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Detection of adherent cells using electrochemical impedance spectroscopy based on molecular recognition of integrin β1
Sensors and Actuators B 149 (2010) 87–93
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Detection of adherent cells using electrochemical impedance spectroscopy based on molecular recognition of integrin 1 Xiangfu Jiang a,b , Liang Tan a,∗ , Baoling Zhang a , Youyu Zhang a , Hao Tang a , Qingji Xie a,∗ , Shouzhuo Yao a a Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research (Ministry of Education of China), College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha 410081, PR China b College of Life Science, Tarim University, Alar 843300, PR China
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Article history: Received 28 February 2010 Received in revised form 8 June 2010 Accepted 11 June 2010 Available online 18 June 2010 Keywords: Integrin 1 Layer-by-layer adsorption Immunoreaction HeLa cells Electrochemical impedance spectroscopy
a b s t r a c t Mouse anti-human integrin 1 monoclonal antibody was assembled on glass carbon electrode by layerby-layer adsorption. The determination of cervical cancer HeLa cells was performed using electrochemical impedance spectroscopy based on the immunoreaction between integrin 1 on cell membrane and the antibody immobilized on the electrode-surface. The factors influencing cell-detection, such as the concentration of gold nanoparticles in the modified film, the drying time of the modified film, the concentration of antibody and the binding time of antibody, were investigated, respectively. Under optimum conditions, the increasing extent of electrode-transfer resistance depended linearly on the cell concentration in the range of 1.0 × 104 to 2.0 × 106 cells mL−1 with a detection limit of 3.5 × 103 cells mL−1 . © 2010 Elsevier B.V. All rights reserved.
1. Introduction The detection of cells can reveal much important information, which is crucial for foundational research of life science, disease prevention and even clinical diagnosis. The monitoring of cell attachment to a surface and their growth is obviously significant in better understanding the cell–substrate interactions and developing cytology-based devices, such as cell biosensor and biochip. Many methods are introduced successfully for this purpose, including photoelectric detection [1], voltammetry [2], surface plasmon resonance analysis [3], fluorescence microscopy [4], and so on. Electrochemical impedance (EI) measurement is a sensitive tool for investigating the changes of interfacial properties at electrode-surfaces. The detection of IgG [5], bacteria [6], DNA [7] and biotin–avidin interaction [8] on electrode-surfaces have been studied by this method. As a noninvasive and sensitive detecting technique for cell adhesion, EI measurement is valid and holds promise for monitoring the morphology, viability, and environmental change of cells. In the last two decades, EI sensing has been employed to monitor many types of cells. Tiruppathi et al. studied endothelial cell shape changes in real time by measuring the changes in electrical impedance to examine the mechanisms
∗ Corresponding authors. Tel.: +86 731 88865515; fax: +86 731 88865515. E-mail address: [email protected] (L. Tan). 0925-4005/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2010.06.026
of alterations in the endothelial barrier function [9]. Xiao et al. investigated the attachment and spreading of fibroblast cells on a gold surface coated with fibronectin or ovalbumin by a modified electric cell–substrate impedance sensor [10]. Yang et al. described a label-free electrochemical impedance immunosensor using an indium–tin oxide interdigitated array micro-electrode for rapid detection of Escherichia coli O157:H7 [11]. Chen et al. developed an electrochemical impedance biosensor for detection of Saccharomyces cerevisiae (yeast cells) by immobilizing cells on a gold-surface modified with an alkanethiolate self-assembled monolayer [12]. Guo et al. monitored the growth of human hepatocarcinoma cell line BEL7404 and evaluated the cytotoxicity of HgCl2 on optically transparent indium–tin oxide electrode [13]. Hao et al. prepared an impedance sensor for quantifying the cell number of leukemia K562A cells based on the immunoreaction of P-glycoprotein [14]. Liu et al. employed a cell-based biosensor with micro-electrode arrays to investigate the culture behavior of human oesophageal cancer cell lines and evaluate the chemosensitivity of cisplatin [15]. Cell adhesion based on molecular interactions plays a critical role in a wide array of biologic processes including hemostasis, the immune response, inflammation embryogenesis and development of neuronal tissue. Cell adhesion molecules (CAMs) mediate the process of cell adhesion through either cell–matrix or cell–cell interactions and they include four major categories: integrins, cadherins, members of the immunoglobulin superfamily and selectins
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[16]. Integrins are transmembrane proteins and heterodimers composed of two kinds of subunits, ␣ and . There are at least 18 ␣ units and 9  units known. The combination of different integrin subunits on the cell surface allow cells to recognize and respond to a variety of different extracellular matrix (ECM) proteins including fibronectin, laminin, collagen and vitronectin [17]. Generally integrins can be divided into more than 20 subfamilies based on  subunits. As the largest subgroup, the 1 subfamily (integrin 1) is important for ECM interactions, focal adhesions, cell polarity, cell shape and cell migration [18]. The layer-by-layer (LbL) assembly technique is an easy and inexpensive process for multilayer formation and allows different types of materials to be incorporated in the film structures [19,20]. This technique was proposed by Decher more than 10 years ago [21] and has been widely employed in many areas for its versatility. Generally, LbL assembly is mainly conducted through covalent interaction or electrostatic adsorption. Many specific species, such as enzyme [22], DNA [23], antibody [24], protein [25], mammalian cells [26] even tissues [27], can be introduced onto the surface with the help of LbL assembly technique, which is conducive to the construction of biosensor. To our best knowledge, to date there are no reports on application of electrochemical impedance method for the detection of adherent cells based on molecular recognition of integrin 1. Here, mouse anti-human integrin 1 monoclonal antibody, as a specific ligand binding integrin 1 on cell membrane, was used to immobilize HeLa cells on the assembled glass carbon electrode. The number of the adherent cells was determined by the electrochemical impedance signals and the relevant optimized conditions for the analysis were discussed. 2. Experimental 2.1. Chemicals and apparatus Mouse anti-human integrin 1 monoclonal antibody (antiCD29 mAb) was purchased from Sizhengbai Biotechnological Co. Ltd. (Beijing, China). Phosphate-buffered saline (PBS, pH 7.4) solution consisting of 87 mmol L−1 NaHPO4 , 14 mmol L−1 KH2 PO4 , 136.8 mmol L−1 NaCl and 2.7 mmol L−1 KCl was used in the experiments. Chitosan (CHIT, from crab shells, >90% deacetylation) was obtained from Shanghai Biochemical Reagents Co. Ltd. (Shanghai, China). Colloidal gold nanoparticles (Au NPs) were prepared according to the literature [28] and stored in a brown glass bottle at 4 ◦ C. The cell-staining experiments were performed with 0.01% acridine orange (AO). All other reagents were of analytical grade. And doubly distilled water was used for all experiments. Electrochemical impedance spectroscopy (EIS) experiments were performed with a CHI660C electrochemical workstation (CH Instruments, Shanghai Chenhua Instruments Inc., China) by using a three-electrode electrolytic cell. Glass carbon electrode (GC, 3 mm in diameter) acted as the working electrode (WE). A KCl saturated calomel electrode (SCE) served as the reference electrode (RE). A platinum plate served as the counter electrode (CE). The sizes of the Au nanoparticles were characterized ex situ by a JEOL-1230 transmission electron microscope (TEM). The images of Au NPs–CHIT film on electrode were obtained with an X-570 scanning electron microscope (SEM, Hitachi, Japan). Atomic force microscope (AFM) measurement was performed on PicoScanTM 2100 (Agilent). The pictures of stained cells were observed on an inverted fluorescence microscope (NIKON Eclipse Ti-S, Japan). 2.2. Cell culture Cervical cancer HeLa cells, which belong to adherent cells, were purchased from XiangYa Central Laboratory of Central South
University, China, and were cultured in RPMI 1640 medium (Gibco) supplemented with newborn calf serum (10%), penicillin (100 U mL−1 ) and streptomycin (100 U mL−1 ) in an incubator (5% CO2 , 37 ◦ C). When the adherent cells spread over the bottom of the culture bottle, they were trypsinized and collected, then washed twice with a sterile pH 7.4 PBS. The sediments were re-suspended in PBS for the following immobilization on the electrode. The density of re-suspended cells was determined with a hematocytometer. 2.3. LbL adsorption on electrode-surface and electrochemical measurement Prior to modification, the glassy carbon electrode was polished with 0.05 m ␣-Al2 O3 power slurries until a mirror shiny surface appeared, and was sonicated sequentially in acetone, HNO3 (1:1, v/v), NaOH (1 mol L−1 ) and doubly distilled water for 3 min. The treated electrode was scanned between −1.0 and 1.0 V versus SCE in 0.5 mol L−1 H2 SO4 aqueous solution for sufficient cycles to obtain reproducible cyclic voltammograms. After the electrode was thoroughly rinsed with doubly distilled water and dried under a stream of nitrogen, as shown in Scheme 1, 3 L of a mixture containing 0.5% chitosan and 2 g mL−1 Au NPs was dropped on the electrode-surface and allowed to dry for 36 h at room temperature. The modified electrode was coated with a drop of 3 L of 2 g mL−1 Au NPs and then was left for 24 h at room temperature. The procedure yielded a robust and strongly adherent composite film. Anti-CD29 mAb was introduced onto the Au NPs–CHIT film modified electrode by dropping 3 L of 20 g mL−1 antibody on the surface and incubating at 4 ◦ C for 24 h. Next, 2% BSA was employed to block the non-specific site. Finally 3 L HeLa cell-PBS suspension was seeded onto the modified electrode-surface and cultured in the incubator for 2 h, which achieved the cell capture based on the immunoreaction between integrin 1 on cell membrane and antibody on the electrode-surface. The electrode was gently washed with PBS after every assembly process. The GC electrodes before and after the film modification and in sequence cell incubation were characterized in 10 mmol L−1 K3 Fe(CN)6 /K4 Fe(CN)6 (1:1) containing 0.1 mol L−1 KCl via EIS at room temperature. For EIS measurements, the working electrode potential was fixed at the formal potential of the ferricyanide/ferrocyanide couple and the amplitude was 5 mV. The impedance spectrograms were collected within the frequency range of 10−1 to 105 Hz. The diameters of Nyquist circles at higher frequencies are obtained by the fitting with ZSimpWin software, Version 3.10. 3. Results and discussion 3.1. Characterization of nanoparticles and the modified electrodes The images of prepared Au NPs and the Au NPs–CHIT film on GC electrode are shown in Fig. 1A and B, respectively. The gold colloids consist of spherical nanoparticles with a size distribution in the range of 10–20 nm. The CHIT gel appeared as if undulant hills. When the NPs were dispersed in the 0.5% CHIT solution, they still kept perfect stability and uniformity. CHIT is the deacetylated derivative of chitin. It has been extensively applied for immobilization of biologic materials, molecular separation, food packaging film, artificial skin, bone substitutes, water treatment, and so on, based on its biocompatibility and biodegradability. CHIT is more useful due to the presence of amino groups that impart a positive charge to the molecule. It is known that Au NPs prepared by Frens method [28] are negatively charged owing to the adsorbed citrate anions. As CHIT in solution is protonated and positively charged, it can be adsorbed onto the surfaces of Au NPs, stabilizing and protecting the nanoparticles.
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Scheme 1. Fabrication and capture processes of the immunosensor for HeLa cells.
Fig. 1. TEM image of Au NPs (A) and SEM image of the Au NPs–CHIT film coated on GC electrode. The scale bars in A and B represent 100 and 500 nm, respectively.
Fig. 2A and B shows AFM images of GC electrode coated with Au NPs–CHIT film and anti-CD29 mAb/Au NPs–CHIT film, respectively. It can be seen that the Au NPs–CHIT film was uniformly spreading on the electrode. The surface became smoother and thicker after
antibody was adsorbed. It looks as if some pits in the Au NPs–CHIT film were filled by the antibody macromolecules. Some researches reported that Au NPs can form covalent bond with S atoms or N atoms of biomolecules [29,30]. Au NPs have been widely used to
Fig. 2. AFM images of GC electrode coated with (A) Au NPs–CHIT film and (B) anti-CD29 mAb/Au NPs–CHIT film.
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construct biosensors, such as enzyme sensor [31], immunosensor [32] and DNA sensor [33], because of their excellent ability to immobilize biomolecules and retain the biocatalytic activities of those biomolecules. So anti-CD29 mAb could be successfully assembled on GC electrode through the binding with Au NPs. 3.2. Impedance characteristics of the modified electrodes EIS is a powerful tool for investigating the interfacial properties of surface-modified electrode. The typical electrochemical interface can be represented as an electrical circuit as shown in the inset of Fig. 3 (Randles and Ershler theoretical model [34]). The equivalent circuit includes four parameters. The ohmic resistance of the electrolyte solution, Rs , the Warburg impedance, Zw , represent bulk properties of the electrolyte solution and diffusion features of the redox probe in solution. The double-layer capacitance, Cdl , and the electron-transfer resistance, Ret , reveal interfacial properties of the electrode, which is highly sensitive to the surface modification. Usually, Ret controls the interfacial electron-transfer rate of the redox probe between the solution and the electrode. In the Nyquist plot of impedance spectroscopy, it equals the semicircle diameter at higher frequencies. Fig. 3 shows the Faradaic impedance spectra, presented as Nyquist plots of the GC electrode before and after the film modification and cell incubation. It is obvious that the semicircle diameter increased by a small value after Au NPs–CHIT film and anti-CD29 mAb were successively immobilized onto the electrodesurface, but the cell capture resulted in a significant augment of the electron-transfer resistance. The stabilization of Au NPs with CHIT has been extensively reported [35,36]. Although CHIT, acting as the insulator, could decrease the electrical conductivity, the electrontransfer between electrochemical probe and electrode might be resumed to a great extent in the presence of the good conductor, gold nanoparticles. Accordingly the Ret value at the electrode modified by Au NPs–CHIT film was only higher by about 105 than that at bare electrode. The subsequent anti-CD29 mAb binding led to another increase of Ret (about 224 ), indicating that the electron-transfer was further hindered for the occupation of some antibody macromolecules at the pits of Au NPs-CHIT film owning
Fig. 3. Nyquist diagrams of electrochemical impedance spectra for [Fe(CN)6 ]3− /[Fe(CN)6 ]4− (10 mmol L−1 , 1:1) in 0.1 mol L−1 KCl at bare GC electrode (a), GC electrode coated with Au NPs–CHIT film (b), anti-CD29 mAb/Au NPs–CHIT film (c) and HeLa cells/anti-CD29 mAb/Au NPs–CHIT film with the cell concentration of 1.0 × 105 cells mL−1 (d).
three-dimensional network structure. As we know, the adsorption of antibody on Au electrode-surface can be easily performed due to the S-Au bonds but the same procedure on GC electrodesurface is relatively difficult. So the immobilization of antibody on GC electrode-surface is commonly achieved by the layer-by-layer adsorption [37], the covalent binding to a self-assembled monolayer (SAM) [14], and so on. Here, the antibody-binding on gold nanoparticles assembled with chitosan were performed. The experimental process was relatively simple and the modification only led to a little Ret , which may be beneficial to the next sensitive detection of cell capture. After the introduction of HeLa cells, the values of Ret rose up from 689 to 1276 . It means that the cells could firmly adhere to the antibody/Au NPs–CHIT film by immunoreaction. As a result, the adhesion of HeLa cells on the electrode led to the decreased active surface area and the barrier effect against the electron communication between the probe and the electrodesurface. 3.3. Condition optimization of the impedance analysis The Ret values depended directly on the interfacial electrontransfer ability. When the CHIT film, an insulation coating, was overlaid on the electrode-surface, it inevitably had negative influence on the electric signals. So some Au NPs were introduced into the CHIT film to improve conductivity of the modified electrode. It is found that the peak-currents of the K3 Fe(CN)6 /K4 Fe(CN)6 probe in cyclic voltammetry measurements increased with augment of the Au NPs-concentration which was lower than 2 g mL−1 . Aggregation of nanoparticles appeared if the content of Au NPs kept on rising, which would seriously affect the stability and uniformity of the film. In our work, the concentration of Au NPs at 2 g mL−1 in the modified film was selected for the stable signals. Besides the concentration of Au NPs, the drying time of the Au NPs–CHIT film on GC electrode at room temperature 298 K was also investigated for EIS measurements. As shown in Fig. 4, the Ret values before and after the film immobilization decreased with the time increasing and reached a plateau at time over 32 h when the electrode-activity still remained high performance. It is possible that many water molecules, which could block electron-transfer from the probe to GC electrode, were involved in the Au NPs–CHIT film when the
Fig. 4. Effect of drying time of Au NPs–CHIT film on the difference of electrontransfer resistance before and after the film immobilization. Results are presented as mean ± SD (error bar) of triplicate experiments.
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Fig. 5. The difference of electron-transfer resistance before and after the adsorption of RPMI 1640 culture medium (a) and the adhesion of 1.0 × 105 cells mL−1 HeLa cells (b) on GC electrode coated with anti-CD29 mAb/Au NPs–CHIT film as well as the difference of electron-transfer resistance before and after the adhesion of 1.0 × 105 cells mL−1 HeLa cells on GC electrode coated with Au NPs–CHIT film (c). Results are presented as mean ± SD (error bar) of triplicate experiments.
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latter was dropped on the electrode-surface. The extent of water evaporation was enhanced with time passing and many passages promoting the electron-transfer would be presented in the modified film. Based on above investigation, the drying time of the modified film at 36 h was selected for all subsequent experiments. In our opinions, some potential factors affecting the Ret measurement in the process of cell capture should be considered. It is found from Fig. 5 that the adsorption of RPMI 1640 culture medium could result in some increases of the electrical conductivity. Based on this fact, a strategy was adopted to eliminate the influence of biomacromolecules in culture medium. After the cells were trypsinized and collected, they were washed and resuspended in pH 7.4 PBS and then were introduced to the modified electrode-surface. A comparison of the adhesion ability of HeLa cells on the electrode-surface modified with and without anti-CD29 mAb was performed. It is found from Fig. 5 that the cell-adhesion on the anti-CD29 mAb/Au NPs–CHIT film could lead to a greater number of Ret . Microscopic fluorescent images of HeLa cells on the different elctrode-surfaces are presented in Fig. 6. After 2-h cell capture and gentle washing with PBS, the amount of adhered cells on the anti-CD29 mAb/Au NPs-CHIT film was larger than that on the Au NPs–CHIT film. In our experiments, the measured cells were re-suspended in pH 7.4 PBS. The adhesion and spreading of cells might be greatly influenced in the absence of culture medium and calf serum containing many nutrient compositions. In this case, some linker molecules capturing cell, such as antibodies, seemed especially important. It can be comprehended that the direct cell-adhesion on the Au NPs–CHIT film was relatively unstable in the absence of anti-CD29 mAb. Fig. 7 shows the effects of antibody concentration and antibody-binding time on the differ-
Fig. 6. Images of stained HeLa cells adhered to anti-CD29 mAb/Au NPs–CHIT film (A) and Au NPs–CHIT film (B) under fluorescence microscope (200×) after 2-h cell capture. The concentration of initially added cell: 1.0 × 105 cells mL−1 .
Fig. 7. (A) Effect of anti-CD29 mAb concentration on the difference of electron-transfer resistance before and after the adhesion of 3.0 × 104 cells mL−1 HeLa cells. The binding time of antibody: 24 h. (B) Effect of binding time of anti-CD29 mAb to Au NPs–CHIT film on the difference of electron-transfer resistance before and after the adhesion of 3.0 × 104 cells mL−1 HeLa cells. The concentration of binding antibody: 20 g mL−1 . Results are presented as mean ± SD (error bar) of triplicate experiments.
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probe. The research result reveals that the increasing extent of Ret was linearly proportional to the amount of HeLa cells in some range. Multidrug resistance is one of the major obstacles to the successful treatment of tumors. It is characterized by a decreased sensitivity of tumor cells not only to the drug employed for chemotherapy but also to a broad spectrum of drugs with neither obvious structural homology nor common targets. Cell adhesionmediated drug resistance is a special mechanism. Damiano demonstrated that ECM protein-mediated cell adhesion conferred a survival advantage for human myeloma cells acutely exposed to cytotoxic drugs by inhibiting drug-induced apoptosis [38]. Aoudjit and Vuori identified integrin 1 signaling as an important survival pathway in drug-induced apoptosis in breast cancer cells [39]. It can be comprehended that the adhesion state of tumor cells and drug-resistance tumor cells is different. Therefore, the expression of integrin 1 molecules on cell membrane of two kinds of tumor cells should be dissimilar since integrin 1 subfamily is closely related to ECM interactions. In our opinions, the detection of this difference may be achieved by the proposed EIS method in future research. Acknowledgements Fig. 8. Calibration curve of the immunosensor for HeLa cells. Results are presented as mean ± SD (error bar) of triplicate experiments.
ence of electron-transfer resistance before and after cell capture with the concentrations of 3.0 × 104 cells mL−1 . An obvious increase in the Ret values with addition of antibody content at the range of 5.0–15 g mL−1 was observed. When the concentration of antibody exceeded 15 g mL−1 , the value of Re basically maintained unchanged. The result indicates that the amount of adhered cells would increase with the antibody increasing, which would effectively reduced passages of electron-transfer. The concentration of antibody at 20 g mL−1 was selected in our work to obtain optimized condition. It is observed that the Ret values expressed a trend of ascending with increase of the antibody-binding time, reaching a plateau at the latter over 24 h. Undoubtedly the high Ret value was related to the stable cell-adhesion which benefited from the abundant fixed-antibodies on GC electrode after enough binding time. In order to obtain maximal signals, we chose the binding time of antibody at 24 h for cell sensing. 3.4. EIS detection of HeLa cells The developed method can be applied to detection of adherent cells. The EIS measurements of the K3 Fe(CN)6 /K4 Fe(CN)6 probe before and after immobilizing HeLa cells with different concentrations were performed and the results are shown in Fig. 8. The Ret value exhibited a linear response with the logarithm of HeLa cells concentration over the range of 1.0 × 104 to 2.0 × 106 cells mL−1 . The linear regression equations was Ret = −694.1 + 252.1 log c (r = 0.994), and the detection limit was 3.5 × 103 cells mL−1 . The measurements were performed three times to assess the reproducibility and precision of the freshly fabricated immunosensor. The RSD of the detection results was all lower than 6%, showing acceptable performance. These imply that the sensitivity of our sensor could bear comparison with that of other detectors for cell or bacteria measurements reported in the literatures [10,13]. 4. Conclusions Mouse anti-human integrin 1 monoclonal antibody was assembled on GC electrode and employed to capture HeLa cells. The cell adhesion based on the reaction between antibody and integrin 1 on cell membrane resulted in significant increase of Ret in EIS measurement using K3 Fe(CN)6 /K4 Fe(CN)6 as electrochemical
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Biographies Xiangfu Jiang is pursuing his M.D. in the Key Laboratory of Chemical Biology & Traditional Chinese Medicine Research (Ministry of Education, China), College of Chemistry and Chemical Engineering, Hunan Normal University. His research interests include nanomaterial constructing, biosensor fabricating, electrochemistry study of cells and biomolecules. Liang Tan is an associate professor in the Key Laboratory of Chemical Biology & Traditional Chinese Medicine Research (Ministry of Education, China), College of Chemistry and Chemical Engineering, Hunan Normal University. He received his Ph.D. degree in 2008 from Hunan University, China. His research interests cover electrochemistry analysis, biosensing and cell study. Baoling Zhang is pursuing her M.D. in the Key Laboratory of Chemical Biology & Traditional Chinese Medicine Research (Ministry of Education, China), College of Chemistry and Chemical Engineering, Hunan Normal University. Her research interests include biosensing and cell study. Youyu Zhang is a professor of Key Laboratory of Chemical Biology & Traditional Chinese Medicine Research (Ministry of Education, China), College of Chemistry and Chemical Engineering, Hunan Normal University. Her research interests include EQCM sensing and FTIR spectroelectrochemistry. Hao Tang is an associate professor of Key Laboratory of Chemical Biology & Traditional Chinese Medicine Research (Ministry of Education, China), College of Chemistry and Chemical Engineering, Hunan Normal University. His research interests include electrochemical detection and biosensing. Qingji Xie is a professor in the Key Laboratory of Chemical Biology & Traditional Chinese Medicine Research (Ministry of Education, China), College of Chemistry and Chemical Engineering, Hunan Normal University. His research is focused on chemo/biosensing and electroanalytical chemistry, including the EQCM, SECM and spectroelectrochemistry characterizations of sensing events. Shouzhuo Yao is a professor in the Key Laboratory of Chemical Biology & Traditional Chinese Medicine Research (Ministry of Education, China), College of Chemistry and Chemical Engineering, Hunan Normal University. He has been appointed as an academician of Chinese Academy of Science in analytical chemistry.
Contents lists available at ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Detection of adherent cells using electrochemical impedance spectroscopy based on molecular recognition of integrin 1 Xiangfu Jiang a,b , Liang Tan a,∗ , Baoling Zhang a , Youyu Zhang a , Hao Tang a , Qingji Xie a,∗ , Shouzhuo Yao a a Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research (Ministry of Education of China), College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha 410081, PR China b College of Life Science, Tarim University, Alar 843300, PR China
a r t i c l e
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Article history: Received 28 February 2010 Received in revised form 8 June 2010 Accepted 11 June 2010 Available online 18 June 2010 Keywords: Integrin 1 Layer-by-layer adsorption Immunoreaction HeLa cells Electrochemical impedance spectroscopy
a b s t r a c t Mouse anti-human integrin 1 monoclonal antibody was assembled on glass carbon electrode by layerby-layer adsorption. The determination of cervical cancer HeLa cells was performed using electrochemical impedance spectroscopy based on the immunoreaction between integrin 1 on cell membrane and the antibody immobilized on the electrode-surface. The factors influencing cell-detection, such as the concentration of gold nanoparticles in the modified film, the drying time of the modified film, the concentration of antibody and the binding time of antibody, were investigated, respectively. Under optimum conditions, the increasing extent of electrode-transfer resistance depended linearly on the cell concentration in the range of 1.0 × 104 to 2.0 × 106 cells mL−1 with a detection limit of 3.5 × 103 cells mL−1 . © 2010 Elsevier B.V. All rights reserved.
1. Introduction The detection of cells can reveal much important information, which is crucial for foundational research of life science, disease prevention and even clinical diagnosis. The monitoring of cell attachment to a surface and their growth is obviously significant in better understanding the cell–substrate interactions and developing cytology-based devices, such as cell biosensor and biochip. Many methods are introduced successfully for this purpose, including photoelectric detection [1], voltammetry [2], surface plasmon resonance analysis [3], fluorescence microscopy [4], and so on. Electrochemical impedance (EI) measurement is a sensitive tool for investigating the changes of interfacial properties at electrode-surfaces. The detection of IgG [5], bacteria [6], DNA [7] and biotin–avidin interaction [8] on electrode-surfaces have been studied by this method. As a noninvasive and sensitive detecting technique for cell adhesion, EI measurement is valid and holds promise for monitoring the morphology, viability, and environmental change of cells. In the last two decades, EI sensing has been employed to monitor many types of cells. Tiruppathi et al. studied endothelial cell shape changes in real time by measuring the changes in electrical impedance to examine the mechanisms
∗ Corresponding authors. Tel.: +86 731 88865515; fax: +86 731 88865515. E-mail address: [email protected] (L. Tan). 0925-4005/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2010.06.026
of alterations in the endothelial barrier function [9]. Xiao et al. investigated the attachment and spreading of fibroblast cells on a gold surface coated with fibronectin or ovalbumin by a modified electric cell–substrate impedance sensor [10]. Yang et al. described a label-free electrochemical impedance immunosensor using an indium–tin oxide interdigitated array micro-electrode for rapid detection of Escherichia coli O157:H7 [11]. Chen et al. developed an electrochemical impedance biosensor for detection of Saccharomyces cerevisiae (yeast cells) by immobilizing cells on a gold-surface modified with an alkanethiolate self-assembled monolayer [12]. Guo et al. monitored the growth of human hepatocarcinoma cell line BEL7404 and evaluated the cytotoxicity of HgCl2 on optically transparent indium–tin oxide electrode [13]. Hao et al. prepared an impedance sensor for quantifying the cell number of leukemia K562A cells based on the immunoreaction of P-glycoprotein [14]. Liu et al. employed a cell-based biosensor with micro-electrode arrays to investigate the culture behavior of human oesophageal cancer cell lines and evaluate the chemosensitivity of cisplatin [15]. Cell adhesion based on molecular interactions plays a critical role in a wide array of biologic processes including hemostasis, the immune response, inflammation embryogenesis and development of neuronal tissue. Cell adhesion molecules (CAMs) mediate the process of cell adhesion through either cell–matrix or cell–cell interactions and they include four major categories: integrins, cadherins, members of the immunoglobulin superfamily and selectins
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[16]. Integrins are transmembrane proteins and heterodimers composed of two kinds of subunits, ␣ and . There are at least 18 ␣ units and 9  units known. The combination of different integrin subunits on the cell surface allow cells to recognize and respond to a variety of different extracellular matrix (ECM) proteins including fibronectin, laminin, collagen and vitronectin [17]. Generally integrins can be divided into more than 20 subfamilies based on  subunits. As the largest subgroup, the 1 subfamily (integrin 1) is important for ECM interactions, focal adhesions, cell polarity, cell shape and cell migration [18]. The layer-by-layer (LbL) assembly technique is an easy and inexpensive process for multilayer formation and allows different types of materials to be incorporated in the film structures [19,20]. This technique was proposed by Decher more than 10 years ago [21] and has been widely employed in many areas for its versatility. Generally, LbL assembly is mainly conducted through covalent interaction or electrostatic adsorption. Many specific species, such as enzyme [22], DNA [23], antibody [24], protein [25], mammalian cells [26] even tissues [27], can be introduced onto the surface with the help of LbL assembly technique, which is conducive to the construction of biosensor. To our best knowledge, to date there are no reports on application of electrochemical impedance method for the detection of adherent cells based on molecular recognition of integrin 1. Here, mouse anti-human integrin 1 monoclonal antibody, as a specific ligand binding integrin 1 on cell membrane, was used to immobilize HeLa cells on the assembled glass carbon electrode. The number of the adherent cells was determined by the electrochemical impedance signals and the relevant optimized conditions for the analysis were discussed. 2. Experimental 2.1. Chemicals and apparatus Mouse anti-human integrin 1 monoclonal antibody (antiCD29 mAb) was purchased from Sizhengbai Biotechnological Co. Ltd. (Beijing, China). Phosphate-buffered saline (PBS, pH 7.4) solution consisting of 87 mmol L−1 NaHPO4 , 14 mmol L−1 KH2 PO4 , 136.8 mmol L−1 NaCl and 2.7 mmol L−1 KCl was used in the experiments. Chitosan (CHIT, from crab shells, >90% deacetylation) was obtained from Shanghai Biochemical Reagents Co. Ltd. (Shanghai, China). Colloidal gold nanoparticles (Au NPs) were prepared according to the literature [28] and stored in a brown glass bottle at 4 ◦ C. The cell-staining experiments were performed with 0.01% acridine orange (AO). All other reagents were of analytical grade. And doubly distilled water was used for all experiments. Electrochemical impedance spectroscopy (EIS) experiments were performed with a CHI660C electrochemical workstation (CH Instruments, Shanghai Chenhua Instruments Inc., China) by using a three-electrode electrolytic cell. Glass carbon electrode (GC, 3 mm in diameter) acted as the working electrode (WE). A KCl saturated calomel electrode (SCE) served as the reference electrode (RE). A platinum plate served as the counter electrode (CE). The sizes of the Au nanoparticles were characterized ex situ by a JEOL-1230 transmission electron microscope (TEM). The images of Au NPs–CHIT film on electrode were obtained with an X-570 scanning electron microscope (SEM, Hitachi, Japan). Atomic force microscope (AFM) measurement was performed on PicoScanTM 2100 (Agilent). The pictures of stained cells were observed on an inverted fluorescence microscope (NIKON Eclipse Ti-S, Japan). 2.2. Cell culture Cervical cancer HeLa cells, which belong to adherent cells, were purchased from XiangYa Central Laboratory of Central South
University, China, and were cultured in RPMI 1640 medium (Gibco) supplemented with newborn calf serum (10%), penicillin (100 U mL−1 ) and streptomycin (100 U mL−1 ) in an incubator (5% CO2 , 37 ◦ C). When the adherent cells spread over the bottom of the culture bottle, they were trypsinized and collected, then washed twice with a sterile pH 7.4 PBS. The sediments were re-suspended in PBS for the following immobilization on the electrode. The density of re-suspended cells was determined with a hematocytometer. 2.3. LbL adsorption on electrode-surface and electrochemical measurement Prior to modification, the glassy carbon electrode was polished with 0.05 m ␣-Al2 O3 power slurries until a mirror shiny surface appeared, and was sonicated sequentially in acetone, HNO3 (1:1, v/v), NaOH (1 mol L−1 ) and doubly distilled water for 3 min. The treated electrode was scanned between −1.0 and 1.0 V versus SCE in 0.5 mol L−1 H2 SO4 aqueous solution for sufficient cycles to obtain reproducible cyclic voltammograms. After the electrode was thoroughly rinsed with doubly distilled water and dried under a stream of nitrogen, as shown in Scheme 1, 3 L of a mixture containing 0.5% chitosan and 2 g mL−1 Au NPs was dropped on the electrode-surface and allowed to dry for 36 h at room temperature. The modified electrode was coated with a drop of 3 L of 2 g mL−1 Au NPs and then was left for 24 h at room temperature. The procedure yielded a robust and strongly adherent composite film. Anti-CD29 mAb was introduced onto the Au NPs–CHIT film modified electrode by dropping 3 L of 20 g mL−1 antibody on the surface and incubating at 4 ◦ C for 24 h. Next, 2% BSA was employed to block the non-specific site. Finally 3 L HeLa cell-PBS suspension was seeded onto the modified electrode-surface and cultured in the incubator for 2 h, which achieved the cell capture based on the immunoreaction between integrin 1 on cell membrane and antibody on the electrode-surface. The electrode was gently washed with PBS after every assembly process. The GC electrodes before and after the film modification and in sequence cell incubation were characterized in 10 mmol L−1 K3 Fe(CN)6 /K4 Fe(CN)6 (1:1) containing 0.1 mol L−1 KCl via EIS at room temperature. For EIS measurements, the working electrode potential was fixed at the formal potential of the ferricyanide/ferrocyanide couple and the amplitude was 5 mV. The impedance spectrograms were collected within the frequency range of 10−1 to 105 Hz. The diameters of Nyquist circles at higher frequencies are obtained by the fitting with ZSimpWin software, Version 3.10. 3. Results and discussion 3.1. Characterization of nanoparticles and the modified electrodes The images of prepared Au NPs and the Au NPs–CHIT film on GC electrode are shown in Fig. 1A and B, respectively. The gold colloids consist of spherical nanoparticles with a size distribution in the range of 10–20 nm. The CHIT gel appeared as if undulant hills. When the NPs were dispersed in the 0.5% CHIT solution, they still kept perfect stability and uniformity. CHIT is the deacetylated derivative of chitin. It has been extensively applied for immobilization of biologic materials, molecular separation, food packaging film, artificial skin, bone substitutes, water treatment, and so on, based on its biocompatibility and biodegradability. CHIT is more useful due to the presence of amino groups that impart a positive charge to the molecule. It is known that Au NPs prepared by Frens method [28] are negatively charged owing to the adsorbed citrate anions. As CHIT in solution is protonated and positively charged, it can be adsorbed onto the surfaces of Au NPs, stabilizing and protecting the nanoparticles.
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Scheme 1. Fabrication and capture processes of the immunosensor for HeLa cells.
Fig. 1. TEM image of Au NPs (A) and SEM image of the Au NPs–CHIT film coated on GC electrode. The scale bars in A and B represent 100 and 500 nm, respectively.
Fig. 2A and B shows AFM images of GC electrode coated with Au NPs–CHIT film and anti-CD29 mAb/Au NPs–CHIT film, respectively. It can be seen that the Au NPs–CHIT film was uniformly spreading on the electrode. The surface became smoother and thicker after
antibody was adsorbed. It looks as if some pits in the Au NPs–CHIT film were filled by the antibody macromolecules. Some researches reported that Au NPs can form covalent bond with S atoms or N atoms of biomolecules [29,30]. Au NPs have been widely used to
Fig. 2. AFM images of GC electrode coated with (A) Au NPs–CHIT film and (B) anti-CD29 mAb/Au NPs–CHIT film.
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construct biosensors, such as enzyme sensor [31], immunosensor [32] and DNA sensor [33], because of their excellent ability to immobilize biomolecules and retain the biocatalytic activities of those biomolecules. So anti-CD29 mAb could be successfully assembled on GC electrode through the binding with Au NPs. 3.2. Impedance characteristics of the modified electrodes EIS is a powerful tool for investigating the interfacial properties of surface-modified electrode. The typical electrochemical interface can be represented as an electrical circuit as shown in the inset of Fig. 3 (Randles and Ershler theoretical model [34]). The equivalent circuit includes four parameters. The ohmic resistance of the electrolyte solution, Rs , the Warburg impedance, Zw , represent bulk properties of the electrolyte solution and diffusion features of the redox probe in solution. The double-layer capacitance, Cdl , and the electron-transfer resistance, Ret , reveal interfacial properties of the electrode, which is highly sensitive to the surface modification. Usually, Ret controls the interfacial electron-transfer rate of the redox probe between the solution and the electrode. In the Nyquist plot of impedance spectroscopy, it equals the semicircle diameter at higher frequencies. Fig. 3 shows the Faradaic impedance spectra, presented as Nyquist plots of the GC electrode before and after the film modification and cell incubation. It is obvious that the semicircle diameter increased by a small value after Au NPs–CHIT film and anti-CD29 mAb were successively immobilized onto the electrodesurface, but the cell capture resulted in a significant augment of the electron-transfer resistance. The stabilization of Au NPs with CHIT has been extensively reported [35,36]. Although CHIT, acting as the insulator, could decrease the electrical conductivity, the electrontransfer between electrochemical probe and electrode might be resumed to a great extent in the presence of the good conductor, gold nanoparticles. Accordingly the Ret value at the electrode modified by Au NPs–CHIT film was only higher by about 105 than that at bare electrode. The subsequent anti-CD29 mAb binding led to another increase of Ret (about 224 ), indicating that the electron-transfer was further hindered for the occupation of some antibody macromolecules at the pits of Au NPs-CHIT film owning
Fig. 3. Nyquist diagrams of electrochemical impedance spectra for [Fe(CN)6 ]3− /[Fe(CN)6 ]4− (10 mmol L−1 , 1:1) in 0.1 mol L−1 KCl at bare GC electrode (a), GC electrode coated with Au NPs–CHIT film (b), anti-CD29 mAb/Au NPs–CHIT film (c) and HeLa cells/anti-CD29 mAb/Au NPs–CHIT film with the cell concentration of 1.0 × 105 cells mL−1 (d).
three-dimensional network structure. As we know, the adsorption of antibody on Au electrode-surface can be easily performed due to the S-Au bonds but the same procedure on GC electrodesurface is relatively difficult. So the immobilization of antibody on GC electrode-surface is commonly achieved by the layer-by-layer adsorption [37], the covalent binding to a self-assembled monolayer (SAM) [14], and so on. Here, the antibody-binding on gold nanoparticles assembled with chitosan were performed. The experimental process was relatively simple and the modification only led to a little Ret , which may be beneficial to the next sensitive detection of cell capture. After the introduction of HeLa cells, the values of Ret rose up from 689 to 1276 . It means that the cells could firmly adhere to the antibody/Au NPs–CHIT film by immunoreaction. As a result, the adhesion of HeLa cells on the electrode led to the decreased active surface area and the barrier effect against the electron communication between the probe and the electrodesurface. 3.3. Condition optimization of the impedance analysis The Ret values depended directly on the interfacial electrontransfer ability. When the CHIT film, an insulation coating, was overlaid on the electrode-surface, it inevitably had negative influence on the electric signals. So some Au NPs were introduced into the CHIT film to improve conductivity of the modified electrode. It is found that the peak-currents of the K3 Fe(CN)6 /K4 Fe(CN)6 probe in cyclic voltammetry measurements increased with augment of the Au NPs-concentration which was lower than 2 g mL−1 . Aggregation of nanoparticles appeared if the content of Au NPs kept on rising, which would seriously affect the stability and uniformity of the film. In our work, the concentration of Au NPs at 2 g mL−1 in the modified film was selected for the stable signals. Besides the concentration of Au NPs, the drying time of the Au NPs–CHIT film on GC electrode at room temperature 298 K was also investigated for EIS measurements. As shown in Fig. 4, the Ret values before and after the film immobilization decreased with the time increasing and reached a plateau at time over 32 h when the electrode-activity still remained high performance. It is possible that many water molecules, which could block electron-transfer from the probe to GC electrode, were involved in the Au NPs–CHIT film when the
Fig. 4. Effect of drying time of Au NPs–CHIT film on the difference of electrontransfer resistance before and after the film immobilization. Results are presented as mean ± SD (error bar) of triplicate experiments.
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Fig. 5. The difference of electron-transfer resistance before and after the adsorption of RPMI 1640 culture medium (a) and the adhesion of 1.0 × 105 cells mL−1 HeLa cells (b) on GC electrode coated with anti-CD29 mAb/Au NPs–CHIT film as well as the difference of electron-transfer resistance before and after the adhesion of 1.0 × 105 cells mL−1 HeLa cells on GC electrode coated with Au NPs–CHIT film (c). Results are presented as mean ± SD (error bar) of triplicate experiments.
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latter was dropped on the electrode-surface. The extent of water evaporation was enhanced with time passing and many passages promoting the electron-transfer would be presented in the modified film. Based on above investigation, the drying time of the modified film at 36 h was selected for all subsequent experiments. In our opinions, some potential factors affecting the Ret measurement in the process of cell capture should be considered. It is found from Fig. 5 that the adsorption of RPMI 1640 culture medium could result in some increases of the electrical conductivity. Based on this fact, a strategy was adopted to eliminate the influence of biomacromolecules in culture medium. After the cells were trypsinized and collected, they were washed and resuspended in pH 7.4 PBS and then were introduced to the modified electrode-surface. A comparison of the adhesion ability of HeLa cells on the electrode-surface modified with and without anti-CD29 mAb was performed. It is found from Fig. 5 that the cell-adhesion on the anti-CD29 mAb/Au NPs–CHIT film could lead to a greater number of Ret . Microscopic fluorescent images of HeLa cells on the different elctrode-surfaces are presented in Fig. 6. After 2-h cell capture and gentle washing with PBS, the amount of adhered cells on the anti-CD29 mAb/Au NPs-CHIT film was larger than that on the Au NPs–CHIT film. In our experiments, the measured cells were re-suspended in pH 7.4 PBS. The adhesion and spreading of cells might be greatly influenced in the absence of culture medium and calf serum containing many nutrient compositions. In this case, some linker molecules capturing cell, such as antibodies, seemed especially important. It can be comprehended that the direct cell-adhesion on the Au NPs–CHIT film was relatively unstable in the absence of anti-CD29 mAb. Fig. 7 shows the effects of antibody concentration and antibody-binding time on the differ-
Fig. 6. Images of stained HeLa cells adhered to anti-CD29 mAb/Au NPs–CHIT film (A) and Au NPs–CHIT film (B) under fluorescence microscope (200×) after 2-h cell capture. The concentration of initially added cell: 1.0 × 105 cells mL−1 .
Fig. 7. (A) Effect of anti-CD29 mAb concentration on the difference of electron-transfer resistance before and after the adhesion of 3.0 × 104 cells mL−1 HeLa cells. The binding time of antibody: 24 h. (B) Effect of binding time of anti-CD29 mAb to Au NPs–CHIT film on the difference of electron-transfer resistance before and after the adhesion of 3.0 × 104 cells mL−1 HeLa cells. The concentration of binding antibody: 20 g mL−1 . Results are presented as mean ± SD (error bar) of triplicate experiments.
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probe. The research result reveals that the increasing extent of Ret was linearly proportional to the amount of HeLa cells in some range. Multidrug resistance is one of the major obstacles to the successful treatment of tumors. It is characterized by a decreased sensitivity of tumor cells not only to the drug employed for chemotherapy but also to a broad spectrum of drugs with neither obvious structural homology nor common targets. Cell adhesionmediated drug resistance is a special mechanism. Damiano demonstrated that ECM protein-mediated cell adhesion conferred a survival advantage for human myeloma cells acutely exposed to cytotoxic drugs by inhibiting drug-induced apoptosis [38]. Aoudjit and Vuori identified integrin 1 signaling as an important survival pathway in drug-induced apoptosis in breast cancer cells [39]. It can be comprehended that the adhesion state of tumor cells and drug-resistance tumor cells is different. Therefore, the expression of integrin 1 molecules on cell membrane of two kinds of tumor cells should be dissimilar since integrin 1 subfamily is closely related to ECM interactions. In our opinions, the detection of this difference may be achieved by the proposed EIS method in future research. Acknowledgements Fig. 8. Calibration curve of the immunosensor for HeLa cells. Results are presented as mean ± SD (error bar) of triplicate experiments.
ence of electron-transfer resistance before and after cell capture with the concentrations of 3.0 × 104 cells mL−1 . An obvious increase in the Ret values with addition of antibody content at the range of 5.0–15 g mL−1 was observed. When the concentration of antibody exceeded 15 g mL−1 , the value of Re basically maintained unchanged. The result indicates that the amount of adhered cells would increase with the antibody increasing, which would effectively reduced passages of electron-transfer. The concentration of antibody at 20 g mL−1 was selected in our work to obtain optimized condition. It is observed that the Ret values expressed a trend of ascending with increase of the antibody-binding time, reaching a plateau at the latter over 24 h. Undoubtedly the high Ret value was related to the stable cell-adhesion which benefited from the abundant fixed-antibodies on GC electrode after enough binding time. In order to obtain maximal signals, we chose the binding time of antibody at 24 h for cell sensing. 3.4. EIS detection of HeLa cells The developed method can be applied to detection of adherent cells. The EIS measurements of the K3 Fe(CN)6 /K4 Fe(CN)6 probe before and after immobilizing HeLa cells with different concentrations were performed and the results are shown in Fig. 8. The Ret value exhibited a linear response with the logarithm of HeLa cells concentration over the range of 1.0 × 104 to 2.0 × 106 cells mL−1 . The linear regression equations was Ret = −694.1 + 252.1 log c (r = 0.994), and the detection limit was 3.5 × 103 cells mL−1 . The measurements were performed three times to assess the reproducibility and precision of the freshly fabricated immunosensor. The RSD of the detection results was all lower than 6%, showing acceptable performance. These imply that the sensitivity of our sensor could bear comparison with that of other detectors for cell or bacteria measurements reported in the literatures [10,13]. 4. Conclusions Mouse anti-human integrin 1 monoclonal antibody was assembled on GC electrode and employed to capture HeLa cells. The cell adhesion based on the reaction between antibody and integrin 1 on cell membrane resulted in significant increase of Ret in EIS measurement using K3 Fe(CN)6 /K4 Fe(CN)6 as electrochemical
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Biographies Xiangfu Jiang is pursuing his M.D. in the Key Laboratory of Chemical Biology & Traditional Chinese Medicine Research (Ministry of Education, China), College of Chemistry and Chemical Engineering, Hunan Normal University. His research interests include nanomaterial constructing, biosensor fabricating, electrochemistry study of cells and biomolecules. Liang Tan is an associate professor in the Key Laboratory of Chemical Biology & Traditional Chinese Medicine Research (Ministry of Education, China), College of Chemistry and Chemical Engineering, Hunan Normal University. He received his Ph.D. degree in 2008 from Hunan University, China. His research interests cover electrochemistry analysis, biosensing and cell study. Baoling Zhang is pursuing her M.D. in the Key Laboratory of Chemical Biology & Traditional Chinese Medicine Research (Ministry of Education, China), College of Chemistry and Chemical Engineering, Hunan Normal University. Her research interests include biosensing and cell study. Youyu Zhang is a professor of Key Laboratory of Chemical Biology & Traditional Chinese Medicine Research (Ministry of Education, China), College of Chemistry and Chemical Engineering, Hunan Normal University. Her research interests include EQCM sensing and FTIR spectroelectrochemistry. Hao Tang is an associate professor of Key Laboratory of Chemical Biology & Traditional Chinese Medicine Research (Ministry of Education, China), College of Chemistry and Chemical Engineering, Hunan Normal University. His research interests include electrochemical detection and biosensing. Qingji Xie is a professor in the Key Laboratory of Chemical Biology & Traditional Chinese Medicine Research (Ministry of Education, China), College of Chemistry and Chemical Engineering, Hunan Normal University. His research is focused on chemo/biosensing and electroanalytical chemistry, including the EQCM, SECM and spectroelectrochemistry characterizations of sensing events. Shouzhuo Yao is a professor in the Key Laboratory of Chemical Biology & Traditional Chinese Medicine Research (Ministry of Education, China), College of Chemistry and Chemical Engineering, Hunan Normal University. He has been appointed as an academician of Chinese Academy of Science in analytical chemistry.