Real-Time Evaluation of Live Cancer Cells by an in Situ Surface

Research Article www.acsami.org

Real-Time Evaluation of Live Cancer Cells by an in Situ Surface Plasmon Resonance and Electrochemical Study Changyu Wu,† Fawad ur Rehman,† Jingyuan Li,‡ Jing Ye,† Yuanyuan Zhang,† Meina Su,† Hui Jiang,† and Xuemei Wang*,† †

State Key Laboratory of Bioelectronics (Chien-Shiung Wu Laboratory), Southeast University, Nanjing 210096, China Laboratory Animal Center, Nantong University, Nantong 226001, China

‡

S Supporting Information *

ABSTRACT: This work presents a new strategy of the combination of surface plasmon resonance (SPR) and electrochemical study for real-time evaluation of live cancer cells treated with daunorubicin (DNR) at the interface of the SPR chip and living cancer cells. The observations demonstrate that the SPR signal changes could be closely related to the morphology and mass changes of adsorbed cancer cells and the variation of the refractive index of the medium solution. The results of light microscopy images and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide studies also illustrate the release or desorption of HepG2 cancer cells, which were due to their apoptosis after treatment with DNR. It is evident that the extracellular concentration of DNR residue can be readily determined through electrochemical measurements. The decreases in the magnitudes of SPR signals were linearly related to cell survival rates, and the combination of SPR with electrochemical study could be utilized to evaluate the potential therapeutic efficiency of bioactive agents to cells. Thus, this label-free, real-time SPR−electrochemical detection technique has great promise in bioanalysis or monitoring of relevant treatment processes in clinical applications. KEYWORDS: surface plasmon resonance, electrochemistry, real-time evaluation, live cells, cancer

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INTRODUCTION

process because of the subsequent removal of the drugs before SPR measurements. Electrochemical techniques are widely used in biomedical applications, such as detection of bioactive molecules21,22 and determination of interactions between bioactive agents and biological supramolecules.23−25 Jiang and co-workers26 proposed a highly sensitive electrochemical method for analyzing daunorubicin (DNR) with a low detection limit of 10 nM and a wide linear range of 10−500 nM. Moreover, analysis of cell features27−30 and evaluation of the therapeutic efficiency of drugs31,32 are also possible because of their simple operation process, fast response speed, high sensitivity, and good accuracy. Zhu and co-workers33 developed an electrochemical cytosensing platform for apoptotic cell detection. This platform presented a significant tool for monitoring the early stages of apoptosis. However, the electrochemical methods were hardly used in label-free and real-time analysis of living cells. Combination of SPR and electrochemical techniques can facilitate the label-free, real-time, and highly sensitive analysis of cells in solution or under physiological conditions.34 SPR can offer the changes in refractive index and other biophysical

Investigation of cells allows us to better understand cell proliferation, function, and death and thereby to improve the diagnosis and treatment of diseases, especially cancer. Dozens of classic and modern techniques have been applied in evaluation of cells, such as 3-(4,5-dimethyl-2-thiazolyl)-2,5diphenyl-2H-tetrazolium bromide (MTT) colorimetric methods, chemical labeling imaging, etc.1−3 The MTT assay and related colorimetric methods are usually used to evaluate the survival rate of cells. However, the colorimetric methods are time-consuming (usually more than 48 h) and cannot be used in a real-time monitoring process.4,5 Fluorescence imaging techniques, which have also been widely used in cell analysis, are strongly reliant on the fluorescent probes.6−8 The surface plasmon resonance (SPR) technique is designed to detect the changes in the refractive index near the surface.9 It is well-known that SPR is a label-free, real-time, sensitive, and fast method for the investigation of the kinetics and affinity constant for molecular interactions,10−13 conformational changes of macromolecules,14,15 etc. Additionally, SPR has been applied in analysis and diagnosis of living cells and clinical samples.16−19 Ona and co-workers20 applied SPR sensors to study the short-term cytotoxicity of anticancer drugs in live cancer cells cultured on a sensor. However, it is not a real-time © 2015 American Chemical Society

Received: August 31, 2015 Accepted: October 22, 2015 Published: October 22, 2015 24848

DOI: 10.1021/acsami.5b08066 ACS Appl. Mater. Interfaces 2015, 7, 24848−24854

Research Article

ACS Applied Materials & Interfaces

Cell morphologies and images were observed via inversed fluorescence microscopy (IX51, Olympus, Tokyo, Japan). The cytotoxicity of DNR to HepG2 cells was assessed with the MTT assay and measured by using a microplate ELISA reader (Bio-Rad550, Bio-Rad Laboratories, Hercules, CA). Cell Culture. HepG2 (human hepatocellular carcinoma) cells were purchased from the Shanghai Institute of Cells of the Chinese Academy of Sciences and cultured in DMEM (high-glucose, Gibco) supplemented with 10% fetal bovine serum (Sigma, St. Louis, MO), 100 units/mL penicillin (Sigma), and 100 mg/mL streptomycin (Sigma) at 37 °C with 5% CO2 in a 95% humidified atmosphere. Preparation of Cell Chips. SPR chips (18 mm × 18 mm, 47 nm Au and 2 nm Cr coated on BK7 glass, Biosensing Instrument Inc.) were tightly placed into the wells of a six-well plate and sterilized using 75% ethanol. Then the six-well plate and SPR chip were rinsed with cell culture medium. The dilution of cells with different concentrations (i.e., 2, 5, 8, and 10 × 105 cells/mL) was added to wells of the six-well plate and covered the surface of the SPR chip. The six-well plate was put in an incubator at 37 °C with 5% CO2 in a 95% humidified atmosphere for various amounts of time (2, 4, and 6 h). The adhesion profile of HepG2 cells on the SPR chip was investigated by using an inverted microscope. After incubation, the suspension was removed. Then wells of a six-well plate with the SPR chip were washed three times with culture medium to remove excess floating cells. SPR chips with cells were carefully taken out of wells by using a fine point tweeze, and their backs were carefully washed with ethanol on a cotton swab and dried with a rubber suction bulb. In Situ EC−SPR Experiments. The dynamic changes in the SPR angle were recorded, and electrochemical signals of DNR residue in medium were collected at the same time. Briefly, the prism surface of the SPR equipment was cleaned by using a cotton applicator dampened with ethanol. A small drop of the refractive index matching liquid was placed near the center of the prism. SPR chips with HepG2 cells were gently positioned onto the center of the prism surface without bubbles being trapped at the sensor−prism interface. The EC−SPR analysis module was slightly released over the SPR chip. Then, 700 μL of culture medium with DNR (0, 1, 5, and 10 μM) was injected into the EC−SPR analysis module with a pipet. The Pt auxiliary electrode and Ag/AgCl reference electrode were placed into the holes of the EC−SPR analysis module, as shown in Figure 1. The dynamic changes in the SPR angle were recorded, and electrochemical signals of the DNR residue in the medium were also collected by using an electrochemical workstation with a cyclic voltammetry (CV) technique over a certain period of time. The experiment was repeated at least three times. The cells were incubated in the buffered medium at room temperature. The initiative potential was at 0 V, while the high and low potentials were separately at 0.8 and −0.8 V, respectively. The scan rate was performed at 0.1 V s−1. The group without incubation with DNR was taken as control, and data for discussion in this paper have been recorded with the control group. In Vitro Cytotoxicity Studies by the MTT Assay. The MTT assay was taken to verify the results of SPR experiments. Cells were trypsinilyzed and seeded onto 96-well plates (3 × 104 cells/well). After incubation for 4 h at room temperature, cells were rinsed in DMEM and incubated with different concentrations of DNR for 2, 4, and 6 h. A MTT solution was added after treatment and the mixture incubated for an additional 4 h. Dimethyl sulfoxide was added to solubilize the formazan crystal, and the optical density (OD) at 490 nm was recorded. Cell Morphological Assessment. Before the cells had been added to the SPR instrument, the morphologies of cells adsorbed on the SPR chip were assessed by optical microscopy. After the EC−SPR experiment (i.e., treated with DNR for 6 h), morphologies of cells were immediately investigated by optical microscopy. Cells treated with DNR for 6 h were also examined with an inverted fluorescence microscope to study the uptake of DNR by HepG2 cells. The fluorescent images of cells after treatment with DNR were excited at 488 nm. The images were captured with a CCD (QCapture, QImaging Corp.).

properties of cells. Electrochemical methods reflect the redox chemistry of the surroundings of cells. The combined techniques can provide multiparametric information about molecules and cells. The combined technique of electrochemical−surface plasmon resonance (EC−SPR) could have great potential applications in monitoring of the survival rate of cells. Robelek and co-workers35 introduced a combination of impedance and SPR to monitor living cells simultaneously. Cui and co-workers36 presented an EC−SPR system to characterize the optical and electrical properties of cells. However, none of these previous studies has attempted to evaluate the treatment efficiency of agents in cells by EC−SPR. Hence, in this study, we aimed to explore the possibility of taking this in situ combination strategy to evaluate live HepG2 cancer cells treated with DNR. The electrochemical technique was applied to monitor in real time the concentration changes of DNR residues outside the cells. The results of the EC−SPR study were confirmed by each method. This label-free, real-time EC− SPR determination has great potential application in monitoring of relevant clinical treatments.

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MATERIALS AND METHODS

Chemicals and Instrumentation. DNR was purchased from Pharmacia Italia, and its stock solution was freshly prepared and stored in the dark at 4 °C. MTT was purchased from Amresco. Other reagents used in this study were of analytical grade. EC−SPR studies were performed on a model BI-2000 instrument with an EC−SPR analysis module (Biosensing Instrument Inc., Tempe, AZ), and the SPR signal was calibrated in refractive index units (RIU) with the ethanol method.37 A 670 nm, 1 mW laser was employed as the light source. Their combination is shown in Figure 1.

Figure 1. Schematic illustrations of combination of SPR and electrochemical techniques in real-time evaluation of live cancer cells after their treatment with DNR.

The SPR instrument was designed to detect changes in the resonance angle with the Kretschmann configuration. Because the shift in the resonance angle is proportional to the amount of mass adsorbed on the surface, monitoring the change in the SPR signal provides an accurate, label-free measurement of the change in mass near the chip surface. An electrochemical workstation (660B, CH Instruments, Inc., Austin, TX) was used to detect the DNR residue outside cells in the culture medium. The SPR chip, placed over the SPR prism, was used as a working electrode. A platinum electrode and a Ag/AgCl electrode were used as the counter and reference electrode, respectively. The geometric size of the working electrode was restricted with a rubber Oring, and the area was set to 180 mm2. The volume of the measurement chamber was 700 μL. 24849


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RESULTS AND DISCUSSION Optimization for the Adhesion of Cell Chips. Initially, the morphologies of HepG2 cells that adhered on the SPR chip were observed on an optical microscope (Figure 2). To

determine the optimal conditions for preparing cell chips, the cell concentration and incubation time were investigated. As shown in Figure 2, the SPR chip was not fully occupied by HepG2 cells at low cell concentrations. However, there were excess cells suspended over the SPR chip at high cell concentrations. The cell concentration was maintained at ∼8 × 105 cells/mL. When the incubation time was 2 h, there were many HepG2 cells suspended and not adhered to the surface of the SPR chip. After being incubated for 4 h, most cells tightly adhered to the SPR chip. Thus, the cellular adhesion time was set to 4 h for the EC−SPR experiments described here. Consequently, the cell chip was prepared with a cell concentration of 8 × 105 cells/mL and incubated for 4 h before being positioned on the prism surface. Real-Time Monitoring of Live HepG2 Cells via EC− SPR. The EC−SPR instrument is schematically shown in Figure 1, and the sensitivity of the instrument was 8.23 × 10−7 RIU (see the Supporting Information). SPR signals were collected continuously while cyclic voltammetry (CV) was applied to determine the relevant DNR residue in the medium. HepG2 cells treated with DNR were monitored with in situ EC−SPR at room temperature in culture medium. The SPR

Figure 2. Optical images of cells adhered to the SPR chip with different cell concentrations and incubation times.

Figure 3. (A) Dynamic plots of magnitudes of SPR signals of HepG2 cells on the SPR chip after incubation with DNR (5 μM) and a control. (B) Partial enlarged view of SPR signals of HepG2 cells on the SPR chip after incubation with DNR (5 μM). (C) Three-dimensional plots of SPR signals of HepG2 cells on the SPR chip after incubation with 1, 5, and 10 μM DNR. The DNR was injected at 0 h, i.e., the start of the EC−SPR experiment. 24850


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Figure 4. (A) Cyclic voltammetric study of DNR after incubation with HepG2 cells for 0, 0.5, 1.0, and 4.5 h. The inset shows the dynamic peak current changes of DNR (i.e., 1, 5, and 10 μM) after incubation with HepG2 cells. (B) Comparison of the CV study (solid line) and scan voltagebased SPR signals (dotted line) of HepG2 cells treated with 10 μM DNR.

cyclic SPR signal was not closed because of the decreased magnitude of the SPR signals. These results are in agreement with previous reports in the literature.36 When potentials were applied to the SPR chip, the electron densities and electroactive species adsorbed on the SPR chip surface varied. The positive potential led to the increase in surface electron intensities and the thickness of the oxidation layer (gold and electroactive species); hence, the magnitudes of the SPR signals increased.36,42 Because cells did not entirely capture the surface of the SPR chip, as shown in Figure 2, the DNR molecule can readily diffuse to the electrode surface through the cell layer. Compared with that of DNR alone, the relevant peaks at approximately −0.6 V of DNR residues outside the cells were found to shift slightly in the positive direction after incubation with HepG2 cells, which did not appear on the cyclic voltammogram of HepG2 cells in medium without DNR (Figure S2). The results of the CV study of DNR residues outside HepG2 cells are shown in Figure 4A and Figure S2. Only one reduction peak, 1c, was recorded immediately after the addition of DNR to culture medium. It is noted that two oxidation peaks, namely, 1a and 2a, appeared after the second scan, and the current of peak 1a was increased, which could be attributed to the reduced products of DNR adsorbed on the SPR chip. When incubated with cells, DNR molecules should be internalized and consumed by HepG2 cells, and the concentration of DNR in the medium decreased, which readily led to a decrease of the current of DNR. The current of peak 1c was found to decrease after the first scan and further decrease to 50% at 1 h for the group treated with 10 μM DNR, as shown in Figure 4A. After a relatively long incubation time, the cells were killed with DNR and detached from the surface of the SPR chip. DNR molecules, which were internalized into cells, were released into the culture medium. At the same time, the surface of the SPR ship was free after the detachment of apoptotic cells, which increase the electrochemically effective area. Thus, the relevant peak current changes with the change in DNR concentration as well as incubation time, which can be further utilized to evaluate the DNR residues in the related interface outside HepG2 cells. Quantitative Analysis of the in Vitro Cytotoxicity by SPR. The MTT assay of in vitro cytotoxicity of DNR to HepG2 cells were taken to confirm the EC−SPR results, as shown in

signals of HepG2 cells with or without DNR treatment were dependent on time and concentration (Figure 3). Magnitudes of SPR signals of DNR treatment groups declined, while the magnitude of the SPR signal of the control group increased as shown in Figure 3A. The slow increase in the magnitude of the SPR signal for the control group was related to the growth of cells and attachment of them to the surface of the SPR chip. The SPR signals of DNR treatment groups with 5 μM shifted approximately −107.913, −238.170, and −350.563 mdeg for 2, 4, and 6 h, respectively. The SPR signals of HepG2 cells treated with DNR (1 and 10 μM) are illustrated in Figure S3 of the Supporting Information. It is evident that SPR could be readily used to evaluate live HepG2 cancer cells treated with DNR and thus monitor the relevant treatment efficiency of DNR in HepG2 cells. Meanwhile, it is observed that SPR signals were also sensitive to changes in mass and refractive index near the interface of the SPR chip (92% over 2 h and decreased to 80% over 4 h after 1 μM DNR treatment. The relative time- and concentration-dependent changes in SPR signals are shown in Figure 5A and Figure S6, respectively. It is evident that the SPR signals were significantly related to the survival rates of HepG2 cells in cultures with different DNR concentrations. As shown in Figure 5B, the decreases in the magnitudes of SPR signals were linearly fitted to cell survival rates, which were expressed via the equation survival rate (%) = 0.0721Δangle (mdeg) + 97.2278, and the adjusted R2 value was 0.9791. Our observations illustrate that the change in the SPR signals pertained to the apoptosis of cells and detachment of them from the interface of the SPR chip. The dynamic SPR signals can be attributed to the living cells, which still adsorbed on the surface of the SPR chip. Herein, the decrease in the magnitudes of the SPR signals in 100 mdeg means that cells in 7.21% had been apoptotic and detached from the SPR chip. These results demonstrate that SPR can be effectively applied in the real-time quantitative evaluation of HepG2 cells. 24852


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groups with higher DNR concentrations declined more rapidly than those with relatively lower concentrations.

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CONCLUSIONS In this study, we have taken the strategy of the EC−SPR study to evaluate live cancer cells treated with DNR on the interface of SPR chips. Signal changes recorded form the SPR resulted from the morphology and mass changes of adsorbed HepG2 cells and refractive index variation of the medium solution. The DNR residue concentration outside the cells in culture medium can be determined by an electrochemical method. The results of light microscopy images and MTT tests also demonstrated the release or desorption of relevant cells, which was caused by the apoptosis of cancer cells after treatment with DNR. The decreases in the magnitudes of SPR signals were linearly related to cell survival rates. Our observations demonstrate that the combination of SPR with an electrochemical study can be utilized to evaluate the treatment efficiency of bioactive agents in cells. Thus, this label-free, real-time EC−SPR method has great potential application in monitoring of relevant clinical treatment processes and pharmaceutical analysis.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.5b08066. Calculated values for the refractive index of ethanol solutions, SPR signals of DNR-treated HepG2 cells with and without electrochemical samplings, scan voltagebased SPR signals, and an in vitro cytotoxicity study using the MTT assay (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Phone/Fax: +86-25-83792177. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is supported by the National High Technology Research & Development Program of China (2015AA020502), the National Natural Science Foundation of China (81325011, 21327902, and 21175020), and the Major Science & Technology Project of Suzhou (ZXY2012028).

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ABBREVIATIONS MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; SPR, surface plasmon resonance; EC, electrochemistry; CV, cyclic voltammetry; DNR, daunorubicin

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REFERENCES

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Real-Time Evaluation of Live Cancer Cells by an in Situ Surface

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