Biomaterials 40 (2015) 80e87
Contents lists available at ScienceDirect
Biomaterials
journal homepage: www.elsevier.com/locate/biomaterials
Fabrication of new single cell chip to monitor intracellular and
extracellular 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 Korea
Department of Chemical & Biomolecular Engineering, Sogang University, 35 Baekbeom-ro, Mapo-gu, Seoul 121-742, Republic of Korea
c
Chemistry Department, Faculty of Science, Assiut University, Assiut 71516, Egypt
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 24 August 2014
Accepted 8 November 2014
Available online 26 November 2014
Probing the local environment of target cells has been considered a challenging task due to the
complexity of living cells. Here, we developed new single cell-based chip to investigate the intracellular
and extracellular redox state of PC12 cells using spectroelectrochemical tool that combined surfaceenhanced Raman scattering (SERS) and linear sweep voltammetry (LSV) techniques. PC12 cells immobilized on gold nanodots/ITO surface were subjected to LSV and their intracellular biochemical changes
were successfully monitored by SERS simultaneously. Moreover, paired gold microelectrodes with
micrometer-sized gap containing hexagonal array of gold nanodots were fabricated to detect electrochemical activity and changes in the redox environment of single PC12 cell based on SERSeLSV tool. This
showed very effective detecting method. The used technology included the utilization of gold nanodots
array inside micro-gap to enhance the Raman signals and the electrochemical activity of single cell. This
could be used as an effective research tool to analyze cellular processes.
© 2014 Elsevier Ltd. All rights reserved.
Keywords:
Intracellular and extracellular redox state
Spectroelectrochemical
Single neural cell-chip
Gold microelectrodes
Gold nanodots array
1. Introduction
A wide range of in-vitro studies have been carried out to
examine the effects of diverse materials (e.g. metal/inorganic
nanoparticles, peptides, anti-cancer drugs or environmental toxins)
on the target cells. The most remarkable advantage of the in-vitro
methods is the ability to analyze wider range of cellular processes,
which is not possible for in-vivo or animal-based test. However, it is
difficult to maintain the biological characteristics of many cells
during analysis compared to analysis of their components. This is
due to the structural/chemical complexity of cells as well as the
difficulties in handling cells [1]. X-ray absorption fine structure
(XAFS) spectra [2,3] have been carried out to study the biological
characteristics of cells by analyzing the intermediate structure of
cellular components. Nevertheless, because of the lengthy time
required for the collection of XAFS data, this method cannot be
easily applied for living cell analysis. Moreover, several studies have
reported IR-spectroelectrochemical (IR-SEC) method as a reliable
* Corresponding author. Department of Chemical & Biomolecular Engineering,
Sogang University, Seoul, Republic of Korea. Tel.: þ82 2 705 8480; fax: þ82 2 3273
0331.
E-mail address: [email protected] (J.-W. Choi).
http://dx.doi.org/10.1016/j.biomaterials.2014.11.023
0142-9612/© 2014 Elsevier Ltd. All rights reserved.
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 cellular
research due to the interference with water in aqueous environments that is not adequate for monitoring living cells [7].
Previously, we have developed various nanopatterned modified
electrodes to investigate the viability of cancer cells after exposure
to different kinds of anti-cancer drugs using cyclic voltammetry
(CV) technique [8e10]. In spite of that, effects of different anticancer drugs cannot be monitored by electrochemical methods,
because the current CV peaks can only indicate the cell viability via
electron transfer between the cells and electrode surface [11e13].
The superiority of the Raman technique is due to the specific
inelastic scattering of photons from chemical bonding in molecules
activated by the light source. Therefore, the biochemical and/or
biological structure of cells can be effectively studied by analyzing
each peak in the Raman spectra, which is unavailable in other optical, biological or electrical methods. Additionally, the weak signals
resulted from Raman scattering can be overcome by using the SERS
technique. Since the signals obtained by the SERS method are
109e15 fold higher than normal Raman, biological molecules can be
readily analyzed with a reduced exposure time to the laser source.
We have previously reported on a nanostructured SERS-active
surface and its application for analysis of the cell intracellular
W.A. El-Said et al. / Biomaterials 40 (2015) 80e87
state [14]. We have also extended this approach to monitor differentiation between live/dead cells, cells in different cell cycle stages
and different kinds of cell lines immobilized on homogeneously
fabricated nanopatterned gold (Au) surface [15]. It was found that
SERS signal obtained from target cells were excellent for monitoring intracellular changes in cellular components [16].
In the current study, we have fabricated Au nanodot array
modified ITO substrate as cell culture system, SERS-active surface
and a working electrode. Also, a new spectroelectrochemical
technique that combined the SERS and voltammetric methods was
developed for analysis of the living cells redox properties.
LSV was used to investigate the biochemical changes in the
intracellular components during the redox process of neural cells
(PC12), while the NIR laser source was simultaneously focused on
the target cell for SERS analysis. Moreover, this SERSeLSV technique was used for simultaneous investigation of single PC12 cell
attached to a micrometer-sized gap between two paired Au microelectrodes. Prior to cell attachment, polystyrene-assisted hexagonal array of Au nanodots was fabricated on the gap between pair
of Au microelectrodes to enhance the Raman signal (Scheme 1).
2. Experimental section
2.1. Materials
81
washed with PBS and were scrapped off the well surface using a cell scraper gently
to prevent foaming. Then, the cell suspension in the well was transferred into a
centrifuge tube and was spin at 5000 rpm for 5 min at 4 C, and any remaining buffer
was removed. 0.25 mL ice cold lysis buffer was added and kept in ice for 20 min. This
cell lysate was centrifuged at 5000 rpm for 5 min at 4 C and the supernatant liquid
was separated.
2.4. Electrochemical measurements
All electrochemical experiments were performed using a potentiostat (CHI-660,
CH Instruments, USA) controlled by general-purpose electrochemical system software. A homemade three-electrode system consisted of Au microelectrode as a
working electrode, platinum wire as counter electrode and Ag/AgCl as the reference
electrode. All electrochemical analyses were carried out to monitor the electrical
properties of living cells and the effect of anti-cancer drugs on their behavior in
normal laboratory conditions. PBS (10 mM, pH 7.4) was used as an electrolyte at a
scan rate of 20 mV/s.
2.5. Raman spectroscopy
Biochemical composition of control PC12 cells and the changes during the redox
processes were investigated by Raman spectroscopy using Raman NTEGRA spectra
(NT-MDT, Russia). The maximum scan-range, XYZ was 100 mm 100 mm 6 mm and
the resolution of the spectrometer in the XY plane was 200 nm and along the Z axis
was 500 nm. Raman spectra were recorded using NIR laser emitting light at 785 nm
wavelength. Ten scans of 5 s from 500 cm1 to 1750 cm1 were recorded and the
mean of these scans was used.
2.6. Fabrication of PS-assisted nanopatterned surface
Polystyrene (PS), dopamine (DA) and phosphate buffered saline (pH 7.4, 10 mM)
were purchased from SigmaeAldrich (St. Louis, MO, USA). All other chemicals used
were obtained commercially as the reagent grade. All aqueous solutions were prepared by using de-ionized water (DIW) that de-ionized with a Millipore Milli-Q
water purifier operating at a resistance of 18MU cm.
PC12 used was derived from rat neural cells (Adrenal medulla) e purchased
from the Korean Cell Line Bank (Seoul, Korea) e and was cultured in PRMI (Invitrogen, Carlsbad, USA) with 10% heat-inactivated fetal bovine serum (FBS; Gibco,
Carlsbad, CA, USA) and 2% antibiotics (streptomycin þ penicillin) (Gibco). The cells
were 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 was
determined with a hemacytometer after trypan blue exclusion.
Monolayer of PS was prepared as described previously [17,18]. PS particles with a
diameter of 100 nm (10 wt % aqueous solution) were mixed with a surfactant
mixture (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 of
NH4OH, H2O2 and H2O (volume ratio 1:1:5) at 80 C for 30 min. The freshly prepared
samples 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 100
and 1000 rpm, at intervals of 100 rpm. The speed was increased steadily from 0 rpm
to 1000 rpm speed and kept constant at a fixed time interval of 30 s before it was
increased to 1000 rpm. The total spinning time was 1 min. The substrate was then
left to dry in the spin coater with a covered lid to maintain a consistent drying
ambient and evaporation rate.
2.3. Cell lysis
2.7. Design and characterization of SERSeLSV system for bulk cells studies
Typically, the cells were allowed to grow for 2 days before devoted to the
preparation of the cell lysate. The growth medium was removed and the cells were
The design of the surface-enhanced Raman spectroscopyelinear sweep voltammetry (SERSeLSV) cell was based on the fabrication of Au nanodots array on the
2.2. Cell culture
Scheme 1. Schematic diagram of the immobilization of a single 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 evaporated on the top of the deposited PS nanospheres mask. After dissolution of the PS
mask in chloroform, an array of ordered round Au nanostructures with lateral dimensions of about 30 nm remained on the ITO substrate (Fig. 1b). A sterile cellchamber with dimensions of 10 mm 10 mm was attached to the Au nanodots/
ITO substrate using polydimethylsiloxane (PDMS) for measurement of the SERS
spectra and LSV behavior of living cells under physiological conditions (37 C temperature and PBS 7.4 pH). This cell culture system was be used as a SERS-active
surface 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 Au
nanodots array inside the microgap. 12 Au microelectrode arrays were patterned on
a 10 mm 10 mm glass substrate, which provided 6 microgaps with width of about
7 mm between each pair of Au microelectrodes. The Au nanodots array was then
fabricated inside these microgaps based on PS nanospheres mask. Au nanodots
formed on the Au microelectrodes as well as on the outside the microelectrodes
array. To localize a single cell over the microgap, a PDMS microchannel (200 mm in
width) was attached. The cells were then transferred onto the chip with new culture
medium through the microchannel inlet. A sterile cell-chamber with dimensions of
8 mm 8 mm was developed to measure the Raman spectra of living single cells
under physiological conditions.
3. Results
A number of studies have reported that the voltammetric
response of cells have a strong relationship with some enzymes in
the cytosol [20,21]. While, others have suggested that the cells
voltammetric behavior may be related to the oxidation of guanine
[22e25]. Moreover, in our previous work, we extracted some redox
enzymes from HeLa cells using 2-D electrophoresis techniques,
including NADH dehydrogenase (ubiquinine) flavoprotein 2,
Quinone oxidoreductase-like (QOH-1), which may be related to the
cell voltammetric behavior [13]. However, more research is needed
to conclusively clarify the voltammetric behavior of the cells. PC12
cells have been reported to secrete the neurotransmitter (DA) [26],
which is an electrochemical active species. Here, we selected PC12
cells as an experimental nerve cell model to investigate the source
of the cell electrochemical activity. Also, a new single cell-chip has
been developed to investigate the Redox states of single cell. This
cell-chip consisted of PDMS micro-channel over six pairs of Au
microelectrodes; moreover, Au nanodots array was fabricated inside the micro-gap between each pair of Au microelectrodes.
3.1. Spectroelectrochemistry of dopamine neurotransmitter
Au nanodots modified ITO substrates were fabricated based on
thermal evaporation of pure Au metal with 30 nm thickness
through polystyrene (PS) monolayer (Fig. 1a), which acted as soft
template. Fig. 1b showed the fabrication of ordered round Au
nanostructures on the ITO substrate after removal of the PS mask.
This substrate was used as a cell culture substrate for utilizing as a
SERS-active surface and for using as the working electrode. The CV
behavior of 50 mM of DA in PBS (pH 7.4) buffer solution within a
potential range from þ0.8 to 0.2 V was shown in Fig. 1c,
demonstrating a reversible redox process with a cathodic peak
at þ0.145 V and an anodic peak at þ0.195 V. Fig. 1fI showed the
Raman spectra of DA, which contained a series of peaks including
peaks at 1270 cm1 (CeO str.), 1358 cm1 (str. catechol), 1443 cm1
(CeN), 1150 and 1599 cm1 (CeC of benzenoid ring) [27].
To identify the species that were produced during the electrochemical redox processes of DA, LSV, oxidation and/or reduction
assays were applied and the Raman spectrum was recorded
simultaneously during each step. Application of the oxidation potential to the DA solution within potential range from 0.2
to þ0.8 V resulted in anodic peak at þ0.195 V (Fig. 1d), and a new
peak near 1565 cm1 was observed in the corresponding Raman
spectra (Fig. 1fII). In contrast, a cathodic peak at þ0.145 V was
observed when reduction potential was applied to the DA solution
in the potential range from þ0.8 to 0.2 V (Fig. 1e), and Raman peak
Fig. 1. (a) SEM image of the PS mask on the ITO substrate. (b) SEM image of Au nanodots array on the ITO substrate. (c) CV of dopamine. (d) LSV oxidation of dopamine. (e) LSV
reduction of dopamine. (f) SERS spectra of dopamine control (black), during oxidation (blue) and during reduction (orange). (For interpretation of the references to color in this
figure legend, the reader is referred to the web version of this article.)
W.A. El-Said et al. / Biomaterials 40 (2015) 80e87
that appeared after the oxidation process (1565 cm1) became a
shoulder peak (Fig. 1fIII).
3.2. Spectroelectrochemical study of the extracellular biochemical
changes of bulk PC12 cells during the redox processes
Fig. 2a showed the CV behavior of PC12 cells in the potential
range from þ0.8 to 0.2 V that demonstrated a quasi-reversible
process with a cathodic peak at þ0.025 V and an anodic peak
at þ0.49 V. To understand the source of electrochemical activity of
the cells and the biochemical changes that occurred during the
redox reactions, SERS spectrum for control PC12 cells were
compared with the SERS spectra during the oxidation and/or
reduction processes; all SERS results were summarized in Table S1.
The Raman spectrum of the control PC12 (Fig. 2dI) showed
peaks of 775 cm1 (Trp, U, C and T), 1001 cm1 (Phe), 1092 cm1
1
(PO
(Phe and Trp), 1230 cm1 (amide III and T),
2 ), 1205 cm
1
857 cm (Tyr), 1490 cm1 (G and A), 1620 cm1 (C]C Trp and Tyr
str.), 1270 cm1 (amide III and catecholamines, e.g. DA) and
1557 cm1 (C]C str. of phenyl group), which is in agreement with
previously reported spectra of cells [28e30].
Fig. 2b depicted the LSV oxidation of PC12 cells in the potential
range from 0.2 to þ0.8 V, which contained an anodic peak
at þ0.4 V. In addition, the number of peaks in Raman spectrum of
PC12 cell changed during the oxidation process. A decrease in the
Raman peak intensities was observed at 1001, 775 cm1 (amino
acids), and 1270 cm1 (DA and amide III). Moreover, the Raman
peaks at 857 and 1557 cm1 disappeared. Also, new Raman peaks at
865, 1405 (A and G), 1544, 1570, and 1602 cm1 appeared, which
83
were assigned to the C]C and/or C]O stretch of the o-quinone
group (A and G) as shown in Fig. 2dII.
Furthermore, when PC12 cells were subjected to a reduction
potential (from þ0.8 to 0.2 V), a cathodic peak at 0.0 V was
observed (Fig. 2c). In comparison with the Raman of PC12 cells
subjected to oxidation, the Raman spectra of PC12 after reduction
process (Fig. 2dIII) increased again (peaks at 1001 cm1) but the
intensity was not as high as that observed in the control cells. At the
same time, intensities of the Raman peaks at 1575 cm1 (Phe) and
1270 cm1 (DA and Amide III) decreased and a new peak at
1226 cm1 (Tyr and Phe) was observed. Furthermore, the peaks at
1720 cm1 (C]O ester) and 1544 cm1 disappeared.
For better understanding of the chemical reactions mechanism
responsible for the electrochemical activity of cells, real time
Raman spectra changes during oxidation and reduction processes
of the living PC12 cells were investigated. For real time monitoring
SERSeLSV approach was used. The LSV assay was performed at a
low scan rate (0.02 V/s) within a potential range from 0.2
to þ0.8 V that corresponded to a total LSV scan time of 50 s. We
then divided the scan range into three regions (corresponding to
15 s) and in each region, we investigated the biochemical changes
using the SERSeLSV assay. Fig. 3a showed the LSV oxidation
behavior of PC12 cell at a scan rate 0.02 V/s, which contained an
oxidation peak at about þ0.39 V. Simultaneously, Raman spectra
readings during each LSV oxidation scan step were 0.2 V
to 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 the
first step when compared to the control PC12 cells. During the
second step, a significant change was observed in the Raman
Fig. 2. (a) CV of PC12 cells. (b) LSV oxidation of PC-12 cells. (c) LSV reduction of PC12 cells. (d) SERS spectra of control PC12 cells (black), during oxidation (orange) and during
reduction (blue). (e) Comparison of the Raman spectra from control PC12 cells and during oxidation and reduction processes. (For interpretation of the references to color in this
figure legend, the reader is referred to the web version of this article.)
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W.A. El-Said et al. / Biomaterials 40 (2015) 80e87
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 during
reduction: 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 to
the web version of this article.)
spectrum due to the oxidation reactions within cell. Finally, Fig. 3c
demonstrated slight changes in the Raman spectrum during the
last step including peaks 875, 1001, 1420, 1475, 1550, 1570 and
1610 cm1.
Moreover, the reduction behavior of PC12 cell within the range
from þ0.8 V to 0.2 V at a scan rate 0.02 V/sec (Fig. 3b) displayed a
reduction peak at about þ0.04 V. Therefore, we selected two regions; the first was from þ0.8 V to þ0.5 V while the second were
from þ0.2 V to 0.1 V. The third step started at 0.14 V and
completed 12 s after the LSV scan finished. Almost no changes were
observed in the Raman spectra during the first step when
compared with the PC12 cell in the oxidation state. Significant
changes in the Raman spectra were observed during the second
step, especially for the Raman peaks at 1001, 1240, 1450 cm1 and
within the range from 1500 to 1620 cm1. Finally, very few changes
were observed in the Raman spectra during the last step (Fig. 3d).
3.3. Spectroelectrochemical study of the intracellular biochemical
changes of PC12 cells during the redox processes
The SERSeLSV technique was also applied to investigate the
biochemical changes in the intracellular compositions during the
redox processes of neural PC12 cell lysates. Fig. S3a showed the CV
behavior of the PC12 cell lysate, which demonstrated anodic and
cathodic current peaks at 0.328 and 0.0 V, respectively. These
values were different when compared with those of living PC12
cells (extracellular). Compared with the Raman spectra of living
PC12 cells, Raman spectra of the lysates of PC12 cells showed a
weak Raman peaks intensity except for the peak corresponding to
the Phe group (1001 cm1). This reflected high concentrations of
protein (Fig. S3dI). However, the Raman spectra of PC12 cell lysates
significantly changed during the oxidation and reduction processes
(Fig. S3b & c). Practical changes were presented in Table S2
including the following changes (Fig. S3dII & III), 671 cm1 (T and
G), 760 cm1 (Trp), 854 cm1 (Trp), 923 cm1 (pro CC str.),
975 cm1 (CeC str. b-sheet ¼ CH bend), 1001 cm1 (Phe), 1125 cm1
(prot. str. CN), 1134 cm1 (Prot), 1207 cm1 (Trp, Phe and Tyr),
1450 cm1 (Prot), and 1625 cm1 (C]C Tyr and Trp).
3.4. Spectroelectrochemical study of single neural cell
SERSeLSV technique was developed to study the biochemical
composition of living single PC12 cells during the redox processes.
In order to evaluate the spectroelectrochemical assay at the single
PC12 cell level (Scheme 1), 12 Au microelectrode arrays were
patterned on a 10 mm 10 mm glass substrate that provided six
microgaps (about seven mm in width) between each microelectrode
pair. 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 buffer
solution and no redox peaks were observed. In contrast, the CV of
single PC12 cell immobilized on microgap between pairs of Au
microelectrodes displayed an anodic peak at about 0.4 V (Fig. 5c).
Fig. 5d showed the LSV oxidation behavior of single PC12 cell
within potential range from 0.2 to þ0.8 V, which contained anodic
peak at about þ0.4 V. Moreover, many peaks in the Raman spectrum were changed relative to the control PC12 single cell (Fig. 5f).
The Raman spectra changes were represented in Table S3, which
W.A. El-Said et al. / Biomaterials 40 (2015) 80e87
85
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) SEM
images 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.
represented decrease in Raman peaks intensities at 720 (A), 760
(Trp), 905 (prot. ring str.), 1001 (Phe), 1060 (str. PO
2 and str. COC),
1093 (lipids: str. CeC, deoxyribose: CeO, CeC str. and str. PO
2 ), 1125
(prot. str. CN), 1175 (prot. Tyr str. CN and CC), 1270 (T, A and amide
III), 1310 (A), 1360 (prot. CH2), 1390 (T, A and G), and 1690 cm1
(amide I and C]C). Additionally, the Raman peaks at 1625 cm1
(C]C, Tyr and Trp), which was observed in control PC12 cells disappeared. Also, peaks at 854 cm1 (prot. Ring br. Tyr), 940
Fig. 5. (a) CV background of microelectrode. (b) CV of single PC12 cell on the microgap between pair of Au microelectrodes. (c) CV of a single PC12 cell on the microgap between pair
Au microelectrodes contain the Au nanodots array. (d) LSV oxidation of a single PC12 cell on the microgap between pair Au microelectrodes. (e) LSV reduction of a single PC12 cell on
the microgap between two Au microelectrodes. (f) SERS spectra of PC12 cells control (black) during oxidation (orange) and reduction (blue). (For interpretation of the references to
color in this figure legend, the reader is referred to the web version of this article.)
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W.A. El-Said et al. / Biomaterials 40 (2015) 80e87
(proteins: a-helix, and deoxy and CeOeC), and 1560 cm1 (proteins: amide II and Trp; C]C and/or C]O stretch of o-quinone
group) shifted to 866, 952 and 1545 cm1, respectively. New Raman
peaks were observed during application of the oxidation voltage,
including Raman peaks at 1330 (G), s1450 (deoxyribose), 1426, 1502
and1595 cm1 (A and G).
In addition, no cathodic peak was observed when PC12 cells
were subjected to reduction potential within potential range
from þ0.8 to 0.2 V (Fig. 5e). However, the Raman spectrum during
the reduction process was different than the Raman spectrum of
the control PC12 or PC12 cells in the oxidation state (Fig. 5f). These
changes in Raman spectra included an increase in the Raman peaks
intensities at 905 (prot. ring str. CC), 1001 (Phe), 1060 (PO
2 str. and
CeO, CeC str.), 1093 (lipids, chain CeC str. deoxyribose: CeO, CeC
str. phosphate: str. PO
2 ), 1125 (CeN str.), 1175 (CeH bend Tyr), 1270
(T, A and amide III), 1310 (A), 1360 (A, G, and prot. CH2 def) and
1390 cm1 (T, A and G). Also, the Raman peak at 1560 cm1 (C]C
and/or C]O stretch of o-quinone group) shifted to 1575 cm1.
Furthermore, new Raman peaks at 1230 (T, A, and amide III), 1426
(G, A and CH def), 1502 cm1 (G and A) were observed. Moreover,
the Raman peaks at 1330 (G), 1595 (A and G), 1625 (C]C Tyr and
Trp) and 1720 cm1 (amide I and C]O ester) disappeared, which
could be related to a reduction of these functional groups.
4. Discussion
Application of the SERSeLSV technique for analysis of complicated system (living cell) was preceded by validation of its ability to
monitor the reaction mechanism of simple oxidation reaction
mechanism of potassium ferrocyanide (Figs. S1 & S2). We then
applied this technique to monitor the oxidation mechanism of DA
neurotransmitter. A new Raman peak at 1565 cm1 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 peak
became shoulder peak during the reduction process. These results
demonstrated that the redox active center of DA was located at its
hydroxyl groups, which is in agreement with the findings of a
previous study [26]. Also, these results confirmed the potential of
using our spectroelectrochemical technique for in-situ monitoring
of the redox mechanism of DA aqueous solution.
Based on our technique, the time needed to achieve sufficient
Raman intensity detection was short (5 s), which will be very useful
for monitoring intermediates/products, generated during cell redox
processes. This technology could be used to overcome the
complicated issues associated with studying the complex components of living cells and to determine changes in the extracellular
and intracellular biochemical compositions during the redox
processes.
Distribution of biopolymers within living PC12 cell using the
SERS technique, which consist of a series of bands corresponding to
all cell biopolymers; nucleic acids (nucleotide and sugar-phosphate
backbone), proteins (amide I and amide III), amino acids (Phe, Tyr
and Trp), lipids (C]C str. and hydrocarbon chains), and carbohydrates (sugars, such as ring CeOeC vibrations). These results
demonstrated that PC12 cells contain different biopolymers that
possessed many electro-active bonds, which could undergo
oxidation and/or reduction processes.
As the source of the electrochemical activity of the cells was not
clear, Raman spectra signals from the cell were investigate during
the oxidation and/or reduction processes and compared to the
signals obtained from the control cells. The SERSeLSV technique
was directly applied to monitor the biochemical composition
changes of bulk living PC12 cells (extracellular) when oxidation and
reduction potentials were applied to the cells.
Different effects of oxidation and reduction potential were
successfully detected by analysis of each peak of the SERS signals.
These effects on cellular biochemical composition were compared
with the changes that occurred during application of the redox
potential to the DA solution. These results demonstrated that PC12
cell contained a Raman peak at 1544 cm1 during the oxidation
process, which disappeared during the reduction process. This
behavior was the same as observed for DA. This signified that DA
somehow plays a role in the electrochemical activity of PC12 cells.
Also, these results (Fig. 2e) demonstrated that not only were DA
peaks involved within the redox processes, but there must have
been a number of other components that played a role in the
electrochemical behavior of PC12 cells.
The intracellular biochemical composition changes during the
redox processes were also investigate and a different behavior was
observed relative to living PC12 cells (extracellular). These results
(Table S1) proved that the responses of the extracellular and
intracellular conductivity were different.
To further confirm the superiority of our newly developed
method, this method was used to monitor the biochemical
composition changes in single PC12 cells. Interestingly, an irreversible oxidation process showing an anodic peak only was
observed from a single PC12 cell, which was significantly distinguished with bulk PC12 cells normally showed quasi-reversible
redox process. This result may be related to the low concentration of electro-active species and a lack of cellecell interactions. The
CV behavior of single PC12 cell immobilized on microgap between
the pair of Au microelectrode containing hexagonal Au nanodots
array showed sharp anodic peak (Fig. 5c). This was significantly
different from the cell on bare Au microelectrode that showed
broad anodic peak (Fig. 5b). These results indicated that Au nanodots array is very effective for the enhancement of the electron
transfer rate. Moreover, the Raman results of this analysis
demonstrated that the changes in the Raman spectra signals of
single PC12 cells during oxidation and/or reduction processes
(Table S2) were more complicated than the changes of bulk PC12
cells. This may be due to the direct effect of voltage on the target
cell.
5. Summary and conclusions
In summary, we reported a new SERSeLSV combined tool to
analyze the intracellular and extracellular state available for both
bulk and single PC12 cell. The use of a transparent and electrical
conductive ITO substrate opens up the possibility of using spectroelectrochemical sensor transduction methods. Also, the use of
the LSV technique allowed us to follow the mechanism of oxidation and reduction systematically as a function of electrode potential, rather than CV curves, which are used to elucidate the
overall reduction mechanism. We applied this SERSeLSV technology to overcome the issues associated with studying the
complex components of living neural PC12 cells and to determine
the extracellular biochemical composition changes during the
redox processes.
Different effects of applied oxidation and reduction potentials
on PC12 cells were successfully detected by analysis of each peak of
the SERS signals. Effects on cellular biochemical composition were
compared with the changes that occurred during application of the
redox potential in a DA solution. These results demonstrate that not
only was DA involved in the redox processes, but many components
could have played a role in the electrochemical behavior of PC12
cells. In addition, the intracellular biochemical compositions
changes during the oxidation and reduction processes were
investigated and under these conditions the cell lysates were
shown to behave differently, compared to living PC12 cells.
W.A. El-Said et al. / Biomaterials 40 (2015) 80e87
Moreover, this SERSeLSV technique was further applied to investigate the cellular biochemical composition changes of single PC12
cell.
In conclusion, based on the combined results described above,
we claim that this new spectroelectrochemical technique is a very
powerful in-situ monitoring tool capable of monitoring changes of
the intracellular biochemical composition of single cell with
respect to their redox environment. Moreover, this technique may
be used to monitor the biochemical changes during cell electrofusion and the electrical stimulation of differentiated neural cells.
In addition, this SERSeLSV technique can be applied for sensitive
in-vitro drug screening with multiple detection and high sensitivity. Thus, this cutting edge technology that combined the SERS
and LSV method can be utilized as a non-invasive and nondestructive tool at various kinds of cellular researches.
Acknowledgment
This work was supported by the Leading Foreign Research
Institute Recruitment Program through the National Research
Foundation of Korea (NRF) funded by the Ministry of Science, ICT &
Future Planning (MSIP) (2013K1A4A3055268) and by the National
Research Foundation of Korea (NRF) grant funded by the Korea
government (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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