Perylene

Article pubs.acs.org/ac

Label-Free Multimodal Protease Detection Based on Protein/ Perylene Dye Coassembly and Enzyme-Triggered Disassembly Yiyang Lin, Robert Chapman, and Molly M. Stevens* Department of Materials, Department of Bioengineering, and Institute for Biomedical Engineering, Imperial College London, Exhibition Road, London SW7 2AZ, United Kingdom S Supporting Information *

ABSTRACT: The development of novel assays for protease sensing plays an important role in clinical diagnostics and therapeutics. Herein, we report a supramolecular platform for label-free protease detection, based on protein/dye self-assembly and enzyme-triggered disassembly. In a typical case, coassembly of protamine sulfate and perylene dye via electrostatic attractions and π−π interactions caused significant colorimetric and fluorescent responses. Subsequent addition of trypsin was found to cleave the amide bonds of protein, triggering the dissociation of protein/dye aggregates and the release of perylene dyes. The enzyme-triggered disassembly was transduced into multiple readouts including absorption, fluorescence, and polarization, which were exploited for trypsin detection and inhibitor testing. This assay was also used for turn-on fluorescence detection of cathepsin B, an enzyme known to be overexpressed in mammalian cancer cells. The integration of supramolecular self-assembly into enzyme detection in this work has provided a novel label-free biosensing platform which is highly sensitive with multimodal readouts. The relative simplicity of the approach avoids the need for time-consuming substrate synthesis, and is also amenable to naked eye detection.

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always requires organic and bioconjugation chemistry to synthesize specific peptide substrates and attach molecular probes to the substrates. Therefore, the development of novel label-free approaches for sensitive protease detection is of great interest. To this end, we have designed a versatile biosensing platform for protease activity detection based on the noncovalent coassembly of protein and perylene bisimide. Protein selfassembly is of great fundamental interest to understand the physicochemical basis of protein−protein interactions and can be used to design new to novel biomaterials for disease diagnostics and therapy.13 However, the concept of integrating organic dyes into protein self-assembly has rarely been reported. On the other hand, perylene bisimide (PBI) derivatives have been extensively used as industrial pigments owing to photo/thermal stability, high fluorescence quantum yield, and inert chemistry.14 More recently, research into PBI derivatives has emerged in various optical and electronic applications including organic photovoltaic devices, field-effect transistors, and n-type semiconductors.14,15 Owing to the hydrophobic effect and π−π interactions, PBI derivatives are mostly water-insoluble and have a strong tendency to aggregate,16 which results in fluorescence quenching in aqueous

roteolytic enzymes or proteases play central roles in most biological processes owing to their ability to regulate the physiological functions of many proteins through initiating hydrolysis at the post-translational level. Aberrant activity of proteases is also central to major human diseases such as cardiovascular, oncologic, neurodegenerative, and inflammatory diseases.1−3 The screening of protease-targeted inhibitors is thus also of high clinical relevance to identify new therapeutic approaches for these diseases. For example, protease inhibitors against matrix metalloproteinases (MMPs) have been proposed to control tumor growth.4 Over the past decade, proteases have mostly been detected using immunoassays which involve antibody or affinity based detection,5 owing to the high sensitivity and specificity of this approach. However, the immunoassay format always requires a process of antibody identification/generation/isolation, as well as potentially complicated bioconjugation for antibody immobilization and fluorophore/chromophore labeling. In addition, such a format cannot be used to directly determine enzymatic activity, but only enzyme concentration. An alternate strategy for protease sensing is based on hydrolysis of the peptide substrate by the target protease. This approach can provide information about enzyme activity and is used in diagnostics and drug discovery.6−8 Assaying proteolytic activity can be diversified by combining short peptide substrates with different detection principles (e.g., fluorescence resonance energy transfer, FRET) and signal-enhancing reporters (e.g., fluorophores and chromophores).9−12 However, this method © XXXX American Chemical Society

Received: February 27, 2014 Accepted: May 27, 2014

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Figure 1. (a) Molecular structure of PBI-Asp; (b) UV−vis and (c) fluorescence spectra of 1.0 μM PBI-Asp solution in 5.0 mM phosphate buffer (pH 8.5). The fluorescence excitation wavelength was 490 nm.

filtered, washed with water and dried under vacuum at 80 °C to give the product.1H NMR (400 MHz, d6-DMSO,) δ: 12.87 (−COOH, 2H), 7.91 (−CH−, 4H), 7.77 (−CH−, 4H), 5.99 (−CH−, 2H), 3.38 (−CH2−, 2H), 2.90 (−CH2−, 2H). FTIR v/cm−1: 3525, 2939, 1750, 1695, 1635, 1590, 1571, 1508, 1435, 1401, 1362, 1341, 1302, 1255, 1172, 1132, 992, 960, 854, 809, 745. Sample Characterization. TEM micrographs were obtained with a JEOL 2000FX (working voltage of 200 kV) instrument by negative-staining method with uranyl acetate solution (1.0 wt %) as the staining agent. One drop of the solution was placed onto a carbon Formvar-coated copper grid for 3−5 min. The excess liquid was sucked away with filter paper. After this, one drop of the staining agent was placed onto the copper grid for 2−5 min. After removing the excess staining agent with filter paper, the sample was dried in air before TEM observation. Fluorescence spectra were recorded on Fluorolog-3 spectrofluorometer. UV−vis absorbance was measured on a PerkinElmer Lambda 25 UV−vis spectrometer with the path length of 1.0 cm. Fluorescence polarization was measured on SpectraMax M5 plate reader and was experimentally calculated from the measurement of fluorescence intensity parallel to the plane of linearly polarized excitation light (I∥) and that perpendicular to the excitation plane (I⊥), which was expressed as P = (I∥ − GI⊥)/(I∥ + GI⊥), wherein G is an instrumental factor. Dynamic light scattering (DLS) and ζ-potential were measured on a Malvern Zetasizer Nano ZS (Malvern, U.K.) instrument with a backscattering detection at 173°, equipped with a He−Ne laser (λ = 632.8 nm). Trypsin Activity Assay. Trypsin stock solutions were prepared in 1.0 mM hydrogen chloride solution and stored at −20 °C. The trypsin activity test was conducted in 384-well plates and monitored via plate reader. In detail, 10 μM PBI-Asp and 15 μg/mL protamine sulfate were dissolved in 5 mM phosphate buffer (pH 8.5) and transferred to 384-well plates. After that, 5 μL of trypsin stock solution was added to reach required enzyme concentrations. The kinetics of enzymatic hydrolysis were monitored by fluorescence intensity (λex = 490 nm, λem = 550 nm) using a plate reader at room temperature. After enzymatic digestion for 3 h, the UV−vis absorbance, fluorescence spectra, and polarization were recorded. Trypsin Inhibitor Test. The trypsin inhibitor, benzamidine hydrochloride, with varying concentrations was preincubated with trypsin at 25 °C for 15 min, and then added into solutions

solution. In the past, this has greatly restricted their applications in biosensing and bioimaging.17−21 Here, a highly fluorescent water-soluble PBI dye was synthesized by attaching multiple charge groups to perylene bisimide.22 Driven by electrostatic attractions and π−π interactions, the protein/PBI dye system self-assembled into soft nanoparticles, which led to a series of optical responses including aggregation-induced fluorescence quenching, polarization, and decrease in UV−vis absorption. The presence of the protease caused hydrolysis of the protein and dissociated the protein/dye self-assembly, which consequently restored PBI absorption/fluorescence emission and decreased its fluorescence polarization. In particular, we have used protamine and polylysine as enzyme substrates to fabricate protease sensors for trypsin and cathepsin B, respectively. In comparison with common protease detection methods, our assay utilizes natural proteins or peptides as substrates which avoids time-consuming substrate synthesis. In addition, the optical probe is integrated into our assay platform through supramolecular self-assembly or noncovalent interactions without the need for complicated fluorophore labeling. The combination of the dynamic nature of noncovalent self-assembly with the possibility to incorporate a variety of molecules that can trigger the optical responses in the protein holds great promise for applications as stimuliresponsive supramolecular systems (e.g., nanocarriers) and biosensors.

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EXPERIMENTAL SECTION Materials. Protamine sulfate from salmon, poly-L-lysine hydrobromide, trypsin from bovine pancreas (TPCK-treated, essentially salt-free, lyophilized powder, ≥10 000 BAEE units/ mg protein), and cathepsin B were purchased from Sigma− Aldrich (U.K.). GRP peptide was provided by GenScript (Piscataway, NJ) and used without further purification. Water was purified using a Millipore filtration system. Synthesis of Water-Soluble PBI Derivative. Asparticacid-functionalized water-soluble perylene bisimide (PBI-Asp) was synthesized according to the literature.22 In detail, 3,4,9,10Perylenetetracarboxylic acid bisanhydride (196 mg, 0.5 mmol), aspartic acid (146 mg, 1.1 mmol), zinc acetate (1.83 mg, 0.05 mmol), and 4.0 g of imidazole were heated at 120 °C for 12 h with stirring under argon atmosphere. The reaction mixture was cooled to 90 °C, and then poured into water in the presence of argon. The mixture was filtered and the filtrate was acidified to pH 2−3 with hydrogen chloride solution. The precipitate was B

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Figure 2. (a) UV−vis and (b) fluorescence spectra of protamine/PBI-Asp. The concentration of PBI-Asp is 10 μM while the protamine concentration is (from top to bottom) 0, 2.0, 4.0, 6.0, 8.0, and 10 μg/mL. The inset in panel (a) shows PBI-Asp solution without (left) and with (right) protamine; the inset in panel (b) shows the same solutions upon UV irradiation (365 nm). (c) Fluorescence intensity of PBI-Asp solution (10 μM) (λex = 490 nm, λem = 550 nm) with different amounts of protamine sulfate. Inset: Intensity profile of PBI-Asp fluorescence when protamine concentration varies from 15 to 120 μg/mL. (d) Fluorescence polarization of protamine/PBI-Asp mixtures, indicating the variations of perylene rotation diffusion rate: φ1 < φ3 < φ2.

aggregated PBI.23 This confirmed the existence of free dye monomer in solution. Meanwhile, the monomeric PBI-Asp displayed a high fluorescence quantum yield (∼50%, Supporting Information) with two emission peaks at 548 and 590 nm (Figure 1c). Protamine sulfate is a small nuclear protein with an argininerich sequence and has the ability to condense plasmid DNA to increase gene therapy transduction rates by both viral and nonviral mediated delivery mechanisms.24 Herein, protamine was chosen as a protease substrate because it is trypsindegradable. Strong interactions between PBI-Asp and protamine sulfate were demonstrated by multiple techniques. As shown in Figure 2a, the absorption intensity of PBI-Asp decreased with the addition of protamine sulfate, suggesting the protein-induced aggregation of PBI-Asp. When the protamine concentration reached 10 μg/mL, the absorption peaks at 534 and 495 nm disappeared completely and a new shoulder attributed to the extended oligomers was observed at 562 nm. The isosbestic point at 552 nm indicated that two species exist in equilibrium between monomers and oligomers. The spectral variations were accompanied by notable changes in the appearances of the solution (Inset in Figure 2a). Simultaneously, when adding protamine sulfate, the fluorescence emission decreased gradually due to the formation of nonemitting aggregates with forbidden low-energy excitonic transitions (Figure 2b).25 The fluorescence emission was largely quenched (∼99.8%) by 15 μg/mL protamine sulfate (Figure 2c), which was noticeable with a conventional UV lamp (inset in Figure 2b). Interestingly, further addition of protamine caused the fluorescence to increase (Inset in Figure 2c), indicating a weakening in the aggregation of PBI-Asp. This

of protamine/PBI-Asp mixture in phosphate buffer (5.0 mM, pH 8.5). The final concentration of PBI-Asp, protamine sulfate, and trypsin was 10 μM, 15 μg/mL, and 10 nM, respectively. The resulting solutions were incubated at room temperature, and the fluorescence intensity (λex = 490 nm, λem = 550 nm) was recorded with time. Cathepsin B Assay. The stock solution of cathepsin B (10 U/mL) was added to activation buffer with a final concentration of 20 mM DTT and 10 mM EDTA and then incubated at 37 °C for 15 min. To the activated enzyme solution, reaction buffer (25 mM sodium acetate, 1 mM EDTA, pH 5.0, prewarmed at 37 °C) and polylysine/PBI-Asp solution (1.0 μg/mL and 1.0 μM) were added. The reaction solution was incubated at 37 °C, and the fluorescence emission from PBI-Asp (λex = 490 nm, λem = 550 nm) was recorded.

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RESULTS AND DISCUSSION Supramolecular Self-Assembly of Protein/Perylene Dye. A water-soluble perylene-derivative named PBI-Asp (Figure 1a) was synthesized through condensation reaction of 3,4,9,10-perylenetetracarboxylic acid bisanhydride and L-aspartic acid. The attachment of aspartic acid to perylene introduced charge repulsion between multiple carboxyl groups and endowed a high solubility in aqueous conditions. Three well-resolved absorption peaks (534, 495, and 466 nm) and a weak broad shoulder around 415 nm were observed in the UV−vis spectrum of PBI-Asp (Figure 1b), which were ascribed to characteristic S0 → S1 transitions with different vibronic structures. The ratio of 0−0 to 0−1 transition in absorption intensity was calculated to be 1.56, being close to the normal Frank−Condon progressions (A0−0/A0−1 ≈ 1.6) for nonC

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Figure 3. (a) ζ-potential of protamine/PBI-Asp with varied protamine concentrations, in which the PBI-Asp concentration is 10 μM. (b) DLS and (c) TEM image of protamine/PBI-Asp with 10 μM PBI-Asp and 15 μg/mL protamine sulfate.

Figure 4. Schematic illustration of guanidinium−carboxylate electrostatic interaction, protamine/PBI-Asp self-assembly into positive and negative nanoparticles, and GRP/PBI-Asp interactions.

self-assembly (Figure 4). As shown in Figure 3a, the ζ-potential decreased from negative (−14 mV) to neutral as the protamine concentration increased, indicating the incorporation of cationic protamine into the protamine/PBI-Asp complex. Zero ζ-potential was achieved with ∼10 μg/mL protamine, consistent with the results of UV−vis absorbance, fluorescence, and polarization (Figure 2). These results suggest the important role of guanidinium−carboxylate charge interaction in protamine/PBI-Asp self-assembly. The formation of 100− 200 nm nanoparticles in protamine/PBI-Asp solution was demonstrated by TEM and DLS (Figure 3b and c, and Supporting Information Figure S1). Owing to the proteinassisted aggregation, PBI-Asp was caged (Figure 4) and its absorption/fluorescence was strongly quenched owing to π−π stacking (Figure 2a and 2b). Meanwhile, molecular aggregation prohibited the rotational diffusion of PBI-Asp and hence intensified its fluorescence polarization (Figure 2d). When the protein concentration exceeded 10 μg/mL, the ζ-potential was reverted to positive (Figure 3a), suggesting the incorporation of excess protamine into the protein/dye complex. The increased positive charges on protamine/PBI-Asp particles led to a loose molecular packing (Figure 4), which restored the absorption and fluorescence of PBI-Asp (Figure 2c and d). Owing to the less dense packing of PBI-Asp inside protamine/PBI-Asp particles, the diffusion rotation of PBI-Asp was enhanced and the fluorescence polarization decreased when the protamine concentration exceeded 10 μg/mL (Figure 2c and d). To further illustrate the factors that contribute to protein/ dye self-assembly, we investigated the coassembly behavior of PBI-Asp and a protamine-analogous peptide. To this end, a short arginine-rich peptide with the sequence of Gly-Arg-ProGly-Arg-Pro-Gly-Arg-Pro (GRP) was synthesized. It is

phenomenon is unexpected and will be discussed in the next section. Fluorescence polarization was further employed to provide information about the protein/dye self-assembly at the molecular level, especially with regard to perylene rotation. In principle, when a dye is excited with polarized light, it will emit fluorescence with the same polarization assuming the dye does not rotate during the lifetime of the excited state. 26 Depolarization occurs when the dye rotates during its emission lifetime. The faster it rotates, the smaller the fluorescence polarization will be. As shown in Figure 2d, the addition of protamine (0−15 μg/mL) greatly enhanced the fluorescence polarization. This suggests that the rotation diffusion of PBIAsp was restricted in the presence of protamine (φ1 > φ2, Figure 2d), owing to the protein-assisted perylene aggregation. Further addition of protamine (15−200 μg/mL) unexpectedly decreased the fluorescence polarization, implying a faster PBIAsp rotation (φ3 > φ2, Figure 2d). This phenomenon coincided with the fluorescence results in Figure 2c, in which a maximum fluorescence quenching effect was observed with 15 μg/mL protamine. We hypothesized that the optical responses (i.e., absorption decrease, emission quenching, and fluorescence polarization) originated from protein-induced dye self-assembly, which were studied by ζ-potential, dynamic light scattering (DLS), and transmission electron microscopy (TEM) (Figure 3). It is known that arginine and aspartic acid can form a salt bridge through guanidinium−carboxylate interaction which is a key stabilizing structural element in natural systems including RNA stem loops25 and zinc finger/DNA complexes.26 In this case, the guanidinium−carboxylate interaction between protamine and PBI-Asp became the main driving force for protein/dye D

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Figure 5. (a) Real-time fluorescence enhancement of protamine/PBI-Asp in the presence of 0−50 nM trypsin; (b) fluorescence spectra after 3 h enzyme digestion. The protein/dye concentration in (a, b) is 15 μg/mL protamine and 10 μM PBI-Asp for 0−10 nM trypsin. (c) Emission intensity of protamine/PBI-Asp after incubation with different amounts of trypsin for 3 h (λex = 490 nm, λem = 550 nm). The protein/dye concentrations in panel (c) are (A) 15 μg/mL protamine and 10 μM PBI-Asp and (B) 2.0 μg/mL protamine and 1.0 μM PBI-Asp. (d−g) Appearance of protamine/ PBI-Asp mixtures before (d, f) and after (e, g) trypsin digestion.

Figure 6. (a) UV−vis spectra and (b) fluorescence polarization of protamine/PBI-Asp solution after incubation with trypsin for 3 h. The results of fluorescence polarization indicate the perylene rotation diffusion φ1 < φ2. The PBI-Asp concentration is 10 μM, and the protamine concentration is 15 μg/mL.

interesting that no significant fluorescence quenching or UV− vis absorption decrease was observed when adding GRP peptide to PBI-Asp solutions (Supporting Information Figure S2). Although GRP is highly positive and will electrostatically interact with peryelene dyes, the short peptide chain makes it less efficient at assembling perylene dyes. This indicates the polymer-analogue structure of the protein is indispensible in promoting the self-assembly of protamine/PBI-Asp (Figure 4). Trypsin Detection Based on Protein/Dye Disassembly. As discussed above, the polymer-analogous structure of protamine plays a crucial role in protamine/PBI-Asp self-

assembly, while small peptides are less efficient at promoting the formation of perylene dye oligomers. It is therefore expected that if protamine is enzymatically cleaved into short peptides, the self-assembled protamine/PBI-Asp nanoparticles will disaggregate and the caged PBI-Asp will be released. Based on this hypothesis, protamine/PBI-Asp self-assembled complexes are expected to be applicable for the detection of protamine-specific proteases. Protamine is an ideal trypsin substrate owing to its argininerich structure. As the most important digestive enzyme, trypsin is secreted by the pancreas and plays a key role in controlling E

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Figure 7. (a) UV−vis absorbance and (b) fluorescence spectra of polylysine/PBI-Asp solution (pH 5.0) for polylysine concentrations from 0 to 2 μg/mL. The inset in panel (b) shows the fluorescence intensity (λex = 490 nm, λem = 550 nm) of PBI-Asp with different amounts of polylysine. (c) Cleavage of polylysine amide bond by CTSB. (d) Fluorescence spectra of polylysine/PBI-Asp mixtures after incubating with cathepsin B for 6 h at 37 °C for CTSB concentrations of 1.0, 5.0, 10, 20, 50, and 200 mU/mL, respectively. The inset in panel (d) gives the fluorescence intensity of polylysine/PBI-Asp for PBI-Asp concentration of 1.0 μM and polylysine concentration of 1.0 μg/mL after incubation with CTSB (λex = 490 nm, λem = 550 nm).

from the enhanced rotational diffusion of PBI-Asp. Similarly, the value of fluorescence polarization was found to plateau with 0.2 nM trypsin. To exclude the contribution of nonspecific interactions between trypsin and protamine/PBI-Asp to fluorescence enhancement, control experiments were conducted by replacing trypsin with different proteins (Supporting Information Figure S3). No significant fluorescence increase was noted in the protamine/PBI-Asp system after incubating with streptavidin, histone H1, lysozyme, cytochrome c, bovine serum albumin, and human serum albumin. In addition, trypsin cleavage-induced disassembly of protamine/PBI-Asp complexes was demonstrated by TEM, wherein no self-assembled particles were observed after trypsin digestion. The drop of light scattering intensity from 162 to 2.80 kcps after trypsin incubation confirmed the dissociation of large particles. The ζ-potential of protamine/PBI-Asp solution after enzyme treatment was found to be −3.64 mV, also indicating the disassembly of protamine/PBI-Asp particles. The detection limit of trypsin by this assay can be further lowered to 0.006 nM when the protamine/PBI-Asp concentration is reduced (Figure 5c and Supporting Information Figure S4). This detection limit is much lower than that of commercial kits using FITC-Casein as a substrate (∼0.5 μg/ mL). Commercial protease-sensing protocols are based on a dye-labeled peptide or protein substrate, which means one amide bond cleavage corresponds to one chromophore or fluorophore release. In our assay, the absorption and fluorescence signals are quenched by the noncovalent selfassembly of protein/perylene dye. This means that in principle

pancreatic exocrine function. It is involved in the digestive enzyme activation cascade, which induces the transformation of other pancreatic proenzymes into their active forms. Changes in trypsin levels are also closely related to the presence of some pancreatic diseases.27,28 Here, the real-time detection of trypsin activity was realized by tracking the fluorescence recovery of protamine/PBI-Asp solution in the presence of trypsin (λex = 490 nm, λem = 550 nm, Figure 5a). A gradual fluorescence increase was observed dependent on the trypsin concentration, with higher enzyme concentrations generating faster fluorescence increments. The fluorescence spectra after 3 h incubation with trypsin were also recorded to evaluate the enzyme activity (Figure 5b). The limit of detection (LOD), defined as the lowest assayed concentration of analyte that yields a signal higher than three times the standard deviation of the background, was determined to be 0.051 nM trypsin which gave a notable fluorescence rise (Figure 5b and c). Importantly, the enzyme activity was detectable directly with the naked eye, both under sunlight and a UV lamp, which is very useful for point-of-care enzyme detection. The enzyme activity could also be measured by UV−vis absorbance and fluorescence polarization. As shown in Figure 6a, the absorption peaks at 495 and 534 nm that were suppressed by protamine sulfate were restored after enzyme incubation. A noticeable increase in the UV−vis absorption was observed with a minimal trypsin concentration of 0.2 nM. The trypsin activity could be also detected by fluorescence polarization. As shown in Figure 6b, trypsin-catalyzed protein digestion caused fluorescence depolarization, which resulted F

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fluorometric responses. The presence of protease specifically hydrolyzed the protein, resulting in the disassembly of supramolecular nanoparticles as detectable by multiple optical readouts (i.e., UV−vis absorbance, fluorescence, and depolarization). This assay was exploited for sensitively detecting trypsin and cathepsin B by using protamine and polylysine respectively as substrates. Because of its inherent modularity and dynamic nature, the developed noncovalent biosensing platform is highly sensitive and can be further explored for the detection of other proteases. We anticipate that the presented concept of integrating natural proteins or polypeptides with synthetic dyes via supramolecular self-assembly will open up a new avenue toward multifunctional biomaterials for smart nanocarriers and biosensors.

the enzymatic digestion of an amide bond is possible to uncage several perylene dyes, which is transduced through multimodal signals. In addition, the high fluorescence quantum yield, large extinction, and strong aggregation-induced quenching of the perylene dye also contribute to the high sensitivity of our assay. Another advantage of this assay is its tunable dynamic detection range. For example, the dynamic detection range shifted from 0.05−10 nM to 0.005−2.0 nM when the PBI-Asp concentration is lowered from 10 to 1.0 μM (Figure 5c). The development of rapid and simple methods for screening chemical libraries of potential protease inhibitors is important in the pharmaceutical industry. For this reason, the developed protamine/PBI-Asp self-assembly system was also used to study the inhibition of trypsin activity by inhibitors. In Supporting Information Figure S6a, the degree of fluorescence enhancement for the ensemble of protamine sulfate and PBI-Asp containing trypsin was retarded by the trypsin inhibitor benzamidine hydrochloride. Based on the plot of the inhibition efficiency versus inhibitor concentration (Supporting Information Figure S5), the IC50 value of benzamidine hydrochloride toward trypsin was estimated to be 5.0 μM (Supporting Information Figure S6b). Label-Free Detection of Cathepsin B. Considering the universality of noncovalent self-assembly in protein/perylene dye systems, we hypothesized that this strategy could be extended to detect different proteases. Of particular interest are disease-related proteases, such as HIV or cancer relevant enzymes. To this end, cathepsin B (CTSB), which is frequently overexpressed in premalignant lesions, was chosen as a target protease. Increased expression of cathepsin B in primary cancers, especially in preneoplastic lesions, suggests that this enzyme might have pro-apoptotic features. For the sensing of cathepsin B, we replaced protamine with polylysine, a highly positive peptide that has been demonstrated to be a CTSB substrate.29,30 Owing to the strong charge interactions, electrostatic self-assembly of polylysine/PBI-Asp was expected. Indeed, upon the addition of polylysine to PBI-Asp, selfassembly was observed by UV−vis absorbance and fluorescence. As before, the absorption peaks at 495 and 533 nm gradually decreased, finally disappearing at 2.0 μg/mL polylysine (Figure 7a). Significant fluorescence quenching (∼98%) was observed in the presence of polylysine (Figure 7b). It was expected that enzymatic cleavage of polylysine amide bonds would disrupt the polymer−analogue structure and reduce the positive charges (via the formation of carboxyl groups), and would consequently trigger the disassembly of polylysine/PBI-Asp (Figure 7c). In our experiment, the enzyme-triggered fluorescence recovery in polylysine/PBI-Asp was recorded after 6 h incubation with CTSB and notable emission increases were observed at enzyme concentrations of 20 mU/mL (Figure 7d). Unlike previous work, in which fluorophore modified polylysines have been used for imaging CTSB, here we provide a simple and effective approach to design a CTSB sensor via a noncovalent strategy without dye labeling.

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ASSOCIATED CONTENT

S Supporting Information *

Measurement of fluorescence quantum yield, TEM image, effects of different proteins to protamine/PBI-Asp fluorescence, comparison of GRP and protamine, and trypsin inhibitor test. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +44 (0)207 594 6804. Fax: +44 (0)207 594 6757. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS M.M.S. thanks the Engineering and Physical Sciences Research Council (EPSRC) grant EP/K020641/1. Thanks to Dr. Roberto de la Rica for help in preparing this manuscript.

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CONCLUSIONS In this work, we have designed a versatile biosensing platform for label-free protease detection through the noncovalent selfassembly of proteins/perylene dye. The driving forces of protein/dye self-assembly were ascribed to electrostatic attractions and π−π interactions, which led to the formation of activatable soft nanoparticles and significant colorimetric and G

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dx.doi.org/10.1021/ac500777r | Anal. Chem. XXXX, XXX, XXX−XXX