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Signal Amplification Cytosensor for Evaluation of Drug-Induced Cancer Cell Apoptosis Yafeng Wu,† Hao Zhou,‡ Wei Wei,† Xin Hua,† Lixin Wang,*,‡ Zhenxian Zhou,§ and Songqin Liu*,† †

State Key Laboratory of Bioelectronics, School of Chemistry and Chemical Engineering, Jiangning District 211189, Nanjing, Jiangsu Province, P. R. China ‡ Medical School, Southeast University, Nanjing, 210096, P. R. China § Nanjing Second Hospital, Nanjing, 210003, P. R. China S Supporting Information *

ABSTRACT: Apoptosis is involved in the pathology of a variety of diseases. The measurement of apoptosis will help us to evaluate the onset of disease and the effect of therapeutic interventions. In addition, the increased demand for understanding the early stages of apoptosis is pushing the envelope for solutions in early instance real-time monitoring of death kinetics. Here we present a novel electrochemiluminescent cytosensing strategy to quantitate apoptotic cell numbers, screen some anticancer drugs, and evaluate their effects on hepatocarcinoma cell line (HepG2) cells by utilizing the human antiphosphatidyl serine antibody (APSA) conjugated Ru(bpy)32+-encapsulated silica nanoparticle (APSASiO2@Ru) as the detection probe. HepG2 cells were easily immobilized on the arginine-glycine-aspartic acid-serine (RGDS)multiwalled carbon nanotubes (RGDS-MWCNTs) nanocomposite by the specific combination of RGD domains with integrin receptors on the cell surface. Then APSA-SiO2@Ru was introduced to the surface of apoptosis cells through the specific interaction between APSA and phosphatidylserine (PS) that distributed on the outer membrane of apoptotic cells. On the basis of the signal amplification of the APSA-SiO2@Ru nanoprobe, the cytosensor could respond as low as 800 cells mL−1, showing very high sensitivity. In addition, the dynamic alterations of surface PS expression on HepG2 cells in response to drugs and the cell heterogeneity were also demonstrated. The strategy presented a promising platform for highly sensitive cytosensing and convenient screening of some clinically available anticancer drugs.

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fluorescent probes or labels for the readout of apoptosis markers in a variety of formats, such as flow cytometry, microscopy, microarray, etc.19−21 Albert van den Berg and coworkers employed quantum dots conjugated-Annexin V for specific targeting of apoptotic cells, in both apoptosis detection and staining of apoptotic “living” cells. Using this approach, they could sensitively detect the staining of apoptotic cells with fluorescence and time-lapse imaging.6 The second approach uses microfluidic devices.22,23 Zhu and co-workers analyzed apoptotic cells in a fabricated microfluidic device by utilizing Annexin V conjugated quantum dots as apoptosis detection probes.23 The third approach is electrochemical detection.24,25 Li’s group reported an electrochemical approach where a helix peptide ferrocene on gold, skillfully designed and immobilized, was applied for detection of apoptosis. Herein we combined a simple electrochemiluminescent transducer with nanobiotechnology to develop a novel method for convenient and sensitive quantification of apoptotic cancer cells. An arginine-glycine-aspartic acid-serine (RGDS) tetrapeptide was employed to functionalize MWCNTs.26,27 The resultant RGDS MWCNTs nanocomposite was then used as a nanoscale anchorage substrate, which effectively captured cells

evelopment of a simple and convenient approach for apoptosis detection has become an area of intensive interest.1−6 Up until now, there are more than 300 different apoptosis-related kits and techniques that were developed for apoptosis detection and quantification. These techniques included detection of surface lipid translocation,7−9 protein release from cell, change of caspase activity,10−12 alteration of mitochondrial integrity and function,13,14 shift in redox state,15 and DNA fragmentation.16 Many of them have high sensitivity and have been successfully implemented in research studying the mechanism of diseases and therapeutics. Nevertheless, the increased demand for understanding the early stages of apoptosis is pushing the envelope for solutions in early instance real-time monitoring of death kinetics. The cell apoptosis process can be divided into a succession of three phases: initiation, decision, and execution. After activation of a death receptor (initiation phase), the complex process is triggered by a series of enzymes (decision phase). Then in the execution phase, cells change at the mitochondrion level at the first and outer membrane and other structures change subsequently. One of the important phenomena of this stage is the translocation of the membrane phosphatidylserine from the inner to the outer leaflet of the plasma membrane.17,18 On the basis of the property, three primary approaches are now available for real-time determination of programmed cell death. The first approach is a spectroscopic technique that relies on © 2012 American Chemical Society

Received: October 13, 2011 Accepted: January 7, 2012 Published: January 7, 2012 1894

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10 μL of 1 mg mL−1 RGDS was immediately dropped on its surface. Then, the MWCNTs/GCE was incubated for 2 h to yield an RGDS-MWCNTs/GCE. Following a thorough rinse with 0.01 M pH 7.4 PBS, 10 μL of apoptotic HepG2 cell suspension at a certain concentration was dropped on the RGDS-MWCNTs/GCE surface. After a careful rinse with 0.01 M pH 7.4 PBS, to remove the nonspecific captured cells, the cytosensor of HepG2/RGDS-MWCNTs/GCE was obtained and used for a subsequent assay. RGDS directly coupled GCE was used to conduct the comparative experiment. ECL Analysis. A volume of 10 μL of APSA-SiO2@Ru was dropped on the surface of HepG2/RGDS-MWCNTs/GCE and incubated at 37 °C for 35 min to obtain APSA-SiO2@Ru/ HepG2/RGDS-MWCNTs/GCE. For the comparative experiment, 10 μL of APSA-SiO2@Ru was dropped on the surface of HepG2/RGDS/GCE and incubated at 37 °C for 35 min to obtain APSA-SiO2@Ru/HepG2/RGDS/GCE. For another comparative experiment, APSA was directly conjugated with NH2-functionalized Ru(bpy)32+ (APSA-Ru) in the presence of 20 mM EDC/NHS at a mole ratio of 1:100, which is the maximum loading capacity of APSA. Then 10 μL of APSA-Ru was dropped on the surface of HepG2/RGDS-MWCNTs/ GCE. The HepG2/RGDS-MWCNTs/GCEs were incubated at 37 °C for 35 min to conjugate Ru on the cell surface (APSARu/HepG2/RGDS-MWCNTs/GCE). After being carefully washed with 0.01 M pH 7.4 PBS, ECL measurements were performed. Monitoring Dynamic Surface Antigen Expression in Response to Drugs. Anticancer drugs such as cisdiamminedichloroplatinum (CDDP), 5-fluorouracil, gemcitabine hydrochloride, and paclitaxel were used as models. The RGDS-MWCNT/GCEs were incubated with drug-treated HepG2 cells for 2 h. After carefully rinsing with PBS, the obtained electrodes were subjected to the nanoprobe-based ECL and fluorescent analysis.

on an electrode surface via specific binding between cell surface integrins and RGD domains. An APSA-SiO2@Ru nanoprobe was then delivered which could readily bind to the outer membrane of apoptotic cells and distinguish the apoptosis from unaffected cells. This led to a highly sensitive cytosensor for quantitation of cell numbers, investigation of the dynamic alterations of surface PS expression on HepG2 cells in response to drugs, and the cell heterogeneity.

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EXPERIMENTAL SECTION The materials and reagents, apparatus, the preparation of SiO2@Ru nanoparticles, and HepG2 cell and culture are shown in the Supporting Information. Preparation of Nanoprobe (APSA-SiO2@Ru). The SiO2@Ru nanoparticles with a diameter of about 50 nm were used (Figure S1 in the Supporting Information). The process for preparation of the nanoprobe is shown in Scheme 1. First, 1 Scheme 1. Schematic for the Preparation of Cytosensor

mL of the above SiO2@Ru nanoparticles suspension was reacted with 5 mL of 5% γ-glycidoxypropyltrimethoxysilane (GPMS) in dry toluene, at room temperature, with continuous stirring overnight. The nanoparticles were then separated by centrifugation and rinsed thoroughly with toluene and ethanol to remove any physically adsorbed GPMS. The particles were dried in a nitrogen atmosphere at 100 °C for 1 h in order to obtain epoxy-functionalized SiO2@Ru nanoparticles. Then, the particles were added to 2 mL of 1 mg mL−1 APSA solution. The reaction was kept at 37 °C for 2 h under stirring. After centrifugation and washing, the APSA modified SiO2@Ru nanoparticles (APSA-SiO2@Ru) were obtained. Finally, the APSA-SiO2@Ru nanoprobes were redispersed in 5 mL of 1% BSA solution for another 30 min to block the excess aminogroup and nonspecific binding sites of the APSA-SiO2@Ru. After centrifugation and washing with PBS, the resultant APSASiO2@Ru nanoprobes were dispersed with 0.02 M of pH 7.4 PBS to a final volume of 2 mL. It was kept at 4 °C for later use.29 As control, anti-CEA modified SiO2@Ru (anti-CEASiO2@Ru) was prepared with a procedure similar to that for the APSA-SiO2@Ru. Preparation of Cytosensor and Cell Capture. Scheme 1 represents the preparation of the cytosensor. A volume of 5 μL of 1 mg mL−1 MWCNTs pretreated with strong acid was dropped on the surface of a clean glassy carbon electrode (GCE) and dried in a desiccator. The MWCNTs/GCE was then immersed in a solution containing 2 mM 1-ethyl-3-(3dimethylaminopropyl) carbodiimide (EDC) and 5 mM Nhydroxysuccinimide (NHS) for 1 h. After the activated MWCNTs/GCE was thoroughly rinsed with deionized water,

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RESULTS AND DISCUSSION Detection Principle and ECL Behavior. Characterization of RGDS-MWCNTs/GCE is shown in the Supporting Information. HepG2 cells were captured on RGDSMWCNT/GCEs through the specific recognition of cell surface integrin to RGDS. Then the resultant HepG2/RGDSMWCNTs/GCE was incubated in APSA-SiO2@Ru suspension to bring nanoprobes to the cell surface through specific immunoreactions. These immunoreactions occurred between APSA, on the nanoprobe, and the corresponding PS, on apoptotic cancer cell surfaces. Upon addition of 2(diisopropylamino)ethylamine (DPEA) into the assay buffer, the electrode exhibited a clear ECL peak at 1.12 V (Figure 1A, curves e and f), which corresponded to the reaction between Ru(bpy)32+ and DPEA (see reaction mechanism in the Supporting Information, Scheme S1). However, no detectable signal was observed on bare GCE, MWCNTs/GCE with RGDS-MWCNTs/GCE, or HepG2/RGDS-MWCNTs/GCE, respectively (Figure 1A, curves a, b, c, and d). Thus, the ECL signal of curves e and f was attributed to APSA-SiO2@Ru on the electrode surface. Furthermore, the ECL intensity increased with the greater apoptotic cancer cell concentrations, which changed from 1 × 105 (Figure 1A, curve e) to 1 × 107 cells mL−1 (Figure 1A, curve f). Thus, the ECL signal was directly related to the coverage of the immobilized APSA-SiO2@Ru, which itself depended on both the expression of PS groups on the apoptotic cancer cell surface and the amount of cells 1895

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The signal reached its maximum at an incubation time of 35 min. A further increase of incubation time resulted in no signal increase, which is attributed to a close-to-completion coupling of the immunoreaction (Figure S2C in the Supporting Information). Therefore, the incubation time of 35 min was used throughout our study. The ECL intensity was observed to be related to pH of the detection solution. At low pH values, the radical cation of the tertiary amine group was difficult to deprotonate into a highreducing free radical intermediate.30 Meanwhile, at high pH values, Ru(bpy)33+ competitively reacted with OH− ions in the system, which reduced the availability of Ru(bpy)33+ and decreased the ECL intensity. In this study, a pH of 7.5 was found to yield the largest ECL response (Figure S2D in the Supporting Information). Cell Detection. Under the optimal conditions, the ECL intensity was proportional to the logarithmic value of the cell concentration, which ranged from 800 to 1.0 × 107 cells mL−1, with a correlation coefficient R of 0.995 (Figure 2). The linear

Figure 1. (A) The ECL curves of (a) GCE, (b) MWCNTs/GCE, (c) RGDS-MWCNTs/GCE, (d) HepG2/RGDS-MWCNTs/GCE, (e) APSA-SiO2@Ru/HepG2/RGDS-MWCNTs/GCE with the HepG2 concentration of 1 × 105 cells mL−1, and (f) APSA-SiO2@Ru/HepG2/ RGDS-MWCNTs/GCE with the HepG2 concentration of 1 × 107 cells mL−1. (B) Comparison of ECL intensity at (1) MWCNT/GCE, (2) HepG2/RGDS-MWCNTs/GCE incubated with anti-CEA-SiO2@ Ru, (3) APSA-Ru/HepG2/RGDS-MWCNT/GCE, (4) APSA-SiO2@ Ru/HepG2/RGDS/GCE, and (5) APSA-SiO2@Ru/HepG2/RGDSMWCNT/GCE. The concentration of HepG2 was 104 and 107 cells mL−1, respectively.

captured on the cytosensor surface. Thus, quantifying the cells could be carried out by monitoring the ECL signal. To verify the specific immunoreaction between APSA and PS, a control experiment was performed by incubation of HepG2/RGDS-MWCNTs/GCE in anti-CEA-SiO2@Ru suspension. No remarkable ECL intensity was observed in comparison with the result obtained in APSA-SiO2@Ru/ HepG2/RGDS-MWCNTs/GCE (Figure 1B, 2). This suggested a good selectivity of the proposed cytosensor and that the signal originated from neither the cross reactions nor the nonspecific adsorption. The much lower ECL intensity was obtained in APSA-Ru/HepG2/RGDS-MWCNTs/GCE (Figure 1B, 3) or APSA-SiO2@Ru/HepG2/RGDS/GCE (Figure 1B, 4) and verified the signal amplification of MWCNTs and APSA-SiO2@Ru nanoprobe. All these results indicated that quantification of the number of cells as well as evaluation of the PS expression on the apoptosis cell surface could be carried out by ECL signal monitoring. Optimization of Detection Conditions. The PS groups expressed on the cell surface depended on the apoptosis time of HepG2 cells with Apoptosis Inducers Kit (C0005). The ECL intensity at the APSA-SiO2@Ru/HepG2/RGDS-MWCNTs/ GCE increased and tended to a steady value after 20 h (Figure S2A in the Supporting Information). That is because the membrane PS has been completely translocated from the inner to the outer leaflet of the plasma membrane. Thus, 20 h was chosen as the apoptotic time. The incubation time was an important parameter for both capturing cells on the RGDS-MWCNTs/GCE and the specific recognition of APSA-SiO2@Ru by PS groups presented on the captured cell surface. With an increased incubation time, the ECL intensity at APSA-SiO2@Ru/HepG2/RGDS-MWCNTs/ GCE also increased and tended to a steady value after 100 min (Figure S2B in the Supporting Information). This indicated a tendency to adequately capture HepG2 cells on the sensor surface. To ensure the adhesion efficiency of HepG2 cells at low concentration, 125 min was chosen as the optimal incubation time of HepG2 cells. The ECL intensity also depended on the incubation time of HepG2/RGDS-MWCNTs/GCE with APSA-SiO2@Ru suspension. The ECL intensity of APSA-SiO2@Ru/HepG2/RGDSMWCNTs/GCE increased initially with the incubation time.

Figure 2. ECL intensity of APSA-SiO 2 @Ru/HepG2/RGDSMWCNTs/GCE obtained with HepG2 cell concentrations of 800, 1 × 103, 1 × 104, 1 × 105, 1 × 106, 5 × 106, and 1 × 107 cells mL−1. Inset: plot of ECL intensity vs logarithm of HepG2 cell numbers.

regression equation is I = 1589 log(ccell/cell mL−1) − 4530, I is ECL intensity, while c is the cell concentration in the suspension. The cytosensor could respond as low as 800 cells mL−1, which was comparable with that of 1500 cells mL−1 for K562 by lectin-based nanoprobes functionalized with ConA and HRP.31 This was also much lower than that of 6000 cells mL−1 at an immunosensor chip for detection of E. coli O157:H7,32 confirming the high sensitivity. In addition, the bound nanoprobes on apoptotic cancer cells can also be determined by Photoluminescence (PL) spectra, which were obtained using an excitation wavelength of 460 nm. The maximum emission peak was observed at about 605 nm (Figure S3 in the Supporting Information). Control experiments were done on a bare microscope slide, MWCNTs/slide, RGDS-MWCNTs/slide, and HepG2/RGDS-MWCNTs/slide, respectively. No absorbance at the same wavelength window was observed. Furthermore, the absorbance of APSA-SiO2@ Ru/HepG2/RGDS-MWCNTs/slide increased with the concentration of apoptotic cancer cells in the range of 800 to 1.0 × 107 cells mL−1, with a correlation coefficient of 0.996. At the cell concentrations of 1.0 × 104 and 1.0 × 107 cells mL−1, the cytosensor showed the relative standard deviation of 4.2% and 3.5%, as examined by five determinations, respectively. Six cytosensors, made independently, showed an acceptable reproducibility with the relative standard deviation of 1.65% and 2.12%, respectively. Thus, the resulting 1896

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The proposed strategy was also available for analyzing the dynamic process of plasma membrane PS redistribution during apoptosis by a time-lapse assay. We analyzed the time-lapse ECL intensity and fluorescence images of cells with CDDP present at a concentration of 2.5 μg mL−1. As shown in Figure

cytosensor showed good performance for detection of cancer cells with a broad detection range, low detection limit, and good reproducibility. Monitoring of Dynamic Surface Antigen Expression in Response to Drugs. The high sensitivity of the proposed cytosensor allowed it to be further used in evaluation of dynamic alterations of surface antigen expression on cells in response to drugs. Drug-treated HepG2 cells could be captured by RGDS-MWCNTs/GCE and monitored by the proposed method, with untreated cells as reference. Figure 3a−d showed

Figure 4. Time-lapse analyzes the nanoprobe stained cells at different time points treated with CDDP at a concentration of 2.5 μg mL−1. (a) Dependence of ECL intensity on the incubation time. Fluorescent images of cells which have been treated with CDDP at a concentration of 2.5 μg mL−1 at the time point of (b) 0, (c) 5, (d) 10, and (e) 20 h, respectively.

4, either the ECL intensity or fluorescent intensity had increased in a time-dependent manner, which indicated that PS was translocated from the inner to outer leaflet of the plasma membrane. The proposed strategy could be further used to test the cell heterogeneity.28 This would reveal some important variations in cell pathway dominated processes, such as apoptosis, for an individual cell. Herein, the fluorescence microscope images of different cells that were treated with CDDP, at a concentration of 2.5 μg mL−1 for 20 h, were presented. From the images in Figure 5, six cells were picked. The assay indicated that the intensity varied a lot from each of the picked cells. Cells numbered 2 and 5 showed comparatively high fluorescence intensity, which may be deduced from the adequate presentation of PS on the membrane of the two cells. Cells numbered 1, 3, 4, and 6 exhibited lower fluorescence intensity as they may be undergoing the early stage of apoptosis. The fluorescence intensity varied a lot across these picked cells, which vividly depicted the single cell heterogeneity during the process of PS externalization when apoptosis was triggered. The results were further confirmed by SEM images of the six picked cells (Figure 5, 1−6). Cells numbered 2 and 5 showed comparatively more nanoparticles on the substrate than cells numbered 1, 3, 4, and 6.

Figure 3. Analysis of cell apoptosis after anticancer drug treatment and nanoprobe immobilization. (a−d) Correlative analysis shows that the percentage of apoptotic cells was in dose-dependent fashion. Cells were incubated with the presence of a CDDP, 5-fluorouracil, gemcitabine, and paclitaxel gradient for 20 h, respectively. Bright field images for cells treated by CDDP with concentrations of (e) 0, (f) 0.5, (g) 1.5, (h) 2.0, and (i) 2.5 μg mL−1 (20 h, 37 °C), respectively.

the proportion of apoptotic cells stimulated by the anticancer drug gradient. The drug-induced apoptosis of cancer cells was observed in a concentration-dependent manner. At the concentration of 2.5 μg mL−1 (20 h, 37 °C), CDDP triggered apoptosis of 95% ± 5.3%. 5-Fluorouracil at 5 μg mL−1 (20 h, 37 °C) induced apoptosis of 86% ± 6.1%. Gemcitabine at 1 mg mL−1 (20 h, 37 °C) induced apoptosis of 75% ± 9.5%. Paclitaxel at 1 mg mL−1 (20 h, 37 °C) induced apoptosis of 92% ± 9.0%. These results revealed that CDDP showed a more potent effect on induction of apoptosis in the HepG2 cells. Figure 3e−i illustrated a series of bright field images for cells treated by CDDP, with concentrations of 0, 0.5, 1.5, 2.0, 2.5 μg mL−1 (20 h, 37 °C), respectively. The cells changed from longstrip to round mean cell apoptosis. For control experiments, antibiotic drugs such as benzylpenicillin, amoxicillin, cefalexin, and norfloxacin treated HepG2 cells (20 h, 37 °C) were used to replace anticancer drug treated HepG2 cells; other steps were the same. No ECL signal was obtained, so antibiotic drugs triggered no cell apoptosis. Therefore, the cytosensor has a good selectivity to platinum anticancer drugs. Thus, the variation of ECL signal after treatment with different drugs allowed investigation of the interaction between the HepG2 cell and drugs.

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CONCLUSIONS In this study, a novel ECL cytosensor is created by dual signal amplification of MWCNTs and APSA-SiO2@Ru nanoprobes, and the resultant cytosensor could respond as low as 800 cells mL−1. The high sensitivity allows the proposed cytosensor to be further used for screening anticancer drugs. Moreover, the proposed method has exhibited the capability of evaluating the 1897

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ACKNOWLEDGMENTS

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REFERENCES

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The project is supported by the National Basic Research Program of China (Grant No. 2010CB732400), the Key Program (Grant No. 21035002) from the National Natural Science Foundation of China, National Natural Science Foundation of China (Grant No. 20875013) and the Key Program (Grant No. BK2010059) from the Natural Science Foundation of Jiangsu Province, Open Foundation from State Key Laboratory of Bioelectronics and Significant Scientific Research Guidance Foundation from Southeast University.

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Figure 5. Cells that were undergoing apoptosis can be discriminated by the nanoprobe. Bright field images (a, b) and fluorescent micrographs (c, d) for cells before (a, c) and after (b, d) treatment with CDDP (2.5 μg mL−1, 20 h), respectively. The different cells were marked as blue dashed circles, and six different cells were picked for analysis (d). (e) Fluorescence intensity analysis of six picked cells. Images 1−6 were the SEM images of six picked cells corresponding to picture d.

dose- and time-dependent effect of different anticancer drugs on the cells. It also allows monitoring the dynamic behavior of PS redistribution from the inner plasma membrane at the single cell level. This capability is proved to be helpful in understanding intracellular enzyme activities, which plays an important role in the apoptosis cascade.33 The proposed strategy also has the ability to test cell heterogeneity. This would reveal some important variations in cell pathway dominated processes, such as apoptosis, for an individual cell. We therefore believe that this method can certainly be used to monitor living cells for real-time studies of biomedical problems, such as anticancer pharmacological kinetics, and may hold potential in deeper cellular biological studies.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in the text. 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] (L.X.W.); [email protected] (S.Q.L.) 1898

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