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Science of Advanced MaterialsVol. 6, pp. 2577–2581, 2014

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Au-Crumpled Graphene Modified Electrode toDetect Neurotransmitters based onSpectroelectrochemical MethodHye-Jun Shin1, Hyeon-Yeol Cho1, Sun Kyung Kim2,3, Hee Dong Jang3,and Jeong-Woo Choi1,2,∗

1Department of Chemical and Biomolecular Engineering, Sogang University, 35 Baekbeom-Ro,Mapo-Gu, Seoul, 121-742, Republic of Korea2Interdisciplinary Program of Integrated Biotechnology, Sogang University, 35 Baekbeom-Ro,Mapo-Gu, Seoul, 121-742, Republic of Korea3Rare Metals Research Center, Korea Institute of Geoscience and Mineral Resources,124 Gwahang-Ro, Yuseong-Gu, Daejeon, 305-350, Republic of Korea

ABSTRACT

Detection of neurotransmitters is one of the most efficient methods for monitoring neurodegenerative diseases.Several electrochemical methods have been developed for neurotransmitter detection with conductive materials.In this study, gold-crumpled graphene (Au-crGr) composite modified electrodes were used to improve electrodeconductivity and affinity with neurotransmitters. Au-crGr composites were synthesized at various componentratios and the structures were confirmed by electron microscopy. After the fabrication of Au-crGr compositemodified electrodes, electrochemical methods and surface-enhanced Raman spectroscopy (SERS) were usedto validate the spectroelectrochemical properties of various electrodes. The highest electrochemical and SERSsignal intensity from a dopamine solution was obtained with Au-crGr composite modified electrodes. Moreover,secreted neurotransmitters from human neuroblastoma cells were successfully detected using the new materialin a spectroelectrochemical method, and these results were confirmed by ultra-performance liquid chromatog-raphy and mass spectrometry. Our newly developed Au-crGr composite modified electrodes can be used as acatecholamine sensor and to study diagnostic methods for neurodegenerative diseases.

KEYWORDS: Au-Crumpled Graphene, Neurotransmitter, Spectroelectrochemical Method, SERS.

1. INTRODUCTIONNeurodegenerative diseases such as Alzheimer’s diseaseand Parkinson’s disease are major impediments to thepromise of a comfortable and undisturbed retirementfor the aged population. Neurodegenerative diseases arecaused by decreased secretion of neurotransmitters includ-ing dopamine (DA)1 and norepinephrine (NE),2 whichplay a key role in brain functions such as memory,awareness, behavior and learning. Researchers have triedto develop monitoring tools to measure the concentra-tion of neurotransmitters from in vitro and in vivo con-ditions after treatment with neurotransmitter precursors.Conventionally, ultra-performance liquid chromatography(UPLC),3 immunohistochemical methods,4 and label-freefluorescence detection methods5 have been used to monitor

∗Author to whom correspondence should be addressed.Email: jwchoi@sogang.ac.krReceived: 9 July 2014Accepted: 20 October 2014

changes in neurotransmitter secretion from dopaminergiccells. They can detect neurotransmitters accurately, butrequire complicated pre-processing and various compo-nents to detect target materials.Electrochemical neurotransmitter detection is another

conventional technique that has gained favor widelybecause of its simplicity and fast analytical capability.To improve its sensitivity, various kinds of conductivematerials have been applied to electrochemical detectiontools, such as gold nanoparticles,6�7 nano-porous goldfilm,8 carbon nanotubes,9 and graphene10�11 modified elec-trodes. Notwithstanding the multitude of materials avail-able, development of new materials is still needed toincrease the sensitivity and stability of electrochemicaldetection, because neurotransmitters in the body exist atlow concentrations, from picomolar to nanomolar scales.In this study, the gold electrode was modified with

gold-crumpled graphene (Au-crGr) composites12 in orderto increase conductivity and neurotransmitter affinity with

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Au-Crumpled Graphene Modified Electrode to Detect Neurotransmitters based on Spectroelectrochemical Method Shin et al.ARTICLE

label-free electrochemical detection methods. Using theAu-crGr composite modified electrode, neurotransmitterssecreted from the SH-SY5Y cells were detectable throughthe electrochemical detection method. The secreted neu-rotransmitters were also measured with UPLC13 and massspectrometry (MS) for confirmation of spectroelectro-chemical results. The schematic diagram below explainsthe application of the newly developed electrode for thespectroelectrochemical detection of secreted neurotrans-mitters from differentiated neuroblastoma cells (Fig. 1).

2. EXPERIMENTAL DETAILS2.1. Synthesis of Au-crGr CompositesAerosol spray pyrolysis, a very convenient method forsingle-step synthesis of graphene-based composite mate-rials was used in this study.14 For the synthesis ofthe Au-crGr composite, a colloidal mixture containinggraphene oxide (GO) colloid and HAuCl4 ·3H2O was usedas a precursor. The precursor was prepared with differentweight ratios of Au/graphene (0.4, 0.6, 0.8, 1.0, 2.0) whilethe concentration of the GO was fixed at 0.5 wt% in the col-loidal mixture. The experimental apparatus for the aerosolspray pyrolysis process consisted of an ultrasonic atomizer,an electrical tubular furnace, and a filter sampler. The ultra-sonic atomizer was used to generate micron-sized dropletsof the GO-Au colloidal precursor. The droplets were thencarried into the furnace by 1.0 L/min of argon. The evapo-ration of water, thermal reduction of GO and Au precursor,and the self-assembly between graphene and Au nanopar-ticles were carried out in series in a tubular furnace. Thesynthesized Au-crGr composites were then collected by aTeflon membrane filter. The details of the preparation ofGO colloid and Au precursor used in the present study havebeen previously described in detail in the literature.12

Fig. 1. Schematic diagram of the spectroelectrochemical detection ofneurotransmitters from cells with the Au-crGr composite modified elec-trode. Working electrode (WE), counter electrode (CE), reference elec-trode (RE).

2.2. Structural Analysis of Au-crGr CompositesThe structure of Au-crGr composites was confirmed byfield emission scanning electron microscopy (FE-SEM;Sirion, FEI) and transmission electron microscopy (TEM;CM12, Philips). FE-SEM images were taken without themetal-coating step.

2.3. Fabrication of Au-crGr Modified ElectrodeThe gold electrode was modified with the Au-crGr condi-tion using cysteamine (1 mM) as a chemical linker. Thechemical linker was first applied to the gold electrode for2 h to create the amino-surface. After washing three timeswith deionized water, Au-crGr composites were immobi-lized on the amino-gold electrode surface with a concen-tration of 0.1 mg/mL for 24 h. Non-immobilized materialswere removed by another washing step. DA and NE wereapplied to the fabricated electrode separately at 1 mM for48 h to confirm immobilization and to study the spectro-electrochemical properties.

2.4. Spectroelectrochemical Detection ofNeurotransmitters

Electrochemical detection of neurotransmitters was con-ducted with a Au-crGr composite modified electrode withcyclic voltammetry (CV) and differential pulse voltam-metry (DPV) using a potentiostat (CHI660a, CHI instru-ment). Surface-enhanced Raman spectroscopy (SERS) wasused for the spectral detection of neurotransmitters witha confocal Raman detection system (NTEGRA spectra,NT-MDT).15

2.5. Cell Culture and DifferentiationThe SH-SY5Y cells were cultured in a cell culture plateand in the presence of CO2 (5%) at 37 �C with Dulbecco’sModified Eagle’s Medium (DMEM) supplemented withfetal bovine serum (FBS, 10%) and penicillin-streptomycin(1%). After seeding in the 6-well cell culture plate, SH-SY5Y cells were cultured in 1% FBS media for 6 daysto differentiate. The cells were then treated with mediacontaining the differentiation factor retinoic acid (RA,10 �M)16 for an additional 6 days. The SH-SY5Y cellswere also treated with glucose (10 mM) and KCl (1 mM)17

to induce neurotransmitter secretion.

2.6. UPLC and MS Analysis for Confirmation ofNeurotransmitter Detection

UPLC (UltiMate3000, Thermo Scientific) and MS [Q-TOF5600+ (AB SCIEX)] were used to detect neurotransmit-ter signals using both qualitative and quantitative analysis.UPLC was conducted under the following conditions;Flow rate: 0.5 ml/min, Column: ACQUITY BEH C18(100× 2�1 mm, 1.7 �m). After UPLC, the sample wasanalyzed with MS to confirm the presence of targetedneurotransmitters.

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Shin et al. Au-Crumpled Graphene Modified Electrode to Detect Neurotransmitters based on Spectroelectrochemical Method

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3. RESULTS AND DISCUSSION3.1. Structural Analysis of Synthesized

Au-crGr CompositesThe Au-crGr composites were synthesized with variousweight ratios of Au and graphene to find an optimizedcondition between conductivity and neurotransmitter affin-ity. The structure and size of Au-crGr composites areshown in Figure 2. The presence of Au nanoparticles inthe Au-crGr composites was confirmed from the TEMimages of selected Au-crGr composites of an Au weightratio of 0.6 wt% (Fig. 2(a)). The SEM image demonstratedthe crumpled-sheet structure of Au-crGr composites andits conductivity (Fig. 2(b)). The number of Au nanoparti-cles increased with increasing Au/graphene ratio (Data notshown).

3.2. Characterization of Au-crGr CompositesThe spectroelectrochemical properties of Au-crGr compos-ites were evaluated using cyclic voltammetry (CV) andSERS. Electrochemical performance of various kinds ofmodified gold substrates were tested with DA solution(10 �M), which was modified with GO and different Auweight ratios of Au-crGr composites (0, 0.4, 0.6, 0.8, 1.0

Fig. 2. TEM (a) and FE-SEM (b) images of the 0.6 wt% Au-crGr com-posite, where GO concentration is 0.5 wt%. Scale bar, 50 nm (left),200 nm (right).

and 2.0) (Fig. 3(a)). The Au-crGr composite with the Auweight ratio of 0.6 wt% showed the highest signal intensity(asterisk) when compared with other modified electrodes.After finding the optimum composition of the materials,two different sizes of gold electrodes were prepared toconfirm the effects of electrode area. Electrochemical sig-nals of the DA solution with different doses were measuredfrom large and small gold electrodes, which were modi-fied with 0.6 wt% Au-crGr composite (Fig. 3(b)). Everytested electrode showed an exponential increase of elec-trochemical signal with DA concentration: These resultswere used to generate a standard curve (R2 > 0�98). TheAu-crGr composite modified small electrodes showed amuch stronger electrochemical signal per unit area com-pared with bare and modified large gold electrodes. Theseresults demonstrated that a 0.6 wt% Au-crGr compositemodified small electrode is more suitable for measuringneurotransmitters at low concentrations.A surface-enhanced Raman scattering signal can be

generated from Au-crGr composite modified electrodes

Fig. 3. Confirmation of electrochemical property of Au-crGr compositemodified electrodes. (a) Oxidation peak currents of the different weightratios of Au-crGr composites in 10 �M dopamine solution. (b) Compar-ison of different electrode properties.

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Au-Crumpled Graphene Modified Electrode to Detect Neurotransmitters based on Spectroelectrochemical Method Shin et al.ARTICLE

by nano-gaps between gold nanoparticles in compositesand the gold electrode surface. As shown in Figure 4,the graphene peak can be confirmed around 1350 cm−1

and 1580 cm−1 by SERS spectra. One peak was aroundthe 1350 cm−1 mean D-band and the other peak wasaround the 1580 cm−1 mean G-band of graphene.18 Ramanpeaks of dopamine and norepinephrine also appeared withgraphene near 1300 cm−1 and 1500 cm−1. The SERS sig-nal overlapped and amplified the signal intensity. In thisway, the strong signal from the Au-crGr composite modi-fied electrode was affected by electrode structure and wassensitive to catecholamine-type neurotransmitters.

3.3. Neurotransmitter Detection in the Cell MediumCell culture medias were collected and used for electro-chemical and component analysis. Using DPV with themodified electrode, the electrochemical signal of secretedneurotransmitters from cells was successfully detected(Fig. 5). The oxidation peak current for neurotransmittersappeared at 0.092 V in the medium sampled after cell cul-turing only, which was similar to the peak potential of DAand NE, respectively. However, there was no oxidation sig-nal for neurotransmitters with the medium sampled before

Fig. 4. Characterization of Au-crGr composite with background, DA,and NE SERS signals.

Fig. 5. Released neurotransmitters detected with DPV. Oxidation peaks(a) and Ip values (b) of before-culture and after-culture media. Data rep-resents mean ± SE of five different experiments.

cell culturing (Fig. 5(a)). The existence of neurotransmit-ters in the cell culture media was confirmed by UPLC andMS (Table I). Both before-culture and after-culture mediaincluded 1% fetal bovine serum, RA, KCl, and glucose inDMEM. In UPLC analysis with before- and after-culturemedia, both the DA and NE peaks appeared from the after-culture medium (data not shown). The concentrations ofDA and NE in the after-culture medium were found to be0.145 mg/mL and 0.042 mg/mL by the qualitative analysiswith MS. The entire process of neurotransmitter detectionwith the spectroelectrochemical method was completedwithin 5 minutes. These results suggest that neurotransmit-ters can be detected by the Au-crGr composite modifiedelectrode, without affecting cell viability or using targetingmaterials such as antibodies or aptamers.

Table I. UPLC and MS of the cell culture media were conducted toconfirm the presence of neurotransmitters. Exact mass information forDA and NE was detected with MS.

Dopamine Norepinephrine

Original MW (g/mol) 153�0869 169�0817Cell media (mg/mL) 0�145 0�042

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ARTICLE

4. CONCLUSIONThis study demonstrates the successful synthesis andapplication of Au-crGr composites to detect secretedneurotransmitters from cells. Various kinds of electrodeconditions for Au-crGr composites were synthesizedto find optimum conditions to detect neurotransmit-ters. The 0.6 wt% Au-crGr composite modified smallgold electrode exhibited outstanding spectroelectrochem-ical properties compared with the other substrates used,and successfully detected neurotransmitters from cell cul-ture medium. These results were confirmed by the con-ventional methods UPLC and MS. In conclusion, ournewly developed Au-crGr composite modified electrodescan be used in biosensors for detecting catecholamine-typeneurotransmitters.

Acknowledgments: This research was supported bythe Leading Foreign Research Institute Recruitment Pro-gram, through the National Research Foundation ofKorea (NRF), funded by the Ministry of Science, ICTand Future Planning (MSIP) (2013K1A4A3055268) andby the National Research Foundation of Korea (NRF)grant funded by the Korea government (MSIP) (no.2014R1A2A1A10051725).

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