Gold Nanoparticles-Based Colorimetric Assay for Cathepsin B Activity

Article pubs.acs.org/ac

Gold Nanoparticles-Based Colorimetric Assay for Cathepsin B Activity and the Efficiency of Its Inhibitors Chan-Jin Kim,† Dong-Ik Lee,‡ Cheonghee Kim,† Kangtaek Lee,† Chang-Ha Lee,† and Ik-Sung Ahn*,† †

Department of Chemical and Biomolecular Engineering, Yonsei University, Seoul 120-749, South Korea Dityrosine Innovation Chemical (D. I. Chemical), Seoul 120-749, South Korea

‡

S Supporting Information *

ABSTRACT: Cathepsin B has been suggested to be a prognostic marker of melanoma, glioma, and a variety of cancers such as brain, breast, colon, esophageal, gastric, lung, ovarian, and thyroid cancers. Cathepsin B inhibitors have also been considered as anticancer drug candidates; hence, there has been a growing need for a probe which enables the selective and simple detection of cathepsin B and its inhibitors. For the purpose of selective assay, a cathepsin B-specific substrate, N,N′-diBoc-dityrosine-glycine-phenylalanine-3-(methylthio)propylamine (DBDY-Gly-Phe-MTPA) was synthesized in this study. Phe-MTPA, which was produced via cathepsin B-catalyzed hydrolysis of DBDY-Gly-Phe-MTPA, allowed aggregation of gold nanoparticles (AuNPs) leading to a color change from red to blue. When tested for cathepsins B, L, and S, this assay method exhibited AuNPs color change only in reaction to cathepsin B. The limits of detection for cathepsin B was 10 and 5 nM in the 1 and 2 h hydrolysis reactions, respectively. The efficiency of cathepsin B inhibitors such as leupeptin, antipain, and chymostatin was easily compared by the degree of color change. Moreover, IC50 values of leupeptin, antipain, and chymostatin were found to be 0.11, 0.48, and 1.78 μM, respectively, which were similar to the results of previous studies. Therefore the colorimetric assay of cathepsin B and cathepsin B inhibitors using DBDY-Gly-Phe-MTPA and AuNPs allowed not only the selective but also the simple assay of cathepsin B and its inhibitors, which was possible with the naked eye.

E

Gold nanoparticles (AuNPs) have received significant attention as chemical and biological sensors because of their simplicity, stability, straightforward synthesis, high surface-tovolume ratio, excellent biocompatibility, and unique optical properties related to the surface plasmon resonance (SPR).26−28 The color change during aggregation (red to blue) or redispersion (blue to red) of AuNPs has made them attractive candidates for colorimetric assays.20−24,26,29,30 Thiol-containing molecules have been frequently used for anchoring AuNPs because thiols (i.e., SH) interact strongly with AuNPs by forming dative bonds, which are also known as coordinate covalent bonds.31 However, the thiol group may inhibit protease activity by breaking disulfide bonds in the protease molecule or forming disulfide bonds with the substrate.21 When the hydrogen of the thiol group was replaced with others, such as an acetyl or methyl group, the comparable binding affinity to the AuNPs was observed without being involved in the disulfide bonding.21,32 In our previous research, a peptide with DBDY and Gly at the P2 and P1 positions, respectively, were found to show selectivity and sensitivity toward cathepsin B among cathepsins B, L, and S.11 Moreover, it has been reported that

leven human cysteine cathepsins (cathepsins B, C, F, H, L, K, O, S, V, W, and X) have played crucial roles in cancer progression, ranging from gene amplification to posttranscriptional modification.1−3 Hence, inhibitors1,2,4 and probes5−7 of cysteine cathepsins have been proposed as anticancer agents. However, it has been reported that a few cathepsins, such as cathepsins L, H, and S, protect normal cells from tumors.1,2,8 Thus, selective inhibitors1,2,8−10 and probes1,5,11,12 targeting each cathepsin are needed. Cathepsin B (EC 3.4.22.1) has been known to play a significant role in tumor formation, growth, invasion, and metastasis.1,3,13,14 Moreover, this protease is related to melanoma, glioma, and a variety of cancers: brain, breast, colon, esophageal, gastric, lung, ovarian, and thyroid.14,15 For these reasons, cathepsin B has been suggested to be a prognostic marker9,15−17 and cathepsin B inhibitors have been considered to be candidates for probable anticancer drugs.3,9,13 Recently, activitybased probes,6 nanosized probes,7 and fluorescent peptide probes11,18,19 have been developed for the selective and sensitive assay of cathepsin B. In spite of the selectivity and the sensitivity toward cathepsin B, these probes require advanced instrumentation unsuitable for simple assay.20−23 Hence, there is a growing need for a probe which enables the detection of cathepsin B activity and permits the screening of cathepsin B inhibitors with the naked eye.20,22,24,25 © 2014 American Chemical Society

Received: December 2, 2013 Accepted: March 18, 2014 Published: March 18, 2014 3825

dx.doi.org/10.1021/ac4039064 | Anal. Chem. 2014, 86, 3825−3833

Analytical Chemistry

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(98%), triethylamine (TEA) (≥99.5%), trifluoroacetic acid (TFA) (99%), N,N-dimethylformamide (DMF) (99.8%), methylene chloride (MC) (≥99.8%), dimethyl sulfoxide-d6 (DMSO-d6) (99.9%), gold(III) chloride trihydrate (≥99.9%), trisodium citrate dihydrate, sodium phosphate monobasic, sodium phosphate dibasic, ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA·Na2·H2O), L-cysteine hydrochloride (L-Cys·HCl) (≥98%), DL-dithiothreitol (DTT) (≥99.5%), N-acetyl-L-cysteine (Ac-L-Cys) (≥99%), cathepsin B and cathepsin L from human liver, and cathepsin S from human spleen were all purchased from Sigma−Aldrich Chemical Company (St. Louis, MO). Acetonitrile and water (HPLC-grade) were purchased from Baker Chemical Company (Phillipsburg, NJ). Sodium chloride (NaCl) and magnesium sulfate (MgSO4) were purchased from Showa Chemical Industry Company (Meguro-ku, Tokyo, Japan). An Advantec PTFE filter membrane with a pore size of 0.2 μm (Advantec, Japan) was used for filtration. Grids for transmission electron microscopy (TEM) were purchased from electron microscopy sciences (Hatfield, PA). Synthesis and Characterization of DBDY-Gly-PheMTPA. For the synthesis of BOC-Phe-MTPA, BOC-Phe-OSu (688.9 mg, 1.9 mmol), MTPA (213.2 μL, 1.9 mmol), and TEA (318.2 μL, 2.3 mmol) were added to DMF at a molar ratio of 1:1:1.2. After the overnight reaction at room temperature, DMF was removed by lyophilization. The residue (BOC-PheMTPA) was dissolved in MC, washed with water 3 times, and dried over anhydrous MgSO4. Through vacuum evaporation of MC, it (BOC-Phe-MTPA, 569.6 mg, 85%) was recovered. Synthesized BOC-Phe-MTPA was dissolved in a mixture of TFA and MC (1:1) and incubated at room temperature for 30 min to remove the BOC group. After the vacuum evaporation of the solvent, the residue (Phe-MTPA, 358.9 mg, 88%) was recrystallized in MC-ethyl ether and recovered by filtration. For the synthesis of BOC-Gly-Phe-MTPA, BOC-Gly-OSu (323.6 mg, 1.19 mmol), Phe-MTPA (300.0 mg, 1.19 mmol), and TEA (199.0 μL, 1.43 mmol) were added to DMF at a molar ratio of 1:1:1.2. After an overnight reaction at room temperature, DMF was removed by lyophilization. The residue (BOC-Gly-Phe-MTPA) was dissolved in MC, washed with water 3 times, and dried over anhydrous MgSO4. Through vacuum evaporation of MC, it (BOC-Gly-Phe-MTPA, 389.5 mg, 80%) was recovered. Synthesized BOC-Gly-Phe-MTPA was dissolved in the mixture of TFA and MC (1:1), and incubated at room temperature for 30 min to remove the BOC group. After the vacuum evaporation of the solvent, the residue (Gly-Phe-MTPA) was recrystallized in MC-ethyl ether and recovered by filtration. The product was purified using a preparative HPLC system equipped with a UV detector (Young Lin Instrument Company, Gyeonggi-do, Korea) and a C-18 column (10 μm particle size, 100 × 21.2 mm, 110 Å pore size, Phenomenex, Torrance, California) and a mixture of water and acetonitrile (3:7) as the eluent. TFA was added beforehand to both water and acetonitrile at a concentration of 0.05%. The flow rate of the eluent and the separation temperature were maintained at 5 mL min−1 and 35 °C, respectively. The product was detected at wavelengths of 210 and 280 nm. Fractions containing Gly-PheMTPA (190.6 mg, 65%) were gathered and lyophilized. The method for the synthesis of DBDY is described in detail in ref 35. To facilitate the binding of Gly-Phe-MTPA, DBDYOSu was prepared from the reaction of DBDY, NHS, and DCC in 1,4-dioxane at the molar ratio of 1.1:1:1.36 After vacuum

phenylalanine (Phe) at the P1′ position further increases the selectivity toward cathepsin B among cathepsins B, L, and S.33 The definition of P1, P2, and P1′ positions, the Schechter and Berger nomenclature, is described in detail in ref 34. Herein, we developed a new probe for the simple and selective colorimetric assay of cathepsin B using citrate-coated (negatively charged) AuNPs (see Scheme 1). DBDY-Gly-Phe Scheme 1. Strategies of the Cathepsin B Assay Method Developed in This Studya

a

(a) Binding of DBDY-Gly-Phe-MTPA, the substrate for cathepsin B, to AuNPs does not cause the aggregation of AuNPs (b) while binding of Phe-MTPA, a product of the cathepsin B-catalyzed hydrolysis reaction, causes aggregation and color change of AuNPs. (c and d) This method can be used for the colorimetric screening of cathepsin B inhibitors. The AuNPs harboring DBDY-Gly-Phe-MTPA and/or PheMTPA are enlarged at the bottom to clearly show the dependence of their color change upon the activity of cathepsin B.

was synthesized as a specific substrate for cathepsin B and conjugated with 3-(methylthio)propylamine (MTPA) for anchoring the AuNPs. The resulting DBDY-Gly-Phe-MTPA is negatively charged, and its binding to the citrate-coated AuNPs should not cause the aggregation of the AuNPs. When it is hydrolyzed by cathepsin B, a positively charged substance, Phe-MTPA, is produced. The binding of Phe-MTPA to the citrate-coated AuNPs may cause AuNPs aggregation and color change. This method can also be used to screen the efficiency of cathepsin B inhibitors by monitoring the degree of color change (see Scheme 1).

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EXPERIMENTAL SECTION Materials. Boc-tyrosine-OH (Boc-Tyr-OH) (98%), BOCphenylalanine-N-hydroxysuccinimide ester (BOC-Phe-OSu) (≥98%), BOC-Glycine-OSu (BOC-Gly-OSu) (≥99%), 3-(Methylthio)propylamine (MTPA) (97%), N,N′-dicyclohexylcarbodiimide (DCC) (99%), N-hydroxysuccinimide (NHS) 3826



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Scheme 2. Synthesis of DBDY-Gly-Phe-MTPA

evaporation of 1,4-dioxane, MC was added subsequently and undissolved N,N′-dicyclohexylurea was removed by filtration. After vacuum evaporation of MC in the filtrate, the residue was dissolved in a mixture of water and acetonitrile (3:7). When the temperature was lowered to −20 °C, DBDY-OSu was partitioned into the acetonitrile layer. The acetonitrile layer was withdrawn and lyophilized. The synthesized DBDY-OSu was stored at −20 °C prior to being used. For the synthesis of DBDY-Gly-Phe-MTPA, DBDY-OSu (210.3 mg, 0.32 mmol), Gly-Phe-MTPA (100.0 mg, 0.32 mmol), and TEA (54.1 μL, 0.39 mmol) were added to DMF at a molar ratio of 1:1:1.2. After 48 h at room temperature, DMF was lyophilized. The residue was then washed with water. The product, DBDY-Gly-Phe-MTPA, was purified by the preparative HPLC system. The conditions of separation and wavelengths for detection were the same as those for the purification of Gly-PheMTPA except that a mixture of water and acetonitrile (35:65) was used as the eluent. The lyophilized DBDY-Gly-Phe-MTPA (145.9 mg, 53%) was stored at −20 °C prior to being used as a substrate for cathepsin B (Scheme 2). Structural identification of Gly-Phe-MTPA and DBDY-GlyPhe-MTPA was based on the following 1H NMR spectra, which were recorded on a Bruker Advance 400-MHz nuclear magnetic resonance spectrometer (Bruker BioSpin Corp., Germany). The 1H NMR spectra of Gly-Phe-MTPA and DBDY-Gly-PheMTPA are shown in the Supporting Information. Preparation and Aggregation Test of Citrate-Coated Gold Nanoparticles. Gold nanoparticles (AuNPs) were synthesized by citrate reduction of HAuCl4 in water.37 HAuCl4 (0.06 g) was first dissolved in 196 mL of deionized water (0.78 mM). This solution was refluxed until boiling, and then 4 mL of 184 mM sodium citrate solution was quickly added under vigorous stirring. The mixture was refluxed again for 30 min, and the color of the solution changed from yellow to wine red. The average size of the AuNPs was found to be

11.6 nm by TEM, which was operated at 200 kV (JEM-2010, JEOL Ltd., Tokyo, Japan). The resulting suspension of AuNPs was stored at 4 °C. AuNPs concentration was determined indirectly by measuring the optical density (OD) at 520 nm (SPECORD 210 PLUS, Analytik Jena, Thuringia, Germany). The extinction coefficient of the 11.6 nm AuNPs used in this study was calculated to be 1.7 × 108 M−1 cm−1.38 For AuNPs, OD520 of 1.0 corresponded to the concentration of 5.9 nM. The stability (i.e., the aggregation) of the AuNPs in the presence of DTT, L-Cys·HCl, and Ac-L-Cys was analyzed by monitoring the color change of the following AuNPs solutions at room temperature: AuNPs (OD520 = 1) in 25 mM phosphate buffer (pH 6.0) containing 50 mM NaCl, 1 mM EDTA, and one of DTT, L-Cys·HCl, and Ac-L-Cys at 1 mM. The effect of Phe-MTPA on the aggregation of AuNPs was investigated by monitoring the changes in color and absorbance spectra of the following AuNPs solutions at room temperature: AuNPs (OD520 = 1) in 25 mM phosphate buffer (pH 6.0) containing 50 mM NaCl, 1 mM EDTA, 1 mM Ac-L-Cys, and Phe-MTPA in the range of 10−50 μM. The stability of AuNPs in the presence of DBDY-Gly-PheMTPA was confirmed by comparing the color and absorbance spectra of the following AuNPs solutions with and without 100 μM DBDY-Gly-Phe-MTPA: AuNPs (OD520 = 1) in 25 mM phosphate buffer (pH 6.0 at 25 °C) containing 50 mM NaCl, 1 mM EDTA, and 1 mM Ac-L-Cys. Activation of Cathepsin B with Ac-L-Cys. To test the feasibility of Ac-L-Cys for cathepsin B activation, the hydrolysis of DBDY-(Gly-INH)2, a sensitive and selective assay material for cathepsin B,11 was performed with cathepsin B, which was preactivated for 10 min with 2 mM Ac-L-Cys. For comparison, cathepsin B preactivated with 2 mM L-Cys was also used. Fluorescence spectra before and after the 1 h reaction were recorded at an excitation wavelength of 320 nm using a luminescence spectrometer (LS 55 Luminescence Spectrometer, 3827



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Figure 1. (a) Colors and (b) absorbance spectra of the AuNPs in the presence of Phe-MTPA at various concentrations. Measurements were taken 10 s after mixing the AuNPs solution [optical density at 520 nm (OD520) = 2] and 50 mM phosphate buffer (pH 6.0) at a volumetric ratio of 1:1. The 50 mM phosphate buffer contained 100 mM NaCl, 2 mM EDTA, 2 mM Ac-L-Cys, and 20−100 μM Phe-MTPA. The incubation temperature was maintained at 25 °C.

PerkinElmer, MA). The fluorescence increased due to the production of fluorescent DBDY. Colorimetric assay for cathepsin B. Cathepsin B solution was prepared in 50 mM phosphate buffer (pH 6.0) containing 100 mM NaCl, 2 mM EDTA, and 2 mM Ac-L-Cys. It was incubated for 10 min at 25 °C for the preactivation of cathepsin B with 2 mM Ac-L-Cys. The hydrolysis reaction (25 °C) was initiated by adding DBDY-Gly-Phe-MTPA prepared in DMF. The stock solution of DBDY-Gly-Phe-MTPA was made in DMF so that the final concentration of DMF in the reaction solution was less than 1% (v/v). The initial concentrations of DBDY-Gly-Phe-MTPA and cathepsin B were 100 μM and 50 nM, respectively. Samples were taken at predetermined time intervals and mixed with the same volume of AuNPs solution (OD520 = 2, 25 °C). The color and UV spectrum of the mixture were recorded 10 s later and the aggregation of AuNPs after the reaction was confirmed by TEM.29 Hydrolysis products were analyzed using an analytical HPLC system equipped with a UV detector (UV 730D, Young Lin Instrument Company). Samples were prepared by terminating the reaction with the addition of 1 M HCl to 2% (v/v). Separation was performed on the C-18 column (5 μm particle size, 250 × 4.6 mm, 80 Å pore size, Phenomenex) using a mixture of 10 mM ammonium bicarbonate and acetonitrile as the eluent. Elution was carried out with a linearly increasing gradient of acetonitrile from 0% to 60% over 15 min. The flow rate of the eluent and the separation temperature were maintained at 1 mL min−1 and 35 °C, respectively. The products were detected at 254 nm. Selectivity of the Assay Method toward Cathepsin B. The selectivity of the assay method toward cathepsin B was investigated by performing the same reaction with cathepsins

Figure 2. Colorimetric assay of cathepsin B using AuNPs and DBDYGly-Phe-MTPA. (a) Color shift and (b) absorbance spectra of the AuNPs were measured 10 s after mixing the AuNPs solution (OD520 = 2), and the reaction solution at a volumetric ratio of 1:1. The reaction time for each sample of the reaction solution is shown in (a) and (b). The difference in the absorbance at 600 nm from the initial value is plotted in (c). A600,t denotes the absorbance measured at the reaction time, t, and the wavelength of 600 nm, while A600,0 denotes the initial absorbance measured at 600 nm. TEM images of the AuNPs before the reaction 1 and after 1 h reaction with cathepsin B (2) are shown in (d). The scale bar is 50 nm. The initial concentrations of DBDY-Gly-PheMTPA and cathepsin B were 100 μM and 50 nM, respectively. The hydrolysis reactions were performed at 25 °C in 50 mM phosphate buffer (pH 6.0), containing 100 mM NaCl, 2 mM EDTA, and 2 mM Ac-L-Cys.

L and S and comparing the results (i.e., colors and UV spectra of the AuNPs solutions) with that of cathepsin B. Solutions of cathepsins L and S were prepared in 50 mM phosphate buffer containing 100 mM NaCl, 2 mM EDTA, and 2 mM Ac-L-Cys. The pH of cathepsins L and S solutions were adjusted to 6.0 and 6.5, respectively. Similar to the assay for cathepsin B, 3828



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Figure 3. Selective colorimetric assay of cathepsin B among cathepsins B, L, and S. The initial concentrations of DBDY-Gly-Phe-MTPA and the cathepsin were 100 μM and 50 nM, respectively. The reactions were performed at 25 °C in 50 mM phosphate buffer containing 100 mM NaCl, 2 mM EDTA, and 2 mM Ac-L-Cys. The pH of the reaction solutions for cathepsins B and L was adjusted to 6.0 while that for cathepsin S was 6.5. All of the color measurements were performed 10 s after mixing the AuNPs solution (OD520 = 2) and the reaction solution at a volumetric ratio of 1:1.

Figure 4. Sensitivity of the assay method to cathepsin B. Changes in the colors and UV spectra of the AuNPs were monitored in samples taken after hydrolysis reactions that lasted (a and b) 1 h and (c and d) 2 h. The concentration of cathepsin B was varied in the range of 0−50 nM. The initial concentration of DBDY-Gly-Phe-MTPA was 100 μM. The hydrolysis reactions were performed at 25 °C in 50 mM phosphate buffer (pH 6.0) with 100 mM NaCl, 2 mM EDTA, and 2 mM Ac-L-Cys.

method for screening the efficiency of these inhibitors was tested by repeating the assay of cathepsin B in the presence of these inhibitors. The initial concentrations of DBDY-Gly-PheMTPA, cathepsin B, and cathepsin B inhibitors were 100 μM, 50 nM, and 0.5 μM, respectively. For colorimetric screening, samples were taken at predetermined time intervals and mixed with the same volume of the AuNPs solution (OD520 = 2, 25 °C). The color change of each mixture was recorded 10 s later. Quantitative evaluation of the inhibition efficiency was made by performing the hydrolysis reaction at various concentrations of the inhibitors for 30 min. The initial concentrations of DBDYGly-Phe-MTPA and cathepsin B were 100 μM and 50 nM, respectively. Ten seconds after mixing the reaction solution and the AuNPs solution (OD520 = 2, 25 °C) at a volumetric ratio of 1:1, the absorbance was measured at 600 nm. Inhibition efficiency defined in eq 1 was then calculated for each inhibitor:

these cathepsins were preactivated with 2 mM Ac-L-Cys at 25 °C for 10 min and the hydrolysis reactions (25 °C) were initiated by adding DBDY-Gly-Phe-MTPA. The initial concentrations of DBDY-Gly-Phe-MTPA and the cathepsin were 100 and 50 nM, respectively. Samples were taken at predetermined time intervals and mixed with the same volume of AuNPs solution (OD520 = 2, 25 °C). The color and UV spectrum of the mixture were recorded 10 s later. Sensitivity of the Assay Method to Cathepsin B. The sensitivity of the assay method to cathepsin B was investigated by varying the concentration of cathepsin B in the range of 0−50 nM. The initial concentration of DBDY-Gly-Phe-MTPA was 100 μM. Samples were collected at predetermined time intervals and mixed with the same volume of AuNP solution (OD520 = 2, 25 °C). The color and absorbance of the mixture at 600 nm were recorded 10 s later. Colorimetric Screening of Cathepsin B Inhibitors. Leupeptin, antipain, and chymostatin were selected and used as the inhibitors of cathepsin B. The feasibility of this assay

Inhibition Efficiency (%) = 3829

A(no inhibitor) − A(inhibitor) A(no inhibitor) − A 0

× 100

(1)



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study they were added after the hydrolysis reaction of DBDYGly-Phe-MTPA (see Scheme 1). Dithiothreitol (DTT) and L-cysteine (L-Cys), which are commonly used as activators of cathepsin B,41 cause aggregation of AuNPs (see Figure S1 of the Supporting Information) due to the two thiol groups in DTT and the positively charged amine group (−NH3+) in L-Cys. In this study, N-acetyl-L-cysteine (Ac-L-Cys) was used instead of DTT and L-Cys to avoid the aggregation of AuNPs (see Figure S1 of the Supporting Information), even though its activation efficiency was about eighty percent that of L-Cys (see Figure S2 of the Supporting Information). Moreover, negatively charged Ac-L-Cys caused the stable suspension of AuNPs in reaction solutions containing highly concentrated salts (e.g., 25 mM phosphate and 50 mM NaCl) due to electrostatic repulsion (see Figure S3 of the Supporting Information). The cleavage site of DBDY-Gly-Phe-MTPA during the cathepsin B-catalyzed hydrolysis was determined from HPLC analysis of the products. The resulting HPLC chromatograms are shown in Figure S4 of the Supporting Information. To guarantee complete conversion, the reaction was performed for up to 6 h. Only the peak attributed to DBDY-Gly [retention time (RT) = 9.9 min] was clearly detectable, while the peak of DBDY-Gly-Phe-MTPA (RT = 14.6 min) disappeared. This indicates that DBDY, Gly, and Phe were recognized by cathepsin B as the P2, P1, and P1′ positions as expected. PheMTPA was therefore the product of the cathepsin B-catalyzed hydrolysis of DBDY-Gly-Phe-MTPA. The peak of Phe-MTPA (RT = 15.1 min) was hardly detectable because of its relatively low absorptivity at 254 nm. Aggregation of AuNPs occurred immediately after being mixed with the solution containing Phe-MTPA (see Figure S5 of the Supporting Information for red shifting in 5 s, which is the color change from red to blue). The gradual binding of the negatively charged Ac-L-Cys, however, caused redispersion

Table 1. Comparison of Assay Methods for Cathepsin B probea DBDY-Gly-Phe-MTPA with AuNPs solution Gly-Arg-Arg-Gly-Lys-Gly-Gly on glycol chitosan nanoparticles DBDY-(Gly-INH)2 Abz-Gly-Ile-Val-Arg-Ala-Lys(Dnp)OH Dnp-Gly-Phe-Arg-Phe-Trp-OH

detection method colorimetric assay fluorometric assay fluorometric assay fluorometric assay fluorometric assay

LOD for cathepsin B

ref

5 nM

b

0.725 nM

7

0.5 nM

11

n.d.c

18

n.d.d

19

a

Abz = ortho-aminobenzoic acid, Dnp = 2,4-dinitrophenyl. bThis study. cLOD for cathepsin B was not separately determined. The concentration range of cathepsin B employed in that study was from 1 to 10 nM. dLOD for cathepsin B was not separately determined. The concentration range of cathepsin B employed in that study was 0.5 to 2 nM.

where A0 is the absorbance of the reaction mixture in the absence of both cathepsin B and inhibitors, A(no inhibitor) is the absorbance of the reaction mixture with cathepsin B but without any inhibitor, and A(inhibitor) is the absorbance of the reaction mixture with both cathepsin and a specific inhibitor.39 A0 was measured 10 s after mixing 50 mM phosphate buffer (pH 6.0, 25 °C) containing 100 mM NaCl, 2 mM EDTA, and 2 mM Ac-L-Cys with the same volume of AuNPs solution (OD520 = 2, 25 °C). IC50, which was defined as the concentration of an inhibitor which achieves 50% inhibition efficiency, was determined from the plot of inhibition efficiency versus inhibitor concentration.

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RESULTS AND DISCUSSION Development of Assay Conditions. Because AuNPs are known to significantly decrease cathepsin B activity,40 in this

Figure 5. Colorimetric screening of cathepsin B inhibitors. Changes in (a) AuNPs color and (b) absorbance at 600 nm were monitored 10 s after mixing the AuNPs solution (OD520 = 2) and the reaction solution at a volumetric ratio of 1:1. The initial concentrations of DBDY-Gly-Phe-MTPA and cathepsin B were 100 μM and 50 nM, respectively. In (a), the initial concentrations of the cathepsin B inhibitors were all adjusted to 0.5 μM. The time in (a) denotes the reaction time for each sample of the reaction solution. P denotes a positive control where the reaction proceeded with cathepsin B in the absence of inhibitors. N denotes a negative control where cathepsin B was not added to the reaction solution. I1, I2, and I3 denote the cathepsin B-catalyzed hydrolysis of DBDY-Gly-Phe-MTPA performed with leupeptin, antipain, and chymostatin, respectively, as the cathepsin B inhibitor. 3830



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of the aggregated AuNPs (see Figure S5 of the Supporting Information for the gradual blue shifting from 5 s to 60 s). The blue color induced by the aggregation of AuNPs was stable for at least 30 s (see Figure S6 of the Supporting Information). Hence, the colorimetric assay (i.e., monitoring of the color and UV absorbance spectrum) was conducted 10 s after mixing the suspension of AuNPs with the reaction solution. The color turned more blue with respect to the increasing concentration of Phe-MTPA, even in the presence of Ac-L-Cys (see Figure 1), which implies the feasibility of this method for the quantitative assay of cathepsin B. Real-Time Colorimetric Assay of Cathepsin B. As expected, DBDY-Gly-Phe-MTPA did not cause any detectable changes in colors and UV spectra of the AuNPs (see Figure S7 of the Supporting Information). However, when it was subject to cathepsin B-catalyzed hydrolysis, the reaction solution containing Phe-MTPA as a product gradually changed color from red to blue (see Figure 2a). In addition, the absorption peak at 520 nm decreased as the reaction progressed, while the absorption peak at 600 nm increased (see Figure 2b). The realtime colorimetric assay of cathepsin B, which was examined by measuring the increase in the absorbance at 600 nm, was found to be feasible, as shown in Figure 2c. The two TEM images shown in Figure 2d confirmed that aggregation of AuNPs was induced by Phe-MTPA, which was a product of cathepsin B-catalyzed hydrolysis of DBDY-Gly-Phe-MTPA. Selective Colorimetric Assay of Cathepsin B among Cathepsins B, L, and S. The selectivity of this assay method toward cathepsin B was tested using cathepsins L and S for comparison. The results are shown in Figure 3. Only cathepsin B resulted in a color change of the AuNPs from red to blue. Therefore, it can be concluded that DBDY-Gly-Phe-MTPA is a specific substrate for cathepsin B, and that this method is selective toward the assay of cathepsin B among cathepsins B, L, and S. Limit of Detection for Cathepsin B. The result of the sensitivity analysis is shown in Figure 4. The color change of AuNPs from red to blue and the increase in the absorbance at 600 nm were clearly detected in the samples collected after the 1 h reaction with 10 and 50 nM cathepsin B (see Figure 4, panels a and b). For samples collected after the 2 h reaction, even 5 nM cathepsin resulted in the obvious color change and increase in absorbance (see Figure 4, panels c and d). Therefore, the limits of detection (LODs) for cathepsin B were 10 and 5 nM when the hydrolysis reaction was performed for 1 and 2 h, respectively. The colorimetric assay method used in this study was less sensitive than fluorescent assay methods because of the sensitivity difference in UV absorbance and fluorescence (see LOD for cathepsin B, as summarized in Table 1). However, this method allows for the simple detection of cathepsin B activities by the naked eye, contrary to other methods that require advanced instrumentation such as a fluorescence spectrometer. Screening of Cathepsin B Inhibitors for Their Inhibition Efficiencies. The feasibility of this method for the colorimetric screening of cathepsin B inhibitors was tested using model cathepsin B inhibitors, leupeptin, antipain, and chymostatin. These inhibitors were pretested for their effect on the stability of AuNPs in the absence of cathepsin B and the reaction buffer. None of the tested inhibitors caused aggregation of AuNPs and changed their color (see Figure S8 of the Supporting Information). As shown in Figure 5, the efficiency of these cathepsin B inhibitors could easily be compared, even with the naked eye: leupeptin (I1) > antipain (I2) > chymostatin (I3).

Figure 6. Quantitative evaluation of inhibition efficiencies of three cathepsin B inhibitors. Changes in AuNPs absorbance at (a) 600 nm were monitored 10 s after mixing the AuNPs solution (OD520 = 2) and the reaction solution at a volumetric ratio of 1:1. Each reaction solution was taken 30 min after initiating the hydrolysis reaction, which was conducted using various concentrations of leupeptin (I1), antipain (I2), and chymostatin (I3). The initial concentrations of DBDY-Gly-Phe-MTPA and cathepsin B were 100 μM and 50 nM, respectively. Inhibition efficiencies of (b) leupeptin, (c) antipain, and (d) chymostatin against cathepsin B were calculated from eq 1 using the data shown in (a).

Equation 1 was used for quantitative evaluation of the inhibition efficiency. Changes in AuNPs solution absorbance at 600 nm were monitored 30 min after the hydrolysis reaction, and the 3831



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results are shown in Figure 6a. Using these results, the inhibition efficiency of each inhibitor was then calculated from eq 1 and plotted in Figure 6b for leupeptin, in Figure 6c for antipain, and in Figure 6d for chymostatin. The following IC50 values, which represent the concentration of inhibitor required for 50% inhibition of cathepsin B activity, are shown in the plots: 0.11 μM for leupeptin, 0.48 μM for antipain, and 1.78 μM for chymostatin, which are similar to those reported in previous studies.42,43

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CONCLUSION In conclusion, we have designed and developed an AuNPsbased colorimetric assay to measure the activity of cathepsin B and the inhibition efficiency of its inhibitors using a cathepsin B-specific substrate, DBDY-Gly-Phe-MTPA. Anchoring DBDYGly-Phe-MTPA, a negatively charged substrate, onto the citratecoated AuNPs did not cause the aggregation of AuNPs due to electrostatic repulsion. When this substrate was hydrolyzed by cathepsin B, a positively charged Phe-MTPA was produced. Its binding to the citrate-coated AuNPs caused the aggregation and color change of AuNPs due to electrostatic attraction. To our knowledge, this is the first application of AuNPs as colorimetric sensors for determining cathepsin B activity. The detection limit for cathepsin B was as low as 5 nM in the 2 h hydrolysis reaction. This method has advantages for the simple assay of both cathepsin B and cathepsin B inhibitors by monitoring color changes.

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ASSOCIATED CONTENT

* Supporting Information S

1

H NMR spectra of the compound and Figures S1−S8. 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: +(82) 2-2123-2752. Fax: +(82) 2-312-6401. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Advanced Biomass R&D Center (ABC) of Global Frontier Project funded by the Ministry of Education, Science and Technology (Grant 20100029734).

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REFERENCES

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Gold Nanoparticles-Based Colorimetric Assay for Cathepsin B Activity

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