Cytosensing and Evaluation of Cell Surface Glycoprotein Based on a

Feb 2, 2009 - On the advantage of electrochemical immunoassay with a signal amplification ... The P-glycoprotein on a single living intact BGC823 cell...
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Langmuir 2009, 25, 3089-3095

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Cytosensing and Evaluation of Cell Surface Glycoprotein Based on a Biocompatible Poly(diallydimethylammonium) Doped Poly(dimethylsiloxane) Film Min-Ling Shao, Hai-Jing Bai, Hong-Lei Gou, Jing-Juan Xu,* and Hong-Yuan Chen Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing UniVersity, Nanjing 210093, China ReceiVed January 3, 2009. ReVised Manuscript ReceiVed January 16, 2009 In this paper, we constructed an interface that not only retains viability of immobilized BGC823 human gastric carcinoma cells (BGC823 cells) but also efficiently resists nonspecific adsorption of the P-glycoprotein antibody and its secondary antibody, which enabled us to sensitively detect the number of cells and P-glycoproteins on the BGC823 cell surface by the immunoassay method. Preparation of the film was quite simple and inexpensive just by spin-coating poly(dimethylsiloxane) (PDMS) doped with poly(diallydimethylammonium) (PDDA) on the surface of gold electrodes. The composite film’s biocompatibility, antinonspecific adsorption ability, and the conductivity for electrochemical probe ([Fe(CN)6]3-/4-) were proved by cell culture experiments, blocking experiments, and electrochemical experiments. Compared with PDMS and PDMS doped with poly(sodium 4-styrenesulfonate) (PSS), the PDMS-PDDA composite film showed a predominant ability to capture cells due to electrostatic reaction between the presence of positively charged PDDA and the negatively charged glycocalyx on the surface of cells. On the advantage of electrochemical immunoassay with a signal amplification path by using biocatalytic precipitation of an insoluble product, differential pulse voltammetry (DPV) measurement based on the changes of electron-transfer resistance was introduced to detect the cell amount and monitor growing states of cells like adhesion, spread, proliferation, and apoptosis on the electrodes. Optimally, signal response was proportional to the logarithm of cell concentration ranging from 1.0 × 103 to 5.0 × 107 cells mL-1 with a detection limit of 7.2 × 102 cells mL-1. On the basis of the special property for resisting nonspecific adsorption of this composite film, an ultraviolet and visible (UV-vis) absorption spectrum with one-step immunoreaction was employed to evaluate the P-glycoprotein on the BGC823 cell surface. The P-glycoprotein on a single living intact BGC823 cell was detected correspondingly to 4.7 × 107 molecules. The work implied that the composite film possessed potential applications for biosensing and convenient evaluation of surface glycoprotein on living cells.

Introduction Recently, much interest has focused on the research of immobilizing living cells within such areas as life science research, toxicity monitoring, clinical diagnostics, and public health protection. Because cell immobilization based on biocompatible materials has great advantages in efficiently keeping the cell concentration, viability, and natural physiological state of cells,1 some research fields like electrochemical sensors, microfluidic technology, and cell patterning have paid more attention to construct all kinds of biomaterials in favor of adhering cells for better research on intact living cells. For example, by constructing a kind of gold nanoparticle-chitosan nanocomposite gel, Ju and co-workers tracked the proliferation and apoptosis of K562 cells on it using an electric impedance sensing system and characterized the viability of cells by voltammetric response.2 The response of AsPC-1 cells to adriamycin was researched by immobilizing cells on mixing colloidal gold nanoparticles in carbon paste.3 Other research groups have researched the effect of exogenous factors including temperature, substrate topography, etc. on living cells in chips fabricated or modified with biocompatible materials.4-6 Moreover, cell immobilization based on biocompatible materials provided a favorable platform for evaluating glycoprotein expression on the living cell surface. Since (1) Choi, J.; Nam, Y.; Fujihira, M. Biotechnol. Bioprocess. Eng. 2004, 9, 76–85. (2) Ding, L.; Hao, C.; Xue, Y. D.; Ju, H. X. Biomacromolecules 2007, 8, 1341–1346. (3) Du, D.; Liu, S.; Chen, J.; Ju, H.; Lian, H.; Li, J. Biomaterials 2005, 26, 6487–6495.

accumulating evidence indicated that abnormal glycoprotein expression was associated with some diseases such as cancers,7 the studies of glycoprotein on the cell surface have become an attractive subject for understanding their role in disease development. Several methods such as flow cytometric assay, highperformance liquid chromatography, and mass spectrometry have been developed for glycoprotein detection.8-10 Although these techniques can reveal molecular details, they usually are timeconsuming and involve an expensive apparatus. Polymerase chain reaction (PCR) achieved more sensitive detection of glycoprotein than protein-based approaches,11 but this strategy could not exclude the possible contamination of the probe. Recently, an electrochemical immunoassay based on cell interface immobilization has been developed to detect cell surface glycoprotein and carbohydrates.12,13 It was a novel methodology for (4) Lucchetta, E. M.; Lee, J. H.; Fu, L. A.; Patel, N. H.; Ismagilov, R. F. Nature. 2005, 434, 1134–1138. (5) Lee, J. N.; Jiang, X. Y.; Ryan, D.; Whitesides, G. M. Langmuir 2004, 20, 11684–11691. (6) Paguirigan, A.; Beebe, D. J. Lab Chip. 2006, 6, 407–413. (7) Barton, A. C.; Davis, F.; Higson, S. P. J. Anal. Chem. 2008, 80, 6198– 6205. (8) Koizumi, S.; Konishi, M.; Ichihara, T.; Wada, H.; Matsukawa, H.; Goi, K.; Mizutani, S. Eur. J. Cancer 1995, 31A, 1682–1688. (9) Qiu, R. Q.; Regnier, F. E. Anal. Chem. 2005, 77, 2802–2809. (10) Kameyama, A.; Kikuchi, N.; Nakaya, S.; Ito, H.; Sato, T.; Shikanai, T.; Takahashi, Y.; Takahashi, K.; Narimatsu, H. Anal. Chem. 2005, 77, 4719–4725. (11) King, M.; Su, W.; Chang, A.; Zuckerman, A.; Pasternak, G. W. Nat. Neurol. 2001, 4, 268–274. (12) Du, D.; Ju, H. X.; Zhang, X. L.; Chen, J.; Cai, J.; Chen, H. Y. Biochemistry 2005, 44, 11539–11545. (13) Cheng, W.; Ding, L.; Lei, J. P.; Ding, S. J.; Ju, H. X. Anal. Chem. 2008, 80, 3867–3872.

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evaluating substances on the surface of living cells because of simplicity and rapidity of detection methods as well as less loss of cell viability and concentration due to immobilizing living cells on biomaterials. An electrochemical enzyme-linked immunoassay was usually used for biochemical analysis by enzymatic amplification of the signals.12-16 Alkaline phosphatase is a stable enzyme with high specific activity toward the hydrolysis of 5-bromo-4-chloro-3indolyl phosphate, which produced an insoluble dimmer bound tightly to the sensor surface. Ruan and co-workers had used enzymatic hydrolysis and polymerization of BCIP as a means of linking the impedance-based sensor response to an enzymelinked immunoassay for Escherichia coli bacteria.17 Precipitation of an insoluble product on a horseradish peroxidase (HRP)modified monolayer electrode and HRP-glucose oxidase bienzyme-layered electrode has been used in development of new electrochemical biosensors to detect hydrogen peroxide and glucose.18 The insoluble precipitation on a modified electrode surface was to block the interfacial electron-transfer feature of the redox probe at the electrode in the solution, and therefore detection sensitivity would be further enhanced. However, some disadvantages such as nonspecific adsorption of film and denaturation of enzyme immobilized on the surface still exist in the process of immunoassay. For an interface of cytosensor with nonspecific adsorption, it does not avoid the trouble of adding the process of blocking nonspecific adsorption and the washing up, which will impair the sensitivity and stability of the method and cell viability. Thus, the construction of an interface that can not only retain viability of immobilized cells but also efficiently resist nonspecific adsorption in the process of immunoreaction is very important for simplifying the assay system and enhancing sensitivity of detection. The physical and chemical properties of a substrate affected the attachment and growth of cells on it.19-22 As a biocompatible,23-26 inexpensive, and durable material, PDMS is used extensively in medical implants, biomedical devices, and fabrication of microsystems.27 The surface chemistry of PDMS has been tailored to improve its bioactivity and enable the adhesion of cells and proteins on the modified PDMS.28,29 Juchniewicz and co-workers reported a porous PDMS coating using water as (14) Duan, C.; Meyerhoff, M. E. Anal. Chem. 1994, 66, 1369–1377. (15) Bauer, C. G.; Eremenko, A. V.; Ehrentreich-For¨ster, E.; Bier, F. F.; Makower, A.; Halsall, H. B.; Heineman, W. R.; Scheller, F. W. Anal. Chem. 1996, 68, 2453–2458. (16) Wang, J.; Pamidi, P. V. A.; Rogers, K. R. Anal. Chem. 1998, 70, 1171– 1175. (17) Ruan, C. M.; Yang, L. J.; Li, Y. B. Anal. Chem. 2002, 74, 4814–4820. (18) Patolsky, F.; Zayats, M.; Katz, E.; Willner, I. Anal. Chem. 1999, 71, 3171–3180. (19) Curtis, A. S. G.; Wilkinson, C. D. W. J. Biomater. Sci., Polym. Ed. 1998, 9, 1313–1329. (20) Tranquillo, R. T. Biochem. Soc. Symp. 1999, 65, 27–42. (21) Thomas, S.; Frederique, K. M.; Stephanie, G. R.; Nicolas, G.; Fabien, S. S.; Catherine, P.; Mathias, B.; Marc, P.; Francois, C.; Franz, B. Biomaterials 2007, 28, 1572–1584. (22) Mann, B. K.; Tsai, A. T.; Scott-Burden, T.; West, J. L. Biomaterials 1999, 20, 2281–2286. (23) Park, J. H.; Park, K. D.; Bae, Y. H. Biomaterials 1999, 20, 943–953. (24) Bordenave, L.; Bareille, R.; Lefebvre, F.; Caix, J.; Baquey, C. J. Biomater. Sci., Polym. Ed. 1992, 3, 409–416. (25) Marois, Y.; Sigot-Luizard, M.-F.; Guidoin, R. ASAIO J. 1999, 45, 272– 280. (26) Sherman, M. A.; Kennedy, J. P.; Ely, D. L.; Smith, D. J. Biomater. Sci., Polym. Ed. 1999, 10, 259–269. (27) Tourovskaia; Barber, T.; Wickes, B. T.; Hirdes, D.; Grin, B.; Castner, D. G.; Healy, K. E.; Folch, A. Langmuir 2003, 19, 4754–4764. (28) Patrito, N.; McCague, C.; Norton, P. R.; Petersen, N. O. Langmuir. 2007, 23, 715–719. (29) Lee, J. H.; Chung, S.; Kim, S. J.; Han, J. Anal. Chem. 2007, 79, 6868– 6873.

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a porogen to adulterate in the PDMS prepolymer.30 De Silva and co-workers recently achieved localized cell attachment by patterning adhesive proteins on PDMS via microcontact printing31 and precision aerosol spraying.32 Garcia and co-workers used the cationic polymer, poly(diallyldimethylammonium chloride) (PDDA), as a surfactant, to coat negatively charged PDMS, which changed the surface charges of this material.33 Due to the bad conductivity and low surface energy,28 PDMS was barely used for the modification of the electrode. To enhance its conductivity, our group previously doped poly(diallyldimethylammonium chloride) (PDDA), a positively charged polymer, into PDMS.34 Herein, we introduced PDMS into the construction of cell-based electrochemical biosensors. Via the doping of PDDA, the PDMS-PDDA film not only made the charge-transfer of the redox probe possible due to the rough and porous morphology but also showed great attached ability for BGC823 cells, which was attributed to the effect of a very high density of positively charged PDDA.35-37 This composite film also efficiently retained the activity of living tumor cells immobilized on the electrode surface because of good biocompatibility of PDMS and PDDA and weak interaction between positive charges on the surface of modified films and the negatively charged glycocalyx on the surface of tumor cells, which provided an environment similar to a native system. In this work, based on cell immobilization on the PDMS-PDDA film, we reported an easy and friendly measurement method with a signal amplification path by using biocatalytic precipitation of an insoluble product to detect cells’ concentration and monitor the adhesion, proliferation, and apoptosis of living cells. Due to the specific property for resisting nonspecific adsorption of PDMS-PDDA film, the P-glycoprotein antibody could only combine with P-glycoprotein on the surface of BGC823 cells. On the basis of this, we developed a novel method for conveniently and sensitively quantifying the glycoprotein expression status on the living cell surface by combining a simple UV measurement with one-step immunoreaction. This work provided an important and easy protocol for quantitative evaluation of the cell surface glycoprotein.

Materials and Methods Materials. Sylgard 184 (PDMS) and curing agent (Si-H group cross-linking agent) were from Dow Corning (Midland, MI). PDDA, Mw ) 100 000-200 000 g mol-1, in 20% aqueous solution and poly(sodium 4-styrenesulfonate) (PSS), Mw ) 70 000 g mol-1, in 30% aqueous solution were purchased from Aldrich. P-Glycoprotein mouse monoclonal antibody (P-gp mAb, 1 mg mL-1) was purchased from Abcam (UK), and the secondary antibody of alkaline phosphatase (AP)-labeled goat antimouse (0.2 mg mL-1) and 5-bromo-4-chloro-3-indolyl phosphate, disodium salt hydrate (BCIP), were obtained from Sigma (USA). Acridine orange (AO) and ethidium bromide (1% w/v, EB) were products from Amresco (USA). All other reagents were of analytical grade. Phosphate buffered saline (PBS) (pH 7.4) contained 137 mM NaCl, 2.7 mM KCl, 87.2 mM (30) Juchniewicz, M.; Stadnik, D.; Biesiada, K.; Olszyna, A.; Chudy, M.; Brzozka, Z.; Dybko, A. Sens. Actuators, B 2007, 126, 68. (31) De Silva, M. N.; Desai, R.; Odde, D. J. Biomed. MicrodeVices 2004, 6, 219–222. (32) De Silva, M. N.; Paulsen, J.; Renn, M. J.; Odde, D. J. Biotechnol. Bioeng. 2006, 93, 919–927. (33) Mora, M. F.; Giacomelli, C. E.; Garcia, C. D. Anal. Chem. 2007, 79, 6675–6681. (34) Zhao, W.; Sun, S. X.; Xu, J. J.; Chen, H. Y.; Cao, X. J.; Guan, X. H. Anal. Chem. 2008, 80, 3769–3776. (35) Jean-Luc, D.; Aurora, D.; Yves-Jacques, S.; Paul, G. R. Biomaterials 1999, 20, 547–559. (36) Lee, M. H.; Brass, D. A.; Morris, R.; Composto, R. J.; Ducheyne, P. Biomaterials 2005, 26, 1721–1730. (37) Van Damme, M. P.; Tiglias, J.; Nemat, N.; Preston, B. N. Anal. Biochem. 1994, 223, 62–70.

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Scheme 1. Mechanism for Preparation and Analysis of the Designed Cytosensor

Na2HPO4 · 12H2O, and 14.1 mM KH2PO4. All aqueous solutions were prepared using ultrapure water (Milli-Q, Millipore). Cell Culture. The BGC823 cell line was kindly provided by the Gulou Hospital, Nanjing, China. BGC823 cells were cultured in a culture bottle with RPMI 1640 medium (GIBCO) supplemented with 10% fetal bovine serum (FBS, GIBCO), penicillin (60 ug mL-1), and streptomycin (100 ug mL-1) at 37 °C in a humidified atmosphere containing 5% CO2. After 72 h, the cells were collected and separated from the medium by centrifugation at 1500 rpm for 5 min and then washed twice with sterile pH 7.4 PBS. The sediment was resuspended in PBS to obtain a homogeneous cell suspension at a certain concentration. The cell number was determined using a PetroffHausser cell counter (USA). Electrode Preparation and Cell Immobilization. Bulk gold electrodes (2 mm diameter) were abraded with fine SiC paper and polished carefully with 0.3 and 0.05 µm alumina slurry and then sonicated in water and absolute ethanol, respectively. PDMS-PDDA electrodes were prepared according to our previous work.34 A mixture of PDDA and PDMS (20% PDDA aqueous solution, PDMS including monomer and curing agent with 10:1 w/w) in a certain weight ratio was dropped on the surface of the electrodes, followed with spin coating at 2000 rpm for 15 min. The structure was cured at 80 °C for 90 min, followed by rinsing thoroughly with pH 7.4 PBS. After that, 20 µL of BGC823 cell suspension at a certain concentration was dropped on the PDMS-PDDA/Au surface and incubated at 37 °C for 2 h. After carefully rinsing with pH 7.4 PBS to remove the noncaptured cells, BGC823/PDMS-PDDA/Au was obtained and used for a subsequent assay (Scheme 1). Enzyme-Amplified Electrochemical Analysis. The BGC823/ PDMS-PDDA/Au was incubated with 10 µg mL-1 of P-gp mAb and 5.0 µg mL-1 of secondary AP-labeled antibody at 37 °C each for 60 min. After washing carefully with pH 7.4 PBS, electrochemical measurements were performed in pH 7.4 PBS containing 6.0 mM [Fe(CN)6]3-, 6.0 mM MgCl2, and 2.0 mM BCIP. DPV measurements were performed from 0 to 0.5 V with a pulse amplitude of 50 mM and width of 50 ms. Cell Proliferation on PDMS-PDDA Film. The same size (5 mm × 6 mm) Au slides coated with PDMS-PDDA film were immersed in 1.0 × 105 cells mL-1 of cell suspension containing RPMI 1640 medium supplemented with 10% FBS, penicillin (60 µg mL-1), and streptomycin (100 µg mL-1) at 37 °C in a humidified atmosphere containing 5% CO2. After the adhered BGC823 cells were cultured for successively different periods, the images of BGC823 cells with different growth periods on the PDMS-PDDA film were recorded by fluorescent staining under an inverted microscope with a magnification of 100× and acquired with a CCD camera. Image-Pro Plus (IPP) software was used to analyze the

amount of cells immobilized on the modified film. The process of assay was the same as that for enzyme-amplified electrochemical analysis. Apparatus. Atomic force microscopy (AFM) experiments were performed in ambient conditions using a molecular imaging pico SPM in contacting mode with a 100 µm scanner. Electrochemical experiments were performed on a CHI 660B electrochemical workstation (Shanghai Chenhua Apparatus Corporation, China). Electrochemical impedance experiments were carried out on a PGSTAT30/FRA2 system (Autolab, Netherlands) in a 10 mM K3Fe(CN)6/K4Fe(CN)6 (1:1) mixture with 0.1 M KCl as supporting electrolyte, using an alternating current voltage of 5.0 mV, within the frequency range of 5 × 102-106 Hz. All experiments were carried out using a conventional three-electrode system comprising a platinum foil as the auxiliary electrode, a saturated calomel electrode as the reference electrode, and the modified gold electrode as the working electrode. Ultraviolet and visible (UV-vis) absorption spectra were recorded with a Lambda 35 UV-vis spectrometer (Perkin-Elmer Instruments).

Results and Discussion Evaluation of PDMS-PDDA Composite Film. PDMS is a useful substrate for medical implants and fabrication of biochips due to its high oxygen permeability, good formability, chemical stability, and optical transparency; however, PDMS is not a good substrate for cell adhesion due to its low surface energy. Herein, a method by doping polyelectrolytes with different charges was adopted to change the surface property of PDMS. It was reported that surface charges played a particularly important role in governing nonspecific cellular adhesion to material substrates, and substrates with positive charges had a significantly higher level for adhesion of cells.36 In our study, the results about cell adhesion ability on PDMS film doping with different electrolytes with different charges were tested, as presented in Figure 1. Although cells could attach to all substrates, there were statistical differences in cell attachment among the substrates. Indeed, the amount of cells attached on the bare PDMS is obviously less than that on PDMS-PDDA film. This could be attributed to electrostatic reaction between the presence of a very high density of positive charges on the surface of modified films doped with PDDA and the negatively charged glycocalyx on the surface of cells.35-37 PSS was an electrolyte with negative charges, and electrostatic repulsion between negative charges of PSS and negatively charged glycocalyx on the surface of cells resulted

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Figure 1. Number of immobilized cells on PDMS-PSS (10:6.5), PDMS, and PDMS-PDDA (10:6.5) film after incubation for 120 min in 1.0 × 106 cells mL-1 of BGC823 cell suspension. At least 10 measurements were taken for each datum in 856.8 µm × 644.8 µm areas, making analysis with IPP software. Error bar represents standard deviation from the mean.

in the least number of adherent cells. Therefore, the PDMS film doped with PDDA had an obvious advantage for immobilizing BGC823 cells. AFM measurement was an effective method to provide surface morphology and phase images. Figure 2 showed the AFM images of the bare PDMS and PDMS-PDDA films on gold slides, respectively. As can be seen, the bare PDMS film is very compact and smooth. With the mixture of PDDA, the surface of the film became rough and porous, which could increase the diffusion of the electrochemical probe [Fe(CN)6]3-/4- and specific surface area of the electrode. It can be seen from Figure 3A that the reduction peak current of [Fe(CN)6]3-/4- increased with the increase of the amount of PDDA. The peak current tended to the maximum when the weight ratio between PDMS and PDDA reached to 10:6.5. More adulterated PDDA did not enhance the response and instead slightly decreased it, which might result from PDMS being partially cured in the process of mixing. With the increase of the amount of PDDA, the hand-mixing process of the PDMS monomer, curing agent, and PDDA became difficult. Thus, a longer mixing time would be required, which caused the mixture to partly cure before spin-coating. Figure 3B showed the number of BGC823 cells (1 × 106 cells mL-1) attached on the PDMS-PDDA film with different adulterated ratios. The amount of attached cells apparently increased with an increase of PDDA due to electrostatic forces. However, the amount of adhered cells greatly decreased when excessive PDDA was used. The reason for this phenomenon partly resulted from the effect of the part curing of PDMS, which led to an uneven mix between PDMS and PDDA. The rough surface of the composite film at a high adulterated ratio might also result in the phenomenon. Some studies had indicated that substrate surface roughness decreased the amount of attached cells.38,39 Therefore, 0.65 was chosen as an optimal ratio between PDMS and PDDA. (38) Andersson, A. S.; Backhed, F.; Euler, A.; Richter-Dahlfors, A.; Sutherland, D.; Kasemo, B. Biomaterials 2003, 24, 3427–3436. (39) Kieswetter, K.; Schwartz, Z.; Hummert, T. W.; Cochran, D. L.; Simpson, J.; Dean, D. D.; Boyan, B. D. J. Biomed. Mater. Res. 1996, 32, 55–63.

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Generally, the transduction principle for detection of BGC823 cells was based on measurements of electron-transfer capability with [Fe(CN)6]3-/4- as a redox probe. After the PDMS-PDDA film was spin-coated in a bare Au electrode surface, the peak current of the redox probe decreased as a result of the hindering effect of the composite film (Figure 4a). The access of the redox probe to the electrode surface would further have been hindered after immobilizing BGC823 cells on the surface of the film, due to the resistance of the cell membrane, causing a further decrease in peak current (Figure 4b). Such an electron-transfer block to the redox probe can be amplified by precipitation on the surface of the modified electrode based on enzymatic reaction. After incubation with P-gp mAb and then the secondary AP-conjugated antibody, the peak current further decreased compared with that of only the BGC823-modified electrode (Figure 4c), which contributed to the precipitation onto the electrode surface produced by the biocatalytic reaction. The precipitation bound tightly to the sensor surface as an electrical barrier could efficiently block the electron-transfer of the redox probe. In the process of electrochemical immunoreaction, nonspecific adsorption of film to antibody usually can not be ignored for an accurate detection. To block the nonspecific binding sites on the surface of the film, the BGC823/PDMS-PDDA/Au was immersed in 1% BSA solution for 30 min. Interestingly, the deviation of five groups of parallel experiments in the presence and absence of BSA was less than 4.7%, which indicated that BSA did not have an apparent effect during the process of blocking over 1 h. In our previous work, we also found that BSA hardly blocked the adsorption of AchE.34 To further examine the nonspecific adsorption of P-gp mAb and AP-labeled antibody on the surface of PDMS-PDDA/Au, they were dropped and incubated for 1 h on the surfaces of PDMS-PDDA/Au and BGC823/PDMS-PDDA/Au under the same experimental conditions step by step. The peak current of [Fe(CN6)]3- almost kept constant on PDMS-PDDA/Au. On the contrary, the peak current would greatly decrease to about 44.6% (average for five repeated experiments) after the same incubation time of P-gp mAb and AP-conjugated antibody on the BGC823/PDMS-PDDA/Au (see Supporting Information, Figure S3). In this work, it was obvious that PDMS-PDDA film possessed a specific property for resisting nonspecific adsorption of BSA, P-gp mAb, and AP-labeled antibody. Thus, it was not necessary to block the nonspecific binding sites with BSA in our research, which was in favor of simplifying the process of experiment and reducing the loss of viability cells. The work also indicated that a combination between cells and the P-gp mAb antibody only depended on the amount of antigen on the surface of tumor cells. Optimization of Detection Conditions. Currently, differential pulse voltammetry (DPV) has been widely used in the immunoassay of cells. However, the DPV method based on the changes of electron-transfer resistance was barely used for detection of cells, to our knowledge. Compared with electrochemical impedance spectroscopy (EIS) measurement,40-43 DPV measurement has a potential advantage for monitoring living cells due to a shorter time of electrical field effect in the process of detection. Herein, faster DPV measurement was adopted to monitor living cells. To evaluate the two detection methods, BGC823/PDMSPDDA/Au electrode was tested in continuous detection 10 times by each detection method in homogeneous solution. From Figure (40) Wegener, J.; Zink, S.; Ros¨en, P.; Galla, H.-J. Pfluegers Arch. Eur. J. Physiol 1999, 437, 925–934. (41) Yang, L. J.; Li, Y. B.; Erf, G. F. Anal. Chem. 2004, 76, 1107–1113. (42) Yang, L.; Li, Y.; Griffis, C. L.; Johnson, M. G. Biosens. Bioelectron. 2004, 19, 1139–1147. (43) Chen, H.; Heng, C. K.; Puiu, P. D.; Zhou, X. D.; Lee, A. C.; Lim, T. M.; Tan, S. N. Anal. Chim. Acta 2005, 554, 52–59.

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Figure 2. AFM images of a bare PDMS film (A) and a PDMS-PDDA film (B) on Au slides.

Figure 3. A: Cyclic voltammograms for 5.0 mM [Fe(CN)6]3-/4- at (a) bare Au and modified electrodes with different adulterated ratio (weight ratio) between PDMS and PDDA; (b) pure PDMS; (c) 10:2; (d) 10:3.5; (e) 10:5; (f) 10:6.5; and (g) 10:8. Scan rate: 50 mV/s. B: The number of BGC823 cells (1 × 106 cells mL-1) attached on the PDMS-PDDA film with different adulterated ratios (10:2, 10:3.5, 10:5, 10:6.5, 10:8, 10: 1). IPP software calculated the amount of cells in the 856.8 µm × 644.8 µm area at least 10 times.

Figure 4. Cyclic voltammograms for 5.0 mM [Fe(CN)6]3-/4- and 2.0 mM BCIP in pH 7.4 PBS at the (a) PDMS-PDDA/Au, (b) BGC823/ PDMS-PDDA/Au, and (c) AP-P-gp-BGC823/PDMS-PDDA/Au modified electrodes. Scan rate: 50 mV/s.

S1 in the Supporting Information, it was not hard to see that resistance greatly changed after 10 measurements in a row and finally reduced to 56.3% by EIS methods. By contrast, the peak current of the modified electrode almost kept a constant value by DPV measurement after 10 measurements. We speculated

that a larger resistance change occurred in the process of EIS measurement resulting from deattachment of some cells from the PDMS-PDDA film due to deterioration of cell vitality under a longer time of electric field effect or accumulation of redox probe within the film. Thus, DPV was used as detection method. The incubation time was an important parameter for both attached cells on the PDMS-PDDA modified film surface and the specific recognition of antibody to P-gp on the captured cell surface. With the incubation time increasing for 106 cells mL-1 of BGC823 cells, the DPV peak current sharply decreased and tended to a steady value after 90 min (Figure 5A), indicating a tendency of thorough attachment of BGC823 cells on the sensor surface. To ensure optimal adhesion efficiency of cells, 120 min was chosen as an optimal incubation time of BGC823 cells. The amount of AP-conjugated antibody and P-gp mAb bounded to BGC823/PDMS-PDDA/Au was also an important parameter for sensitive and reproducible assays of concentration and growing states of cells. With the increasing concentration of P-gp mAb and AP-conjugated antibody in the incubation solution, both of the DPV peak currents of the obtained APP-gp-BGC823/PDMS-PDDA/Au in PBS containing BCIP decreased linearly and then tended to a constant value after 10 µg mL-1 of P-gp mAb and 5.0 µg mL-1 of AP-conjugated antibody, respectively. Therefore, the optimal concentrations for

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Figure 6. A: DPV curves of AP-P-gp-BGC823/PDMS-PDDA/Au obtained with BGC823 cell concentrations of 1 × 103, 1 × 104, 1 × 105, 1 × 106, 1 × 107, and 5 × 107 cells mL-1 (from f to a). Inset: Linear relationship between reduction of peak current of [Fe(CN)6]3- and logarithm of BGC823 cell concentration. B: DPV curves of AP-P-gpBGC823/PDMS-PDDA/Au obtained with BGC823 cell concentration of 1 × 105 cells mL-1 for different incubation times: 4 h, 8 h, 24 h, 48 h, 72 h, 96 h (from a to f). Inset: Relationship between the reduction of peak current of [Fe(CN)6]3- and the incubation time of BGC823 cells on the surface of a modified electrode. Figure 5. Dependence of DPV peak currents of enzyme-catalyzed analysis on incubation time in 106 cells mL-1 of BGC823 cell suspension (A), concentrations of P-gp mAb (2), and secondary AP-conjugated antibody (9) (B), incubation time in P-gp mAb (b) and AP-conjugated antibody (1) (C), and different reaction time for AP-P-gp-BGC823/PDMS-PDDA/ Au in PH 7.4 PBS containing 6.0 mM [Fe(CN)6]3-, 6.0 mM MgCl2, and 2.0 mM BCIP (D). When one parameter changes, the others are under their optimal conditions.

P-gp mAb and AP-conjugated antibody were 10 µg mL-1 and 5.0 µg mL-1, respectively. Other factors influencing the immunoreaction and immunoassay included the incubation time of P-gp mAb and APconjugated antibody and reaction time between AP and BCIP. With an increase of the incubation time, the DPV response decreased and tended to a minimum at 60 min. A longer incubation time did not reduce the response. It can be seen from Figure 5D that the DPV peak current sharply decreased and tended to a steady value after the reaction time for 5 min, indicating the amount of precipitate accumulated with time on the modified electrode through a biocatalytic reaction process. Thus, the optimal incubation and detection time were 60 and 5 min, respectively. Detection of BGC823 Cells. The electrochemical signal was directly related to the amount of cells attached on the surface of the modified electrode. With an increasing concentration of BGC823 cells for their immobilization, the obtained AP-P-gpBGC823/PDMS-PDDA/Au showed a decreasing peak current of [Fe(CN)6]3- (Figure 6A), indicating a higher amount of BGC823 cells were immobilized to the surface of the modified electrode. The peak current was proportional to the logarithmic value of the cell concentration ranging from 1.0 × 103 to 5.0 × 107 cells mL-1 with a correlation coefficient R of 0.997 (inset in Figure 6A, n ) 6). The detection limit was 7.2 × 102 cells mL-1 based on the slope of the linear plot and 10 times the value of standard deviation, which was lower than those of 6 × 103 cells mL-117 and 7.1 × 103 cells mL-1 at an impedance sensor for K562 A cells44 and 1.0 × 104 cells mL-1 at a piezoelectric immunosensor for Salmonella.45 The method also showed comparable sensitivity with that of 8.71 × 102 cells mL-1 at an impedance sensor for K562.2 Therefore, the PDMS-PDDA film could be used for detection of BGC823 cells due to the simple (44) Hao, C.; Yan, F.; Ding, L.; Xue, Y. D.; Ju, H. X. Electrochem. Commun. 2007, 9, 1359–1364. (45) Wong, Y. Y.; Ng, S. P.; Ng, M. H.; Si, S. H.; Yao, S. Z.; Fung, Y. S. Biosens. Bioelectron. 2002, 17, 676–684.

fabrication process, low detection limit, and broad detection range. The decrease of the peak current was related not only to the amount of cells immobilized on PDMS-PDDA/Au but also to the states of adhesion cells. Therefore, besides the detection of cell concentration, the method could also be used to monitor growing conditions of cells on the electrode surface and indirectly evaluate biocompatibility of the film. It can be seen from Figure S4 in the Supporting Information that BGC823 cells were capable of not only adhering to the PDMS-PDDA film but also spreading and proliferating on the film. After incubation for 8 h, the attaching round cells almost adhered and spread to irregular shapes on the surface of the film. BGC823 cells apparently proliferated after 24 h and spread evenly over the entire surface after 48 h. During these periods, the cells had a good viability as evidenced by observing cell morphology and fluorescent staining. However, a longer incubation time caused a loss of normal cell characteristics and viability, just as showed by images e and f of Figure S4 (Supporting Information). After 72 h, adhered cells shrinked and tended to round, and some cells even died presenting the red fluorescent. The average number of adhered cells could be calculated on the same size areas (856.8 um × 644.8 um) of the film during the different periods by IPP software analysis. The number of cells on the electrode surface with 2 mm diameter was estimated as 1443 cells mm-2, 1421 cells mm-2, 3126 cells mm-2, 3614 cells mm-2, 3495 cells mm-2/28 cells (dead cells) mm-2, and 3097 cells mm-2/324 cells (dead cells) mm-2, respectively. By DPV measurement, the corresponding process could be well showed on DPV curves. Compared to curve a (Figure 6B), the peak current of b apparently decreased with an approximate amount of cells, resulting from cell spread. With an increasing incubation of BGC823/PDMS-PDDA/Au, the process of electron transfer would become more and more difficult, as a result of the cell proliferation on the surface, which introduced a great barrier for the electrochemical process (Figure 6B, curve c and d). The peak current drastically decreased after the incubation time of 72 h, and this change might be related to the apoptosis of cells since the amount of live cells after 72 h was less than that after incubation of 48 h. Thus, it is feasible to monitor states of adherent cells by this method and to evaluate indirectly compatibility of composite films. Detection of Surface P-Glycoprotein of BGC823 Cells. The P-glycoprotein on the surface of BGC823 tumor cells could specifically combine with P-gp mAb antibody. We found that PDMS-PDDA film had a good ability for resisting nonspecific

Cytosensing of Cell Surface Glycoprotein

adsorption of P-gp mAb in the process of DPV measurement. Therefore, the decreasing concentration of P-gp mAb in the solution rested with the amount of P-glycoprotein on the surface of tumor cells. For evaluating the amount of P-glycoprotein on the cell surface, UV-vis absorption spectroscopy was employed. As can be seen in Figure S5 (see Supporting Information), the P-gp solution has a distinct surface plasmon absorption band at about 276 nm. The standard curve of peak absorbance vs concentrations of P-gp was drawn (Figure S5, A, inset, Supporting Information). PDMS-PDDA and BGC823/PDMS-PDDA modified Au slides were incubated in the P-gp solution of 10 µg mL-1 for 1 h, respectively. The UV-visible absorption spectra of P-gp were recorded, respectively. The absorbance of P-gp solution incubated on PDMS-PDDA film for 1 h kept a constant value (curve a) while it decreased on BGC823/PDMS-PDDA film (curve b). Due to the difference of peak absorbance, the loading of P-gp antibody on the BGC823/PDMS-PDDA film was calculated as 2.94 µg mL-1. By IPP analysis software, the number of immobilized cells on the surface film with 90 mm2 was calculated as 8.80 × 104. Therefore, the amount of glycoprotein on each BGC823 cell could be calculated correspondingly to 4.7 × 107 molecules.

Conclusions The PDMS-PDDA film showed excellent biocompatible property for living cells on surface. Positively charged PDDA was doped into a PDMS film, which not only improved attachment ability of cells but also enhanced charge-transfer of the redox probe to the electrode surface, and the composite film could be simply and reproducibly prepared with low cost and in a short amount of time. DPV measurement based on signal amplification was first introduced for detecting concentration, proliferation, and apoptosis of tumor cells. The method showed a wide linear

Langmuir, Vol. 25, No. 5, 2009 3095

range and low detection limit for quantification of cells. Due to the special property for resisting nonspecific adsorption of film, P-glycoprotein on the surface of a single living BGC823 cell was qualified by a simple UV-vis measurement. This technique provided a new avenue for evaluating the glycoprotein expression on other tumor cell surfaces by modification of the electrode with PDMS-PDDA film. In brief, an interface that not only retains viability of immobilized BGC823 human gastric carcinoma cells (BGC823 cells) but also efficiently resists nonspecific adsorption of the P-glycoprotein antibody and its secondary antibody was constructed by spin-coating PDMS doped with PDDA on the surface of gold slices. It enabled us to sensitively detect the number of cells and P-glycoproteins on the BGC823 cell surface by the immunoassay method. Acknowledgment. This work was supported by the National Natural Science Foundation (No. 20890020, 20775033), the National Natural Science Funds for Creative Research Groups (20821063), the program for New Century Excellent Talents in University (NCET), and the 973 Program (2007CB936404, 2006CB93301) of China. Supporting Information Available: Nyquist diagrams of electrochemical impedance spectra and DPV diagrams of BGC823/ PDMS-PDDA/Au in continuous detection 10 times in homogeneous solution (Figure S1) as well as comparison diagrams of results by two kinds of methods (Figure S2). Ratios of peak currents of [Fe(CN6)]3on AP-P-gp-BGC823/PDMS-PDDA/Au or AP-P-gp-PDMS-PDDA/ Au to that on PDMS-PDDA/Au (Figure S3). Fluorescent images of BGC823 cells cultivated on PDMS-PDDA film at different growing periods (Figure S4). UV-visible absorption spectra about evaluating P-gp expression on the cell surface (Figure S5). This material is available free of charge via the Internet at http://pubs.acs.org. LA9000158