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Biomaterials 40 (2015) 80e87

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Biomaterials

journal homepage: www.elsevier .com/locate/biomateria ls

Fabrication of new single cell chip to monitor intracellular andextracellular redox state based on spectroelectrochemical method

Waleed Ahmed El-Said a, c, Tae-Hyung Kim b, Yong-Ho Chung b, Jeong-Woo Choi a, b, *

a Interdisciplinary Program of Integrated Biotechnology, Sogang University, 35 Baekbeom-ro, Mapo-Gu, Seoul 121-742, Republic of Koreab Department of Chemical & Biomolecular Engineering, Sogang University, 35 Baekbeom-ro, Mapo-gu, Seoul 121-742, Republic of Koreac Chemistry Department, Faculty of Science, Assiut University, Assiut 71516, Egypt

a r t i c l e i n f o

Article history:Received 24 August 2014Accepted 8 November 2014Available online 26 November 2014

Keywords:Intracellular and extracellular redox stateSpectroelectrochemicalSingle neural cell-chipGold microelectrodesGold nanodots array

* Corresponding author. Department of ChemicalSogang University, Seoul, Republic of Korea. Tel.: þ820331.

E-mail address: jwchoi@sogang.ac.kr (J.-W. Choi).

http://dx.doi.org/10.1016/j.biomaterials.2014.11.0230142-9612/© 2014 Elsevier Ltd. All rights reserved.

a b s t r a c t

Probing the local environment of target cells has been considered a challenging task due to thecomplexity of living cells. Here, we developed new single cell-based chip to investigate the intracellularand extracellular redox state of PC12 cells using spectroelectrochemical tool that combined surface-enhanced Raman scattering (SERS) and linear sweep voltammetry (LSV) techniques. PC12 cells immo-bilized on gold nanodots/ITO surface were subjected to LSV and their intracellular biochemical changeswere successfully monitored by SERS simultaneously. Moreover, paired gold microelectrodes withmicrometer-sized gap containing hexagonal array of gold nanodots were fabricated to detect electro-chemical activity and changes in the redox environment of single PC12 cell based on SERSeLSV tool. Thisshowed very effective detecting method. The used technology included the utilization of gold nanodotsarray inside micro-gap to enhance the Raman signals and the electrochemical activity of single cell. Thiscould be used as an effective research tool to analyze cellular processes.

© 2014 Elsevier Ltd. All rights reserved.

1. Introduction

A wide range of in-vitro studies have been carried out toexamine the effects of diverse materials (e.g. metal/inorganicnanoparticles, peptides, anti-cancer drugs or environmental toxins)on the target cells. The most remarkable advantage of the in-vitromethods is the ability to analyze wider range of cellular processes,which is not possible for in-vivo or animal-based test. However, it isdifficult to maintain the biological characteristics of many cellsduring analysis compared to analysis of their components. This isdue to the structural/chemical complexity of cells as well as thedifficulties in handling cells [1]. X-ray absorption fine structure(XAFS) spectra [2,3] have been carried out to study the biologicalcharacteristics of cells by analyzing the intermediate structure ofcellular components. Nevertheless, because of the lengthy timerequired for the collection of XAFS data, this method cannot beeasily applied for living cell analysis. Moreover, several studies havereported IR-spectroelectrochemical (IR-SEC) method as a reliable

& Biomolecular Engineering,2 705 8480; fax: þ82 2 3273

method for the analysis of heme proteins; including myoglobin,hemoglobin, cytochrome c3 and the cytochrome bc1 complex[4e6]. Notwithstanding, IR-SEC cannot be utilized in cellularresearch due to the interference with water in aqueous environ-ments that is not adequate for monitoring living cells [7].

Previously, we have developed various nanopatterned modifiedelectrodes to investigate the viability of cancer cells after exposureto different kinds of anti-cancer drugs using cyclic voltammetry(CV) technique [8e10]. In spite of that, effects of different anti-cancer drugs cannot be monitored by electrochemical methods,because the current CV peaks can only indicate the cell viability viaelectron transfer between the cells and electrode surface [11e13].

The superiority of the Raman technique is due to the specificinelastic scattering of photons from chemical bonding in moleculesactivated by the light source. Therefore, the biochemical and/orbiological structure of cells can be effectively studied by analyzingeach peak in the Raman spectra, which is unavailable in other op-tical, biological or electrical methods. Additionally, theweak signalsresulted from Raman scattering can be overcome by using the SERStechnique. Since the signals obtained by the SERS method are109e15 fold higher than normal Raman, biological molecules can bereadily analyzed with a reduced exposure time to the laser source.

We have previously reported on a nanostructured SERS-activesurface and its application for analysis of the cell intracellular

W.A. El-Said et al. / Biomaterials 40 (2015) 80e87 81

state [14]. We have also extended this approach to monitor differ-entiation between live/dead cells, cells in different cell cycle stagesand different kinds of cell lines immobilized on homogeneouslyfabricated nanopatterned gold (Au) surface [15]. It was found thatSERS signal obtained from target cells were excellent for moni-toring intracellular changes in cellular components [16].

In the current study, we have fabricated Au nanodot arraymodified ITO substrate as cell culture system, SERS-active surfaceand a working electrode. Also, a new spectroelectrochemicaltechnique that combined the SERS and voltammetric methods wasdeveloped for analysis of the living cells redox properties.

LSV was used to investigate the biochemical changes in theintracellular components during the redox process of neural cells(PC12), while the NIR laser source was simultaneously focused onthe target cell for SERS analysis. Moreover, this SERSeLSV tech-nique was used for simultaneous investigation of single PC12 cellattached to a micrometer-sized gap between two paired Au mi-croelectrodes. Prior to cell attachment, polystyrene-assisted hex-agonal array of Au nanodots was fabricated on the gap between pairof Au microelectrodes to enhance the Raman signal (Scheme 1).

2. Experimental section

2.1. Materials

Polystyrene (PS), dopamine (DA) and phosphate buffered saline (pH 7.4, 10 mM)were purchased from SigmaeAldrich (St. Louis, MO, USA). All other chemicals usedwere obtained commercially as the reagent grade. All aqueous solutions were pre-pared by using de-ionized water (DIW) that de-ionized with a Millipore Milli-Qwater purifier operating at a resistance of 18MU cm.

2.2. Cell culture

PC12 used was derived from rat neural cells (Adrenal medulla) e purchasedfrom the Korean Cell Line Bank (Seoul, Korea) e and was cultured in PRMI (Invi-trogen, Carlsbad, USA) with 10% heat-inactivated fetal bovine serum (FBS; Gibco,Carlsbad, CA, USA) and 2% antibiotics (streptomycin þ penicillin) (Gibco). The cellswere maintained under standard cell culture conditions (37 �C in a humidified of 5%CO2). The medium was changed every two days and the number of cells wasdetermined with a hemacytometer after trypan blue exclusion.

2.3. Cell lysis

Typically, the cells were allowed to grow for 2 days before devoted to thepreparation of the cell lysate. The growth medium was removed and the cells were

Scheme 1. Schematic diagram of the immobilization of a single

washed with PBS and were scrapped off the well surface using a cell scraper gentlyto prevent foaming. Then, the cell suspension in the well was transferred into acentrifuge tube andwas spin at 5000 rpm for 5min at 4 �C, and any remaining bufferwas removed. 0.25 mL ice cold lysis buffer was added and kept in ice for 20 min. Thiscell lysate was centrifuged at 5000 rpm for 5 min at 4 �C and the supernatant liquidwas separated.

2.4. Electrochemical measurements

All electrochemical experiments were performed using a potentiostat (CHI-660,CH Instruments, USA) controlled by general-purpose electrochemical system soft-ware. A homemade three-electrode system consisted of Au microelectrode as aworking electrode, platinumwire as counter electrode and Ag/AgCl as the referenceelectrode. All electrochemical analyses were carried out to monitor the electricalproperties of living cells and the effect of anti-cancer drugs on their behavior innormal laboratory conditions. PBS (10 mM, pH 7.4) was used as an electrolyte at ascan rate of 20 mV/s.

2.5. Raman spectroscopy

Biochemical composition of control PC12 cells and the changes during the redoxprocesses were investigated by Raman spectroscopy using Raman NTEGRA spectra(NT-MDT, Russia). Themaximum scan-range, XYZ was 100 mm� 100 mm� 6 mmandthe resolution of the spectrometer in the XY plane was 200 nm and along the Z axiswas 500 nm. Raman spectra were recorded using NIR laser emitting light at 785 nmwavelength. Ten scans of 5 s from 500 cm�1 to 1750 cm�1 were recorded and themean of these scans was used.

2.6. Fabrication of PS-assisted nanopatterned surface

Monolayer of PS was prepared as described previously [17,18]. PS particles with adiameter of 100 nm (10 wt % aqueous solution) were mixed with a surfactantmixture (Triton-X and methanol in a volume ratio of 1:400) in a ratio of 1:1 (v/v).The ITO substrates were cut into 10 mm � 10 mm and heated in aqueous solution ofNH4OH, H2O2 and H2O (volume ratio 1:1:5) at 80 �C for 30 min. The freshly preparedsamples were used after dry under N2 just before deposition of the PS particles.Seven mL of the diluted PS solution mentioned above was applied onto the ITO,which spread all over the substrate using the spin coating method over large areas[19] to achieve large monolayer coverage. The spin speed was varied between 100and 1000 rpm, at intervals of 100 rpm. The speed was increased steadily from 0 rpmto 1000 rpm speed and kept constant at a fixed time interval of 30 s before it wasincreased to 1000 rpm. The total spinning time was 1 min. The substrate was thenleft to dry in the spin coater with a covered lid to maintain a consistent dryingambient and evaporation rate.

2.7. Design and characterization of SERSeLSV system for bulk cells studies

The design of the surface-enhanced Raman spectroscopyelinear sweep vol-tammetry (SERSeLSV) cell was based on the fabrication of Au nanodots array on the

cell on the microgap between pairs of Au microelectrodes.

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ITO substrate through PS mask (Fig. 1a). An Au layer of thickness 30 nm was evap-orated on the top of the deposited PS nanospheres mask. After dissolution of the PSmask in chloroform, an array of ordered round Au nanostructures with lateral di-mensions of about 30 nm remained on the ITO substrate (Fig. 1b). A sterile cell-chamber with dimensions of 10 mm � 10 mm was attached to the Au nanodots/ITO substrate using polydimethylsiloxane (PDMS) for measurement of the SERSspectra and LSV behavior of living cells under physiological conditions (37 �C tem-perature and PBS 7.4 pH). This cell culture system was be used as a SERS-activesurface as well as the working electrode.

2.8. Design SERSeLSV system for single cell studies

Scheme 1 shows the fabrication of the cell-based chip on patterned the Aunanodots array inside the microgap. 12 Au microelectrode arrays were patterned ona 10 mm � 10 mm glass substrate, which provided 6 microgaps with width of about7 mm between each pair of Au microelectrodes. The Au nanodots array was thenfabricated inside these microgaps based on PS nanospheres mask. Au nanodotsformed on the Au microelectrodes as well as on the outside the microelectrodesarray. To localize a single cell over the microgap, a PDMS microchannel (200 mm inwidth) was attached. The cells were then transferred onto the chip with new culturemedium through the microchannel inlet. A sterile cell-chamber with dimensions of8 mm � 8 mm was developed to measure the Raman spectra of living single cellsunder physiological conditions.

3. Results

A number of studies have reported that the voltammetricresponse of cells have a strong relationship with some enzymes inthe cytosol [20,21]. While, others have suggested that the cellsvoltammetric behavior may be related to the oxidation of guanine[22e25]. Moreover, in our previous work, we extracted some redoxenzymes from HeLa cells using 2-D electrophoresis techniques,including NADH dehydrogenase (ubiquinine) flavoprotein 2,Quinone oxidoreductase-like (QOH-1), which may be related to thecell voltammetric behavior [13]. However, more research is neededto conclusively clarify the voltammetric behavior of the cells. PC12cells have been reported to secrete the neurotransmitter (DA) [26],which is an electrochemical active species. Here, we selected PC12

Fig. 1. (a) SEM image of the PS mask on the ITO substrate. (b) SEM image of Au nanodots areduction of dopamine. (f) SERS spectra of dopamine control (black), during oxidation (blufigure legend, the reader is referred to the web version of this article.)

cells as an experimental nerve cell model to investigate the sourceof the cell electrochemical activity. Also, a new single cell-chip hasbeen developed to investigate the Redox states of single cell. Thiscell-chip consisted of PDMS micro-channel over six pairs of Aumicroelectrodes; moreover, Au nanodots array was fabricated in-side the micro-gap between each pair of Au microelectrodes.

3.1. Spectroelectrochemistry of dopamine neurotransmitter

Au nanodots modified ITO substrates were fabricated based onthermal evaporation of pure Au metal with 30 nm thicknessthrough polystyrene (PS) monolayer (Fig. 1a), which acted as softtemplate. Fig. 1b showed the fabrication of ordered round Aunanostructures on the ITO substrate after removal of the PS mask.This substrate was used as a cell culture substrate for utilizing as aSERS-active surface and for using as the working electrode. The CVbehavior of 50 mM of DA in PBS (pH 7.4) buffer solution within apotential range from þ0.8 to �0.2 V was shown in Fig. 1c,demonstrating a reversible redox process with a cathodic peakat þ0.145 V and an anodic peak at þ0.195 V. Fig. 1fI showed theRaman spectra of DA, which contained a series of peaks includingpeaks at 1270 cm�1 (CeO str.), 1358 cm�1 (str. catechol), 1443 cm�1

(CeN), 1150 and 1599 cm�1 (CeC of benzenoid ring) [27].To identify the species that were produced during the electro-

chemical redox processes of DA, LSV, oxidation and/or reductionassays were applied and the Raman spectrum was recordedsimultaneously during each step. Application of the oxidation po-tential to the DA solution within potential range from �0.2to þ0.8 V resulted in anodic peak at þ0.195 V (Fig. 1d), and a newpeak near 1565 cm�1 was observed in the corresponding Ramanspectra (Fig. 1fII). In contrast, a cathodic peak at þ0.145 V wasobserved when reduction potential was applied to the DA solutionin the potential range fromþ0.8 to�0.2 V (Fig.1e), and Raman peak

rray on the ITO substrate. (c) CV of dopamine. (d) LSV oxidation of dopamine. (e) LSVe) and during reduction (orange). (For interpretation of the references to color in this

W.A. El-Said et al. / Biomaterials 40 (2015) 80e87 83

that appeared after the oxidation process (1565 cm�1) became ashoulder peak (Fig. 1fIII).

3.2. Spectroelectrochemical study of the extracellular biochemicalchanges of bulk PC12 cells during the redox processes

Fig. 2a showed the CV behavior of PC12 cells in the potentialrange from þ0.8 to �0.2 V that demonstrated a quasi-reversibleprocess with a cathodic peak at þ0.025 V and an anodic peakat þ0.49 V. To understand the source of electrochemical activity ofthe cells and the biochemical changes that occurred during theredox reactions, SERS spectrum for control PC12 cells werecompared with the SERS spectra during the oxidation and/orreduction processes; all SERS results were summarized in Table S1.

The Raman spectrum of the control PC12 (Fig. 2dI) showedpeaks of 775 cm�1 (Trp, U, C and T), 1001 cm�1 (Phe), 1092 cm�1

(PO2�), 1205 cm�1 (Phe and Trp), 1230 cm�1 (amide III and T),

857 cm�1 (Tyr), 1490 cm�1 (G and A), 1620 cm�1 (C]C Trp and Tyrstr.), 1270 cm�1 (amide III and catecholamines, e.g. DA) and1557 cm�1 (C]C str. of phenyl group), which is in agreement withpreviously reported spectra of cells [28e30].

Fig. 2b depicted the LSV oxidation of PC12 cells in the potentialrange from �0.2 to þ0.8 V, which contained an anodic peakat þ0.4 V. In addition, the number of peaks in Raman spectrum ofPC12 cell changed during the oxidation process. A decrease in theRaman peak intensities was observed at 1001, 775 cm�1 (aminoacids), and 1270 cm�1 (DA and amide III). Moreover, the Ramanpeaks at 857 and 1557 cm�1 disappeared. Also, newRaman peaks at865, 1405 (A and G), 1544, 1570, and 1602 cm�1 appeared, which

Fig. 2. (a) CV of PC12 cells. (b) LSV oxidation of PC-12 cells. (c) LSV reduction of PC12 cellreduction (blue). (e) Comparison of the Raman spectra from control PC12 cells and during ofigure legend, the reader is referred to the web version of this article.)

were assigned to the C]C and/or C]O stretch of the o-quinonegroup (A and G) as shown in Fig. 2dII.

Furthermore, when PC12 cells were subjected to a reductionpotential (from þ0.8 to �0.2 V), a cathodic peak at 0.0 V wasobserved (Fig. 2c). In comparison with the Raman of PC12 cellssubjected to oxidation, the Raman spectra of PC12 after reductionprocess (Fig. 2dIII) increased again (peaks at 1001 cm�1) but theintensity was not as high as that observed in the control cells. At thesame time, intensities of the Raman peaks at 1575 cm�1 (Phe) and1270 cm�1 (DA and Amide III) decreased and a new peak at1226 cm�1 (Tyr and Phe) was observed. Furthermore, the peaks at1720 cm�1 (C]O ester) and 1544 cm�1 disappeared.

For better understanding of the chemical reactions mechanismresponsible for the electrochemical activity of cells, real timeRaman spectra changes during oxidation and reduction processesof the living PC12 cells were investigated. For real time monitoringSERSeLSV approach was used. The LSV assay was performed at alow scan rate (0.02 V/s) within a potential range from �0.2to þ0.8 V that corresponded to a total LSV scan time of 50 s. Wethen divided the scan range into three regions (corresponding to15 s) and in each region, we investigated the biochemical changesusing the SERSeLSV assay. Fig. 3a showed the LSV oxidationbehavior of PC12 cell at a scan rate 0.02 V/s, which contained anoxidation peak at about þ0.39 V. Simultaneously, Raman spectrareadings during each LSV oxidation scan step were �0.2 Vto þ0.1 V, þ0.16 V to þ0.46 V and þ0.5 V to þ0.8 V (Fig. 3a).

There was almost no change in the Raman spectrum during thefirst step when compared to the control PC12 cells. During thesecond step, a significant change was observed in the Raman

s. (d) SERS spectra of control PC12 cells (black), during oxidation (orange) and duringxidation and reduction processes. (For interpretation of the references to color in this

Fig. 3. Real time monitoring of the effect of the redox potential on the biochemical composition of living PC12 cell: (a) LSV oxidation for PC12 cells. (b) LSV reduction for PC12 cells.Scan rate at 0.02 V/s. (c) Real time Raman spectra of PC12 cells during oxidation: step A (black), step B (red) and step C (blue). (d) Real time Raman spectra of PC12 cells duringreduction: step A (black), step B (red) and step C (blue). Exposure time 15 s for each step. (For interpretation of the references to color in this figure legend, the reader is referred tothe web version of this article.)

W.A. El-Said et al. / Biomaterials 40 (2015) 80e8784

spectrum due to the oxidation reactions within cell. Finally, Fig. 3cdemonstrated slight changes in the Raman spectrum during thelast step including peaks 875, 1001, 1420, 1475, 1550, 1570 and1610 cm�1.

Moreover, the reduction behavior of PC12 cell within the rangefrom þ0.8 V to �0.2 V at a scan rate 0.02 V/sec (Fig. 3b) displayed areduction peak at about þ0.04 V. Therefore, we selected two re-gions; the first was from þ0.8 V to þ0.5 V while the second werefrom þ0.2 V to �0.1 V. The third step started at �0.14 V andcompleted 12 s after the LSV scan finished. Almost no changes wereobserved in the Raman spectra during the first step whencompared with the PC12 cell in the oxidation state. Significantchanges in the Raman spectra were observed during the secondstep, especially for the Raman peaks at 1001, 1240, 1450 cm�1 andwithin the range from 1500 to 1620 cm�1. Finally, very few changeswere observed in the Raman spectra during the last step (Fig. 3d).

3.3. Spectroelectrochemical study of the intracellular biochemicalchanges of PC12 cells during the redox processes

The SERSeLSV technique was also applied to investigate thebiochemical changes in the intracellular compositions during theredox processes of neural PC12 cell lysates. Fig. S3a showed the CVbehavior of the PC12 cell lysate, which demonstrated anodic andcathodic current peaks at 0.328 and 0.0 V, respectively. Thesevalues were different when compared with those of living PC12cells (extracellular). Compared with the Raman spectra of livingPC12 cells, Raman spectra of the lysates of PC12 cells showed aweak Raman peaks intensity except for the peak corresponding to

the Phe group (1001 cm�1). This reflected high concentrations ofprotein (Fig. S3dI). However, the Raman spectra of PC12 cell lysatessignificantly changed during the oxidation and reduction processes(Fig. S3b & c). Practical changes were presented in Table S2including the following changes (Fig. S3dII & III), 671 cm�1 (T andG), 760 cm�1 (Trp), 854 cm�1 (Trp), 923 cm�1 (pro CC str.),975 cm�1 (CeC str. b-sheet¼ CH bend),1001 cm�1 (Phe),1125 cm�1

(prot. str. CN), 1134 cm�1 (Prot), 1207 cm�1 (Trp, Phe and Tyr),1450 cm�1 (Prot), and 1625 cm�1 (C]C Tyr and Trp).

3.4. Spectroelectrochemical study of single neural cell

SERSeLSV technique was developed to study the biochemicalcomposition of living single PC12 cells during the redox processes.In order to evaluate the spectroelectrochemical assay at the singlePC12 cell level (Scheme 1), 12 Au microelectrode arrays werepatterned on a 10 mm � 10 mm glass substrate that provided sixmicrogaps (about seven mm inwidth) between eachmicroelectrodepair. Inside these microgaps, Au nanodots arrays were produced.Single cells were then immobilized over the microgaps (Fig. 4).

Fig. 5a showed the CV of the Au microelectrodes in PBS buffersolution and no redox peaks were observed. In contrast, the CV ofsingle PC12 cell immobilized on microgap between pairs of Aumicroelectrodes displayed an anodic peak at about 0.4 V (Fig. 5c).

Fig. 5d showed the LSV oxidation behavior of single PC12 cellwithin potential range from�0.2 toþ0.8 V, which contained anodicpeak at about þ0.4 V. Moreover, many peaks in the Raman spec-trumwere changed relative to the control PC12 single cell (Fig. 5f).The Raman spectra changes were represented in Table S3, which

Fig. 4. Fabrication of the nanodot array inside the microgap as cell-based chip for single cell studies (a) SEM image of the microgap between pair of Au microelectrodes. (b & c) SEMimages of Au nanodots array inside the microgap between pair of Au microelectrodes. (d) AFM image of PC12 cell on the microgap between pair Au microelectrodes.

W.A. El-Said et al. / Biomaterials 40 (2015) 80e87 85

represented decrease in Raman peaks intensities at 720 (A), 760(Trp), 905 (prot. ring str.), 1001 (Phe), 1060 (str. PO2

� and str. COC),1093 (lipids: str. CeC, deoxyribose: CeO, CeC str. and str. PO2

�), 1125(prot. str. CN), 1175 (prot. Tyr str. CN and CC), 1270 (T, A and amide

Fig. 5. (a) CV background of microelectrode. (b) CV of single PC12 cell on the microgap betweAumicroelectrodes contain the Au nanodots array. (d) LSV oxidation of a single PC12 cell on tthe microgap between two Au microelectrodes. (f) SERS spectra of PC12 cells control (black)color in this figure legend, the reader is referred to the web version of this article.)

III), 1310 (A), 1360 (prot. CH2), 1390 (T, A and G), and 1690 cm�1

(amide I and C]C). Additionally, the Raman peaks at 1625 cm�1

(C]C, Tyr and Trp), which was observed in control PC12 cells dis-appeared. Also, peaks at 854 cm�1 (prot. Ring br. Tyr), 940

en pair of Au microelectrodes. (c) CV of a single PC12 cell on the microgap between pairhe microgap between pair Aumicroelectrodes. (e) LSV reduction of a single PC12 cell onduring oxidation (orange) and reduction (blue). (For interpretation of the references to

W.A. El-Said et al. / Biomaterials 40 (2015) 80e8786

(proteins: a-helix, and deoxy and CeOeC), and 1560 cm�1 (pro-teins: amide II and Trp; C]C and/or C]O stretch of o-quinonegroup) shifted to 866, 952 and 1545 cm�1, respectively. New Ramanpeaks were observed during application of the oxidation voltage,including Ramanpeaks at 1330 (G), s1450 (deoxyribose),1426,1502and1595 cm�1 (A and G).

In addition, no cathodic peak was observed when PC12 cellswere subjected to reduction potential within potential rangefromþ0.8 to�0.2 V (Fig. 5e). However, the Raman spectrum duringthe reduction process was different than the Raman spectrum ofthe control PC12 or PC12 cells in the oxidation state (Fig. 5f). Thesechanges in Raman spectra included an increase in the Raman peaksintensities at 905 (prot. ring str. CC), 1001 (Phe), 1060 (PO2

� str. andCeO, CeC str.), 1093 (lipids, chain CeC str. deoxyribose: CeO, CeCstr. phosphate: str. PO2

�), 1125 (CeN str.), 1175 (CeH bend Tyr), 1270(T, A and amide III), 1310 (A), 1360 (A, G, and prot. CH2 def) and1390 cm�1 (T, A and G). Also, the Raman peak at 1560 cm�1 (C]Cand/or C]O stretch of o-quinone group) shifted to 1575 cm�1.Furthermore, new Raman peaks at 1230 (T, A, and amide III), 1426(G, A and CH def), 1502 cm�1 (G and A) were observed. Moreover,the Raman peaks at 1330 (G), 1595 (A and G), 1625 (C]C Tyr andTrp) and 1720 cm�1 (amide I and C]O ester) disappeared, whichcould be related to a reduction of these functional groups.

4. Discussion

Application of the SERSeLSV technique for analysis of compli-cated system (living cell) was preceded by validation of its ability tomonitor the reaction mechanism of simple oxidation reactionmechanism of potassium ferrocyanide (Figs. S1 & S2). We thenapplied this technique to monitor the oxidation mechanism of DAneurotransmitter. A new Raman peak at 1565 cm�1 assigned to C]C and/or C]O stretch of o-quinone group (product of oxidized DA)was observed during the oxidation process of DA and this peakbecame shoulder peak during the reduction process. These resultsdemonstrated that the redox active center of DA was located at itshydroxyl groups, which is in agreement with the findings of aprevious study [26]. Also, these results confirmed the potential ofusing our spectroelectrochemical technique for in-situ monitoringof the redox mechanism of DA aqueous solution.

Based on our technique, the time needed to achieve sufficientRaman intensity detectionwas short (5 s), which will be very usefulfor monitoring intermediates/products, generated during cell redoxprocesses. This technology could be used to overcome thecomplicated issues associated with studying the complex compo-nents of living cells and to determine changes in the extracellularand intracellular biochemical compositions during the redoxprocesses.

Distribution of biopolymers within living PC12 cell using theSERS technique, which consist of a series of bands corresponding toall cell biopolymers; nucleic acids (nucleotide and sugar-phosphatebackbone), proteins (amide I and amide III), amino acids (Phe, Tyrand Trp), lipids (C]C str. and hydrocarbon chains), and carbohy-drates (sugars, such as ring CeOeC vibrations). These resultsdemonstrated that PC12 cells contain different biopolymers thatpossessed many electro-active bonds, which could undergooxidation and/or reduction processes.

As the source of the electrochemical activity of the cells was notclear, Raman spectra signals from the cell were investigate duringthe oxidation and/or reduction processes and compared to thesignals obtained from the control cells. The SERSeLSV techniquewas directly applied to monitor the biochemical compositionchanges of bulk living PC12 cells (extracellular) when oxidation andreduction potentials were applied to the cells.

Different effects of oxidation and reduction potential weresuccessfully detected by analysis of each peak of the SERS signals.These effects on cellular biochemical composition were comparedwith the changes that occurred during application of the redoxpotential to the DA solution. These results demonstrated that PC12cell contained a Raman peak at 1544 cm�1 during the oxidationprocess, which disappeared during the reduction process. Thisbehavior was the same as observed for DA. This signified that DAsomehow plays a role in the electrochemical activity of PC12 cells.Also, these results (Fig. 2e) demonstrated that not only were DApeaks involved within the redox processes, but there must havebeen a number of other components that played a role in theelectrochemical behavior of PC12 cells.

The intracellular biochemical composition changes during theredox processes were also investigate and a different behavior wasobserved relative to living PC12 cells (extracellular). These results(Table S1) proved that the responses of the extracellular andintracellular conductivity were different.

To further confirm the superiority of our newly developedmethod, this method was used to monitor the biochemicalcomposition changes in single PC12 cells. Interestingly, an irre-versible oxidation process showing an anodic peak only wasobserved from a single PC12 cell, which was significantly distin-guished with bulk PC12 cells normally showed quasi-reversibleredox process. This result may be related to the low concentra-tion of electro-active species and a lack of cellecell interactions. TheCV behavior of single PC12 cell immobilized on microgap betweenthe pair of Au microelectrode containing hexagonal Au nanodotsarray showed sharp anodic peak (Fig. 5c). This was significantlydifferent from the cell on bare Au microelectrode that showedbroad anodic peak (Fig. 5b). These results indicated that Au nano-dots array is very effective for the enhancement of the electrontransfer rate. Moreover, the Raman results of this analysisdemonstrated that the changes in the Raman spectra signals ofsingle PC12 cells during oxidation and/or reduction processes(Table S2) were more complicated than the changes of bulk PC12cells. This may be due to the direct effect of voltage on the targetcell.

5. Summary and conclusions

In summary, we reported a new SERSeLSV combined tool toanalyze the intracellular and extracellular state available for bothbulk and single PC12 cell. The use of a transparent and electricalconductive ITO substrate opens up the possibility of using spec-troelectrochemical sensor transduction methods. Also, the use ofthe LSV technique allowed us to follow the mechanism of oxida-tion and reduction systematically as a function of electrode po-tential, rather than CV curves, which are used to elucidate theoverall reduction mechanism. We applied this SERSeLSV tech-nology to overcome the issues associated with studying thecomplex components of living neural PC12 cells and to determinethe extracellular biochemical composition changes during theredox processes.

Different effects of applied oxidation and reduction potentialson PC12 cells were successfully detected by analysis of each peak ofthe SERS signals. Effects on cellular biochemical composition werecompared with the changes that occurred during application of theredox potential in a DA solution. These results demonstrate that notonly was DA involved in the redox processes, butmany componentscould have played a role in the electrochemical behavior of PC12cells. In addition, the intracellular biochemical compositionschanges during the oxidation and reduction processes wereinvestigated and under these conditions the cell lysates wereshown to behave differently, compared to living PC12 cells.

W.A. El-Said et al. / Biomaterials 40 (2015) 80e87 87

Moreover, this SERSeLSV technique was further applied to inves-tigate the cellular biochemical composition changes of single PC12cell.

In conclusion, based on the combined results described above,we claim that this new spectroelectrochemical technique is a verypowerful in-situ monitoring tool capable of monitoring changes ofthe intracellular biochemical composition of single cell withrespect to their redox environment. Moreover, this technique maybe used to monitor the biochemical changes during cell electro-fusion and the electrical stimulation of differentiated neural cells.In addition, this SERSeLSV technique can be applied for sensitivein-vitro drug screening with multiple detection and high sensi-tivity. Thus, this cutting edge technology that combined the SERSand LSV method can be utilized as a non-invasive and non-destructive tool at various kinds of cellular researches.

Acknowledgment

This work was supported by the Leading Foreign ResearchInstitute Recruitment Program through the National ResearchFoundation of Korea (NRF) funded by the Ministry of Science, ICT &Future Planning (MSIP) (2013K1A4A3055268) and by the NationalResearch Foundation of Korea (NRF) grant funded by the Koreagovernment (MSIP) (No. 2014R1A2A1A10051725).

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.biomaterials.2014.11.023.

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