1. Introduction
Proteases play an unprecedented role in our everyday lives by enabling and facilitating numerous essential biochemical processes. Beside human metabolic pathways, where three main proteases including trypsin, chymotrypsin, and pepsin are produced, they are also indispensable in immune functions, as well as cell division, blood clotting, and protein recycling processes [
1]. By monitoring their activities, some complex biochemical actions inside the cells have been clarified and explained.
As the functionalities of particular proteases are usually interconnected, simultaneous detection of more than one enzyme species might be necessary for the unambiguous elucidation of some metabolic mechanisms or the detection of known processes connected with proteolytic cascades [
2,
3,
4]. Although a broad scope of tools and applications [
5,
6] for sensitive and selective in vitro, as well as in vivo detection of different proteases has already been reported, individual techniques could be rarely combined for the purpose of synchronous detection of diverse enzymes. Various uptakes due to different pH of media, incubation parameters, as well as mutual undesired interactions causing spectral interferences are just a few of the potential drawbacks that one might frequently have faced.
Even though, some fluorescence based sensing techniques (e.g., Förster resonance energy transfer—FRET) enable detection of more than one enzyme species at the same time, they have mostly been used for single enzyme screening [
7,
8,
9]. Consequently, only a few examples of multi-dye fluorescent probes for simultaneous monitoring of various caspases [
10,
11], caspases in combination with metalloproteinases [
12,
13], or various proteases [
14] have been reported until now.
While soluble protease probes are predominant in assay kits, as well as abundantly described in the literature, their immobilized analogues have been reported only sporadically. Two such studies, dealing with fluorescently labelled proteins covalently bound to a solid support, were published by Trzcinska et al. [
15,
16]. Although, the authors highlighted efficiency, simplicity, and potential versatility of developed sensors, they were constructed in a way to monitor only one protease at a time.
Considering various surfaces suitable for the attachment of peptide probes, superficial modification of carriers with poly(ethylene glycol) spacers seems to be one of the primary choices [
17]. On the other hand, different materials including gold nanoparticles (AuNPs) have been used as solid supports as well. Despite the fact that reported AuNP based sensors [
18,
19] enable simultaneous sensing of different proteases, the availability of materials for flat applications with respect to reasonable price remains challenging.
Immobilized peptides were also used as protease substrates in assay kits comprised of liquid crystals. The enzymatic disruption of the peptide chains resulted in a homeotropic orientation of LCs, and a consequent change of optical signals. Although the authors demonstrated very low detectable concentrations of trypsin and chymotrypsin, the system was unable to distinguish between both proteases [
20].
Notwithstanding the above-mentioned examples of the solid support-based multi-enzyme detection, the complexity and sophistication of some of the already established conceptual models could partially or even completely suppress their larger adoption and practical implementation. Therefore, the idea of immobilized probes aimed at simultaneous detection of various proteases still requires fundamental innovations and further development.
Here we report a new concept for synchronous detection of two model proteases in a relatively wide concentration range. Our main focus was to develop a robust and reliable methodology that is simple to use and requires no advanced equipment, skills, or techniques. Due to its flexibility, availability, and scalability, the presented sensor herein might be of great added value in various scientific fields including biochemistry, biology, pharmacy, and medicine.
2. Materials and Methods
Solid-phase synthesis was performed on four different resins including Rink Amide PEGA resin (0.35 mmol/g, Novabiochem, Germany), TentaGel XV RAM resin (0.22 mmol/g, Rapp Polymere GmbH, Tübingen, Germany), H-Rink amide ChemMatrix resin (0.40–0.60 mmol/g, Merck KGaA, Darmstadt, Germany) and Polystyrene Wang resin (0.9 mmol/g, AAPPTec, Louisville, KY, USA). The chemicals and solvents were obtained from the available commercial sources. 7-Diethylaminocoumarin-3-carboxylic acid (DEAC) was synthesized according to a literature procedure [
21].
2.1. Synthesis and Evaluation of Immobilized Peptides
The appropriate solid support was first placed into 5 or 10 mL plastic syringe (B. Braun Melsungen AG, Melsungen, Germany) equipped with a sintered plastic filter (Torviq, Tucson, AZ, USA), then prewashed with dichloromethane (5 times), and subsequently reacted with suitable reagents. Resins with Rink linker were pretreated with 50% piperidine in DMF before the first reaction step. The transformations were carried out on a Titer Plate Shaker (Thermo Fisher Scientific, Waltham, MA, USA), while after completion of the reactions, the resins were washed with dimethylformamide (10 times) and dichloromethane (10 times), using a Domino Block (Torviq, Niles, MI, USA) and a vacuum pump (KNF Neuberger Inc., Trenton, NJ, USA). The complete synthetic pathway is presented in the
Supplementary Materials (Figures S1 and S2).
After each reaction step, a small portion of embedded peptide was chemically cleaved from the carrier using 50% trifluoroacetic acid (TFA) in dichloromethane (DCM). Volatile liquids were evaporated under a stream of nitrogen, while the resulting sticky residue was treated with acetonitrile, then diluted with ultrapure water (Mili-Q Water Purification System, Progard T3, Merck Millipore, Darmstadt, Germany), filtered, and subsequently analyzed by LC-MS carried out on an UHPLC chromatograph (Acquity), using X-select C-18 column (Waters, Borehamwood, UK). Ten millimolar ammonium acetate in ultrapure water and acetonitrile (gradient 20–80% during the first 3 min) was used as a mobile phase, while its flowrate was set to 600 µL/min. Detection was performed by a photodiode array detector (Waters, UK) and a single-quadrupole mass spectrometer (Waters, UK). Characterization of the intermediates can be found in the
Supplementary Materials (Figures S3–S12).
2.2. Loading, Yield Determination, and Storage (Rink Amide PEGA Resin)
After the first reaction step (binding of PEG spacer), the precisely weighed amount of approximately 100 mg of the resin was treated with 50% TFA in dichloromethane (5 × 5 mL). Fractions of the cleaved compound were joined and the volatile liquids were evaporated under a stream of nitrogen. The sticky residue was dissolved in deuterated DMSO and an NMR spectrum using an internal standard (2-propanol) was measured for the purpose of compound quantification. The residual solid support left over after TFA cleavage was intensively washed with DCM (10 times) and diethyl ether (10 times), subsequently dried under a stream of nitrogen, and finally weighed. Taking into account the quantity of cleaved compound, as well as the mass of dried resin, a loading of 0.20 mmol/g was established.
The final trypsin and chymotrypsin probes anchored to Rink Amide PEGA resin were also subjected to the above described procedure of loading determination. Considering a final loading of 0.11 mmol/g for the former and 0.15 mmol/g for the latter, the corresponding probes were synthesized in good to very good overall yields of 55% and 75% after 11 and 13 reaction steps, respectively (
Figures S1 and S2). The probable cause of the considerably lower yield for the trypsin linker in comparison with the chymotrypsin could potentially have originated from the use of fluorinated chemicals during removal of the 4-methyltrityl protecting group from the lysine sidechain, and consequent damage of solid support.
The final resin-anchored probes were intensively washed with DCM (5 times), then dried under a stream of nitrogen until constant weight, and finally stored at −80 °C.
2.3. Characterization of the Probes
For the purpose of characterization, the trypsin probe TP (substrate for trypsin) was synthesized on Polystyrene Wang resin, while TentaGel XV RAM resin was utilized for the chymotrypsin probe CP (substrate for chymotrypsin) preparation. The individual compounds were then chemically cleaved from the solid support using a few portions (5 × 5 mL) of 50% trifluoroacetic acid (TFA) in DCM. The obtained fractions were combined and the volatile liquids were evaporated under a stream of nitrogen, providing sticky residue that was further dissolved in acetonitrile (4 mL), diluted with ultrapure water (6 mL), and filtered through a syringe filter (0.22 µm, HPST, Prague, Czech Republic). The resulting sample (10 mL) was purified on reverse-phase C-18 semi-preparative HPLC column (YMC—Actus Pro 12 mm S—5 µm 100 × 20 mm, Kyoto, Japan) with a gradient of 10 mM solution of ammonium acetate in ultrapure water and acetonitrile, using a flowrate of 15 mL/min. The collected fractions were combined and concentrated in vacuo using a rotary evaporator (Buchi R-215 Rotavapor, Marshall Scientific, Flawil, Switzerland), while the aqueous solution of ammonium acetate was removed by freeze-drying on lyophilizer (ScanVac Coolsafe 110-4, LaboGene, Lillerød, Denmark). The obtained dry colorful powder was characterized using LC-MS, NMR (ECX500, JEOL Resonance, Tokyo, Japan) and HRMS (Dionex Ultimate 3000, Orbitrap Elite high-resolution mass spectrometer, Thermo Fisher Scientific, Waltham, MA, USA) analyses. Because of stability reasons, the isolated compounds were kept in a freezer (Thermo Fisher Scientific, Waltham, MA, USA) at −80 °C.
The quantum yields of the synthesized probes (
Figures S13 and S16) were calculated by the standard procedure (Recording Fluorescence Quantum Yields—HORIBA Scientific) using fluorescein in 0.1 M NaOH and Rhodamine B in distilled water as references for the trypsin probe (
TP) and the chymotrypsin probe (
CP), respectively. The fluorescence measurements were performed with a Cary Eclipse Fluorescence Spectrophotometer (Agilent Technologies, Santa Clara, CA, USA).
2.4. Biological Testing
2.4.1. Reconstitution of Enzymes
Both, trypsin (bovine pancreas, TPCK treated, ≥10,000 BAEE units/mg protein) and α-chymotrypsin (bovine pancreas, TLCK treated, type VII, ≥40 units/mg protein) were ordered from Sigma-Aldrich (Steinheim, Germany) in the form of lyophilized white powders. The appropriate amount of individual enzyme was weighed into a 1.5 mL Eppendorf safe-lock plastic tube (Hamburg, Germany), reconstituted in 1 mM HCl in ultrapure water, properly aliquoted, and stored at −80 °C. The content of a particular Eppendorf tube with reconstituted enzyme was used within a few hours after its first defrosting.
2.4.2. In-Solution Enzyme Assays
Isolated probe was dissolved in 0.1 M Tris-HCl buffer (1.8 mL, pH = 8.0), and then the appropriate aliquot of the corresponding enzyme reconstituted in 1 mM HCl (200 µL) was added. The enzymatic cleavage was monitored by LC-MS. In all cases, the corresponding masses of cleaved fragments were unambiguously confirmed (
Figures S19–S22).
2.4.3. On-Resin Enzyme Assays
The process of on-resin enzyme testing is presented in
Figure 1. The corresponding dried resin (2.0–2.1 mg) was weighed into 2 mL Eppendorf safe-lock plastic tubes using an analytical balance (XPE26, Mettler Toledo, Maharashtra, India). Afterwards, the appropriate volume (900 µL for the dye releasing studies; 800 µL for all other experiments) of Tris-HCl buffer was added (
Figure 1A) and a solid support was mechanically crushed into smaller bits using a spatula (
Figure 1B). In the next step, a suitable volume (100 µL for the dye releasing studies; 200 µL for all other experiments) of 1 mM HCl in ultrapure water, with or without enzyme was added (
Figure 1C). The obtained heterogeneous system was shaken (ν = 210 min
−1) on a horizontal shaker (Benchmark Scientific, Sayreville, NJ, USA) during the incubation in the incubator (Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C (
Figure 1D). After incubation was completed, the content of an Eppendorf tube was sucked up into a syringe (
Figure 1E), and subsequently filtered into a single-use plastic fluorimeter cuvette (Sigma-Aldrich, Milano, Italy) (
Figure 1F). Finally, the fluorescence response of the sample prepared in this way was measured with a Cary Eclipse Fluorescence Spectrophotometer (Agilent Technologies, Santa Clara, CA, USA).
All fluorescence data were obtained in three independent parallels, while for each individual set of measurements, an average value as well as the standard deviation were calculated and are graphically presented.
2.5. Stability Testing of Resin-Anchored Probes
To examine the stability of freshly synthesized materials, suitable dried resin (2.0−2.1 mg) was weighed into several Eppendorf tubes. Some parallels were stored at −80 °C, while the others were kept at laboratory temperature in darkness. After the appropriate time period, Tris-HCl buffer (800 μL) and 1 mM HCl (200 μL) were added to the individual tubes and the samples obtained in this way were then incubated at 37 °C for 15 min. After completion of the incubation process, the residual solid support was filtered out and the fluorescence response of the released fluorophore in the resulting filtrate was measured.
4. Conclusions
In conclusion, a methodology for synchronous detection of two model proteases, namely trypsin and chymotrypsin, based on single excitation–double emission fluorescence-monitored enzymatic cleavage of target resin-bound peptides, was successfully established and verified. Among all the examined solid supports, Rink Amide PEGA resin was recognized as the best matrix for probe immobilization and subsequent implementation of on-resin biological assays. The developed methodology was found reliable for unambiguous detection of a single or both enzymes in a mixture, throughout the specified concentration range. Taking into account the simple and relatively inexpensive multi-step synthesis as well as the high purity (>90%) of the final materials, the presented approach, herein, could be easily scaled-up, potentially verified by simple modification of the amino acid sequences, and finally applied to selective detection of different proteases.