochim. Biophys. Acta

Anal. Chim. Acta

Biosens. Bioelectron.

Arch. Biochem. Biophys.

J. Biol. Chem.

J. Biol. Chem.

Functional nanoprobes for ultrasensitive detection of biomolecules

Chem. Soc. Rev.

Proteolytic enzymes, past and future

Proc. Natl. Acad. Sci. USA

Targeting proteases: successes, failures and future prospects

Nat. Rev. Drug Discov.

Proteases as therapeutics

Biochem. J.

Proteases and disease

Proteom. Clin. Appl.

Electrochemical determination of trypsin using a heptapetide substrate self–assembled on a gold electrode

Microchim. Acta