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A novel sensitive sensor for serotonin based on high-quality of AuAg nanoalloy encapsulated graphene electrocatalyst
Author’s Accepted Manuscript A Novel Sensitive Sensor for Serotonin Based on High-quality of AuAg Nanoalloy Encapsulated Graphene Electrocatalyst Tran Duy Thanh, Jayaraman Balamurugan, Hoa Van Hien, Nam Hoon Kim, Joong Hee Lee www.elsevier.com/locate/bios
PII: DOI: Reference:
S0956-5663(17)30324-X http://dx.doi.org/10.1016/j.bios.2017.05.014 BIOS9726
To appear in: Biosensors and Bioelectronic Received date: 28 February 2017 Revised date: 26 April 2017 Accepted date: 5 May 2017 Cite this article as: Tran Duy Thanh, Jayaraman Balamurugan, Hoa Van Hien, Nam Hoon Kim and Joong Hee Lee, A Novel Sensitive Sensor for Serotonin Based on High-quality of AuAg Nanoalloy Encapsulated Graphene E l e c t r o c a t a l y s t , Biosensors and Bioelectronic, http://dx.doi.org/10.1016/j.bios.2017.05.014 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A Novel Sensitive Sensor for Serotonin Based on High-quality of AuAg Nanoalloy Encapsulated Graphene Electrocatalyst Tran Duy Thanha, Jayaraman Balamurugana, Hoa Van Hiena, Nam Hoon Kima, Joong Hee Leea,b* a
Advanced Materials Institute of BIN Convergence Technology (BK21 plus Global), Dept. of BIN
Convergence Technology, Chonbuk National University, Jeonju, Jeonbuk 54896, Republic of Korea. b
Carbon Composite Research Centre, Department of Polymer & Nanoscience and Technology,
Chonbuk National University Jeonju, Jeonbuk 54896, Republic of Korea.
*Corresponding author:
Tel.: 82-63-270-2342; Fax: 82-63-270-2341. E-mail address: [email protected] (Joong Hee Lee)
Abstract A high quality graphene-encapsulated AuAg alloy (AuAg-GR) nanohybrid with homogeneous structure and good reproducibility over a desired area was successfully fabricated. Taking benefits of the unique architecture, such nanohybrid was employed as an efficient electrocatalytst for sensing application. The AuAg-GR based sensor could sensitively detected neurotransmitter serotonin (5-HT) with wide linear detection range (2.7 nM to 4.82 M), very low detection limit (1.6 nM), negligible interference, and excellent reproducibility. In addition, AuAg-GR based sensor accurately determined 5-HT in human serum samples. This is due to the enhanced catalytic activity of GR nanosheets-encapsulated AuAg nanostructures, which possessed well monodispersion of AuAg alloy, greater electrochemical active sites, and good charge transfer 1
possibility. The obtained results imply that such nanohybrid is a potential candidate for synthesizing electrochemical sensors in requirement of high sensitivity, long-term stability, and good reproducibility. Keywords: Gold-silver alloy, graphene, encapsulation, electrocatalysts, serotonin, electrochemical sensors.
1.
Introduction Serotonin (5-hydroxytryptamine, 5-HT) is a crucial monoamine neurotransmitter existing in
the central nervous system for regulating mood, sleep, emesis, sexuality, appetite, and pain (Xue et al., 2014; Artigas et al., 1985). The irregular 5-HT concentration is related with several disorders, including depression, anxiety, and migraines, as well as toxic and potentially fatal effects (Xue et al., 2014; Artigas et al., 1985; Goyal et al., 2008). In addition, 5-HT altered levels have been reported to be associated with sudden death symptom of infant and an influence on phenomenon of natural ageing (Paterson et al., 2008). This implies that it is highly important to quickly detect 5-HT for health care and clinical treatment. However, 5-HT concentration from body fluid is 15 nM (Artigas et al., 1985) and from urine is around 295-687 nM (Huang et al., 2012); thus, the detection of 5-HT is often applied by specific techniques which are complicated strategies, high cost, and time consuming (Tekes, 2008; Umeda et al., 2005). In this regard, high quality electrocatalyst based electrochemical sensor would be a valuable clinical diagnostic tool to permit the simple, sensitive, accurate, rapid, and reliable detection of low 5-HT levels. The intense and ongoing research of novel electrocatalytic nanomaterials with specific size, shape, and self-assembled architecture is academically and commercially attractive in the 2
development of advanced functional materials (Guo and Wang, 2011; Lu et al., 2007; Chaudhuri and Paria, 2012; Lin et al., 2012). Considering outstanding catalytic properties of AuAg alloys and the specific traits of graphene (GR), such as high specific surface area, exceptional electronic conductivity, high thermal conductivity, mechanical strength, flexibility, chemical inertness, corrosion resistance, and biocompatibility, the hybridization of AuAg alloys with GR has yielded very interesting and varied applications (Roy et al., 2013; Zhu et al., 2011; Wang et al., 2013; Chen et al., 2011; Duan et al., 2015; Zhao et al., 2015; Shi et al., 2014; Neppolian et al., 2014; Atar et al., 2015), consequently enhanced catalytic activity, more active surface area, and good stability are being demonstated for such hydrid catalysts compared to monometallic nanoparticles (NPs) or monometallic NPs/GR counterparts. In bioanalytical applications, the interesting unique properties of AuAg alloy NPs including superior conductivity, excellent catalytic activity, and high chemical stability (Shi et al., 2014; Neppolian et al., 2014; Atar et al., 2015), have led to the potential development of electrocatalyst for electrochemical sensor (Pruneanu et al., 2012; Wu et al., 2013; Kim et al., 2013; Hutter et al., 2001). In this context, low Ag content along with its distribution and homogenization has strong influence on the enhanced catalytic performance of AuAg nanostructures (Stewart et al., 2008; Saha et al., 2012; Bjerneld et al., 2003; Zhang et al., 2011; Kim et al., 2005; Yang et al., 2008). In addition, the hybridization of AuAg alloy NPs with GR significantly improves the catalytic activity because the large surface area of GR can efficiently support the adsorption of organic molecules through π–π stacking and electrostatic interactions (Dutta et al., 2013; Yin et al., 2013; Villacorta et al., 2016; Ling et al., 2010; Kim et al., 2013). And an enhancement of active surface area can be more pronounced with the formation of metal NP wrapped GR nanostructures due to prevention of the aggregation and dissolution possibility of NPs (Gupta et al., 2013). Also, this unique architecture demonstrated that Gr can 3
also be an efficient conductive medium bridging for accelerating electron transter ability, thereby enhancing sensitivity of the sensor. A great challenge of AuAg-GR synthesis still lies in the requirements of purity, stability, uniformity of large area, reproducibility, relative simplicity, and easy storage. Wet-chemical syntheses have been commonly employed for metal alloy NPs/GR nanohybrid synthesis because of its simplicity, cost effective, applicable possibility for diverse types of metals (Shi et al., 2014; Neppolian et al., 2014; Atar et al., 2015; Pruneanu et al., 2012; Wu et al., 2013). However, certain impurities still remain due to the use of an external organic stabilizer, reducing agent, and polymer-based binder for synthesis and attachment on the electrode carrier during application, significantly impart the negative effects on the material’s activity. Furthermore, the common use of chemically synthesized GR (obtained from an environmentally hazardous synthesis procedure) with its large amount of defects, the restacking phenomenon, and low layer reproducibility also reduces the charge transfer properties, working stability and reproducibility of the prepared nanohybrid materials (Kim et al., 2013). Therefore, the development of a simple, clean, and eco-friendly synthetic approach for the AuAg alloy NPs/GR nanohybrid based electrocatalyst having unique nanoarchitectures, high quality and controllability, is necessary to enhance sensitivity and stability of sensor to date. In this work, we proposed a high quality of AuAg alloy NPs (12.5 nm) encapsulated in GR nanosheets with uniform structures and good reproducibility (in the number of layers during the fabrication process and homogeneous transducer coverage) as a potential electrocatalyst for electrochemical sensor. This electrocatalyst provided excellent catalytic activity and long-term stability for 5-HT detection at very low concentration.
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2. 2.1
Experimental Reagents and solutions Silver nitrate (AgNO3, 99%), hydrogen tetrachloroaurate (HAuCl4∙3H2O, 99.9%),
serotonin (5-hydroxytryptamine, 5-HT, 98%), uric acid (UA, 99%), glucose ( 99.5%), ascorbic acid (AA, 99.5%), sodium dihydrogen phosphate (NaH2PO4.H2O, 98%) and sodium monohydrogen phosphate (Na2HPO4, 99%) were purchased from Sigma Aldrich Chemicals Co. (USA). Ferric chloride (III) (FeCl3, 98%), potassium chloride (KCl, > 99.5%) and acetic acid (CH3COOH, 99.7%) were purchased by Samchun Co. (Korea). Indium doped tin-oxide (ITO) conducting glass was obtained from HS technologies (Korea). Uncoated copper foil (25 μm thickness) was delivered from Alfa Aesar Co. (USA). Ultra-pure water was provided by an EYELA Still Ace SA-2100E1 filter (Tokyo Rikakikai Co., Japan) throughout all experiments. 2.2
Synthesis of AuAg-GR nanohybrid The procedure to synthesize AuAg-GR nanohybrid began with the deposition of Au and Ag
on a Cu substrate by dipping in 1 mM HAuCl4 for 3 min and then in 2 mM AgNO3 solution for 5 min. The Cu substrate was subsequently dried and then loaded into a quartz tube of a CVD system. The quartz tube was heated at 1000 oC for 25 min under atmospheric pressure consistent with H2, Ar, and CH4 which flowed at 50, 900, and 2 sccm followed by a rapid cooling to room temperature. The AuAg-GR/Cu was then kept floating in a 0.25 M FeCl3 solution to completely etch the Cu. The obtained AuAg-GR film was carefully rinsed with distilled water, and was then transferred on to a target substrate. Finally, the AuAg-GR deposited substrate was dried at 60 oC for 2 h in a vacuum oven. The same procedure was also used to produce pristine GR (without involving Au3+ and Ag+ sources), Au-GR (without involving Ag+ source), and Ag-GR (without
5
involving Au3+ source). Scheme 1 schematically illustrates the synthesis procedure as well as applications of the AuAg-GR nanohybrid. 2.3
Material Characterization The morphology of the as-synthesized materials was studied by field-emission scanning
electron microscopy (FE-SEM) and energy dispersive X-ray analysis (EDAX) on a Supra 40 VP instrument (Zeiss, Germany). The ImageJ software (National Institutes of Health, USA) was used to calculate the size distribution of AuAg NPs at the AuAg-GR hybrid. Transmission electron microscopy (TEM) measurements were carried out on an H-7650 (Hitachi Ltd., Japan) microscope at 120 kV in Jeonju center of KBSI. Raman spectra were recorded by a HORIBA Jobin Yvon (HORIBA Scientific, USA). X-ray photoelectron spectroscopy (XPS) measurements were analyzed using a Theta Probe (Thermo Fisher Scientific Inc., USA). Atomic force microscopy (AFM) scans were performed on a Park NX10 (Park System Co., Korea). 2.4 Electrochemical characterization All electrochemical experiments were characterized with an electrochemical analyzer CHI 660D workstation (CH Instruments Inc., USA). A three-electrode electrochemical system consisting of an AuAg-GR modified ITO working electrode (AuAg-GR/ITO), a Pt wire counter electrode, and Ag/AgCl as a reference electrode was used. The catalytic performance of the AuAg-GR/ITO was evaluated towards HT-5 detection using cyclic voltammetry (CV) and amperometry techniques in 0.1 M phosphate-buffered saline (PBS) solution (pH = 7.4), which was saturated nitrogen by purging with nitrogen gas for 30 min. Amperometric measurements were performed by continuous addition of known 5-HT concentrations into a PBS solution under constant stirring, and current response was recorded when it reached the steady state. The effects of interference by several materials, including uric acid (UA), glucose, potassium ion (K+), 6
chloride ion (Cl-), and ascorbic acid (AA), on 5-HT detection were also explored through this amperometric technique. To evaluate the ability of the AuAg-GR/ITO as a potential diagnostic tool, different 5-HT concentrations in human serum samples were detected. In a particular procedure, 0.1 ml of blood serum was diluted in 25 ml of 0.1M PBS solution. Subsequently, the solution was spiked with three different known 5-HT concentrations. The recoveries of the 5-HT concentrations were carried out by measuring the current response at +0.4 V relative to the Ag/AgCl electrode.
Scheme 1. Fabrication and application of the AuAg-GR nanohybrid material. 3.
Results and discussion
3.1
Morphological studies
Figs. S1a and b show the optical images of the successfully synthesized AuAg-GR nanohybrid film with a large area and transparent possibility. Insights into the morphological and structural features of nanohybrid are provided by FE-SEM analysis. The FE-SEM images (Fig. 1a and b) 7
show that the large area AuAg-GR nanohybrid with a high density of the AuAg alloy NPs, which uniformly disperse within GR nanosheets. Interestingly, the high-magnification FE-SEM images are evidenced the encapsulation of the AuAg alloy NPs in GR nanosheets (Inset of Fig. b). This architecture is further clearly illustrated in Fig. 1c. The average diameter of AuAg NPs is around 12.5 nm along with particle range of 8-20 nm (Fig. S2). The formation of small and uniformly sized AuAg NPs resulted from the growth of the GR nanosheets which encapsulate alloy NPs during the CVD process at high temperature, leading to the prohibition of nanoparticle aggregation. This can induce enhanced interactions between GR and alloy NPs, thereby accelerating the electron transfer possibility (Liu et al., 2014). It also improved the active sites and nanohybrid’s stability by obstructing the aggregation and leaching potential of alloy NPs during operating applications (Gu et al., 2013; Wu et al., 2012; Fan et al., 2015).
Fig. 1 (a, b) FE-SEM images of the AuAg-GR nanohybrid. (c) The simulated morphology of the AuAg-GR nanohybrid. (d, e) TEM image of the AuAg-GR nanohybrid at different magnifications. 8
(f) HR-TEM images of the AuAg-GR hybrid (Inset: SAED of the AuAg-GR nanohybrid and HRTEM of a AuAg nanoparticle ). The morphology of pristine GR, Ag-GR, Au-GR, and other nanohybrids with shorter and longer deposition times in the Au3+ source for a comparison. The FE-SEM images of these samples indicate bigger particle sizes and low uniformity of metal NPs (Figs. S3 and S4). The obtained morphological results imply that the present synthesis procedure can effectively fabricate high quality, large area AuAg-GR nanohybrid films in the absence of an organic surfactant and reducing agent during the synthesis process, as well as without the use of a polymer (such as PMMA) for transfer. Further, TEM images of the AuAg-GR nanohybrid show the successful attachment of alloy NPs within the GR structure with additional features regarding size and dispersion state of the AuAg NPs. Lower magnification TEM images reveal the high density and homogeneous dispersion of AuAg NPs within GR nanosheets (Figs. 1d and e). High resolution TEM (HR-TEM) images provide additional confirmation that the AuAg NPs are encapsulated in few carbon layers (Fig. 1f and Fig. S5). The d-spacing at (111) planes of an individual NPs is 0.23 nm, revealing the formation of AuAg alloy nanostructure (Inset of Fig. 1f) (Zou et al., 2016). In addition, the selected area electron diffraction (SAED) shows a defected characteristic of GR nanosheets as well as the multicrystalline nature of AuAg NPs (Inset of Fig. 1f). High-angle annular dark-field scanning TEM (HAADF-STEM) and EDAX elemental mapping pattern was carried out to further characterize the surface morphology and positional distribution of carbon, Au and Ag in the nanohybrid. The HAADF-STEM (Fig. 2a) and EDAX area color mapping (Figs. 2b, c, and d) verify that the Au and Ag have an alloy nanostructure attaching within a large area GR structure. Furthermore, the HAADF-STEM (Fig. 2e) and EDAX elemental mapping of a 9
selected alloy nanoparticle (Figs. 2f, g, h, and k) confirm over the formation of a unique nanostructure in which AuAg NPs are wrapped by GR layer. An EDAX analysis shows the chemical composition of the AuAg-GR nanohybrid with Au/Ag atom ratio of 9/1.
Fig. 2 (a) Low resolution STEM-HADDF image of AuAg-GR. (b, c, and d) The whole area mapping analysis of the nanohybrid in a dark field showing the distribution of (b) C, (c) Au and (d) Ag. (e) High resolution STEM-HADDF image of an AuAg nanoparticle and the corresponding elemental color maps of (f) C, (g) Au, (h) Ag.
3.2 AFM, Raman, and XPS analysis
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The AFM technique was analyzed to provide characteristic in terms of the distribution, density of the NPs, and the roughness of the as-synthesized nanohybrid (Fig. S6a). The results obtained assumed that a high density of AuAg NPs was generated. Besides, a crumbled surface can be seen clearly, indicating highly rough characteristics of the nanohybrid consistent with the root mean square (RMS) roughness, around 8.4 nm, which is higher than that of other samples (Fig. S6b). A high roughness value is thought to impart important effects on the electrochemical sensing applications due to facilitattion of mass transfer ability (Leem et al., 2015; Yang et al., 2013). Raman analysis is a non-destructive and useful technique to recognize the structural features, including defects, layer number and interactions between metal/graphene nanohybrids. Figure S7 depicts the Raman spectra of the GR and AuAg-GR nanohybrid. The main features of the Raman spectrum for the GR are 2D, G, and D bands, located at 2707, 1577, and 1346 cm−1, respectively (Ferrari et al., 2006). The intensity ratios of the 2D peak to G peak (I2D/IG) and D peak to G peak (ID/IG) were found to be around 0.85 and 0.16, suggesting several layered features with a minor amount of defects on the GR surface. Compared to the pristine GR, the positions of the 2D, G, and D peaks of the AuAg-GR nanohybrid shift to slightly lower values for the wavelength. Furthermore, there was a significant increase in the ID/IG ratio (1.1) and a decrease in the I2D/IG ratio (0.44), which might be due to the incorporation of defects into the GR structure (Haniff et al., 2015). As the surface of the GR supports does contain certain defects, there might be strong chemical interactions between the deposited metal catalysts and the GR supports due to donation and transfer of electrons from metal to GR (Zheng et al., 2015).
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The surface composition and electronic structures of the AuAg-GR catalysts were analyzed by the XPS technique (Fig. 3a). Typical XPS spectra of the GR and AuAg-GR display the presence of the C1s core-level spectra at around 283.8 eV, which are correlated to the carbon element of the GR. The C1s core-level spectrum of the AuAg-GR could be reasonably deconvoluted into 3 peaks at 284.6, 285.4, and 286.5 eV, consistent with the binding energies of C=C, C-C, and C-O bonds, respectively (Fig. 3b) (Yang et al., 2015). The presence of the C-C and C-O binding energies indicates a significantly defective structure of the GR in the AuAg-GR nanohybrid (Szwarckopf et al., 2004). A defected GR structure, such as this, has been demonstrated to effectively improve the electrocatalytic activity of nanohybrid materials in electrochemical applications (Pumera, 2013). At the same time, the presence of two additional peaks of Au4f and Ag3d binding energies informs the successful introduction of Au and Ag metals within the GR structures. The Au4f spectrum consists of two pairs of doublets (Fig. 3c). The most intense doublet at 83.8 and 87.5 eV are contributed to Au 4f7/2 and Au 4f5/2, while the other weak doublet at 85.0 and 88.8 eV corresponds to Au3+4f7/2 and Au3+4f5/2 (Liu et al., 2014). The high resolution Ag3d spectrum is comprised of two components of metallic Ag 3d and Ag+3d (Fig. 3d) (An et al., 2012), which were deconvoluted into four peaks at 367.6, 373.9, 369.9, and 375.5 eV, correlating with Ag 3d5/2, Ag+3d5/2, Ag03d3/2, and Ag+3d3/2, respectively. The presence of Au3+ and Ag+ peaks might result from the charge transfer between metal parts with the GR structure (Giovannetti et al., 2008; Ren et al., 2010) as well as the slight oxidation of nanoparticles upon exposure to air (Xu et al., 2014). The electrocatalytic performance of metals is increased with the ionized state of metal atoms (Gougis et al., 2014).
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Fig. 3 (a) XPS spectra of the GR and AuAg-GR nanohybrid. High resolution XPS spectra of (b) C1s, (c) Au4f and (d) Ag3d.
3.3 Electrochemical sensing application The measurement of 5-HT is of importance because it can provide supporting information to understand the role of 5-HT in neurological disordered syndromes, thereby improving clinical treatment efficiency. For this purpose, the electrocatalytic behaviors of the AuAg-GR nanohybrid were applied to detect 5-HT in 0.1 M PBS solutions (pH =7.4) and clinical samples. Figure 4a displays CV responses of the AuAg-GR/ITO in 0.1 M PBS with absence and presence of 217 M 5-HT. The CV curve of AuAg-GR shows two peak appearing at +0.45 and +0.24 in a blank BPS solution, coressponding to the oxidation and reduction process of silver part in the alloy material, 13
respectively. The anode scan shows a high peak current observed at +0.4 V correlating with the addition of 217 M 5-HT, suggesting that electroactive 5-HT molecules are quickly adsorbed and then oxidized at the AuAg-GR/ITO surface.
Fig. 4 (a) CVs of AuAg-GR/ITO in the absence and presence of 217 M 5-HT in 0.1 M PBS solution at can rate of 50 mV·s−1. (b) CVs of bare ITO, GR/ITO, Au-GR/ITO, AuAg/ITO, and AuAg-GR/ITO in the presence of 217 M 5-HT in a 0.1 M PBS solution at can rate of 50 mV·s−1. (c) The amperometric measurement of AuAg-GR/ITO towards different concentrations of 5-HT at AuAg-GR/ITO. (d) The fitting curve of amperometric current responses vs 5-HT concentration.
Furthermore, it can be seen that the largest catalytic currents and earliest onset potentials were observed at the AuAg-GR/ITO compared with GR/ITO, Au-GR/ITO, and AuAg/ITO, 14
demonstrating much better catalytic activity of the AuAg-GR/ITO toward 5-HT than the other modified ITO electrodes (Fig. 4b and S8). This good performance can be attributed to the formation of AuAg alloy NPs encapsulated GR architecture, which produces high density and uniform dispersion of small AuAg alloy NPs due to prohibition of aggregation and dissolurion possibility, a significant alternation of electronic configuration of AuAg alloy because of atomic scale mixing effect, and high interactions between AuAg alloy and Gr nanosheets. As a result, the excellent catalytic activity, high electroactive surface area, and an improved charge transfer efficiency can be obtained at the AuAg-GR nanohybrid. The CV measurements of the AuAg-GR nanohybrid towards 5-HT detection upon different scan rates were carried out (Fig. S9). The increase of peak current along with the increased scan rate confirms a diffusion controlled mechanism of 5-HT oxidation occurring at the AuAg-GR/ITO electrode. Detection of 5-HT at the AuAg-GR/ITO was further studied by amperometric current-time measurements at +0.4 V (Fig. 4c). The oxidation currents increased gradually during successive additions of 5-HT into the stirred PBS solution. The corresponding calibration curve shows a linear detection range of 5-HT from 2.7 nM to 4.82 M (R = 0.9813) with a sensitivity of 0.766 AμM−1cm−2 (Fig. 4d). Furthermore, the limit of detection (LOD) of the AuAg-GR/ITO was found, based on the signal-to-noise ratio (S/N) of 3, to be 1.6 nM. The AuAg-GR/ITO showed higher sensitivity and much lower LOD than those of bare ITO, GR/ITO, Au-GR/ITO, and AuAg/ITO (Fig. S10). The linear detection range and LOD value of this sensor are highly comparable to those of previous reports either high-performance liquid chromatography (HPLC) method (Tekes, 2008; Umeda et al., 2005) or electrochemical sensors, as indicated in Table S1. The detection of 5-HT in PBS solution has also been analyzed by high-performance liquid chromatography (HPLC) method as a comparison (Fig. S11). The obtained results demonstrated 15
that the electrochemical 5-HT analysis with the AuAg-GR/ITO electrode is not only simple, fast, and cost effective but also good sensitivity and satisfactory LOD, which are comparative to HPLC method. The selectivity, stability, and reproducibility are also the most important analytical factors for 5HT detection. As shown in Fig. 5a, the first successive injection of 54 M 5-HT produced a remarkable increase in the current. In contrast, the subsequent additions of 0.2 mM glucose, 0.2 mM K+ and Cl-, 2 M UA, and 2 M AA only produced minor amperometric responses. This implies that AuAg-GR/ITO can successfully avoid different interferents, indicating excellent selectivity toward 5-HT detection. Besides, Fig. S12 showed the CV curves of PBS solution containing 5-HT and the mixture of 5-HT, glucose, KCl, UA and AA. It can be seen the welldefined current peak of 5-HT oxidation with the same intensity in both cases, further demonstrating effective detection of 5-HT with neglected interference from glucose, UA, KCl, and AA. Also, the fast response time of the AuAg-GR/ITO was found to be around 5 s (Inset of Fig. 5b).
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Fig. 5 (a) Amperometric response of the AuAg-GR/ITO towards 54 M 5-HT in the presence of various interferents (0.2 mM glucose, 0.2 mM K+ and Cl-, 2 M UA, and 2 M AA), (b) amperometric stability of the AuAg-GR/ITO towards 54 M 5-HT in 0.1 M PBS solution over a running time of 1500 s (inset: amperometric time response of the AuAg-GR/ITO), (c) reproducibility of the AuAg-GR/ITO with 8 different electrodes, (d) applicable ability of AuAgGR/ITO toward different 5-HT concentrations in human serum–spiked PBS solutions.
The working stability of the AuAg-GR/ITO based sensor were evaluated by amperometric response with presence of 54 M 5-HT (Fig. 5b). As a result, the current response remained at 95% of its initial value after 1000 seconds and 90% of its initial value after 1500 seconds. In addition, to evaluate the reproducibility of the AuAg-GR/ITO, eight different proposed electrodes 17
were employed to determine 5-HT (217 M) under the same operating conditions (Fig. 5c). The obtained results showed an acceptable reproducibility with an RSD% of 5.8%. The storage stability of the AuAg-GR/ITO was also demonstrated with current retention of 91% after 15 days and of 88% after 19 days, implying relative long-term storage ability of the studied sensor for sensing application (Fig. S13). In order to evaluate whether the proposed sensor may detect 5HT in an actual sample, several human serum samples were used for recovery experiments. The sensor displayed a good percentage recovery with the addition of three known concentrations of 5-HT (27, 54, and 81 M) into human serum-spiked PBS solutions (Fig. 5d). The recoveries and RSD% were found to be in the range of 91.2–98.1% and 2.5–5.7% (Table S2), respectively, which implies that our sensor shows potential for 5-HT detection in real samples. The present AuAgGR/ITO based sensor together with an available HPLC was evaluated for 5-HT detection in human urine samples by the standard addition method. Our sensor has proven to be robust and comparable accuration to detect 5-HT in comparision with HPLC analysis (Table S3). 4. Conclusion A novel and high quality electrocatalyst of uniform AuAg NPs (12.5 nm) encapsulated in GR nanosheets has been prepared towards 5-HT electrochemical detection using electroless deposition followed by CVD method for the first time The nanohybrids with homogeneous structure and good reproducibility can be synthesized over a desired area without requirement of any external organic stabilizers, reducing agents, or polymer-based binders, thereby achieving high purity and quality of the final material. The potential application of the AuAg-GR nanohybrid based binder-free sensor was well demonstrated with extreme sensitivity, a long linear detection range, very low LOD (1.6 nM), and stability towards 5-HT determination in PBS solution. Furthermore, it also displayed high accuracy in the detection of 5-HT in real samples. 18
We believe that such nanohybrid is an effective alternative for high quality fabrication of robust and reproducible sensor for electrochemical sensing application. Acknowledgements This study was supported by the Basic Research Laboratory Program (2014R1A4A1008140) and Nano-Material Technology Development Program (2016M3A7B4900117) through the National Research Foundation (NRF) funded by the Ministry of Science, ICT & Future Planning of Republic of Korea.
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Highlight
High quality of AuAg NPs-GR hybrid was successfully synthesized using electroless deposition followed by chemical vapor deposition.
It demonstrated excellent catalytic activity towards serotonin (5-HT) with wide linear 22
detection range and low detection limit.
The AuAg-GR based sensor displayed good reproducibility and stability.
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PII: DOI: Reference:
S0956-5663(17)30324-X http://dx.doi.org/10.1016/j.bios.2017.05.014 BIOS9726
To appear in: Biosensors and Bioelectronic Received date: 28 February 2017 Revised date: 26 April 2017 Accepted date: 5 May 2017 Cite this article as: Tran Duy Thanh, Jayaraman Balamurugan, Hoa Van Hien, Nam Hoon Kim and Joong Hee Lee, A Novel Sensitive Sensor for Serotonin Based on High-quality of AuAg Nanoalloy Encapsulated Graphene E l e c t r o c a t a l y s t , Biosensors and Bioelectronic, http://dx.doi.org/10.1016/j.bios.2017.05.014 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A Novel Sensitive Sensor for Serotonin Based on High-quality of AuAg Nanoalloy Encapsulated Graphene Electrocatalyst Tran Duy Thanha, Jayaraman Balamurugana, Hoa Van Hiena, Nam Hoon Kima, Joong Hee Leea,b* a
Advanced Materials Institute of BIN Convergence Technology (BK21 plus Global), Dept. of BIN
Convergence Technology, Chonbuk National University, Jeonju, Jeonbuk 54896, Republic of Korea. b
Carbon Composite Research Centre, Department of Polymer & Nanoscience and Technology,
Chonbuk National University Jeonju, Jeonbuk 54896, Republic of Korea.
*Corresponding author:
Tel.: 82-63-270-2342; Fax: 82-63-270-2341. E-mail address: [email protected] (Joong Hee Lee)
Abstract A high quality graphene-encapsulated AuAg alloy (AuAg-GR) nanohybrid with homogeneous structure and good reproducibility over a desired area was successfully fabricated. Taking benefits of the unique architecture, such nanohybrid was employed as an efficient electrocatalytst for sensing application. The AuAg-GR based sensor could sensitively detected neurotransmitter serotonin (5-HT) with wide linear detection range (2.7 nM to 4.82 M), very low detection limit (1.6 nM), negligible interference, and excellent reproducibility. In addition, AuAg-GR based sensor accurately determined 5-HT in human serum samples. This is due to the enhanced catalytic activity of GR nanosheets-encapsulated AuAg nanostructures, which possessed well monodispersion of AuAg alloy, greater electrochemical active sites, and good charge transfer 1
possibility. The obtained results imply that such nanohybrid is a potential candidate for synthesizing electrochemical sensors in requirement of high sensitivity, long-term stability, and good reproducibility. Keywords: Gold-silver alloy, graphene, encapsulation, electrocatalysts, serotonin, electrochemical sensors.
1.
Introduction Serotonin (5-hydroxytryptamine, 5-HT) is a crucial monoamine neurotransmitter existing in
the central nervous system for regulating mood, sleep, emesis, sexuality, appetite, and pain (Xue et al., 2014; Artigas et al., 1985). The irregular 5-HT concentration is related with several disorders, including depression, anxiety, and migraines, as well as toxic and potentially fatal effects (Xue et al., 2014; Artigas et al., 1985; Goyal et al., 2008). In addition, 5-HT altered levels have been reported to be associated with sudden death symptom of infant and an influence on phenomenon of natural ageing (Paterson et al., 2008). This implies that it is highly important to quickly detect 5-HT for health care and clinical treatment. However, 5-HT concentration from body fluid is 15 nM (Artigas et al., 1985) and from urine is around 295-687 nM (Huang et al., 2012); thus, the detection of 5-HT is often applied by specific techniques which are complicated strategies, high cost, and time consuming (Tekes, 2008; Umeda et al., 2005). In this regard, high quality electrocatalyst based electrochemical sensor would be a valuable clinical diagnostic tool to permit the simple, sensitive, accurate, rapid, and reliable detection of low 5-HT levels. The intense and ongoing research of novel electrocatalytic nanomaterials with specific size, shape, and self-assembled architecture is academically and commercially attractive in the 2
development of advanced functional materials (Guo and Wang, 2011; Lu et al., 2007; Chaudhuri and Paria, 2012; Lin et al., 2012). Considering outstanding catalytic properties of AuAg alloys and the specific traits of graphene (GR), such as high specific surface area, exceptional electronic conductivity, high thermal conductivity, mechanical strength, flexibility, chemical inertness, corrosion resistance, and biocompatibility, the hybridization of AuAg alloys with GR has yielded very interesting and varied applications (Roy et al., 2013; Zhu et al., 2011; Wang et al., 2013; Chen et al., 2011; Duan et al., 2015; Zhao et al., 2015; Shi et al., 2014; Neppolian et al., 2014; Atar et al., 2015), consequently enhanced catalytic activity, more active surface area, and good stability are being demonstated for such hydrid catalysts compared to monometallic nanoparticles (NPs) or monometallic NPs/GR counterparts. In bioanalytical applications, the interesting unique properties of AuAg alloy NPs including superior conductivity, excellent catalytic activity, and high chemical stability (Shi et al., 2014; Neppolian et al., 2014; Atar et al., 2015), have led to the potential development of electrocatalyst for electrochemical sensor (Pruneanu et al., 2012; Wu et al., 2013; Kim et al., 2013; Hutter et al., 2001). In this context, low Ag content along with its distribution and homogenization has strong influence on the enhanced catalytic performance of AuAg nanostructures (Stewart et al., 2008; Saha et al., 2012; Bjerneld et al., 2003; Zhang et al., 2011; Kim et al., 2005; Yang et al., 2008). In addition, the hybridization of AuAg alloy NPs with GR significantly improves the catalytic activity because the large surface area of GR can efficiently support the adsorption of organic molecules through π–π stacking and electrostatic interactions (Dutta et al., 2013; Yin et al., 2013; Villacorta et al., 2016; Ling et al., 2010; Kim et al., 2013). And an enhancement of active surface area can be more pronounced with the formation of metal NP wrapped GR nanostructures due to prevention of the aggregation and dissolution possibility of NPs (Gupta et al., 2013). Also, this unique architecture demonstrated that Gr can 3
also be an efficient conductive medium bridging for accelerating electron transter ability, thereby enhancing sensitivity of the sensor. A great challenge of AuAg-GR synthesis still lies in the requirements of purity, stability, uniformity of large area, reproducibility, relative simplicity, and easy storage. Wet-chemical syntheses have been commonly employed for metal alloy NPs/GR nanohybrid synthesis because of its simplicity, cost effective, applicable possibility for diverse types of metals (Shi et al., 2014; Neppolian et al., 2014; Atar et al., 2015; Pruneanu et al., 2012; Wu et al., 2013). However, certain impurities still remain due to the use of an external organic stabilizer, reducing agent, and polymer-based binder for synthesis and attachment on the electrode carrier during application, significantly impart the negative effects on the material’s activity. Furthermore, the common use of chemically synthesized GR (obtained from an environmentally hazardous synthesis procedure) with its large amount of defects, the restacking phenomenon, and low layer reproducibility also reduces the charge transfer properties, working stability and reproducibility of the prepared nanohybrid materials (Kim et al., 2013). Therefore, the development of a simple, clean, and eco-friendly synthetic approach for the AuAg alloy NPs/GR nanohybrid based electrocatalyst having unique nanoarchitectures, high quality and controllability, is necessary to enhance sensitivity and stability of sensor to date. In this work, we proposed a high quality of AuAg alloy NPs (12.5 nm) encapsulated in GR nanosheets with uniform structures and good reproducibility (in the number of layers during the fabrication process and homogeneous transducer coverage) as a potential electrocatalyst for electrochemical sensor. This electrocatalyst provided excellent catalytic activity and long-term stability for 5-HT detection at very low concentration.
4
2. 2.1
Experimental Reagents and solutions Silver nitrate (AgNO3, 99%), hydrogen tetrachloroaurate (HAuCl4∙3H2O, 99.9%),
serotonin (5-hydroxytryptamine, 5-HT, 98%), uric acid (UA, 99%), glucose ( 99.5%), ascorbic acid (AA, 99.5%), sodium dihydrogen phosphate (NaH2PO4.H2O, 98%) and sodium monohydrogen phosphate (Na2HPO4, 99%) were purchased from Sigma Aldrich Chemicals Co. (USA). Ferric chloride (III) (FeCl3, 98%), potassium chloride (KCl, > 99.5%) and acetic acid (CH3COOH, 99.7%) were purchased by Samchun Co. (Korea). Indium doped tin-oxide (ITO) conducting glass was obtained from HS technologies (Korea). Uncoated copper foil (25 μm thickness) was delivered from Alfa Aesar Co. (USA). Ultra-pure water was provided by an EYELA Still Ace SA-2100E1 filter (Tokyo Rikakikai Co., Japan) throughout all experiments. 2.2
Synthesis of AuAg-GR nanohybrid The procedure to synthesize AuAg-GR nanohybrid began with the deposition of Au and Ag
on a Cu substrate by dipping in 1 mM HAuCl4 for 3 min and then in 2 mM AgNO3 solution for 5 min. The Cu substrate was subsequently dried and then loaded into a quartz tube of a CVD system. The quartz tube was heated at 1000 oC for 25 min under atmospheric pressure consistent with H2, Ar, and CH4 which flowed at 50, 900, and 2 sccm followed by a rapid cooling to room temperature. The AuAg-GR/Cu was then kept floating in a 0.25 M FeCl3 solution to completely etch the Cu. The obtained AuAg-GR film was carefully rinsed with distilled water, and was then transferred on to a target substrate. Finally, the AuAg-GR deposited substrate was dried at 60 oC for 2 h in a vacuum oven. The same procedure was also used to produce pristine GR (without involving Au3+ and Ag+ sources), Au-GR (without involving Ag+ source), and Ag-GR (without
5
involving Au3+ source). Scheme 1 schematically illustrates the synthesis procedure as well as applications of the AuAg-GR nanohybrid. 2.3
Material Characterization The morphology of the as-synthesized materials was studied by field-emission scanning
electron microscopy (FE-SEM) and energy dispersive X-ray analysis (EDAX) on a Supra 40 VP instrument (Zeiss, Germany). The ImageJ software (National Institutes of Health, USA) was used to calculate the size distribution of AuAg NPs at the AuAg-GR hybrid. Transmission electron microscopy (TEM) measurements were carried out on an H-7650 (Hitachi Ltd., Japan) microscope at 120 kV in Jeonju center of KBSI. Raman spectra were recorded by a HORIBA Jobin Yvon (HORIBA Scientific, USA). X-ray photoelectron spectroscopy (XPS) measurements were analyzed using a Theta Probe (Thermo Fisher Scientific Inc., USA). Atomic force microscopy (AFM) scans were performed on a Park NX10 (Park System Co., Korea). 2.4 Electrochemical characterization All electrochemical experiments were characterized with an electrochemical analyzer CHI 660D workstation (CH Instruments Inc., USA). A three-electrode electrochemical system consisting of an AuAg-GR modified ITO working electrode (AuAg-GR/ITO), a Pt wire counter electrode, and Ag/AgCl as a reference electrode was used. The catalytic performance of the AuAg-GR/ITO was evaluated towards HT-5 detection using cyclic voltammetry (CV) and amperometry techniques in 0.1 M phosphate-buffered saline (PBS) solution (pH = 7.4), which was saturated nitrogen by purging with nitrogen gas for 30 min. Amperometric measurements were performed by continuous addition of known 5-HT concentrations into a PBS solution under constant stirring, and current response was recorded when it reached the steady state. The effects of interference by several materials, including uric acid (UA), glucose, potassium ion (K+), 6
chloride ion (Cl-), and ascorbic acid (AA), on 5-HT detection were also explored through this amperometric technique. To evaluate the ability of the AuAg-GR/ITO as a potential diagnostic tool, different 5-HT concentrations in human serum samples were detected. In a particular procedure, 0.1 ml of blood serum was diluted in 25 ml of 0.1M PBS solution. Subsequently, the solution was spiked with three different known 5-HT concentrations. The recoveries of the 5-HT concentrations were carried out by measuring the current response at +0.4 V relative to the Ag/AgCl electrode.
Scheme 1. Fabrication and application of the AuAg-GR nanohybrid material. 3.
Results and discussion
3.1
Morphological studies
Figs. S1a and b show the optical images of the successfully synthesized AuAg-GR nanohybrid film with a large area and transparent possibility. Insights into the morphological and structural features of nanohybrid are provided by FE-SEM analysis. The FE-SEM images (Fig. 1a and b) 7
show that the large area AuAg-GR nanohybrid with a high density of the AuAg alloy NPs, which uniformly disperse within GR nanosheets. Interestingly, the high-magnification FE-SEM images are evidenced the encapsulation of the AuAg alloy NPs in GR nanosheets (Inset of Fig. b). This architecture is further clearly illustrated in Fig. 1c. The average diameter of AuAg NPs is around 12.5 nm along with particle range of 8-20 nm (Fig. S2). The formation of small and uniformly sized AuAg NPs resulted from the growth of the GR nanosheets which encapsulate alloy NPs during the CVD process at high temperature, leading to the prohibition of nanoparticle aggregation. This can induce enhanced interactions between GR and alloy NPs, thereby accelerating the electron transfer possibility (Liu et al., 2014). It also improved the active sites and nanohybrid’s stability by obstructing the aggregation and leaching potential of alloy NPs during operating applications (Gu et al., 2013; Wu et al., 2012; Fan et al., 2015).
Fig. 1 (a, b) FE-SEM images of the AuAg-GR nanohybrid. (c) The simulated morphology of the AuAg-GR nanohybrid. (d, e) TEM image of the AuAg-GR nanohybrid at different magnifications. 8
(f) HR-TEM images of the AuAg-GR hybrid (Inset: SAED of the AuAg-GR nanohybrid and HRTEM of a AuAg nanoparticle ). The morphology of pristine GR, Ag-GR, Au-GR, and other nanohybrids with shorter and longer deposition times in the Au3+ source for a comparison. The FE-SEM images of these samples indicate bigger particle sizes and low uniformity of metal NPs (Figs. S3 and S4). The obtained morphological results imply that the present synthesis procedure can effectively fabricate high quality, large area AuAg-GR nanohybrid films in the absence of an organic surfactant and reducing agent during the synthesis process, as well as without the use of a polymer (such as PMMA) for transfer. Further, TEM images of the AuAg-GR nanohybrid show the successful attachment of alloy NPs within the GR structure with additional features regarding size and dispersion state of the AuAg NPs. Lower magnification TEM images reveal the high density and homogeneous dispersion of AuAg NPs within GR nanosheets (Figs. 1d and e). High resolution TEM (HR-TEM) images provide additional confirmation that the AuAg NPs are encapsulated in few carbon layers (Fig. 1f and Fig. S5). The d-spacing at (111) planes of an individual NPs is 0.23 nm, revealing the formation of AuAg alloy nanostructure (Inset of Fig. 1f) (Zou et al., 2016). In addition, the selected area electron diffraction (SAED) shows a defected characteristic of GR nanosheets as well as the multicrystalline nature of AuAg NPs (Inset of Fig. 1f). High-angle annular dark-field scanning TEM (HAADF-STEM) and EDAX elemental mapping pattern was carried out to further characterize the surface morphology and positional distribution of carbon, Au and Ag in the nanohybrid. The HAADF-STEM (Fig. 2a) and EDAX area color mapping (Figs. 2b, c, and d) verify that the Au and Ag have an alloy nanostructure attaching within a large area GR structure. Furthermore, the HAADF-STEM (Fig. 2e) and EDAX elemental mapping of a 9
selected alloy nanoparticle (Figs. 2f, g, h, and k) confirm over the formation of a unique nanostructure in which AuAg NPs are wrapped by GR layer. An EDAX analysis shows the chemical composition of the AuAg-GR nanohybrid with Au/Ag atom ratio of 9/1.
Fig. 2 (a) Low resolution STEM-HADDF image of AuAg-GR. (b, c, and d) The whole area mapping analysis of the nanohybrid in a dark field showing the distribution of (b) C, (c) Au and (d) Ag. (e) High resolution STEM-HADDF image of an AuAg nanoparticle and the corresponding elemental color maps of (f) C, (g) Au, (h) Ag.
3.2 AFM, Raman, and XPS analysis
10
The AFM technique was analyzed to provide characteristic in terms of the distribution, density of the NPs, and the roughness of the as-synthesized nanohybrid (Fig. S6a). The results obtained assumed that a high density of AuAg NPs was generated. Besides, a crumbled surface can be seen clearly, indicating highly rough characteristics of the nanohybrid consistent with the root mean square (RMS) roughness, around 8.4 nm, which is higher than that of other samples (Fig. S6b). A high roughness value is thought to impart important effects on the electrochemical sensing applications due to facilitattion of mass transfer ability (Leem et al., 2015; Yang et al., 2013). Raman analysis is a non-destructive and useful technique to recognize the structural features, including defects, layer number and interactions between metal/graphene nanohybrids. Figure S7 depicts the Raman spectra of the GR and AuAg-GR nanohybrid. The main features of the Raman spectrum for the GR are 2D, G, and D bands, located at 2707, 1577, and 1346 cm−1, respectively (Ferrari et al., 2006). The intensity ratios of the 2D peak to G peak (I2D/IG) and D peak to G peak (ID/IG) were found to be around 0.85 and 0.16, suggesting several layered features with a minor amount of defects on the GR surface. Compared to the pristine GR, the positions of the 2D, G, and D peaks of the AuAg-GR nanohybrid shift to slightly lower values for the wavelength. Furthermore, there was a significant increase in the ID/IG ratio (1.1) and a decrease in the I2D/IG ratio (0.44), which might be due to the incorporation of defects into the GR structure (Haniff et al., 2015). As the surface of the GR supports does contain certain defects, there might be strong chemical interactions between the deposited metal catalysts and the GR supports due to donation and transfer of electrons from metal to GR (Zheng et al., 2015).
11
The surface composition and electronic structures of the AuAg-GR catalysts were analyzed by the XPS technique (Fig. 3a). Typical XPS spectra of the GR and AuAg-GR display the presence of the C1s core-level spectra at around 283.8 eV, which are correlated to the carbon element of the GR. The C1s core-level spectrum of the AuAg-GR could be reasonably deconvoluted into 3 peaks at 284.6, 285.4, and 286.5 eV, consistent with the binding energies of C=C, C-C, and C-O bonds, respectively (Fig. 3b) (Yang et al., 2015). The presence of the C-C and C-O binding energies indicates a significantly defective structure of the GR in the AuAg-GR nanohybrid (Szwarckopf et al., 2004). A defected GR structure, such as this, has been demonstrated to effectively improve the electrocatalytic activity of nanohybrid materials in electrochemical applications (Pumera, 2013). At the same time, the presence of two additional peaks of Au4f and Ag3d binding energies informs the successful introduction of Au and Ag metals within the GR structures. The Au4f spectrum consists of two pairs of doublets (Fig. 3c). The most intense doublet at 83.8 and 87.5 eV are contributed to Au 4f7/2 and Au 4f5/2, while the other weak doublet at 85.0 and 88.8 eV corresponds to Au3+4f7/2 and Au3+4f5/2 (Liu et al., 2014). The high resolution Ag3d spectrum is comprised of two components of metallic Ag 3d and Ag+3d (Fig. 3d) (An et al., 2012), which were deconvoluted into four peaks at 367.6, 373.9, 369.9, and 375.5 eV, correlating with Ag 3d5/2, Ag+3d5/2, Ag03d3/2, and Ag+3d3/2, respectively. The presence of Au3+ and Ag+ peaks might result from the charge transfer between metal parts with the GR structure (Giovannetti et al., 2008; Ren et al., 2010) as well as the slight oxidation of nanoparticles upon exposure to air (Xu et al., 2014). The electrocatalytic performance of metals is increased with the ionized state of metal atoms (Gougis et al., 2014).
12
Fig. 3 (a) XPS spectra of the GR and AuAg-GR nanohybrid. High resolution XPS spectra of (b) C1s, (c) Au4f and (d) Ag3d.
3.3 Electrochemical sensing application The measurement of 5-HT is of importance because it can provide supporting information to understand the role of 5-HT in neurological disordered syndromes, thereby improving clinical treatment efficiency. For this purpose, the electrocatalytic behaviors of the AuAg-GR nanohybrid were applied to detect 5-HT in 0.1 M PBS solutions (pH =7.4) and clinical samples. Figure 4a displays CV responses of the AuAg-GR/ITO in 0.1 M PBS with absence and presence of 217 M 5-HT. The CV curve of AuAg-GR shows two peak appearing at +0.45 and +0.24 in a blank BPS solution, coressponding to the oxidation and reduction process of silver part in the alloy material, 13
respectively. The anode scan shows a high peak current observed at +0.4 V correlating with the addition of 217 M 5-HT, suggesting that electroactive 5-HT molecules are quickly adsorbed and then oxidized at the AuAg-GR/ITO surface.
Fig. 4 (a) CVs of AuAg-GR/ITO in the absence and presence of 217 M 5-HT in 0.1 M PBS solution at can rate of 50 mV·s−1. (b) CVs of bare ITO, GR/ITO, Au-GR/ITO, AuAg/ITO, and AuAg-GR/ITO in the presence of 217 M 5-HT in a 0.1 M PBS solution at can rate of 50 mV·s−1. (c) The amperometric measurement of AuAg-GR/ITO towards different concentrations of 5-HT at AuAg-GR/ITO. (d) The fitting curve of amperometric current responses vs 5-HT concentration.
Furthermore, it can be seen that the largest catalytic currents and earliest onset potentials were observed at the AuAg-GR/ITO compared with GR/ITO, Au-GR/ITO, and AuAg/ITO, 14
demonstrating much better catalytic activity of the AuAg-GR/ITO toward 5-HT than the other modified ITO electrodes (Fig. 4b and S8). This good performance can be attributed to the formation of AuAg alloy NPs encapsulated GR architecture, which produces high density and uniform dispersion of small AuAg alloy NPs due to prohibition of aggregation and dissolurion possibility, a significant alternation of electronic configuration of AuAg alloy because of atomic scale mixing effect, and high interactions between AuAg alloy and Gr nanosheets. As a result, the excellent catalytic activity, high electroactive surface area, and an improved charge transfer efficiency can be obtained at the AuAg-GR nanohybrid. The CV measurements of the AuAg-GR nanohybrid towards 5-HT detection upon different scan rates were carried out (Fig. S9). The increase of peak current along with the increased scan rate confirms a diffusion controlled mechanism of 5-HT oxidation occurring at the AuAg-GR/ITO electrode. Detection of 5-HT at the AuAg-GR/ITO was further studied by amperometric current-time measurements at +0.4 V (Fig. 4c). The oxidation currents increased gradually during successive additions of 5-HT into the stirred PBS solution. The corresponding calibration curve shows a linear detection range of 5-HT from 2.7 nM to 4.82 M (R = 0.9813) with a sensitivity of 0.766 AμM−1cm−2 (Fig. 4d). Furthermore, the limit of detection (LOD) of the AuAg-GR/ITO was found, based on the signal-to-noise ratio (S/N) of 3, to be 1.6 nM. The AuAg-GR/ITO showed higher sensitivity and much lower LOD than those of bare ITO, GR/ITO, Au-GR/ITO, and AuAg/ITO (Fig. S10). The linear detection range and LOD value of this sensor are highly comparable to those of previous reports either high-performance liquid chromatography (HPLC) method (Tekes, 2008; Umeda et al., 2005) or electrochemical sensors, as indicated in Table S1. The detection of 5-HT in PBS solution has also been analyzed by high-performance liquid chromatography (HPLC) method as a comparison (Fig. S11). The obtained results demonstrated 15
that the electrochemical 5-HT analysis with the AuAg-GR/ITO electrode is not only simple, fast, and cost effective but also good sensitivity and satisfactory LOD, which are comparative to HPLC method. The selectivity, stability, and reproducibility are also the most important analytical factors for 5HT detection. As shown in Fig. 5a, the first successive injection of 54 M 5-HT produced a remarkable increase in the current. In contrast, the subsequent additions of 0.2 mM glucose, 0.2 mM K+ and Cl-, 2 M UA, and 2 M AA only produced minor amperometric responses. This implies that AuAg-GR/ITO can successfully avoid different interferents, indicating excellent selectivity toward 5-HT detection. Besides, Fig. S12 showed the CV curves of PBS solution containing 5-HT and the mixture of 5-HT, glucose, KCl, UA and AA. It can be seen the welldefined current peak of 5-HT oxidation with the same intensity in both cases, further demonstrating effective detection of 5-HT with neglected interference from glucose, UA, KCl, and AA. Also, the fast response time of the AuAg-GR/ITO was found to be around 5 s (Inset of Fig. 5b).
16
Fig. 5 (a) Amperometric response of the AuAg-GR/ITO towards 54 M 5-HT in the presence of various interferents (0.2 mM glucose, 0.2 mM K+ and Cl-, 2 M UA, and 2 M AA), (b) amperometric stability of the AuAg-GR/ITO towards 54 M 5-HT in 0.1 M PBS solution over a running time of 1500 s (inset: amperometric time response of the AuAg-GR/ITO), (c) reproducibility of the AuAg-GR/ITO with 8 different electrodes, (d) applicable ability of AuAgGR/ITO toward different 5-HT concentrations in human serum–spiked PBS solutions.
The working stability of the AuAg-GR/ITO based sensor were evaluated by amperometric response with presence of 54 M 5-HT (Fig. 5b). As a result, the current response remained at 95% of its initial value after 1000 seconds and 90% of its initial value after 1500 seconds. In addition, to evaluate the reproducibility of the AuAg-GR/ITO, eight different proposed electrodes 17
were employed to determine 5-HT (217 M) under the same operating conditions (Fig. 5c). The obtained results showed an acceptable reproducibility with an RSD% of 5.8%. The storage stability of the AuAg-GR/ITO was also demonstrated with current retention of 91% after 15 days and of 88% after 19 days, implying relative long-term storage ability of the studied sensor for sensing application (Fig. S13). In order to evaluate whether the proposed sensor may detect 5HT in an actual sample, several human serum samples were used for recovery experiments. The sensor displayed a good percentage recovery with the addition of three known concentrations of 5-HT (27, 54, and 81 M) into human serum-spiked PBS solutions (Fig. 5d). The recoveries and RSD% were found to be in the range of 91.2–98.1% and 2.5–5.7% (Table S2), respectively, which implies that our sensor shows potential for 5-HT detection in real samples. The present AuAgGR/ITO based sensor together with an available HPLC was evaluated for 5-HT detection in human urine samples by the standard addition method. Our sensor has proven to be robust and comparable accuration to detect 5-HT in comparision with HPLC analysis (Table S3). 4. Conclusion A novel and high quality electrocatalyst of uniform AuAg NPs (12.5 nm) encapsulated in GR nanosheets has been prepared towards 5-HT electrochemical detection using electroless deposition followed by CVD method for the first time The nanohybrids with homogeneous structure and good reproducibility can be synthesized over a desired area without requirement of any external organic stabilizers, reducing agents, or polymer-based binders, thereby achieving high purity and quality of the final material. The potential application of the AuAg-GR nanohybrid based binder-free sensor was well demonstrated with extreme sensitivity, a long linear detection range, very low LOD (1.6 nM), and stability towards 5-HT determination in PBS solution. Furthermore, it also displayed high accuracy in the detection of 5-HT in real samples. 18
We believe that such nanohybrid is an effective alternative for high quality fabrication of robust and reproducible sensor for electrochemical sensing application. Acknowledgements This study was supported by the Basic Research Laboratory Program (2014R1A4A1008140) and Nano-Material Technology Development Program (2016M3A7B4900117) through the National Research Foundation (NRF) funded by the Ministry of Science, ICT & Future Planning of Republic of Korea.
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Highlight
High quality of AuAg NPs-GR hybrid was successfully synthesized using electroless deposition followed by chemical vapor deposition.
It demonstrated excellent catalytic activity towards serotonin (5-HT) with wide linear 22
detection range and low detection limit.
The AuAg-GR based sensor displayed good reproducibility and stability.
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