Ultrasensitive fluorescent detection of trypsin on the basis of surfactant–protamine assembly with tunable emission wavelength

Abstract

A facile and ultrasensitive fluorometric assay for trypsin detection was successfully established on the basis of surfactant/protamine/fluorescent hydrophobic dye assemblies. The supramolecular micellar-type assemblies were obtained by mixing the positively charged protein protamine as the substrate of trypsin with the anionic surfactant sodium dodecyl sulfate (SDS) at a level lower than its critical micelle concentration (CMC), and their apolar interiors could sequester several types of the representative hydrophobic fluorescent dyes such as Nile red, pyrene, and coumarin 6. It was found that the supramolecular assemblies exhibited a high specificity towards trypsin and its stability had a significant effect on the fluorescent intensity of the sequestered dyes. The addition of trypsin into the assembly system led to the dissociation of the assemblies and the decrease of the fluorescent intensity of the dye, which was exploited for trypsin detection and inhibitor screening. A detection level down to 0.044 ng mL⁻¹ was obtained for trypsin. Moreover, the emission wavelength and detection range could be tuned by simply varying the type of fluorescent dye.

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Introduction

Trypsin is an important digestive enzyme produced by the pancreas; the enzyme promotes other pancreatic proenzymes to their active forms and controls pancreatic exocrine function.¹ It is also employed in proteomics, particularly in mass spectroscopy (MS)-based proteomics,^2,3 to degrade proteins into peptides. Trypsin deficiency or mutated trypsin can cause pancreatic diseases, such as meconium ileus and hereditary pancreatitis.^4,5 The clinical relevance of trypsin for the diagnosis and treatment of diseases has drawn considerable attention to the rapid detection and activity monitoring of this enzyme.

Numerous methods for trypsin detection are available, including enzyme linked immunosorbent assay,⁶ MS,⁷ gel electrophoresis,⁸ radioimmunoassay,⁹ electrochemical assay,¹⁰ quartz crystal microbalance (QCM) sensing,¹¹ surface-enhanced Raman scattering (SERS),¹² and optical detection using nanomaterials^13–15 and organic fluorescent probes.^16–18 Among them, the fluorescent approach has attracted more attention because of its low-cost, operational simplicity, high sensitivity, and real-time detection. This technique is somewhat limited by the fluorogenic substrates, usually the labeled molecular beacons or peptides, which were expensive and difficult to synthesize.

Self-assembling and self-organizing methodologies have been extensively used to construct the fluorescent chemosensors.¹⁹ Following these strategies, the subunits can be separately designed and synthesized, and then easily combine to produce the sensing system. The assay of trypsin activity has been achieved by integrating different substrates with signal-enhancing reporters (e.g., aggregation-induced emission (AIE) molecules,^20,21 conjugated polymers,^22–24 quantum dots^25,26 or fluorescent dye derivatives^16–18) through non-covalent interaction. These popular methods are simple, flexible and sensitive; however, the preparation of these fluorescent reporters still involved laborious synthesis procedures.

Supramolecular assemblies from surfactants are of interest because of their container properties in aqueous solutions.²⁷ Depending on the micellar form template provided by surfactant, the fluorescent reporter and the substrate do not interact directly and the communication between them is ensured only by their spatial closeness.¹⁹ The metal ions^27,28 and proteins pattern sensing^29,30 have been realized based on the surfactants–ligands and surfactants–polymers assemblies, respectively. However, there are few reports related to enzyme assaying.

In the present study, a facile fluorometric system was constructed for trypsin activity assay and inhibitor screening. The design rationale is illustrated in Scheme 1. The positively charged protamine was designed not only as the substrate of trypsin, but also as the building block of the supramolecular assemblies. Protamine would form micellar-type assemblies with anionic surfactant sodium dodecyl sulfate (SDS) at a level lower than the critical micelle concentration (CMC). The combination should provide supramolecular assemblies with apolar interiors that could sequester hydrophobic dye (Nile red, pyrene, or coumarin 6) molecules. The hydrophobic dye was used as the reporter and converted binding events into fluorescent signals.

Scheme 1 Scheme of the SDS/protamine/dye assembly and the disassembly induced by trypsin-catalyzed hydrolysis.

The hydrolysis of protamine into short peptides was facilitated by trypsin addition; consequently, the SDS/protamine/dye assemblies disassembled, and the fluorescence intensity reduced obviously as the hydrophobic dye was released. In the presence of inhibitors, protamine hydrolysis would be retarded and a slight fluorescence intensity decrease would be observed. Thus, the assembly system could also be used to screen the inhibitors of trypsin.

This supramolecular assembly fluorescence system shows several advantages: first, the realization and optimization of florescent sensing system are quite easy to realize. Second, the present design strategy exhibits a wide applicability to various targets. The system could be applied to fabricate new sensors that target different molecules by replacing the receptor units with the appropriate surfactant. Third, a variety of fluorophores could be used to work as the fluorescent reporter. Thus, the emission and the detection wavelengths of the sensing system are tunable.

Materials and methods

Materials

Trypsin (1: 2500) from porcine pancreas, SDS, protamine sulfate from salmon, bovine serum albumin (BSA), papain, pepsin and the trypsin inhibitors from soybean were purchased from Sangon Biotech Co., Ltd (Shanghai, China). Nile red (NR), coumarin 6 and pyrene were provided by Sigma-Aldrich (U. K.). Peptide Arg₈ was obtained from GL Biochem (Shanghai) Ltd (China). All reagents were of analytical grade, and solutions were prepared using Milli-Q water (electric resistance > 18.3 MΩ). Enzyme assays were performed in Tris–HCl buffer solution (10 mM, pH = 8.0).

CMC of SDS

To determine the CMC of SDS, SDS was dissolved in Tris–HCl buffer to obtain a series of solutions with concentrations from 5 × 10⁻⁶ M to 1.5 × 10⁻² M. The hydrophobic dye was dissolved in acetone and then added to each of the above SDS solutions to a final concentration of 1 μM. The mixtures were sonicated for 30 min to equilibrate the dye and the micelles and evaporate the acetone. Finally, 2 mL of the mixtures was transferred to a cuvette for fluorescence experiments.

Preparation of SDS/protamine/dye assembly

To optimize its concentration, protamine was dissolved in Tris–HCl buffer to obtain a series of solutions, and then a fixed concentration of SDS was mixed with the above-mentioned protamine solutions to a final protamine concentration of 5 × 10⁻⁶ to 1 mg mL⁻¹. Dye encapsulation and fluorescence measurements were conducted in accordance with a similar process to determine the CMC of SDS.

Trypsin detection with assembly system

The assembly system was composed of 0.3 mM SDS, 60 μg mL⁻¹ protamine, and 1 μM NR. The pH exerted a negligible effect on the fluorescence intensity of the supramolecular assemblies (Fig. S1†). As reported previously,²⁰ the optimal pH for analyzing trypsin activity is approximately 8.0. Thus, the pH was set to 8.0 in our assay.

For trypsin activity assay, different amounts of trypsin (1 × 10⁻⁸ to 0.05 mg mL⁻¹) were added to the solutions of supramolecular assemblies and the fluorescence intensities of the mixture were measured every minute during hydrolysis (up to 30 min).

Other proteins, including BSA, papain, pepsin and lysozyme, were also tested under the same conditions to investigate the selectivity of the system towards trypsin.

Fluorometric inhibitor screening. Inhibitors were pre-incubated at varying concentrations (0.005, 0.01, 0.02, 0.03, 0.04, 0.05, and 0.06 mg mL⁻¹) with trypsin (50 μg mL⁻¹) in Tris–HCl buffer solution at 37 °C for 10 min. The mixtures were added to the supramolecular assembly solutions, and the resulting solutions were incubated at 37 °C for 30 min. Fluorescence spectra were obtained, and the fluorescence intensities at 630 nm were monitored.

Inhibition efficiency was estimated as follows:

Inhibition efficiency = (F_i − F_n)/(F₀ − F_n) × 100

where F_i and F_n refer to the fluorescence intensity at 630 nm of solutions containing SDS, protamine, and trypsin in the presence of different inhibitor concentrations and in the absence of inhibitors, respectively. F₀ refers to the fluorescence intensity of the assembly at 630 nm in the absence of trypsin and inhibitors.

Characterizations

Fluorescence emission spectra were recorded on a Fluoromax-4 spectrometer (HORIBA Scientific, USA) (λ_ex = 545 nm for NR, λ_ex = 339 nm for pyrene, and λ_ex = 440 nm for coumarin 6, respectively). All measurements were conducted in triplicate and the average of such values was reported. Transmission electron microscopy (TEM) was performed on a JEM-1200EX (JEOL, Japan) operating at 80 kV in bright-field mode.

Results and discussion

Optimization of SDS/protamine/dye assembly system

The CMC of SDS was determined by a fluorescence technique using NR as a probe. At concentrations near or above the CMC of the surfactant, the emission maximum for NR was centered upon 630 nm (Fig. S2†). Plotting the emission peak intensity at 630 nm as a function of logarithm surfactant concentration yielded a sigmoidal curve (Fig. 1). The CMC of SDS in Tris–HCl buffer was measured to be 0.93 mM by the intersection point. The measured CMC of SDS in Tris–HCl buffer was lower than the reported CMC value in water (∼8.3 mM (ref. 31)), which may be ascribed to environmental conditions, such as pH or ionic strength. The concentration below CMC (0.3 mM) was selected for the following experiment.

Fig. 1 Plot of the I₆₃₀ of NR emission spectra as a function of the logarithm concentration of SDS in Tris–HCl buffer (10 mM, pH = 8.0).

The critical aggregation concentration (CAC) of surfactants decreases when polyelectrolytes act as counterions.^29,30 Protamine is a highly cationic protein that possesses a high content of basic amino acids, about 50% of which are arginine residues. This attribute renders protamine an attractive and widely used substrate for trypsin-like serine proteases,^10,14,20 cleaving polypeptide mainly at the carboxyl side of lysine or arginine. In the present study, protamine was used as the substrate of trypsin, which could form assemblies with SDS. The fluorescence property of protamine itself with NR was investigated. The peak intensities of the protamine solutions remained about the same with increasing protamine concentration (Fig. 2). This result indicates that protamine would not form assemblies or aggregation by itself.

Fig. 2 Plot of the I₆₃₀ as a function of logarithm concentrations of protamine in SDS–protamine and protamine solutions. The inset is the TEM image of the assemblies.

To obtain the assemblies of SDS–protamine and avoid the formation of SDS micelles, the concentration of SDS was set to lower than its CMC (e.g., 0.3 mM). Interaction between protamine and SDS were demonstrated by fluorescence enhancement and TEM images. When protamine was added to the SDS–NR solution, the fluorescence emission gradually increased with increasing protamine concentration. This result indicates that the supramolecular assemblies of SDS–protamine may be produced through electrostatic interaction (Fig. 2). TEM images show the presence of spherical nanoparticles with a diameter of ∼70 nm in the SDS–protamine solution (inset in Fig. 2). This result confirms the formation of micellar-type assemblies. To obtain stable assemblies, the critical concentration of protamine was determined to be 0.06 mg mL⁻¹ from the second inflection point. The supramolecular assembly system composed of 0.3 mM SDS, 60 μg mL⁻¹ protamine and 1 μM NR was selected for the following examination.

Protamine could be enzymatically cleaved into fragments with decreased charge and molecular weight. Hence, protein length may be a key factor contributing to supramolecular assembly formation. Oligo–polyarginines (such as Arg₅,³² Arg₆,¹³ or Arg₈ (ref. 33)) could also be used as the substrate of trypsin. In this study, the coassembly behavior of SDS and the arginine-rich peptide Arg₈ also was tested.

In contrast to the peak intensity of 0.3 mM SDS–NR, the peak intensity of the SDS/Arg₈/NR solution enhanced by about 10 fold when the concentration of Arg₈ exceeded 0.2 mg mL⁻¹, whereas that of the SDS/protamine/NR solution enhanced by about 16 fold with the same concentration of protamine (Fig. S3†). Compared with the SDS/protamine/NR system, the SDS/Arg₈/NR system showed less fluorescence increases. In addition, the critical concentration of Arg₈ was increased to 0.2 mg mL⁻¹, which corresponded to 0.3 mM SDS.

The peptide Arg₈ was less efficient than protamine in promoting the formation of supramolecular assemblies possibly because of the shorter chain of the former than the later peptide. However, Arg₈ demonstrated a remarkable fluorescence enhancement effect. Whether Arg₈ could be used as a receptor unit needs further verification.

Fluorometric trypsin activity assay

To ensure the feasibility of the supramolecular assemblies of SDS/protamine/NR for trypsin activity assay, Fig. 3a shows the fluorescence spectral changes of supramolecular assemblies as a function of trypsin catalytic hydrolyzing time. Upon the addition of trypsin (0.05 mg mL⁻¹) to the supramolecular assemblies and equilibration at 37 °C, the fluorescence intensity of the assemblies gradually decreased as the hydrolysis reaction time was prolonged and then leveled off after 30 min of incubation. The peak emission intensity of the assembly system decreased by approximately 90%. This phenomenon could be qualified for fluorescence reporting.

Fig. 3 (a) Time-dependent fluorescence quenching of SDS/protamine/NR assemblies in the presence of trypsin (0.05 mg mL⁻¹). Inset: photographs were taken upon UV lamp excitation at 365 nm. (b) Quenching efficiency of assemblies as a function of trypsin concentration. The inset corresponds to the linear plots of the detection.

To clarify the contribution of electrostatic interaction between protein and SDS/protamine/NR to fluoresce quenching, control experiments were conducted. The fluorescence emission spectra of NR and SDS/NR responding to various protein with different pI values, including BSA (pI 4.7–5.3), lysozyme (pI 11), pepsin (pI 1), papain (pI 8.75), protamine (pI 10–12) and trypsin (pI 10.5), were investigated. All of the fluorescence intensities were quite low in protein/NR systems (Fig. S4a†) except that BSA induced certain fluorescence enhancement, this might be ascribed to the fact that the hydrophobic pocket of BSA could sequester NR and thus the fluorescence intensity increased.³⁰ Positively charged lysozyme induced obvious enhancement as protamine (Fig. S4b†), which might due to the electrostatic interaction as these two proteins had similar pI values, and papain induced slightly increment with lower pI value. However, trypsin induced no changes in the fluorescence emission of SDS–NR. Hence, electrostatic binding was not the sole driving force for the formation of assembly, the structure of protein might also play important role.

We hypothesized that the emission quenching originated from trypsin-trigged disassembly, which were studied by ζ-potential and TEM. The ζ-potential decreased from negative (−15.7 mV) to nearly neutral (−3.91 mV) as the protamine concentration increased (Table S1†). After enzyme treatment, the ζ-potential was found to be −16.6 mV, close to the value of SDS, suggesting the dissociation of SDS/protamine/NR assembly. In addition, trypsin cleavage-induced disassembly was also confirmed by TEM images, wherein no self-assembly particles were observed after trypsin digestion (Fig. S5†).

As shown in Fig. 3b, the quenching efficiency of the assemblies at 630 nm obviously increased with increasing trypsin concentration until a plateau was reached after 1 μg mL⁻¹. The inset is a calibration curve that corresponds with the analysis of trypsin. A linear relationship between quenching efficiency and trypsin concentration was observed over the enzyme concentration of 0.01–0.1 ng mL⁻¹. The limit of detection (LOD) was calculated to be 0.044 ng mL⁻¹ (S/N = 3). A comparison between the proposed method and other reported methods for trypsin determination in detection limit and linear range was summed up in Table 1. It could be seen from Table 1 that the sensitivity of this proposed method was the lowest among the best trypsin assays reported.^{12,14,17,18,34}

Table 1 Comparison of the analytical data of some reported methods for the determination of trypsin

Method	Linear range	Detection limit	Reference
Electrochemistry	5–150 ng mL^−1	1.8 ng mL^−1	10
SERS	0.1–10 000 ng mL^−1	0.1 ng mL^−1	12
Colorimetric	—	1.6 ng mL^−1	14
Fluorimetry	0.1–1000 ng mL^−1	0.048 ng mL^−1	15
Fluorimetry	—	0.5 μg mL^−1	16
Fluorimetry	0–60 mU mL^−1	0.5 mU mL^−1	17
Fluorimetry	0.005–2 nM	0.006 nM	18
Fluorimetry	1.25–375 nM	0.42 nM	25
Fluorimetry	0.01–100 μg mL^−1	2 ng m^−1 (86 pM)	34
Fluorimetry	0.01–0.1 ng mL^−1	0.044 ng mL^−1	This work

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Protamine could form assemblies with different kinds of reporters, such as organic fluorescent probes (pyrene derivative,¹⁶ benzoperylene,¹⁷ perylene¹⁸), nanoclusters (DNA hosted Cu nanoclusters¹⁵) and quantum dots,²⁶ via electrostatic interactions to monitor the trypsin activity. Compared with these reports, our system achieved a lower or comparable detection limit without synthetic process.

The fluorescent response was examined using Arg₈. Interestingly, no significant fluorescence quenching (∼10%) was observed when 0.05 mg mL⁻¹ trypsin was added to the SDS–Arg₈ solution (Fig. S6†). The SDS/Arg₈/NR system was not practical for trypsin activity sensing. In some studies, oligo–polyarginines (such as Arg₅,³² Arg₆ (ref. 13) or Arg₈ (ref. 33)) were utilized to assay trypsin enzymatic activity as the substrate of trypsin with conjugated polymers or AIE molecules. Moreover, the five-mer peptide (i.e., Arg₅) was reported to be of critical length for trypsin-catalyzed hydrolysis with anionic conjugated polyelectrolyte (PPESO₃).²³ In our system, however, although Arg₈ was highly positive and could electrostatically interact with SDS, the short peptide chain rendered Arg₈ inefficient at sensing trypsin. These results indicate the polymer-analogue structure of the protamine is indispensible in our sensing system.

To demonstrate the potential of the SDS/protamine/NR system in monitoring trypsin activity in real time, we studied the fluorescence quenching of the assembly system at 630 nm as a function of trypsin concentration (Fig. 4). A gradual fluorescence decrease dependent on trypsin concentration was observed.

Fig. 4 Real-time fluorescence intensity changes of SDS/protamine/NR assemblies at different trypsin concentrations.

Fluorescent sensing systems with tunable detection wavelengths might be desirable for specific use.³⁵ Commonly used fluorescence probes pyrene and coumarin 6 (ref. 13 and 14) were selected as the reporter unit for this study. Consistent with the use of NR as the probe, both of the fluorescence intensities of the assemblies with pyrene and coumarin 6 gradually decreased with increasing trypsin concentration (Fig. S7†). The emission maxima for coumarin 6 and pyrene were 502 nm and 391 nm, respectively. Compared with that of NR, the linear range of determination of trypsin widened with pyrene and coumarin 6 from 0.01 ng mL⁻¹ to 0.5 ng mL⁻¹ for both dyes (Fig. 5). Pyrene and coumarin 6 could achieve good detection sensitivity, as evidenced by the respective LODs of 0.09 ng mL⁻¹ and 0.48 ng mL⁻¹, which were higher than that of NR.

Fig. 5 Calibration curves corresponding to the analysis of trypsin with different fluorescent dyes.

These results demonstrate that the modular nature of the assemblies enabled the tuning of the emission wavelength and detection range by simply varying the type of reporter in the disassembly-based sensing. With this strategy, the preparation and optimization of the fluorescent system were easily realized. All three components of the assemblies could be naturally changed, and an optimum combination for specific use exists. For example, pyrene and coumarin 6 were inferior to NR as the fluorescent reporter, and Arg₈ was a less efficient substrate than protamine. The surfactant also played a significant role; further work is now under way along these lines.

Fluorometric inhibitor screening

Developing rapid and simple methods for screening chemical libraries of potential trypsin inhibitors is important in the pharmaceutical industry. The developed self-assembly system was also used to study the inhibition of trypsin activity with inhibitors from soybean. Emission intensity was obviously much higher in the system with inhibitors than in the reference system without inhibitor (Fig. 6a). The inhibition ability of inhibitors was described by an IC₅₀ value, which refers to the inhibitor concentration required for 50% inhibition of enzyme activity. On the basis of the plot of the inhibition efficiency versus inhibitor concentration, the IC₅₀ value of the inhibitors toward trypsin was estimated to be 0.02 mg mL⁻¹ (Fig. 6b). The results showed the usefulness of this fluorometric assay for screening trypsin inhibitors.

Fig. 6 (a) Fluorescence emission spectra of SDS/protamine/NR assemblies, with trypsin, and with trypsin and inhibitor. (b) Plot of the inhibition efficiency of tacrine toward trypsin versus inhibitor concentration.

The quenching efficiencies of the supramolecular assemblies in the presence of other proteins, such as papain, pepsin, lysozyme and BSA, were recorded to investigate the selectivity of this fluorometric trypsin assay. The concentration of each enzyme was maintained at 0.05 mg mL⁻¹, and each solution was incubated for 30 min under the same conditions as those for trypsin. The peak emission intensity of the supramolecular assemblies at 630 nm was reduced largely only after trypsin addition (Fig. 7). The intensities were only slightly reduced or increased in the presence of other proteins. The sensing system exhibited a high specificity toward trypsin.

Fig. 7 Quenching efficiency of formed assemblies at 630 nm with different proteins. The concentration of each protein was 0.05 mg mL⁻¹.

Conclusions

We successfully constructed a simple biosensing platform for trypsin and its inhibitor screening with the supramolecular assemblies of SDS/protamine/dye. Protamine formed micellar-type assemblies with SDS at a level lower than the CMC. Organic synthesis procedure was avoided with the micellar form template which enabled the transduction mechanism between protamine and the dye. The enzyme-triggered disassembly was converted into fluorescence quenching, which allowed the ultrasensitive assay of trypsin with a LOD as low as 0.044 ng mL⁻¹. The small peptide substrates Arg₈ that have been used to characterize trypsin activity may not be as effective as protein substrate. The dynamic nature of the supramolecular assemblies enabled the tuning of the emission wavelength and detection range by simply varying the type of hydrophobic dye. Furthermore, this fluorometric assay may be used to screen trypsin inhibitors. This modular construction allowed substituting one component to satisfy a wide range of desired specifications.

Acknowledgements

We gratefully acknowledge the financial support from the Nature Science Foundation of China (Grant No. 51303124), Natural Science Foundation of Shanxi (Grant No. 2013021009-2), and International Scientific and Technological Cooperation Projects of Shanxi Province (Grant No. 2014081007-2).

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Footnote

† Electronic supplementary information (ESI) available: The fluorescence enhancement of SDS–protamine and SDS–Arg₈ with increasing concentrations of protamine and Arg₈ respectively; fluorescence emission spectra of SDS–Arg₈–NR and SDS–Arg₈–NR + trypsin; fluorescence emission spectra of different hydrophobic dyes in SDS/protamine assemblies. See DOI: 10.1039/c6ra19220c

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