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Sensors and Actuators B 153 (2011) 398–403

Contents lists available at ScienceDirect

Sensors and Actuators B: Chemical

journa l homepage: www.e lsev ier .com/ locate /snb

etection of EGFR on living human gastric cancer BGC823 cells using surfacelasmon resonance phase sensing

angfang Liua,1, Jingyu Zhangb,1, Yan Denga, Daqian Wanga, Youyong Lub, Xinglong Yua,∗

State Key Laboratory of Precision Measurement and Instruments, Dept. of Precision Instruments, Tsinghua University, Beijing 100084, PR ChinaLaboratory of Molecular Oncology, Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Peking University School of Oncology, Beijing Cancerospital & Institute, Beijing 100142, PR China

r t i c l e i n f o

rticle history:eceived 22 March 2010eceived in revised form 26 October 2010ccepted 8 November 2010vailable online 13 November 2010

eywords:

a b s t r a c t

Label-free and real-time information acquisition of molecular phenotype and its function on living cellsplays a significant role in disease diagnosis and drug development. In this paper, SPR phase sensingwas applied to monitor the interactions between EGFR antibody, EGFR1, and membrane proteins EGFRon living human gastric cancer BGC823 cells. When 50 �g/mL EGFR1 was added onto the fixed cellschip and the living cells chip, a significant difference in the binding amount could be observed fromthe immunofluorescence images. Quantitative results were obtained by following SPR detection, whichwere 722 RU and 438 RU, respectively. On the same living cells chip, SPR detection also showed markedly

urface plasmon resonance (SPR)mmunofluorescence imageiving cellsGC823pidermal growth factor receptor (EGFR)

different results of cellular responses when it was stimulated by EGFR1 at different concentrations, suchas adhesion and/or morphology variation, revealing the EGFR1’s cytotoxic effect on the BGC823 cells. Theresults demonstrate SPR phase sensing is capable of real-time detection of molecular interactions andcellular responses on living cells, and suggest that further studies on the mechanism and the techniquemay allow SPR sensing become a powerful tool not only for the basic research of cell biology, but also for

ug de

medical diagnosis and dr

. Introduction

Researching molecular reactions on living cells is a challengeut a precondition for further cellomics research, as a living cell

s fundamentally different from an in vitro solution which is araditional and widely used biochemical study vector [1]. Live-ell sensing has become a hot research topic, and there are manylectrical and optical methods that provide measurement of cel-ular morphology, cellular metabolism, and cells behavior, such aslectrical impedance [2,3], scanning electrochemistry [4], and fluo-escence resonance energy transfer (FRET) [5]. But live-cell sensings still in urgent need for stable, real-time and label-free tools

hich can provide more information. Sensors based on surfacelasmon resonance (SPR) have been regarded as one of the bestools for label-free and real-time detection, and they are widelysed in molecular interaction analysis. Benefiting from the high

ensitivity to small changes in refractive index occurring on theurface of metal film, SPR becomes one of the most promising can-idates among optical detection methods for living cell sensing [6].ecently, combining SPR technique with living cells sensing has

∗ Corresponding author. Tel.: +86 10 62790982; fax: +86 10 62784691.E-mail address: jyxyxl@mail.tsinghua.edu.cn (X. Yu).

1 The first and second author contributed equally to this work.

925-4005/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.snb.2010.11.005

velopment.© 2010 Elsevier B.V. All rights reserved.

become a new research trend. It was reported that SPR could detectcell–ligand interactions [7], adhesion of immobilized cells [8], anddownstream cellular responses to extra stimulation, such as toxicelements [9], odorant molecule [10], and cell-reactive antigen [11].

Cells sense and respond to their extracellular environmentlargely depending on the function of receptors, transporters, andchannels imbedded in cell membrane. Abnormal expression of cer-tain membrane proteins may be associated with the production,growth, and metastasis of tumor cell [12]. Therefore, study onthe function of these membrane proteins on cancer cells plays asignificant role in cancer markers screening and anti-cancer drugdevelopment. Epidermal growth factor receptor (EGFR) is one offour member of the ErbB family of tyrosine kinase growth factorreceptors, and is very important in cell growth and differentiation[13]. EGFR is highly overexpressed in numerous types of humancancers, including stomach, lung, and head-neck cancers, and actsas a strong prognostic factor [14,15], making it an attractive tar-get for developments of anti-cancer therapeutic agents [13]. Manykinds of monoclonal antibodies to EGFR can interact with the extra-cellular domain of EGFR preventing its activation, and are widely

researched as anti-cancer agents owing to their property of induc-ing tumor cell death [16].

In this paper, SPR sensing based on phase modulation inter-ference was used to detect the interaction between monoclonalantibody EGFR1 and membrane protein EGFR on human gastric

F. Liu et al. / Sensors and Actuators B 153 (2011) 398–403 399

ccts

2

bantsre

aimiFti

ϕ

n

wna

Sib

lictS

affl

has been previously described in detail [19].In the experimental setup, the collimated light passed through

an electro-optical crystal (CASTECH Inc., China) after reflectedfrom a ZF5 prism (Rayleigh Instrument Co., Ltd., China). Theelectro-optical crystal was used as a phase modulation element

Fig. 1. Phase difference between p- and s-polarized components.

ancer BGC823 cells. Different cellular responses of living BGC823ells to the stimulation of EGFR1’s binding at different concentra-ion were also obtained. Experiment results showed that SPR phaseensing can satisfy the detection requirement of living cells.

. Principle

SPR is interpreted as an optical phenomenon that can be excitedy p polarization component of the light. When SPR is excited,n evanescent field with a penetration depth of a few hundredanometers will propagate into the sensing layer [17]. The refrac-ive index changes in this layer will cause both intensity and phasehift of p-component of the reflected light while s-componentemains approximately constant. Thus the s-component can bexploited as the reference signal.

As shown in Fig. 1, the phase difference ϕ between s-componentnd p-component, which originates from the change of refractivendex in sensing layer, can be acquired through phase detection

ethods, either in spatial phase modulation interference [18], orn time domain phase modulation interference [19]. According toresnel formula and equivalent refractive index model of SPR [20],he relationship between the phase difference ϕ and the refractivendex of sensing layer n satisfies Eqs. (1) and (2):

= −2arctg(n4

p sin2 � − n2pn2

ef)1/2

n2ef

cos �(1)

ef =(

n2ε′m

n2 + ε′m

)1/2

(2)

here np is the refractive index of the prism, � is the incident angle,ef is the equivalent refractive index of gold film and sensing layer,nd ε′

m is the real part of complex refractive index of the gold film.Eqs. (1) and (2) indicate that the phase difference signal of

PR represents the refractive index change of the sensing layern real-time, which is most useful for the characterization of theiomolecular interactions within this layer.

The principle of membrane protein interactions detection oniving cells based on SPR sensing is shown in Fig. 2. Living cells aremmobilized on SPR chip surface coated with poly-l-lysine, and thehip is placed on a coupling prism in the Kretschmann configura-ion. Collimated light with wavelength of 650 nm is used to excite

PR.

In this paper, human gastric cancer cell line BGC823 was useds model system. When the cell comes into contact with the sur-ace of SPR chip, the membrane proteins will adhere to the surfaceattening the cell. This deformation allows for an increase in the

Fig. 2. Scheme of living cells detection based on SPR sensing.

number of membrane proteins that are in close proximity of thesensing layer [21]. Once these proteins interact with correspond-ing ligands in the sample solution that induce the changes in cellmorphology, refractive index of the sensing layer changes, leadingto a phase difference variation �ϕ, which can be obtained by SPRsensing.

3. Experiments

3.1. Materials and reagents

1 × 105 gastric cancer BGC823 cells were seeded to each chip(16 mm × 16 mm). EGFR1, one monoclonal antibody to EGFR, waspurchased from Abcam Ltd. (Hong Kong, China). 20 mM HEPES(Solarbio, China) and 10% FBS (Thermo Scientific, USA) were 1:1mixed and selected as the buffer to keep pH value in SPR detec-tion process. 4% paraformaldehyde was used to fix the cells. Goatanti-mouse IgG/FITC (Santa Cruz, USA) was used as the second anti-body in the immunofluorescence experiment, and Hoechst33342and DAPI (Sigma–Aldrich Co., USA) were used as DNA staining.

3.2. Experimental setup

A novel SPR system based on time domain phase modulationinterference was utilized. The configuration is shown in Fig. 3, and

Fig. 3. Configuration scheme of experimental setup: (1) He–Ne laser, (2) pinhole(10 �m), (3) collimator, (4) 1/4 wave plate, (5) rectangular diaphragm, (6) triangleprism, (7) micro-array sensing chip, (8) flow cell, (9) beam expander, (10) electro-optical crystal, (11) polarizing prism, (12) CCD camera, (13) computer and (14)electro-optical crystal driver.

400 F. Liu et al. / Sensors and Actuators B 153 (2011) 398–403

FN

tprcippbpltTwbwTwttR(

Gv

3

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Fig. 5. The nonspecific binding signal detected when the blocked chip (1) and un-

ig. 4. SPR phase signal calibration curve to refractive index units using 0.01–3.0%aCl solutions.

o introduce additional phase shifts between s-component and-component. Five-step phase shift modulation and Stoilov algo-ithm were used to resolve the phase difference information, whichould characterize the variation of refractive index caused by bio-nteractions. Our CCD camera has a maximum acquisition rate of 5ictures per second. Before detections, the chip was placed on therism and the corresponding refractive index matching fluid shoulde filled between them. A temperature control device was used torovide stable detection temperature and 37 ◦C environment for

iving cells survival. To determine the sensitivity of this SPR sys-em, standardized experiments of NaCl solution were carried out.he SPR phase difference between pure water and NaCl solutionith concentration of 0.01–3% were detected. The relationships

etween refractive index and the concentration of NaCl solutionere obtained from the Handbook of Chemistry and Physics [22].

hrough the conversion, the experiment data and fitting curveere shown in Fig. 4, which represents the relationship between

he variation of refractive index and SPR phase signal. Therefore,he sensitivity of this SPR system can be calculated as 0.018◦/RU.U represents response unite, and is defined as 1 RU = 1 × 10−6 RIUrefractive index units).

Laser confocal scanning microscope (LCSM) (SP5, Leica,ermany) was used in immunofluorescence experiments to pro-ide visual information for comparison with SPR signal.

.3. Preparation of cells chip

Glass ZF5 was chosen for the chip’s substrate. After beingleaned by piranha solution, two layers including 2 nm-thickhromium and 45 nm-thick gold were subsequently evaporatednto one surface of the substrate. Then, to promote cellular adhe-ion, poly-l-lysine was used to coat the gold surface, by immersinghe chip into 0.02 mg/mL poly-l-lysine overnight. BGC823 cellsere seeded on the gold/poly-l-lysine surface, and maintained inulbecco’s Modified Eagle Medium (DMEM) supplemented with% FBS at 37 ◦C in a 5% CO2 incubator for overnight.

During SPR chip preparation, a blocking measurement shoulde taken to reduce nonspecific binding. In preparation of proteinhip, BSA is usually used as the blocking protein, but in cells chip’sreparation BSA is not needed. There are many proteins and car-

ohydrates in cell culture solution—DMEM/FBS. They can bind tohe surface of chip in cells culture process acting as blocking pro-eins. Fig. 5 represents the non-specific binding signal when ann-blocked chip and a blocked chip were exposed to 50 �g/mLGFR1 solution for 75 min which was diluted by HEPES/FBS buffer.

blocked chip (2) were exposed to 50 �g/mL EGFR1 solution for 75 min.

The un-blocked chip was the chip coated with poly-l-lysine, whilethe blocked chip was obtained by immersing the un-blocked chipinto DMEM/FBS solution overnight. Curve 1 in Fig. 5 reaches 657 RUat the end of the detection while curve 2 reaches 130 RU. Accordingto this figure, it is indicated that after the process of chip prepara-tion, nonspecific binding signal will be reduced remarkably in thedetection process.

Besides living cells chip, fixed cells chip was also prepared asa control object, by immersing living BGC823 cells chip into 4%paraformaldehyde at room temperature for 10 min.

3.4. Immunofluorescence detection

Immunofluorescence experiments were used to provide visualinformation for comparison with SPR signal. Immunofluorescenceexperiment of living cells chip and fixed cells chip were followedaccordingly: after the preparation, the cells chip was incubatedwith 50 �g/mL EGFR1 for 3 h at 37 ◦C. 40 �g/mL goat anti-mouseIgG conjugated with FITC was used as the secondary antibody, andthen added and kept for 1 h at 37 ◦C. Finally, a 5 min room tem-perature incubation with 10 �g/mL Hoechst33342 was performedfor staining of the cell nucleus. In fixed cells immunofluores-cence experiment, DAPI was used as nucleus staining instead ofHoechst33342. After each step, the chip was washed by PBS forthree times. Images were captured with LCSM and 63-fold oilimmersion lens to observe the location and expression level of EGFRon BGC823 cells, which is shown in Fig. 6.

3.5. SPR detection of living cells chip and fixed cells chip

For the purpose of detecting different response of living cells andfixed cells exposed to EGFR1, a living cells chip and a fixed cells chipprepared under the same condition were chosen for SPR detection.EGFR1 solution was diluted into 50 �g/mL using HEPES/FBS buffer.Before it was added onto the fixed cells chip or the living cells chip,HEPES/FBS buffer was maintained in flow cell for baseline drawing.

◦

Then monitoring was carried out for 25 min at 37 C. The interactioninformation of two different chips were obtained and shown inFig. 7.

F. Liu et al. / Sensors and Actuators B 153 (2011) 398–403 401

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3c

dt

Fl

4.1. Immunofluorescence detection of EGFR’s binding to EGFR

The confocal immunofluorescence images in Fig. 6 indicate thatEGFR1 specifically interacts with EGFR both on living cells and fixed

ig. 6. Confocal image of immunofluorescence experiment (a) living cells chipgG/FITC–EGFR1–EGFR. (For interpretation of the references to color in this figure le

.6. Long-time SPR detection of EGFR1’s stimulation on livingells chip

This experiment used a flow cell with two channels. After base-ine drawing, 100 �g/mL EGFR1 was added to the detecting channelf the living BGC823 cells chip. EGFR1 solution was replaced by theEPES/FBS buffer after 50 min. In the reference channel, HEPES/FBSuffer without EGFR1 was maintained throughout data collection.n extended period of time (75 min) SPR monitoring was made toetect the cellular response and to study the effect of EGFR1. SPRignals in central area of detecting channel and reference channelre shown in Fig. 8. Before and after the monitoring, micrographsf cell morphology changes in two channels were obtained usingiological microscopy (Tokyo Union, Japan) equipped with a CCDamera (Olympus, Japan) and 20-fold objective, which are alsohown in Fig. 8.

.7. SPR detection of EGFR1 at different concentrations on livingells chip

For further study on EGFR1’s effect to living BGC823 cells, SPRetection on the same chip using EGFR1 solutions at the concen-rations of 0 �g/mL (HEPES/FBS buffer), 20 �g/mL, 50 �g/mL, and

ig. 7. SPR kinetic response of interactions between 50 �g/mL EGFR1 and EGFR oniving and fixed BGC823 cells.

fixed cells chip. Blue: cells nucleus; green: binding chains of goat anti-mouse, the reader is referred to the web version of the article.)

100 �g/mL were carried out respectively. Four different real-timecurves were obtained and shown in Fig. 9.

4. Results and discussions

Fig. 8. (a) Living BGC823 cells response to 100 �g/mL EGFR1’s stimulation (detec-tion curve) and HEPES/FBS buffer (reference curve). Micrographs of cell morphologyobtained (b) before and (c) after 100 �g/mL EGFR1’s stimulation in detection chan-nel, (d) before and (e) after exposing to HEPES/FBS buffer in reference channel. Scalebars represent 50 �m.

402 F. Liu et al. / Sensors and Actuato

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ig. 9. SPR detection of living BGC823 cells response to EGFR1’s stimulation withoncentrations of (1) 0 �g/mL (HEPES/FBS buffer), (2) 20 �g/mL, (3) 50 �g/mL, (4)00 �g/mL.

ells, which is located on BGC823 cell’s membrane. Under the sameonditions, the living cells chip bound less amount of EGFR1 thanhe fixed cells chip. The possible reason might be that the mono-ntibody of EGFR promotes EGFR internalization in living cells,hich ultimately decreases the number of EGFR on the living cell

urface [23]. To a certain extent, this result reveals the differenceetween detection in vivo and in vitro.

.2. Different response between living cells chip and fixed cellship

The results of EGFR1 interacting with living BGC823 cells andxed BGC823 cells are shown in Fig. 7. When EGFR1 solution was

njected, SPR signals rise for both living cells chip and fixed cellship. But after 5 min, the signal of living cells chip achieves 438 RUhen starts to decline gradually; while the signal of fixed cells chipncreases continuously to 722 RU and kept approximately constant.y comparing two curves in Fig. 7, it is clear that the rising signalsepresent the interaction processes between antibody EGFR1 andGFR on BGC823 cells. The declining signal may associated withhe downstream response of living cells, which was caused by thetimulation of EGFR1’s binding. In this experiment, the drop of SPRignal caused by the downstream cellular response was 243 RU.hough the fixed cells were already dead, the membrane proteinsGFR still showed the binding capability with EGFR1. However,inding to EGFR1 would not induce the response of fixed cells.

.3. Detection of EGFR1 function on living BGC823 cells

Real-time monitoring was carried out on the stimulation ofGFR1 to living BGC823 cells. In Fig. 8(a), curve 1 represents thetate change of living cells in reference channel without the stim-lation of EGFR1; and SPR response value is about −75 RU after5 min of monitoring. This behavior can be explained by the differ-nce between detection environment and cell culture environment,hich may lead to a little change of the cells state. In curve 2,

onstant growth can be obtained after the injection of EGFR1, and400 RU is acquired when t = 800 s. Then the signal begins to declineradually. According to Fig. 8(b) and (c), we observe that the shapesf most cells changed from fusiform to round after the 100 �g/mL

GFR1’s stimulation. In Fig. 8(d) and (e), there were little change inells states before and after detection. Comparing Fig. 8(c) with (e),t is indicated that EGFR1 has the function of affecting cells adhesionnd/or morphology.

rs B 153 (2011) 398–403

Based on the experiments above, it is confirmed that EGFR1 canbind to EGFR on living BGC823 cells, and cause changes in cellsmorphology. In further study, it is indicated that different concen-trations of EGFR1 were associated with different cells responses,as shown in Fig. 9. When the concentration of sample solution was0 �g/mL (HEPES/FBS buffer), there is little change in SPR signal ofcurve 1. In curve2, after the injection of EGFR1, SPR signal increasesto 107 RU and finally declines to 37 RU. The result suggests that20 �g/mL EGFR1 led to very small influence on living cells. In curve3 and curve 4, SPR signal of 50 �g/mL EGFR1 rose to 700 RU at firstthen declined to 424 RU, while 100 �g/mL EGFR1 caused increaseof SPR signal to 1335 RU in about 7 min and then down to 682 RU.According to Fig. 9, it is shown that the higher of the concentrationof EGFR1, the greater the fraction of rounded cells and the largerthe decrease in observed SPR signal.

In summary, the stimulation of EGFR1 on BGC823 cells decreasescell adhesion and increases cell rounding in a concentration depen-dant process. The decrease in number of cells adhered and theincrease in cell rounding lead to the decrease in effective refractiveindex in the SPR sensing layer [24], and then affect the SPR signal.Other contributions to SPR signal could originate from cytoskeletalrearrangements which cause the density or mass variation withinthe evanescent filed [25,26].

5. Conclusions

This paper introduced the SPR phase sensing into moleculardetection on living cells, and characterized the interaction betweenEGFR1 and membrane protein EGFR on human gastric cancer cellline BGC823. Immunofluorescence detection demonstrated thatEGFR1 binds to the membrane protein EGFR on both living cellsand fixed cells, while SPR detection could give quantitative result ontheir binding process and showed a significant difference. In livingcells chip, cellular responses were occurred owing to EGFR1’s stim-ulation, which was verified by optical microscopy. Further analysisindicated that EGFR1 might lead to the cytoskeletal rearrangement,reduce cells adhesion force, and influence living BGC823 cells’ sur-vival properties. The experiment results suggest that SPR phasesensing can match the requirements of molecular interaction andfunction detection on living cells. Further studies may allow SPRsensing to become a powerful tool not only for the basic research ofcell biology, but also for medical diagnosis and drug development.

Acknowledgements

This work was funded by NSFC Grant no. 30970757. The authorswould like to thank Wei Zuo and Zhongwei Li in Prof. Ye-GuangChen’s group (from Dept. of Biological Sciences and Biotechnology,Tsinghua University) for cells culture.

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Biographies

Fangfang Liu studied instrumentation at Tsinghua University in Beijing, China,where she received the bachelor’s degree in July 2005. Currently, she is workingon her PhD in the group of Prof. Yu at the Department of Precision Instrumentsand Mechanology, Tsinghua University. Her research is focused on surface plasmonresonance biosensor detection.

Jingyu Zhang got his PhD in Chinese Academy of Sciences in 2005 and now he isan assistant professor of Molecular Oncology, Peking University School of Oncology,Beijing Cancer Hospital & Institute. Till date, five papers were published as firstauthor in peer-reviewed journals. His current projects mainly involved in screeningmolecular markers for tumor early diagnosis and potential targets for anti-tumordrug.

Yan Deng is an associate professor in Dept. of Precision Instruments and Mechanol-ogy, Tsinghua University. She is carrying out the research and teaching workon structural dynamic test technology, virtual instrumentation, life science mea-surement technology and instruments. Her research interests are biomolecularinteraction detection and surface plasmon resonance sensing technique.

Daqian Wang studied at Beijing Institute of Technology, China, where he receivedthe bachelor’s degree in July 2007. Currently, he is working on his PhD in the groupof Prof. Yu at the Department of Precision Instruments and Mechanology, TsinghuaUniversity. His research is focused on surface plasmon resonance detection.

Youyong Lu was appointed as professor and director of laboratory of molecularoncology in Peking University School of Oncology, Beijing Cancer Hospital/Institutein 1994. His research interests are including the genetic alterations of multiple-stepcarcinogenesis in the gastric cancer; characterization of gene and protein expressionprofiling in the neoplastic disease progression; cellular and molecular mecha-nism on the anti-carcinogenesis of garlic. He has published 150 original research

articles.

Xinglong Yu is a professor of precision instruments, in Tsinghua University. He pub-lished more than 70 scientific papers in referred journals and in the proceedings ofinternational conferences. His activities are focused on the biomolecular interactiondetection and surface plasmon resonance sensing technique.