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Electrochemiluminescent sensing for caspase-3 activity based on Ru(bpy)32+-doped silica nanoprobe Yong-Ping Dong, Gang Chen, Ying Zhou, and Jun-Jie Zhu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b04379 • Publication Date (Web): 05 Jan 2016 Downloaded from http://pubs.acs.org on January 10, 2016

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Analytical Chemistry

Electrochemiluminescent sensing for caspase-3 activity based on Ru(bpy)32+-doped silica nanoprobe Yong-Ping Donga,b, Gang Chena, Ying Zhoua,b, and Jun-Jie Zhua,* a

State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, China

b

School of Chemistry and Chemical Engineering, Anhui University of Technology, Maanshan 243002, China

E-mail: [email protected]

ACS Paragon Plus Environment

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ABSTRACT: Caspase-3 is one of the most frequently activated cysteine proteases during the apoptosis process and has been identified as a well-established cellular marker of apoptosis. In this study, a novel approach for the sensitive determination of caspase-3 activity was proposed using

electrochemiluminescence

(ECL)

of

Ru(bpy)32+-doped

silica

(Ru@SiO2)

with

tripropylamine (TPA) as coreactant. A nanocomposite containing gold nanoparticles (AuNPs), poly(dimethyldiallyl ammonium chloride) (PDDA), and multi-walled carbon nanotubes (CNTs) was fabricated as an ECL platform. The biotinylated DEVD-peptide (biotin-Gly-Asp-Gly-AspGlu-Val-Asp-Gly-Cys) was immobilized on the nanocomposite surface via the strong bonding interaction between AuNPs and the thiol group. Then the streptavidin-modified Ru(bpy)32+doped silica (Ru@SiO2-SA) was immobilized on the ECL platform via the specific interaction between biotin and streptavidin to generate ECL signal. Caspase-3 can specifically recognize and cleave the N-terminus of DEVD, leading to the loss of the biotin label and the decrease of ECL intensity to determine the activity of caspase-3. The results revealed a new ECL avenue for the sensitive and specific monitor of caspase-3, and the platform could be utilized to evaluate anticancer drugs. KEYWORDS: caspase-3; electrochemiluminescence; Ru(bpy)32+-doped silica; apoptosis


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INTRODUCTION Apoptosis is an evolutionarily conserved and highly regulated process resulting in cell death. The process of apoptosis is controlled partially by a series of aspartate-specific cysteinedependent proteases that are activated specifically in apoptotic cells.1,2 Among apoptotic protease, caspase-3 is the most frequently activated cysteine protease and has been identified as a key mediator of apoptosis of mammalian cells.3 Therefore, highly sensitive and specific detection of caspase-3 activity has become an important subject in apoptosis diagnosis. Caspase-3 can specifically cleave the N-terminus of the tetrapeptide Asp-Glu-Val-Asp (DEVD), which has been used to develop caspase-specific probes for the analysis of caspase-3 activity, including chemiluminescent detection,3 western blot assay,4 colorimetric assay,5 fluorescence resonance energy transfer-based fluorometric assay,6,7 flow cytometric-based analysis,8 and electrochemical methods.9,10 However, most of these techniques either are laborious and time-consuming or require high technical expertise and precise. Electrochemiluminescence (ECL) is a powerful analytical technique due to its high sensitivity, wide dynamic concentration response range, as well as its potential and spatial controlment.11 ECL biosensor has been widely used in DNA analysis,12 immunoassay,11,13 and cancer cell detection.14-16 Among many of ECL systems, the Ru(bpy)32+ based ECL is most famous and can be used for detecting various samples. The immobilization of Ru(bpy)32+ onto a solid electrode surface is important for Ru(bpy)32+ ECL applications because it can simplify experimental design and reduce the consume of luminescent reagent. Since it was first synthesized by Tan’s group, Ru(bpy)32+-doped silica (Ru@SiO2) has become an ideal tag in bioanalysis because silica nanoparticls have been demonstrated to be a good matrix for immobilizing a high concentration of Ru(bpy)32+ to promote ECL detection.17-21 During past few decades, carbon nanotubes and gold nanoparticles have been extensively used as the matrix in ECL biosensor due to their


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excellent conductivity, large surface area, good biocompatibility.22, 23 Therefore, the combination of Ru@SiO2 with AuNPs and CNTs will construct many kinds of ECL biosensors with high sensitivity and good selectivity. Since caspase family of proteases has been implicated as therapeutic targets for the control of inappropriate apoptosis, the development of the assay methods should significantly facilitate apoptosis-mediated drug discovery and therapeutic applications. However, to the best of our knowledge, there has no report about the sensitive and specific ECL detection of caspase-3 activity to evaluate the medical value of anticancer drugs. Herein, we developed a novel ECL sensing platform to monitor the caspase-3 activity by combining the ECL intensity change of Ru@SiO2/TPA system with the specific recognition and cleavage of a biotinylated DEVDpeptide (biotin-Gly-Asp-Gly-Asp-Glu-Val-Asp-Gly-Cys) by caspase-3. The fabrication process of ECL sensor is shown in Scheme 1. Firstly, the glassy carbon electrode (GCE) was modified with nanomaterals composed of AuNPs/PDDA/CNTs. The biotinylated DEVD peptides were linked to the modified GCE surface by Au-S bond between the thiol group of the terminal cysteine residue and AuNPs. The streptavidin-modified Ru@SiO2 was captured via the specific interaction between biotin of the peptide and streptavidin, leading to ECL emission in the presence of TPA. When the sensor was immersed into the apoptotic cell lysates containing active caspase-3, the biotinylated DEVD peptides were specifically recognized and cleaved, which resulted in the decreased binding sites for streptavidin modified Ru@SiO2 and weak ECL emission. The ECL intensity was proportional to the amount of Ru@SiO2 on the sensor, corresponding to the uncleaved biotin-DEVD peptide, which in turn depended on the activity of caspase-3 in cell lysates. Consequently, the caspase-3 activity can be indirectly evaluated by measuring the ECL intensity.


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Scheme 1. Schematic representation of the construction of ECL probe (A) and the detection of caspase-3 activity (B). EXPERIMENTAL SECTIONS Materials and Reagents The biotinylated DEVD-peptide (biotin-Gly-Asp-Gly-Asp-Glu-Val-Asp-Gly-Cys) (1092.13 g/mol, 96.60% purity) was purchased from GL Biochem. Ltd. (Shanghai, China). Streptavidin was obtained from Shanghai Sangon Biotechnology Co. Ltd. (Shanghai, China). Multi-walled carbon nanotubes (CNTs, CVD method, purity >95%, diameter 10-20 nm, length 0.5-2.0 µm) were purchased from Nanoport. Co. Ltd (Shenzhen, China). Chloroauric acid (HAuCl4·4H2O) was obtained from Shanghai Chemical Reagent Co. Ltd. (Shanghai, China). Tris(2carboxyethyl)phosphine hydrochloride (TCEP), poly(dimethyldiallyl ammonium chloride)


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(PDDA), 6-mercapto-1-hexanol (MCH), Ru(bpy)3Cl2·6H2O, tripropylamine (TPA), bovine serum album (BSA), and 3-aminopropyltriethoxysilane (APTES) were purchased from SigmaAldrich. Apoptosis Inducers Kit (C0005) and Caspase-3 Inhibitor (Ac-DEVD-CHO) were purchased from Beyotime Institute of Biotechnology (Haimen, China). Caspase-3 cell activity detection kit was purchased from Nanjing Keygen Biotech. Co. Ltd. (Jiangsu, China). Tetraethylorthosilicate (TEOS) was obtained from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). All other reagents were of analytical grade. All aqueous solutions were prepared with ultrapure water purified by Millipore-Q System. Apparatus Scanning electron micrographs (SEM) were measured on an S-4800 scanning electron microscope. Transmission electron micrographs (TEM) were measured on a JEOL JEM 200CX transmission electron microscope, using an accelerating voltage of 100 kV. The ECL emission measurements were conducted on a model MPI-M electrochemiluminescence analyzer (Xi’An Remax Electronic Science & Technology Co. Ltd., China). Cyclic voltammograms (CVs) and electrochemical impedance spectroscopy (EIS) were performed with an Autolab electrochemical analyzer (Eco. Chemie., Netherlands) with a conventional three-electrode system comprised of a saturated calomel electrode (SCE) as the reference electrode, a platinum wire as the auxiliary electrode, and the modified GCE as the working electrode. Supporting electrolyte: 0.1 mol L-1 KCl solution containing K3[Fe(CN)6]/K4[Fe(CN)6] (5 mM, 1:1), scan rate: 100 mV s-1, frequency range: 0.01 Hz-100 kHz, and potential amplitude: 5 mV. UV–vis spectra were recorded on a UV3600 spectrophotometer (Shimadzu, Kyoto, Japan). Zeta Potential was tested on a Nano-z Zeta Potential Analyzer. Synthesis of Ru@SiO2 Nanoparticles


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Ru@SiO2 nanoparticles were synthesized according to the previous literatures.19,20 First, 1.77 mL of Triton X-100 were mixed with 7.5 mL of cyclohexane, 1.8 mL of n-hexanol, and 340 µL of Ru(bpy)32+ (40mM) solution in water. After 30 min stirring, 100 µL of TEOS was added into the mixture. Then, a polymerization reaction was initiated by adding 60 µL of NH3·H2O (~30 wt%). The solution was stirred for 24 h in the dark to obtain Ru@SiO2 nanoparticles, which were isolated by acetone, and followed by centrifuging and washing with ethanol and ultrapure water several times to remove the residual surfactant molecules and extra Ru(bpy)32+. The precipitation was dispersed with ethanol to a final volume of 5 mL. Modification of Ru@SiO2 Nanoparticles with Streptavidin The modification of streptavidin (SA) on the Ru@SiO2 nanoparticles was carried out according to the previously report with some modifications.21 1.0 mL of 2.0 mg mL-1 Ru@SiO2 was diluted with ethanol to 5.0 mL and 400 µL of APTES was added in the solution. After being stirred for 4 h, the mixture was centrifuged and washed with ethanol several times to remove the excess APTES, and the amino-terminated Ru@SiO2 nanoparticles were obtained. Subsequently, the amino-terminated Ru@SiO2 nanoparticles reacted with glutaraldehyde solution (5.0 mL, 5%) at 37°C for 4 h under stirring. Then, the mixture was centrifuged and washed three times with water to remove excess glutaraldehyde. The precipitation was dispersed into 2.0 mL of ultrapure water. After that, 200 µL of 1.0 mg mL-1 SA was added into the suspension, and followed by stirring for 4h under 37 °C water bath. The SA modified Ru@SiO2 nanoparticles (Ru@SiO2-SA) were obtained after the mixture was centrifuged and washed with PBS (pH 7.4, 0.01 mol L-1). At last, the Ru@SiO2-SA nanoparticles were redispersed in 1.0 mL of pH 7.4 PBS containing 1% BSA and stored at 4 °C for use. Preparation of AuNPs/PDDA/CNTs Nanomaterials


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First, the colloidal AuNPs with 13 nm diameter were prepared from HAuCl4 aqueous solution and sodium citrate according to the previous protocol.22 Then, CNTs were treated with 3:1 H2SO4/HNO3 using sonication for 4 h to obtain carboxylic group-functionalized CNTs, which was dispersed in ultrapure water to a concentration of ~1.0 mg mL-1.23 Next, 1.0 mL of the carboxylated CNTs solution was dispersed into 5 mL 1.2 wt% PDDA aqueous solution and sonicated for 1 h to obtain a homogenous black suspension. The residual PDDA was removed by high-speed centrifugation (21000 rpm, 20 min), and the complex was washed with ultrapure water for four times to obtain PDDA functionalized CNTs. Subsequently, 1.0 mL PDDA functionalized CNTs were dispersed into 5.0 mL of as-prepared colloidal AuNPs and sonicated for 10 min. After centrifugation, AuNPs/PDDA/CNTs composites were obtained, which were further washed with water and dispersed in water with a concentration of ~1.0 mg mL-1. Cell Culture and Apoptosis Assay Human chronic myeloid leukemia (CML) cell K562 were cultured in a flask in RPMI 1640 medium (Keygen Biotech, Jiangsu, China) containing 10% fetal calf serum, 80 µg mL-1 streptomycin and 80 µg mL-1 penicillin in an incubator (5% CO2, 37 °C). At the logarithmic growth phase, 6 mL of K562 cells (2×106 cells mL-1) was treated with Apoptosis Inducers Kit (1 µL mL-1) for 0, 2, 4, 6, 8, 10, 12, and 14 h respectively. Then K562 cells were collected and washed twice with PBS, and lysed in 200 µL of ice-cold Western and IP lysis solution for 30 min. Then, the cell suspension was centrifuged at 13,000 rpm for 15 min at 4°C. The supernatant was then used as the cell extract containing caspase-3 for analysis. In a well of a 96-well plate, 50 µL of cell extract, 50 µL of 2×Reaction Buffer containing 0.5 µL of 0.1 mol L-1 DTT, and 5 µL caspase-3 substrate with the chromophoric group were mixed and incubated at 37 °C for 4 h. Spectrophotometry was then performed to detect the chromophoric group. The caspase-3 stock solution was diluted with desired concentrations in the reaction buffer solution.


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Electrode Modification Prior to modification, glassy carbon electrode (GCE, 3 mm in diameter) was polished sequentially with 0.3 µm and 0.05 µm alumina power, followed by ultrasonic cleaning with ethanol and ultrapure water, and rinsing thoroughly with ultrapure water and drying under a N2 flow. Subsequently, 6 µL of AuNPs/PDDA/CNTs suspension was dropped on the pretreated GCE surface. After drying in air, 10 µL of biotin-DEVD peptide (5 mM) was activated with 1.5 µL TCEP (10 mM) in acetate buffer (pH 5.2) for 1 h to prevent terminal cysteine from forming disulphide bonds. Then, 10 µL of the activated biotin-DEVD was coated on the AuNPs/PDDA/CNTs modified GCE surface, which was allowed to incubate for 12 h at 4 °C in a 100 % moisture-saturated environment. After that, the electrode was immersed into 1 mM MCH for 1 h to remove the nonspecific adsorption, and then was washed with PBS (pH 7.4). The peptide-modified electrode was then dunked into 100 µL cell lysates containing active caspase-3 for 1 h at 37 °C and washed twice with PBS, and last incubated with 10 µL Ru@SiO2-SA for 2 h to form biotin-streptavidin bio-affinity complexes at 37°C. During the caspase-3 sensing process, 100 µL cell lysates was substituted with 100 µL caspase-3 working solution. The biosensor was washed with PBS to remove the non-specific absorption of the ECL probe before ECL measurements. ECL Detection The ECL measurements were performed in a 5 mL homemade quartz cell with the modified glassy carbon electrode as working electrode at room temperature in 0.1 mol L-1 pH 7.4 PBS containing 0.1 mol L-1 TPA. The continuous potential scanning was applied on the working electrode for electrochemical measurements from -1.2 to 1.2 V at a scanning rate of 100 mV s-1. The ECL signal was recorded by the MPI-M electrochemiluminescence analyzer and the voltage of the photomultiplier tube (PMT) was set at −600 V during the detection.


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RESULTS AND DISCUSSION Characterization of ECL Sensing Platform

Figure 1. TEM images of CNTs (A), AuNPs/PDDA/CNTs (B). SEM images of CNTs (C), AuNPs/PDDA/CNTs (D). Carbon nanotubes exhibit unique physical and chemical properties and have been widely used as carriers to load numerous biomacromolecules, including DNA, RNA, and proteins.24-25 Recently, carbon nanotubes have often been applied to fabricate ECL platform.18,28 Gold nanoparticles (AuNPs) have also been extensively used in ECL biosensors due to their good biocompatibility, excellent electrocatalytic activity, and large surface area.29-31 In the present work, CNTs were used as the substrate to fabricate the biosensor for caspase-3. PDDA was used to change the negatively charged CNTs to positively charged state, which could make nanocomposites connect with negatively charged AuNPs to further facilitate the immobilization


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of biomolecule on the ECL platform. The thiol group of the terminal cysteine residue of the peptide could be bound to AuNPs surface via the Au-S bond, as a result, the peptide was modified on the ECL platform successfully. The nanocomposites could not only enhance the ECL intensity by improving the charge transfer rate and increase the load of the peptide, but also improve the biocompatibility and retain the bioactivity of peptide. Transmission electron microscope (TEM) and scanning electron microscope (SEM) were used to characterize the morphologies of CNTs and AuNPs/PDDA/CNTs composite (Figure 1). The as-prepared carboxylated CNTs show a homogeneous and good dispersion (Figure 1A and 1C). After the positively charged PDDA modified CNTs (PDDA/CNTs) were formed, negatively charged AuNPs could be electrostatically attached on the positively charged surface of the PDDA/CNTs (Figure 1B and 1D). 60

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Figure 2. (A) Zeta potentials of CNTs, PDDA/CNTs, AuNPs, AuNPs/PDDA/CNTs (B) UV-vis absorption spectra of CNTs, PDDA/CNTs, AuNPs, AuNPs/PDDA/CNTs. In order to support this speculation, Zeta potentials of the nanocomposites were recorded as shown in Figure 2A. It can be concluded that the negatively charged carboxylated CNTs and AuNPs can not form nanocomposite due to the electrostatic repulsion. When PDDA is modified on CNTs, the composite is positively charged and the stable nanocomposite containing CNTs,


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PDDA and AuNPs can be formed through electrostatic interaction. The preparation process of AuNPs/PDDA/CNTs was also characterized by UV-vis absorption spectra (Figure 2B). The typical surface plasma resonance absorption of AuNPs at 520 nm reveals the successfully preparation

of

AuNPs/PDDA/CNTs.

The

above

results

prove

the

formation

of

AuNPs/PDDA/CNTs nanocomposite. The uniformly distributed AuNPs on the modified electrode can be used to immobilize the biotinylated DEVD-peptide via Au-S bonds. Characterization of Ru@SiO2 Probe Silica nanoparticles is a good matrix for immobilizing a high concentration of Ru(bpy)32+ to promote ECL detection.30 As a result, Ru(bpy)32+-doped silica nanoparticles have become ideal tags in bioanalytical applications because some functional groups can be easily introduced to the surface of Ru@SiO2 nanoparticles.19, 32-34 In the present work, Ru@SiO2 nanoparticles was used as probes to generate ECL signal with TPA. W/O microemulsion method was employed to synthesize Ru@SiO2 nanoparticles according to the literature.21 The morphology of Ru@SiO2 nanoparticles was characterized by TEM as shown in Figure 3A. The synthesized Ru@SiO2 nanoparticles were spherical, monodisperse, and uniform-sized with a diameter of ~70 nm. The process of modification of streptavidin on Ru@SiO2 nanoparticles is illustrated in Scheme 1A and characterized by FTIR spectroscopy as shown in Figure 3B. The strong peak at 1096 cm-1 can be attributed to the stretching vibration of Si-O-Si.18,35 After coupling of streptavidin on Ru@SiO2 nanoparticles, two new absorption bands appear at 1659 and 1533 cm-1, which correspond to amide bands I (C=O stretching) and II (N-H bending) and are consistent with the pure streptavidin. The results indicate that streptavidin can be successful modified on the Ru@SiO2 nanoparticles.


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Figure 3. (A) TEM image of Ru@SiO2 nanoparticles. (B) FTIR spectra of streptavidin (red), Ru@SiO2 nanoparticles (black), Ru@SiO2-SA (blue). (C) Evolution of Zeta potential of the Ru@SiO2-SA coupling process. (D) ECL curves of Ru@SiO2-SA modified electrode. Additionally, the process of coupling was also confirmed by Zeta Potential. The values of the Zeta voltage of Ru@SiO2, Ru@SiO2-NH2, Ru@SiO2-SA are shown in Figure 3C. After the Ru@SiO2 nanoparticles are functionalized with amino-groups, the negatively charged Ru@SiO2 nanoparticles change to the positively charged state, which demonstrates the successful reaction between APTES and the hydroxy groups of Ru@SiO2 surface. When Ru@SiO2 nanoparticles are coupled with streptavidin via the linker of glutaraldehyde, the Zeta potential of Ru@SiO2


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become negatively charged again because streptavidin is positively charged at neutral pH. The above results demonstrate the successful modification of Ru@SiO2. ECL behavior of Ru@SiO2-SA modified GCE was investigated in the presence of 0.1 mol L-1 TPA as shown in Figure 3D. No ECL peak is obtained at the bare GCE. At the Ru@SiO2-SA modified electrode, one strong anodic ECL can be obtained at 1.1 V, revealing that Ru@SiO2-SA can react with TPA to generate ECL and act as nanoprobe for the ECL bioassay. Electrochemical Characterization of ECL Sensor

A 40 20

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a b c d e f

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0.0

-0.2

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Z'/Ohm

Figure 4. Cyclic voltammograms (CVs) (A) and Electrochemical impedance spectroscopy (EIS) (B) for the ECL sensing platform during the fabrication process: (a) bare GCE, (b) AuNPs/PDDA/CNTs/GCE, (c) biotin-DEVD peptide/AuNPs/PDDA/CNTs/GCE, (d) MCH/ biotin-DEVD

peptide/

AuNPs/PDDA/CNTs/GCE,

(e)

MCH/

biotin-DEVD

peptide/AuNPs/PDDA/CNTs/GCE after cleavage by active caspase-3, (f) Ru@SiO2/MCH/ biotin-DEVD peptide/AuNPs/PDDA/CNTs/GCE after cleavage by active caspase-3. The active caspase-3 was obtained from K562 cells incubated with 1µL mL-1 apoptosis inducer for 12 h. After the immobilization of the biotinylated DEVD-peptide on the AuNPs/PDDA/CNTs modified GCE, the modified electrode was first immersed into the 6-mercapto-1-hexanol (MCH) aqueous solution to block the residual active sites. Subsequently, the above electrode was dunked


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into 100 µL of cell lysates containing active caspase-3 which could selectively cleave the Nterminus of DEVD-peptide. The cleaved biotin label could diffuse away from the electrode surface. And then, the Ru@SiO2-SA signaling probe could effectively recognize the uncleaved biotin-DEVD through the biotin-avidin interaction. The assembly process of ECL sensor was characterized by cyclic voltammograms (CVs) as shown in Figure 4A. Compared with the bare GCE, the peak current increases significantly and the peak potential difference between the anodic and the cathodic peaks decreases after the modification of AuNPs/PDDA/CNTs composites because of the excellent conductivity of the composites. The peak current gradually decreases after the modification of biotin-DEVD peptide and MCH, and then increases again when the peptides are recognized and cleaved by the active caspase-3. At last, the capture of the Ru@SiO2-SA leads to the decrease of the peak current due to the electrostatic repulsive between the ECL probe and [Fe(CN)6]3-/4-. Electrochemical impedance spectroscopy (EIS) was also used to confirm the assembly process, and the results are consistent with the CV results as shown in Figure 4B. ECL Detection of Caspase-3 Activity from K562 Cells by Apoptosis Inducer

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Figure 5. (A) ECL measurements for captured Ru@SiO2-SA on the MCH/ biotin-DEVD peptide/ AuNPs/PDDA/CNTs/GCE after being immersed in apoptotic cell lysates for different


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times. K562 cells were incubated with apoptosis inducer for 12 h. (B) Relationship between the ECL intensity and the immersed time. K562 cells were chosen as a model and exposed to apoptosis inducer for the activation of caspase-3. To detect the caspase-3 activation, the time-dependent cleavage of biotin-DEVD peptide by active caspase-3 in the lysates from K562 cells treated with apoptosis inducer for 12 h was investigated by ECL method and shown in Figure 5. According to the strategy listed in Scheme 1, when the immersion time of ECL sensor in caspase-3 increased, more residual peptides can be cleaved and less ECL probes are attached on the sensor, leading to the decreased ECL signal. Figure 5 shows that the ECL intensities decrease gradually with increasing immersion time from 0 to 90 min. When the immersion time is over 60 min, the decreased ECL signal begins to level off. Thus, 60 min of immersion time in active caspase-3 lystates can effectively cleave biotin-DEVD peptide. Marnett et al. reported that caspase-3 activation is time-depended.4 In this work, the time course of caspase-3 activation by apoptosis inducer was investigated and shown in Figure 6. In the absence of apoptosis inducer, caspase-3 is not activated, leading to a maximum ECL intensity. After the K562 cells are incubated with apoptosis inducer, the ECL intensities decrease gradually with the increase of incubation time and reach a minimum value at 12 h. Further increase incubation time, the ECL intensities begin to increase. It was reported that B-cell lymphoma-2 (Bcl-2) family is a critical regulatory factor in response to apoptosis. Bcl-2 expression affects cation transport in mitochondria and protects against organelle dysfunction induced by several apoptotic stimuli. In cells overexpressing Bcl-2 or the closely related protein will block apoptotic response and decrease caspase-3 activity.36 Therefore, when K562 cell is incubated in apoptosis


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inducer for more than 12 h, apoptotic response will be blocked, resulting in the decrease of caspase-3 activity. As a result, 12 h of incubation time is chosen.

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Figure 6. (A) ECL measurements for caspase-3 activity after the K562 cells were incubated with apoptosis inducer for different times: 0, 2, 4, 6, 8, 10, 12 and 14 h. (B) Relationship between the ECL intensity of the sensor and the apoptosis inducer incubation time. To confirm that the decrease in ECL intensity is derived from the activation of caspase-3 in apoptosis, the activity of caspase-3 was detected by colorimetric method using a caspase-3 cellular activity assay kit under the same condition. It can be found in Figure 7 that the activity of caspase-3 reaches the maximum when the K562 cells are incubated with apoptosis inducer for 12 h. Further increase incubation time, the activity of caspase-3 decreases. The results of colorimetric method are in good accordance with the results of ECL detection, suggesting that the proposed ECL method can be successfully used to monitor caspase-3 activity during cell apoptosis.


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Figure 7. (A) caspase-3 colorimetric kit for caspase-3 activity after the K562 cells were incubated with apoptosis inducer for different times: 0, 2, 4, 6, 8, 10, 12 and 14 h. (B) Relationship between the caspase-3 activity and the apoptosis inducer incubation time. In order to further assess the specificity of the ECL sensor, caspase-3 inhibitor Ac-DEVD-CHO was used to inhibit the activity of caspase-3. As shown in Figure 8, the ECL intensity is much higher in the presence of Ac-DEVD-CHO compared to the absence of Ac-DEVD-CHO. The results further indicate that the obtained ECL intensity is related to the caspase-3 activity and the ECL sensor can monitor caspase-3 activity during the cell apoptosis.

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Figure 8. ECL measurements for caspase-3 inhibition by Ac-DEVD-CHO. K562 cells were preincubated with 25 µM Ac-DEVD-CHO for 30 min. Detection of Caspase-3 Activity with ECL Method In order to make sure that the proposed strategy can be quantitatively used to evaluate caspase3 activity, ECL curves upon analyzing caspase-3 at different concentrations were recorded as shown in Figure 9A. The ECL signals decrease in response to caspase-3 with a mounting concentration within the range from 0.2 to 200 pg/mL. A well linear response between ECL intensity and caspase-3 concentration can be obtained as shown in Figure 9B. The detection limit is determined as 0.07 pg/mL (3σ), which is more sensitive compared with other reports.9,37,38 To evaluate the specificity of the method, several proteins such as thrombin, trypsin, and carboxypeptidase have been explored. The ECL results depict that these proteins can not influence ECL intensity, implying the high selectivity of the sensor. 6000 0 pg/mL 0.2 pg/mL 2 pg/mL 5 pg/mL 20 pg/mL 120 pg/mL 200 pg/mL

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Figure 9. (A) ECL measurements of caspase-3 at different concentrations. (B) The linear relationship between the ECL intensity and the caspase-3 concentrations. Detection of Caspase-3 activity from K562 Cells Induced by Anticancer Drugs Using ECL Sensor


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The possibility of this strategy for assay caspase-3 activity in response to anticancer drugs was evaluated. Nilotinib is a useful drug in the treatment of imatinib-refractory chronic myeloid leukemia (CML) by inducing growth arrest and apoptosis of CML cells. Thus, nilotinib was often used as a model antileukemia drug to induce the apoptosis of K562 cells.39, 40 In the present work, nilotinib is chosen as a model drug to induce the apoptosis of K562 cells in the ECL detection of caspase-3. Figure 10 shows the concentration dependence for caspase-3 activity from K562 cells induced by different concentrations of nilotinib in DMSO solution for 12h. In the absence of nilotinib, the ECL intensity is at its maximum corresponding to the lowest caspase-3 activity. While increasing the concentration of nilotinib from 5 to 10 µM, the ECL intensities decrease gradually, and then increase from 10 to 40 µM. The results indicate that the proposed strategy can effectively monitor caspase-3 activity from k562 cells induced by anticancer drugs with a great sensitivity.

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Figure 10. Concentration dependence of caspase-3 activity by nilotinib. (A) ECL measurements for caspase-3 activity after the K562 cells were incubated with the following concentration of nilotinib for 12 h. (B) Relationship between the ECL intensity and the concentrations of nilotinib. CONCLUSIONS


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In summary, we developed a novel ECL sensing platform to monitor caspase-3 activity during cell apoptosis via combining the cleavage of biotin-DEVD peptide by active caspase-3 with the specific interaction between biotin of the peptide and streptavidin. The streptavidin-modified Ru(bpy)32+-doped silica nanoparticles were applied in the field of ECL sensing for caspase-3 for the first time, which obtained high sensitivity and stability for cytosensing. Using the sensor, the anticancer drug nilotinib for the treatment of CML were successfully evaluated. This sensing platform presents a promising application in cancer research, such as the evaluating of drug efficacy and the monitoring of cancer treatment. Corresponding Author E-mail: [email protected].

ACKNOWLEDEMENT

Y. P. Dong. and G. Chen. contributed equally to this work. This research was supported by National Natural Science Foundation of China (Nos 21335004, 21427807, 21575002), Natural Science Foundation of Anhui Province (No. 1408085MF114), China Postdoctoral Science Foundation (No. 2013M541637), and Jiangsu Province Postdoctoral Science Foundation (No. 1302126C). REFERENCES (1) Zhang, J. J.; Zheng, T. T.; Cheng, F. F.; Zhu, J. J. Chem. Commun. 2011, 47, 1178-1180. (2) Hengartner, M. O. Nature 2000, 407, 770-776. (3) Huang, X. Y.; Liang, Y. R.; Ruan, L. G.; Ren, J. C. Anal. Bioanal. Chem. 2014, 406, 56775684.


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Electrochemiluminescent Sensing for Caspase-3 ... - ACS Publications

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