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Facile preparation of electroactive graphene derivative and its potential application in electrochemical detection
Accepted Manuscript Title: Facile preparation of electroactive graphene derivative and its potential application in electrochemical detection Author: Hsiao-Chien Chen Hung-Wei Yang Kuang-Hsuan Yang Ching-Hsiang Chen Chung-Che Hou Yi-Ming Tu PII: DOI: Reference:
S0925-4005(16)31461-7 http://dx.doi.org/doi:10.1016/j.snb.2016.09.044 SNB 20909
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
10-5-2016 26-8-2016 8-9-2016
Please cite this article as: Hsiao-Chien Chen, Hung-Wei Yang, Kuang-Hsuan Yang, Ching-Hsiang Chen, Chung-Che Hou, Yi-Ming Tu, Facile preparation of electroactive graphene derivative and its potential application in electrochemical detection, Sensors and Actuators B: Chemical http://dx.doi.org/10.1016/j.snb.2016.09.044 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 proof before it is published in its final 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.
Facile preparation of electroactive graphene derivative and its potential application in electrochemical detection
Hsiao-Chien Chen a, Hung-Wei Yang b, Kuang-Hsuan Yang c,*, Ching-Hsiang Chen d, Chung-Che Hou e , Yi-Ming Tu e
a
Department of Biochemistry and Molecular Cell Biology, School of Medicine, College of
Medicine, Taipei Medical University, 250, Wuxing St., Taipei 11031, Taiwan. b
Institute of Medical Science and Technology, National Sun Yat-sen University, 70, Lienhai Rd.,
Kaohsiung 80424, Taiwan. c
Department of Food and Beverage Management, Vanung University, 1, Van Nung Rd.,
Shuei-Wei Li, Chung-Li City 32061, Taiwan. d
Sustainable Energy Development Center, National Taiwan University of Science and
Technology, 43, Sec. 4, Keelung Rd., Taipei 106, Taiwan. e
Department of Chemical and Materials Engineering, Chang Gung University, 259 Wen-Hwa
1st Rd., Tao-Yuan 33302, Taiwan.
* Corresponding author: Tel: + 886-3-4515811 ext. 84609; Fax: + 886-3-4628015 E-mail addresses: [email protected] (K.-H. Yang)
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Highlights 1. Facile preparation of graphene derivative 2. Highly conjugated level of graphene derivative 3. Hydrogen peroxide and choline biosensors.
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ABSTRACT
A one-step process combining chemical modification and exfoliation for obtaining a graphene (Gs) derivative from pristine graphite is conducted through Friedel–Crafts chemical acylation. This electrophilic substitution reaction results in the carboxylic group (–COOH) of niacin transforming into an acylium ion (–C+=O), which is an active species that replaces the sp2 C–H of graphite. In addition, highly viscous polyphosphoric acid under mechanical stirring was used to exert strong shear stress on and consequently exfoliate the graphite layers. Raman spectra and absorption spectra demonstrate that the obtained graphene-1-one-pyridine (GsNc) has a highly conjugated structure that facilitates electron transfer in electrochemical processes. This novel GsNc possess high sensitivity toward H2O2 (1157.1 μA mM–1 cm–2) within a linear concentration range of 1 μM to 5 mM. The proposed enzyme-free H2O2 sensor exhibits high selectivity and excellent stability. Furthermore, a choline biosensor is developed for detecting H2O2 released in bioreactions. In this biosensor, positively charged GsNc provides optimal conditions for immobilizing choline oxidase (ChOx) through electrostatic interactions. This immobilization retains 83.4% of ChOx bioactivity and is thus excellent for choline determination. This modified electrode has high potential for application in oxidase-based biosensors.
Keywords: graphene; modification; hydrogen peroxide; choline; sensor
Running header: Electroactive graphene in the application of electrochemical sensor
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1. Introduction
Choline is synthesized by organism but additional quantities from other nutrients are essential. It is used in the synthesis of constructional components in cell membranes [1]. In addition, choline maintains the central nervous system and is essential for brain development in people of all ages [2]. A clinical study reported that choline deficiency increases the risk of neurodegenerative disorders such as Alzheimer’s and Parkinson’s diseases [3]. Therefore, determining choline and in biological samples and food is crucial. So far, numerous methods, such as those based on colorimetry [4], chemiluminescence [5], high-performance liquid chromatography, and electrochemistry [6, 7], have been developed for choline determination. Of these, the electrochemical technique has several advantages, such as high sensitivity and rapid, specific, and real-time detection [7-9]. Electrochemical choline biosensors are fabricated using choline oxidase (ChOx) modified electrodes. Because ChOx can identify choline quickly and produce hydrogen peroxide (H2O2) in the presence of oxygen, most choline biosensors function on the basis of H2O2 detection [10, 11]. Currently, electrodes used in the electrochemical detection of H2O2 are of two types: enzymatic and enzyme-free. Although enzymes possess high sensitivity and specificity, loss of content and degradation lead to decreased enzyme activity with time [12, 13]. Enzyme-free electrodes fabricated using polymers, metallic particles, or metallic oxides can preclude the problem of stability [14, 15]. The simplest alternative to using enzymatic electrodes is to electrocatalyze H2O2 directly at working potentials of 0.5–0.7 V. However, determination accuracy may be compromised at such high potentials [16].
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Graphene sheet (Gs) is a one-atom-thick sheet of hexagonally arrayed sp2-bonded carbon atoms packed in a two-dimensional honeycomb structure [17]. Owing to this unique honeycomb structure, Gs inherently possesses a high surface area for loading enzymes or metallic nanoparticles and high conductivity for electron transfer; moreover, the structure imparts excellent optical properties [18]. These favorable characteristics can enhance the performance of electrochemical sensors. Therefore, considerable efforts have been made to develop high-quality Gs [19]. Generally, Gs is obtained from graphite through liquid-phase or mechanical exfoliation [19, 20]. Further, Gs is often functionalized with hydrophilic groups to achieve excellent suspension in aqueous solution. The most widely used chemical modification of Gs to obtain a water-dispersible graphene oxide (GO) is Hummers’ method [21]. In this method, natural graphite is treated with a strong acid to generate various oxygen-containing groups such as hydroxyl groups, epoxy rings, and carboxylic groups. These groups provide active sites for further modification. However, the Hummers’ method also increases the number of defect structures and reduces conductivity, and GO preparation is a complex and time-consuming process. Carbon materials modified through Friedel–Crafts chemical acylation have been demonstrated to retain their inherent conjugated structure, thus making them suitable candidates for use in electrochemical sensors [22]. Furthermore, enzyme-free H2O2 sensors with high selectivity have been reported, and probes have been fabricated using polymers with an imine structure [23, 24]. The imine structure is sensitive to H2O2 in the presence of a carboxylic group. The combination of modified carbon materials and polymers containing an imine structure improves sensor performance in H2O2 detection, but an imine structure on the main chain of the insulating polymer hinders electron transfer.
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In this study, a simple one-step process combining exfoliation and modification to obtain water-dispersible Gs was conducted through Friedel–Crafts acylation. Through electrophilic aromatic substitution, niacin was directly bonded on Gs to form Gs-1-one-pyridine (GsNc). This novel nanocomposite has a highly conjugated structure and a H2O2-sensitive imine structure. Furthermore, a choline biosensor in which ChOx is immobilized through electrostatic interactions was developed.
2. Materials and methods
2.1. Materials
Niacin, Gs, choline, phosphorus pentoxide (P2O5), dopamine (DA) bovine serum albumin (BSA) and acetic acid (99.7%) were purchased Sigma–Aldrich. Polyphosphoric acid (PPA), glucose, uric acid (UA), ascorbic acid (AA) and H2O2 (30%) were from Showa. Choline oxidase (ChOx) from Alcaligenes with an activity of 13 U mg–1 was from Sigma. The supporting electrolyte consisted 0.2 M phosphate buffer solution (PBS). Deionized (DI) water was used in all experiments.
2.2. Preparation of GsNc
The preparation of GsNc was based on Friedel–Crafts chemical acylation. 50 mg of graphite, 150 g of PPA, 5 g of P2O5 and 3 g of niacin were placed in 250 mL flask and stirred mechanically at 130 °C under nitrogen for 24 h. Then the reactor was cooled down to room
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temperature under mechanical stirring. The viscous solution was diluted by large amount of DI water. After filtering through 0.1 μm PVDF membranes and washing by methanol and DI water 3 times, the filter cake was dispersed in DI water for further analyzing.
2.3. Preparation of the GsNc/GC electrode
The solid content (5 mg mL–1) of GsNc aqueous solution including 0.1 % of chitosan for improving adhesion was prepared. 5 µL of the solution was dropped onto the glass carbon (GC) electrode (0.071 cm2) to form a GsNc/GC electrode after drying at 50oC for 2 h.
2.4. Preparation of the ChOx/GsNc/GC electrode
The immobilization of ChOx on the GsNc was according by electrostatic interaction. 10 mg of ChOx was added into 1 ml of GsNc solution (solid content 2 mg mL–1) for mixing 15, 30, 45, 60, 75 and 90 min. Then, the free ChOx was removed by high-speed centrifugation (1000 rpm). The precipitate (ChOx/GsNc) was re-dispersed in 1 ml of aqueous solution including 0.1 % of chitosan. 5 µL of the solution was dropped onto the GC electrode to form a ChOx/GsNc/GC electrode.
2.5. Equipment and measurements
Absorption spectra were recorded on a UV2450 spectrophotometer (Shimadzu). The characteristic of GsNc were performed by Fourier transform infrared (FT-IR) spectra obtaining
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from Bruker-Tensor 27 spectrometer at a spectral resolution of 8 cm–1 and Raman spectra obtaining from UniRAM system by excitation wavelength of 532 nm as integrated by Uninanotech Co., Ltd. The exfoliated GsNc were confirmed by X-ray diffraction (XRD) patterns on a Rigaku D/Max-2B with nickel-filtered Cu-Ka radiation at a scanning rate of 1° min–1 and by atomic force microscopy (AFM, Digital Instrument D3100). Field-emission scanning electron microscopy (FE-SEM) was performed by using a Hitachi S-5000 system. Hall effect system was performed using a HL5500PC system from ACCENT company. Electrochemical measurements were performed on a CHI 660A electrochemical workstation (CH Instruments, USA) with a three-electrode system consisting of the GsNc/GC electrode, a platinum plate electrode, and an Ag/AgCl electrode as the working, counter, and reference electrodes, respectively. All electrochemical measurements were performed in 40 mL of 0.2 M phosphate buffer solution containing 0.1 ml of acetic acid at 25 °C.
3. Results and discussion
3.1. Characteristics of GsNc
The Friedel–Crafts chemical acylation of graphite through covalent bonding accompanied by exfoliation was performed in a single step (Scheme 1). The carboxylic acid of niacin was transferred to the acylium ion, an active group that substituted the sp2 C–H of graphite through electrophilic substitution. At the meantime, the solvent of PPA with high viscosity was used as the catalyst and provides strong shear stress under mechanical stirring, resulting in exfoliation of Ga. Also, the grafted pyridine on the graphene/graphite improved the dispersion and prevented
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them restacking. This chemical modification resulted in stable dispersion of GsNc in the aqueous solution (Fig. 1a, inset); this phenomenon could be demonstrated by the present charge of GsNc. Imine structures can be protonated in the presence of an acid solution. Hence, GsNc was protonated through PPA during chemical modification and purification to form a =N+– structure. This protonated imine structure exhibited a positive charge of 30.5 mV according to zeta potential measurement. Electrical repulsion prevented GsNc repacking, and GsNc dispersed well in the aqueous solution. The absorption spectrum of GsNc exhibited an absorption peak at 233 nm, corresponding to the π–π* transition of the aromatic C=C band [25]; however, no absorbance peak was observed for graphite owing to precipitation at the bottom (Fig. 1a). Compared with the absorption peak of GO obtained through the Hummers’ method, that of GsNc red-shifted by approximately 7 nm. In addition, the absorption band increased evidently at higher wavelengths, in which pure niacin exhibited a narrow band with the maximum absorption peak at 282 nm which could not contribute the higher absorption band at the long wavelength region, meaning GsNc possessed of highly conjugated level compared to GO [26,27]. In addition, the electron mobility of GsNc, which was associated with the conjugated level and conductivity was measured by Hall effect system. The electron mobility of GsNc was 2565 cm2 V–1 s–1, higher than 184 cm2 V–1 s–1 of GO. The high preservation of conjugated structure can be attributed to the Friedel–Crafts acylation. In the previous literature, the reactive site for the electrophilic aromatic substitution of 2-methylnaphthalene occurs at the structure with high electron density and the yield is approximately 80% whereas the other 20% occurs at other positions, indicating that the most of conjugated structure does not be destroyed based on this reaction [28]. Fig. 1b shows the Fourier-transform infrared spectra (FT-IR) of graphite, niacin, and GsNc. The characteristic band of graphite at 1532 cm–1 was assigned to the stretching vibration of C=C
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(νC=C). The characteristic bands of niacin were observed at 3069 cm–1 (unsaturated νC-H), 2821 cm–1 (νO-H when the hydrogen bond was present), 2433 cm–1 (νN+-H hydrogen bond salt), 1918 cm–1 (νC=N+), 1705 cm–1 (νC=O of carboxylic acid), 1596 cm–1 (νC=N and νC=C), 1420 cm–1 (symmetric stretching vibration of COO– salt), and 1313 cm–1 (νC-N and O–H deformation vibration). After Friedel–Crafts chemical acylation, peaks attributed to the carboxylic acid group at 2821, 2433, 1918, and 1420 cm–1 were not observed for GsNc, in which the broad band in the wavenumber region of 2400-2500 cm-1 could be the stretching vibration of =N+-H from the protonation of imine structure [23,29], indicating that the acid group reacted during the acylation. Instead, additional peaks at 3021 cm–1 (unsaturated νC-H), 1613 cm–1 (νC=O of ketone group), and 1530 cm–1 (νC=C and νC=N) were generated, confirming that GsNc was prepared according to the proposed mechanism. The characteristic G and D bands of graphene, which are defined by a graphitized structure and local defects at the edges of graphene and graphite platelets, can be seen in their Raman spectra (Fig. 1c). The G and D bands of graphite occur at 1336 and 1570 cm–1, respectively. Compared with the G band of graphite, that of GsNc was red-shifted to 1575 cm–1. Moreover, the intensity ratio of G to D bands (ID/IG) for GsNc was 0.85. Although this value was higher than the pure graphene without chemical modification [30,31], the ratio of ID/IG was smaller than graphene derivatives which were obtained by chemical functionalization [32,33]. The observations evidenced that the number of defects in GsNc, which were generated at the edges of small sheets and were covalently bonded to inside plane of graphene [28], was lower than that in other graphene derivatives. Furthermore, GsNc showed the smaller intensity ratio of ID/IG, the much higher electron mobility and the higher absorption band absorption band of GsNc owns higher absorption at the long wavelength region. These results were referred to the lower defect
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level of GsNc which was obtained via Friedel-Crafts chemical acylation. Based on these advantages, the prepared GsNc was favorable to development of electrochemical sensor which was involved in electron transfer.
Scheme 1 and Fig. 1 are here.
Further evidence for the exfoliation of graphene was obtained through X-ray diffraction (XRD) and atomic force microscopy (AFM). The effect of reaction time to exfoliation was demonstrated by measuring XRD. Primitive graphite exhibited two diffraction peaks at 26.7° and 54.8°, which are characteristic of typical crystalline graphite (Fig. 2a). The former peak decreased and shifted to lower angle accompanying with the decrease of latter peak after reacting 12 h, meaning that the heavily packing graphene of graphite was loosened. Furthermore, the former peak was further decreased and shifted to 25.4° which was attributed to the graphene structure [34], while the latter peak was disappeared completely after reacting 24 h. This feature did not change clearly after 36 h of reaction, indicating the optimal condition for preparation of GsNc was 24 h. This peak disappeared completely meaning that the heavy packing layers of graphite were exfoliated during Friedel–Crafts chemical acylation. The exfoliating process was attributed to the highly viscous PPA which exerted strong shear stress under mechanical stirring to loosen the heavily packing structure of graphite. Moreover, the bonded molecules at the edge of graphite forming a steric hinder to avoid the loose graphite repacking. The exfoliated GsNc layer was imaged through AFM (Fig. 2b). The thickness of exfoliated GsNc was approximately 1 nm, suggesting that monolayer GsNc was obtained. The fluctuation of AFM image might be attributed to the wrinkle-like structure of GsNc, which was corresponding to the structural
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feature of graphene. These observations confirmed that GsNc preparation and simultaneous graphite exfoliation was realized successfully through Friedel–Crafts chemical acylation.
Fig. 2 is here.
3.2. Electrochemical behavior of GsNc/GC electrode
Fig. 3a shows the cyclic voltammograms (CVs) of graphite/GC and GsNc/GC electrodes in phosphate-buffered saline (PBS) at pH 6.4 (adjusted using acetic acid). No redox peak corresponding to the graphite/GC electrode was observed. By contrast, redox peaks corresponding to the GsNc/GC electrode were observed at 0.08 V (Epa) with the anodic current of –4.9 µA and –0.08 V (Epc) with the cathodic current of 11.1 µA at a scan rate (υ) of 0.05 V s–1. It has been reported that imine structure is possessed of electroactive property due to the presence of lone pair of electrons. This electrochemical behavior is considered to consist of a single electron transfer within
.N.
and
.N+
[23]. The bonded molecules to Gs of GsNc
own the pyridine ring with imine structure. Therefore, the redox peaks of GsNc was contributed to the presence of imine structure. The peak-to-peak separation of the GsNc/GC electrode was 0.16 V, which was close to that for an electrochemically reversible reaction because of the highly conjugated structure of GsNc. To further investigate the electrochemical behavior of the GsNc/GC electrode, CVs at υ ranging from 0.05 to 1 V s–1 were investigated (Fig. 3b). With an increase in υ, the oxidative and reductive peaks did not clearly shift toward positive and negative potentials, respectively, implying that fast electron transfer occurred between the imine structure and electrode. This
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result was attributed to the presence of highly conjugated Gs, which improved electron transfer from the imine structure to the electrode. In addition, as υ increased, the peak current increased proportional to υ1/2, indicating that the electrochemical behavior of the GsNc/GC electrode belonged diffusion-controlled process (Fig. 3c) [35].
Fig. 3 is here.
3.3. GsNc/GC electrode–based H2O2 sensor
The electrochemical behavior of the GsNc/GC electrode changed in the presence of 0.2 mM H2O2 (Fig. 3a) at pH 6.4 of PBS buffer adjusted by acetic acid. The reduction current increased with decreasing oxidation current. Meanwhile, acetic acid was used as the reaction medium in this detection mechanism toward to H2O2. The peroxy acid was formed while H2O2 was added into the PBS buffer at pH 6.4. The peroxy further oxidized the imine structure of GsNc to formed N-oxide structure. This oxidized N-oxide structure of GsNc could be reverted by electrochemical reduction, resulting in increasing the reduction current [23]. Meanwhile, the oxidation to GsNc resulted in the decrease of the amount of imine structure for electrochemical oxidation, thus decreasing the oxidation current. At a potential of –0.3 V, the reduction current increased by nearly 8.7 µA. Therefore, the current response of the GsNc/GC electrode in the presence of H2O2 therefore indicates that the modified electrode is sensitive to H2O2. Fig. 4a illustrates the current–time plot for the GsNc/GC electrode corresponding to successive additions of H2O2 at −0.3 V. The current increased clearly with increasing H2O2 concentration at pH 6.4 PBS. However, this oxidative reaction did not happen in the absence of
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acetic acid (pH 7.0). Therefore, the current did not response while H2O2 was added into PBS buffer at pH 7.0. This observation is consistent with an earlier report that materials with coexistence of imine structure and carboxylic group are sensitive to H2O2 [24]. In addition, the GsNc/GC electrode exhibited a very rapid response to H2O2. The response time which was defined as the time to achieve 90% of steady-state current was less than 2.7 s. This rapid response was attributed to the presence of highly conjugated GsNc based on the results of Raman spectra, absorption spectra and electron mobility. The GsNc/GC electrode showed a linear response to H2O2 in the range of 1 μM–5 mM, with a sensitivity of 1157.1 μA mM–1 cm–2. The detection limit was 0.57 μM at a signal-to-noise ratio of 3. Comparing with H2O2 sensors based on graphene derivatives-modified electrodes was listed in Table 1. The proposed GsNc/GC electrode showed the high sensitivity and rapid response time toward to H2O2. The selectivity of the proposed GsNc/GC electrode–based H2O2 sensor was tested by injecting AA (0.025 mM), UA (0.3 mM), BSA (70 g L–1), DA (50 nM), and H2O2 (1 mM). As shown in Fig. 4b, no current response was observed after injecting these compounds except after the addition of H2O2. According to a previous study, the potentials required to electrocatalyze DA, AA, and UA are 0.2, 0.25, and 0.54 V, respectively [41]. In this study, the detecting potential was negative (−0.3 V), thus precluding the influence of such potentially interfering species. Therefore, the GsNc/GC electrode possessed high selectivity. Further, the stability of the GsNc/GC electrode was examined (Fig. 4c). In contrast to the behavior of enzyme-based sensors, which are often stored at 4 °C to retain their activities, the GsNc/GC electrode showed a variation of less than 5% in the response current to 1 mM of H2O2 even after storage in a saturated N2 atmosphere at room temperature for 10 days. It could be expected that the GsNc
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was more resistant to heat because the modifying process was performed at high temperature (130 °C). These results evidence that the proposed sensitive probe possesses high stability.
Fig. 4 and Table 1 are here.
3.4. ChOx/GsNc/GC electrode–based choline biosensor
H2O2 is also a byproduct of bioreactions catalyzed by various oxidase enzymes. To test the proposed GsNc in an oxidase-based biosensor, choline detection was performed by monitoring H2O2 produced in a choline oxidase (ChOx)–choline reaction [42].
Choline + 2O2 + H2O
Betaine + 2H2O2
(1)
The isoelectric point of ChOx is 4.8 [43]; thus, ChOx exhibits a negative charge at pH 6.5. Therefore, it can be adsorbed on positively charged GsNc through electrostatic interactions. Fig. 5a shows that the reaction time dominated the amount of ChOx immobilized on suspended GsNc. The quantity of immobilized ChOx was estimated through the examination of free ChOx by using a hydrogen peroxide assay kit in the presence of 1 mM choline after separating ChOx/GsNc and free GsNc. Herein, fresh ChOx was used as a reference. The optimal time for loading ChOx on GsNc was 60 min. The results revealed that 3.7 mg of ChOx (48.8 U) could be loaded on 1 mg of GsNc, indicating that prepared GsNc had a high surface area. In addition, the bioactivity of immobilized ChOx on the suspended ChOx/GsNc composite was estimated using a hydrogen peroxide assay kit in the presence of 1 mM choline, and approximately 83.4% of its
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bioactivity was retained. The high degree of bioactivity implies that the immobilization method based on electrostatic interactions could prevent the denaturation of ChOx. Similar to the method adopted for evaluating H2O2 detection, successive choline addition to the ChOx/GsNc/GC electrode in a saturated oxygen environment was performed at an applied potential of −0.3 V; the corresponding current–time curve is presented in Fig. 5b. The curve indicates that response currents were generated, suggesting that the ChOx/GsNc nanocomposite has strong biocatalytic activity toward choline. The response time to choline was approximately 3.9 s. This rapid response was attributed to fast electron transfer afforded by highly conjugated GsNc. The ChOx/GsNc/GC electrode responded linearly to choline concentration in the range 20 µm–2.5 mM with a sensitivity of 39.6 μA mM–1 cm–2 (Fig. 5b, inset). This detection range is suitable for the physiological choline concentration range (10–50 µM) [44]. The comparison of the performance of various choline biosensors indicated the proposed choline biosensor based on ChOx/GsNc/GC electrode exhibited the wider liner range and rapid response time due to the highly sensitive to the produced H2O2 from the choline-ChOx bioreaction (Table 2). In addition, the immobilization of ChOx on GsNc through electrostatic interactions retained the bioactivity of ChOx, which was associated with the amount of H2O2 produced. Further, the interfering test of choline biosensor based on ChOx/GsNc/GC electrode was performed by adding 100 μL of glucose (5 mM), UA (0.3 mM), AA (0.025 mM) and BSA (70 g L–1) (Fig. 5c). Excepting to the choline, no response current was observed after injecting glucose, UA, AA and BSA, meaning that the proposed choline biosensor owned high selectivity due to the negative working potential. This implies that the proposed biosensor can be an accurate and feasible sensor for choline detection in individual samples. Furthermore, the ChOx/GsNc/GC electrode retained 85.1% of
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its bioactivity toward choline after storage at 4 °C for 2 weeks, indicating that immobilized ChOx on GsNc is stable.
Fig. 5 and Table 2 are here.
4. Conclusion
We have successfully synthesized a novel graphene derivative through a one-step process that combines chemical modification and exfoliation. GsNc synthesized in this study possessed a high conjugation level, thus exhibiting rapid response in electrochemical redox reactions. Moreover, GsNc was sensitive to H2O2 in the presence of acetic acid. The proposed GsNc/GC electrode–based H2O2 sensor exhibited excellent performance in terms of sensitivity, selectivity, response time, and stability. Furthermore, a choline biosensor was developed after immobilizing ChOx on the GsNc/GC electrode through electrostatic interactions. The nondestructive immobilization preserved enzyme bioactivity. Therefore, novel GsNc prepared in this study could provide a platform for developing oxidase-based biosensors.
Acknowledgements The authors thank the Vanung University and Ministry of Science and Technology (MOST) of ROC (MOST 104-2221-E-238-007 and MOST 104-2622-E-238-004-CC3) for their financial support.
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Kuang-Hsuan Yang received the PhD degree of Chung Cheng Institute of Technology at National Defense University in 2003 and continued his postgraduate studies at the Department of Applied Science of Living in Chinese Culture University. He was a professor at the Department of Materials Science and Engineering, Vanung University. Currently he had transferred to Department of Food and Beverage Management at the same university. His research interests are noble nanoparticles, carbon materials, electrochemical sensor, SERS and food nutrition.
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Figure Captions
Scheme 1. Schematic diagram of preparing GsNc via chemical modification and exfoliation.
Fig. 1. (a) The absorption spectra of graphite, niacin, GsNc and GO in aqueous solution. Inset: Photo images of graphite, GsNc and GO in aqueous solution. (b) FT-IR spectra of graphite, niacin and GsNc. (c) Raman spectra graphite and GsNc.
Fig. 2. (a) XRD spectra of graphite and GsNc from different reaction time. (b) AFM image of the GsNc. Inset: The section analysis of one of the sheets.
Fig. 3. (a) CVs of graphite/GC and GsNc/GC (with and without H2O2) electrodes in 0.2M PBS at pH 6.4. (b) CVs of GsNc/GC electrode at various scan rates. (c) Linear relationship of peak currents vs. . 1/2
Fig. 4. (a) Current-time plot of GsNc/GC electrode with successive addition of increasing [H 2O2]. Inset: Linear dependences of response currents vs. [H2O2]. (b) Influence of interfering substances on the response currents of AA –1
(0.025 mM), UA (0.3 mM), BSA (70 g L ) and DA (50 nM) to the GsNc/GC electrode before the addition of 1mM H2O2. (c) Variation of current responses to 1 mM H2O2 versus storage time under saturated N2 and atmosphere at room temperature.
Fig. 5. (a) The quantity of immobilized ChOx on GsNc. (b) Current-time plot of ChOx/GsNc/GC electrode with successive addition of increasing [Choline] under saturated oxygen. Inset: Linear dependences of response currents –1
vs. [Choline]. (c) The interfering test of glucose (5 mM), UA (0.3 mM), AA (0.025 mM) and BSA (70 g L ) to the response of choline (1 mM) based on ChOx/GsNc/GC electrode.
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Table 1. Comparison of the performance of various H2O2 sensors. Electrode AuPd@GR/ITO AuNPs/N-GQDs/GC MNRS@NG/GS/GC Ag-AlOOH-rGO/GC His–CuNPs/RGO GsNc/GC
Linear range (mM) 0.005~11.5 0.00025~13.3 0.002~0.24 0.005~4.2 0.001~5.0 0.001~5.0
Sensitivity (µA mM–1cm–2) 186.9 186.2 707.9 115.4 424.4 1157.1
Response time (s) 3 5 5 2.7
Ref. [36] [37] [38] [39] [40] This work
Table 2. Comparison of the performance of various choline biosensors. Electrode CHOD/HRP/Fc-CRGO/GC PVA/Aunanorods/ChOx/Pt ChOx/IL/MWCNT/GC ChOx/MWCNT/ZrO2NP/GC PDDA/ChOx/ZnO/MWCNT/PG ChOx/GsNc/GC a
µA mM–1
Linear range (mM) 0.001~0.4 0.02~0.4 0.0069~0.67 0.00005~0.2 0.001~0.8 0.02~2.5
Sensitivity (µA mM–1cm–2) 15.7 2.59a 178 39.6
25
Response time (s) 8 8 4 3.9
Ref. [45] [46] [47] [48] [49] This work
Scheme 1.
26
Fig. 1
27
Fig. 2
28
Fig. 3
29
Fig. 4
30
Fig. 5
31
S0925-4005(16)31461-7 http://dx.doi.org/doi:10.1016/j.snb.2016.09.044 SNB 20909
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
10-5-2016 26-8-2016 8-9-2016
Please cite this article as: Hsiao-Chien Chen, Hung-Wei Yang, Kuang-Hsuan Yang, Ching-Hsiang Chen, Chung-Che Hou, Yi-Ming Tu, Facile preparation of electroactive graphene derivative and its potential application in electrochemical detection, Sensors and Actuators B: Chemical http://dx.doi.org/10.1016/j.snb.2016.09.044 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 proof before it is published in its final 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.
Facile preparation of electroactive graphene derivative and its potential application in electrochemical detection
Hsiao-Chien Chen a, Hung-Wei Yang b, Kuang-Hsuan Yang c,*, Ching-Hsiang Chen d, Chung-Che Hou e , Yi-Ming Tu e
a
Department of Biochemistry and Molecular Cell Biology, School of Medicine, College of
Medicine, Taipei Medical University, 250, Wuxing St., Taipei 11031, Taiwan. b
Institute of Medical Science and Technology, National Sun Yat-sen University, 70, Lienhai Rd.,
Kaohsiung 80424, Taiwan. c
Department of Food and Beverage Management, Vanung University, 1, Van Nung Rd.,
Shuei-Wei Li, Chung-Li City 32061, Taiwan. d
Sustainable Energy Development Center, National Taiwan University of Science and
Technology, 43, Sec. 4, Keelung Rd., Taipei 106, Taiwan. e
Department of Chemical and Materials Engineering, Chang Gung University, 259 Wen-Hwa
1st Rd., Tao-Yuan 33302, Taiwan.
* Corresponding author: Tel: + 886-3-4515811 ext. 84609; Fax: + 886-3-4628015 E-mail addresses: [email protected] (K.-H. Yang)
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Highlights 1. Facile preparation of graphene derivative 2. Highly conjugated level of graphene derivative 3. Hydrogen peroxide and choline biosensors.
2
ABSTRACT
A one-step process combining chemical modification and exfoliation for obtaining a graphene (Gs) derivative from pristine graphite is conducted through Friedel–Crafts chemical acylation. This electrophilic substitution reaction results in the carboxylic group (–COOH) of niacin transforming into an acylium ion (–C+=O), which is an active species that replaces the sp2 C–H of graphite. In addition, highly viscous polyphosphoric acid under mechanical stirring was used to exert strong shear stress on and consequently exfoliate the graphite layers. Raman spectra and absorption spectra demonstrate that the obtained graphene-1-one-pyridine (GsNc) has a highly conjugated structure that facilitates electron transfer in electrochemical processes. This novel GsNc possess high sensitivity toward H2O2 (1157.1 μA mM–1 cm–2) within a linear concentration range of 1 μM to 5 mM. The proposed enzyme-free H2O2 sensor exhibits high selectivity and excellent stability. Furthermore, a choline biosensor is developed for detecting H2O2 released in bioreactions. In this biosensor, positively charged GsNc provides optimal conditions for immobilizing choline oxidase (ChOx) through electrostatic interactions. This immobilization retains 83.4% of ChOx bioactivity and is thus excellent for choline determination. This modified electrode has high potential for application in oxidase-based biosensors.
Keywords: graphene; modification; hydrogen peroxide; choline; sensor
Running header: Electroactive graphene in the application of electrochemical sensor
3
1. Introduction
Choline is synthesized by organism but additional quantities from other nutrients are essential. It is used in the synthesis of constructional components in cell membranes [1]. In addition, choline maintains the central nervous system and is essential for brain development in people of all ages [2]. A clinical study reported that choline deficiency increases the risk of neurodegenerative disorders such as Alzheimer’s and Parkinson’s diseases [3]. Therefore, determining choline and in biological samples and food is crucial. So far, numerous methods, such as those based on colorimetry [4], chemiluminescence [5], high-performance liquid chromatography, and electrochemistry [6, 7], have been developed for choline determination. Of these, the electrochemical technique has several advantages, such as high sensitivity and rapid, specific, and real-time detection [7-9]. Electrochemical choline biosensors are fabricated using choline oxidase (ChOx) modified electrodes. Because ChOx can identify choline quickly and produce hydrogen peroxide (H2O2) in the presence of oxygen, most choline biosensors function on the basis of H2O2 detection [10, 11]. Currently, electrodes used in the electrochemical detection of H2O2 are of two types: enzymatic and enzyme-free. Although enzymes possess high sensitivity and specificity, loss of content and degradation lead to decreased enzyme activity with time [12, 13]. Enzyme-free electrodes fabricated using polymers, metallic particles, or metallic oxides can preclude the problem of stability [14, 15]. The simplest alternative to using enzymatic electrodes is to electrocatalyze H2O2 directly at working potentials of 0.5–0.7 V. However, determination accuracy may be compromised at such high potentials [16].
5
Graphene sheet (Gs) is a one-atom-thick sheet of hexagonally arrayed sp2-bonded carbon atoms packed in a two-dimensional honeycomb structure [17]. Owing to this unique honeycomb structure, Gs inherently possesses a high surface area for loading enzymes or metallic nanoparticles and high conductivity for electron transfer; moreover, the structure imparts excellent optical properties [18]. These favorable characteristics can enhance the performance of electrochemical sensors. Therefore, considerable efforts have been made to develop high-quality Gs [19]. Generally, Gs is obtained from graphite through liquid-phase or mechanical exfoliation [19, 20]. Further, Gs is often functionalized with hydrophilic groups to achieve excellent suspension in aqueous solution. The most widely used chemical modification of Gs to obtain a water-dispersible graphene oxide (GO) is Hummers’ method [21]. In this method, natural graphite is treated with a strong acid to generate various oxygen-containing groups such as hydroxyl groups, epoxy rings, and carboxylic groups. These groups provide active sites for further modification. However, the Hummers’ method also increases the number of defect structures and reduces conductivity, and GO preparation is a complex and time-consuming process. Carbon materials modified through Friedel–Crafts chemical acylation have been demonstrated to retain their inherent conjugated structure, thus making them suitable candidates for use in electrochemical sensors [22]. Furthermore, enzyme-free H2O2 sensors with high selectivity have been reported, and probes have been fabricated using polymers with an imine structure [23, 24]. The imine structure is sensitive to H2O2 in the presence of a carboxylic group. The combination of modified carbon materials and polymers containing an imine structure improves sensor performance in H2O2 detection, but an imine structure on the main chain of the insulating polymer hinders electron transfer.
6
In this study, a simple one-step process combining exfoliation and modification to obtain water-dispersible Gs was conducted through Friedel–Crafts acylation. Through electrophilic aromatic substitution, niacin was directly bonded on Gs to form Gs-1-one-pyridine (GsNc). This novel nanocomposite has a highly conjugated structure and a H2O2-sensitive imine structure. Furthermore, a choline biosensor in which ChOx is immobilized through electrostatic interactions was developed.
2. Materials and methods
2.1. Materials
Niacin, Gs, choline, phosphorus pentoxide (P2O5), dopamine (DA) bovine serum albumin (BSA) and acetic acid (99.7%) were purchased Sigma–Aldrich. Polyphosphoric acid (PPA), glucose, uric acid (UA), ascorbic acid (AA) and H2O2 (30%) were from Showa. Choline oxidase (ChOx) from Alcaligenes with an activity of 13 U mg–1 was from Sigma. The supporting electrolyte consisted 0.2 M phosphate buffer solution (PBS). Deionized (DI) water was used in all experiments.
2.2. Preparation of GsNc
The preparation of GsNc was based on Friedel–Crafts chemical acylation. 50 mg of graphite, 150 g of PPA, 5 g of P2O5 and 3 g of niacin were placed in 250 mL flask and stirred mechanically at 130 °C under nitrogen for 24 h. Then the reactor was cooled down to room
7
temperature under mechanical stirring. The viscous solution was diluted by large amount of DI water. After filtering through 0.1 μm PVDF membranes and washing by methanol and DI water 3 times, the filter cake was dispersed in DI water for further analyzing.
2.3. Preparation of the GsNc/GC electrode
The solid content (5 mg mL–1) of GsNc aqueous solution including 0.1 % of chitosan for improving adhesion was prepared. 5 µL of the solution was dropped onto the glass carbon (GC) electrode (0.071 cm2) to form a GsNc/GC electrode after drying at 50oC for 2 h.
2.4. Preparation of the ChOx/GsNc/GC electrode
The immobilization of ChOx on the GsNc was according by electrostatic interaction. 10 mg of ChOx was added into 1 ml of GsNc solution (solid content 2 mg mL–1) for mixing 15, 30, 45, 60, 75 and 90 min. Then, the free ChOx was removed by high-speed centrifugation (1000 rpm). The precipitate (ChOx/GsNc) was re-dispersed in 1 ml of aqueous solution including 0.1 % of chitosan. 5 µL of the solution was dropped onto the GC electrode to form a ChOx/GsNc/GC electrode.
2.5. Equipment and measurements
Absorption spectra were recorded on a UV2450 spectrophotometer (Shimadzu). The characteristic of GsNc were performed by Fourier transform infrared (FT-IR) spectra obtaining
8
from Bruker-Tensor 27 spectrometer at a spectral resolution of 8 cm–1 and Raman spectra obtaining from UniRAM system by excitation wavelength of 532 nm as integrated by Uninanotech Co., Ltd. The exfoliated GsNc were confirmed by X-ray diffraction (XRD) patterns on a Rigaku D/Max-2B with nickel-filtered Cu-Ka radiation at a scanning rate of 1° min–1 and by atomic force microscopy (AFM, Digital Instrument D3100). Field-emission scanning electron microscopy (FE-SEM) was performed by using a Hitachi S-5000 system. Hall effect system was performed using a HL5500PC system from ACCENT company. Electrochemical measurements were performed on a CHI 660A electrochemical workstation (CH Instruments, USA) with a three-electrode system consisting of the GsNc/GC electrode, a platinum plate electrode, and an Ag/AgCl electrode as the working, counter, and reference electrodes, respectively. All electrochemical measurements were performed in 40 mL of 0.2 M phosphate buffer solution containing 0.1 ml of acetic acid at 25 °C.
3. Results and discussion
3.1. Characteristics of GsNc
The Friedel–Crafts chemical acylation of graphite through covalent bonding accompanied by exfoliation was performed in a single step (Scheme 1). The carboxylic acid of niacin was transferred to the acylium ion, an active group that substituted the sp2 C–H of graphite through electrophilic substitution. At the meantime, the solvent of PPA with high viscosity was used as the catalyst and provides strong shear stress under mechanical stirring, resulting in exfoliation of Ga. Also, the grafted pyridine on the graphene/graphite improved the dispersion and prevented
9
them restacking. This chemical modification resulted in stable dispersion of GsNc in the aqueous solution (Fig. 1a, inset); this phenomenon could be demonstrated by the present charge of GsNc. Imine structures can be protonated in the presence of an acid solution. Hence, GsNc was protonated through PPA during chemical modification and purification to form a =N+– structure. This protonated imine structure exhibited a positive charge of 30.5 mV according to zeta potential measurement. Electrical repulsion prevented GsNc repacking, and GsNc dispersed well in the aqueous solution. The absorption spectrum of GsNc exhibited an absorption peak at 233 nm, corresponding to the π–π* transition of the aromatic C=C band [25]; however, no absorbance peak was observed for graphite owing to precipitation at the bottom (Fig. 1a). Compared with the absorption peak of GO obtained through the Hummers’ method, that of GsNc red-shifted by approximately 7 nm. In addition, the absorption band increased evidently at higher wavelengths, in which pure niacin exhibited a narrow band with the maximum absorption peak at 282 nm which could not contribute the higher absorption band at the long wavelength region, meaning GsNc possessed of highly conjugated level compared to GO [26,27]. In addition, the electron mobility of GsNc, which was associated with the conjugated level and conductivity was measured by Hall effect system. The electron mobility of GsNc was 2565 cm2 V–1 s–1, higher than 184 cm2 V–1 s–1 of GO. The high preservation of conjugated structure can be attributed to the Friedel–Crafts acylation. In the previous literature, the reactive site for the electrophilic aromatic substitution of 2-methylnaphthalene occurs at the structure with high electron density and the yield is approximately 80% whereas the other 20% occurs at other positions, indicating that the most of conjugated structure does not be destroyed based on this reaction [28]. Fig. 1b shows the Fourier-transform infrared spectra (FT-IR) of graphite, niacin, and GsNc. The characteristic band of graphite at 1532 cm–1 was assigned to the stretching vibration of C=C
10
(νC=C). The characteristic bands of niacin were observed at 3069 cm–1 (unsaturated νC-H), 2821 cm–1 (νO-H when the hydrogen bond was present), 2433 cm–1 (νN+-H hydrogen bond salt), 1918 cm–1 (νC=N+), 1705 cm–1 (νC=O of carboxylic acid), 1596 cm–1 (νC=N and νC=C), 1420 cm–1 (symmetric stretching vibration of COO– salt), and 1313 cm–1 (νC-N and O–H deformation vibration). After Friedel–Crafts chemical acylation, peaks attributed to the carboxylic acid group at 2821, 2433, 1918, and 1420 cm–1 were not observed for GsNc, in which the broad band in the wavenumber region of 2400-2500 cm-1 could be the stretching vibration of =N+-H from the protonation of imine structure [23,29], indicating that the acid group reacted during the acylation. Instead, additional peaks at 3021 cm–1 (unsaturated νC-H), 1613 cm–1 (νC=O of ketone group), and 1530 cm–1 (νC=C and νC=N) were generated, confirming that GsNc was prepared according to the proposed mechanism. The characteristic G and D bands of graphene, which are defined by a graphitized structure and local defects at the edges of graphene and graphite platelets, can be seen in their Raman spectra (Fig. 1c). The G and D bands of graphite occur at 1336 and 1570 cm–1, respectively. Compared with the G band of graphite, that of GsNc was red-shifted to 1575 cm–1. Moreover, the intensity ratio of G to D bands (ID/IG) for GsNc was 0.85. Although this value was higher than the pure graphene without chemical modification [30,31], the ratio of ID/IG was smaller than graphene derivatives which were obtained by chemical functionalization [32,33]. The observations evidenced that the number of defects in GsNc, which were generated at the edges of small sheets and were covalently bonded to inside plane of graphene [28], was lower than that in other graphene derivatives. Furthermore, GsNc showed the smaller intensity ratio of ID/IG, the much higher electron mobility and the higher absorption band absorption band of GsNc owns higher absorption at the long wavelength region. These results were referred to the lower defect
11
level of GsNc which was obtained via Friedel-Crafts chemical acylation. Based on these advantages, the prepared GsNc was favorable to development of electrochemical sensor which was involved in electron transfer.
Scheme 1 and Fig. 1 are here.
Further evidence for the exfoliation of graphene was obtained through X-ray diffraction (XRD) and atomic force microscopy (AFM). The effect of reaction time to exfoliation was demonstrated by measuring XRD. Primitive graphite exhibited two diffraction peaks at 26.7° and 54.8°, which are characteristic of typical crystalline graphite (Fig. 2a). The former peak decreased and shifted to lower angle accompanying with the decrease of latter peak after reacting 12 h, meaning that the heavily packing graphene of graphite was loosened. Furthermore, the former peak was further decreased and shifted to 25.4° which was attributed to the graphene structure [34], while the latter peak was disappeared completely after reacting 24 h. This feature did not change clearly after 36 h of reaction, indicating the optimal condition for preparation of GsNc was 24 h. This peak disappeared completely meaning that the heavy packing layers of graphite were exfoliated during Friedel–Crafts chemical acylation. The exfoliating process was attributed to the highly viscous PPA which exerted strong shear stress under mechanical stirring to loosen the heavily packing structure of graphite. Moreover, the bonded molecules at the edge of graphite forming a steric hinder to avoid the loose graphite repacking. The exfoliated GsNc layer was imaged through AFM (Fig. 2b). The thickness of exfoliated GsNc was approximately 1 nm, suggesting that monolayer GsNc was obtained. The fluctuation of AFM image might be attributed to the wrinkle-like structure of GsNc, which was corresponding to the structural
12
feature of graphene. These observations confirmed that GsNc preparation and simultaneous graphite exfoliation was realized successfully through Friedel–Crafts chemical acylation.
Fig. 2 is here.
3.2. Electrochemical behavior of GsNc/GC electrode
Fig. 3a shows the cyclic voltammograms (CVs) of graphite/GC and GsNc/GC electrodes in phosphate-buffered saline (PBS) at pH 6.4 (adjusted using acetic acid). No redox peak corresponding to the graphite/GC electrode was observed. By contrast, redox peaks corresponding to the GsNc/GC electrode were observed at 0.08 V (Epa) with the anodic current of –4.9 µA and –0.08 V (Epc) with the cathodic current of 11.1 µA at a scan rate (υ) of 0.05 V s–1. It has been reported that imine structure is possessed of electroactive property due to the presence of lone pair of electrons. This electrochemical behavior is considered to consist of a single electron transfer within
.N.
and
.N+
[23]. The bonded molecules to Gs of GsNc
own the pyridine ring with imine structure. Therefore, the redox peaks of GsNc was contributed to the presence of imine structure. The peak-to-peak separation of the GsNc/GC electrode was 0.16 V, which was close to that for an electrochemically reversible reaction because of the highly conjugated structure of GsNc. To further investigate the electrochemical behavior of the GsNc/GC electrode, CVs at υ ranging from 0.05 to 1 V s–1 were investigated (Fig. 3b). With an increase in υ, the oxidative and reductive peaks did not clearly shift toward positive and negative potentials, respectively, implying that fast electron transfer occurred between the imine structure and electrode. This
13
result was attributed to the presence of highly conjugated Gs, which improved electron transfer from the imine structure to the electrode. In addition, as υ increased, the peak current increased proportional to υ1/2, indicating that the electrochemical behavior of the GsNc/GC electrode belonged diffusion-controlled process (Fig. 3c) [35].
Fig. 3 is here.
3.3. GsNc/GC electrode–based H2O2 sensor
The electrochemical behavior of the GsNc/GC electrode changed in the presence of 0.2 mM H2O2 (Fig. 3a) at pH 6.4 of PBS buffer adjusted by acetic acid. The reduction current increased with decreasing oxidation current. Meanwhile, acetic acid was used as the reaction medium in this detection mechanism toward to H2O2. The peroxy acid was formed while H2O2 was added into the PBS buffer at pH 6.4. The peroxy further oxidized the imine structure of GsNc to formed N-oxide structure. This oxidized N-oxide structure of GsNc could be reverted by electrochemical reduction, resulting in increasing the reduction current [23]. Meanwhile, the oxidation to GsNc resulted in the decrease of the amount of imine structure for electrochemical oxidation, thus decreasing the oxidation current. At a potential of –0.3 V, the reduction current increased by nearly 8.7 µA. Therefore, the current response of the GsNc/GC electrode in the presence of H2O2 therefore indicates that the modified electrode is sensitive to H2O2. Fig. 4a illustrates the current–time plot for the GsNc/GC electrode corresponding to successive additions of H2O2 at −0.3 V. The current increased clearly with increasing H2O2 concentration at pH 6.4 PBS. However, this oxidative reaction did not happen in the absence of
14
acetic acid (pH 7.0). Therefore, the current did not response while H2O2 was added into PBS buffer at pH 7.0. This observation is consistent with an earlier report that materials with coexistence of imine structure and carboxylic group are sensitive to H2O2 [24]. In addition, the GsNc/GC electrode exhibited a very rapid response to H2O2. The response time which was defined as the time to achieve 90% of steady-state current was less than 2.7 s. This rapid response was attributed to the presence of highly conjugated GsNc based on the results of Raman spectra, absorption spectra and electron mobility. The GsNc/GC electrode showed a linear response to H2O2 in the range of 1 μM–5 mM, with a sensitivity of 1157.1 μA mM–1 cm–2. The detection limit was 0.57 μM at a signal-to-noise ratio of 3. Comparing with H2O2 sensors based on graphene derivatives-modified electrodes was listed in Table 1. The proposed GsNc/GC electrode showed the high sensitivity and rapid response time toward to H2O2. The selectivity of the proposed GsNc/GC electrode–based H2O2 sensor was tested by injecting AA (0.025 mM), UA (0.3 mM), BSA (70 g L–1), DA (50 nM), and H2O2 (1 mM). As shown in Fig. 4b, no current response was observed after injecting these compounds except after the addition of H2O2. According to a previous study, the potentials required to electrocatalyze DA, AA, and UA are 0.2, 0.25, and 0.54 V, respectively [41]. In this study, the detecting potential was negative (−0.3 V), thus precluding the influence of such potentially interfering species. Therefore, the GsNc/GC electrode possessed high selectivity. Further, the stability of the GsNc/GC electrode was examined (Fig. 4c). In contrast to the behavior of enzyme-based sensors, which are often stored at 4 °C to retain their activities, the GsNc/GC electrode showed a variation of less than 5% in the response current to 1 mM of H2O2 even after storage in a saturated N2 atmosphere at room temperature for 10 days. It could be expected that the GsNc
15
was more resistant to heat because the modifying process was performed at high temperature (130 °C). These results evidence that the proposed sensitive probe possesses high stability.
Fig. 4 and Table 1 are here.
3.4. ChOx/GsNc/GC electrode–based choline biosensor
H2O2 is also a byproduct of bioreactions catalyzed by various oxidase enzymes. To test the proposed GsNc in an oxidase-based biosensor, choline detection was performed by monitoring H2O2 produced in a choline oxidase (ChOx)–choline reaction [42].
Choline + 2O2 + H2O
Betaine + 2H2O2
(1)
The isoelectric point of ChOx is 4.8 [43]; thus, ChOx exhibits a negative charge at pH 6.5. Therefore, it can be adsorbed on positively charged GsNc through electrostatic interactions. Fig. 5a shows that the reaction time dominated the amount of ChOx immobilized on suspended GsNc. The quantity of immobilized ChOx was estimated through the examination of free ChOx by using a hydrogen peroxide assay kit in the presence of 1 mM choline after separating ChOx/GsNc and free GsNc. Herein, fresh ChOx was used as a reference. The optimal time for loading ChOx on GsNc was 60 min. The results revealed that 3.7 mg of ChOx (48.8 U) could be loaded on 1 mg of GsNc, indicating that prepared GsNc had a high surface area. In addition, the bioactivity of immobilized ChOx on the suspended ChOx/GsNc composite was estimated using a hydrogen peroxide assay kit in the presence of 1 mM choline, and approximately 83.4% of its
16
bioactivity was retained. The high degree of bioactivity implies that the immobilization method based on electrostatic interactions could prevent the denaturation of ChOx. Similar to the method adopted for evaluating H2O2 detection, successive choline addition to the ChOx/GsNc/GC electrode in a saturated oxygen environment was performed at an applied potential of −0.3 V; the corresponding current–time curve is presented in Fig. 5b. The curve indicates that response currents were generated, suggesting that the ChOx/GsNc nanocomposite has strong biocatalytic activity toward choline. The response time to choline was approximately 3.9 s. This rapid response was attributed to fast electron transfer afforded by highly conjugated GsNc. The ChOx/GsNc/GC electrode responded linearly to choline concentration in the range 20 µm–2.5 mM with a sensitivity of 39.6 μA mM–1 cm–2 (Fig. 5b, inset). This detection range is suitable for the physiological choline concentration range (10–50 µM) [44]. The comparison of the performance of various choline biosensors indicated the proposed choline biosensor based on ChOx/GsNc/GC electrode exhibited the wider liner range and rapid response time due to the highly sensitive to the produced H2O2 from the choline-ChOx bioreaction (Table 2). In addition, the immobilization of ChOx on GsNc through electrostatic interactions retained the bioactivity of ChOx, which was associated with the amount of H2O2 produced. Further, the interfering test of choline biosensor based on ChOx/GsNc/GC electrode was performed by adding 100 μL of glucose (5 mM), UA (0.3 mM), AA (0.025 mM) and BSA (70 g L–1) (Fig. 5c). Excepting to the choline, no response current was observed after injecting glucose, UA, AA and BSA, meaning that the proposed choline biosensor owned high selectivity due to the negative working potential. This implies that the proposed biosensor can be an accurate and feasible sensor for choline detection in individual samples. Furthermore, the ChOx/GsNc/GC electrode retained 85.1% of
17
its bioactivity toward choline after storage at 4 °C for 2 weeks, indicating that immobilized ChOx on GsNc is stable.
Fig. 5 and Table 2 are here.
4. Conclusion
We have successfully synthesized a novel graphene derivative through a one-step process that combines chemical modification and exfoliation. GsNc synthesized in this study possessed a high conjugation level, thus exhibiting rapid response in electrochemical redox reactions. Moreover, GsNc was sensitive to H2O2 in the presence of acetic acid. The proposed GsNc/GC electrode–based H2O2 sensor exhibited excellent performance in terms of sensitivity, selectivity, response time, and stability. Furthermore, a choline biosensor was developed after immobilizing ChOx on the GsNc/GC electrode through electrostatic interactions. The nondestructive immobilization preserved enzyme bioactivity. Therefore, novel GsNc prepared in this study could provide a platform for developing oxidase-based biosensors.
Acknowledgements The authors thank the Vanung University and Ministry of Science and Technology (MOST) of ROC (MOST 104-2221-E-238-007 and MOST 104-2622-E-238-004-CC3) for their financial support.
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Kuang-Hsuan Yang received the PhD degree of Chung Cheng Institute of Technology at National Defense University in 2003 and continued his postgraduate studies at the Department of Applied Science of Living in Chinese Culture University. He was a professor at the Department of Materials Science and Engineering, Vanung University. Currently he had transferred to Department of Food and Beverage Management at the same university. His research interests are noble nanoparticles, carbon materials, electrochemical sensor, SERS and food nutrition.
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Figure Captions
Scheme 1. Schematic diagram of preparing GsNc via chemical modification and exfoliation.
Fig. 1. (a) The absorption spectra of graphite, niacin, GsNc and GO in aqueous solution. Inset: Photo images of graphite, GsNc and GO in aqueous solution. (b) FT-IR spectra of graphite, niacin and GsNc. (c) Raman spectra graphite and GsNc.
Fig. 2. (a) XRD spectra of graphite and GsNc from different reaction time. (b) AFM image of the GsNc. Inset: The section analysis of one of the sheets.
Fig. 3. (a) CVs of graphite/GC and GsNc/GC (with and without H2O2) electrodes in 0.2M PBS at pH 6.4. (b) CVs of GsNc/GC electrode at various scan rates. (c) Linear relationship of peak currents vs. . 1/2
Fig. 4. (a) Current-time plot of GsNc/GC electrode with successive addition of increasing [H 2O2]. Inset: Linear dependences of response currents vs. [H2O2]. (b) Influence of interfering substances on the response currents of AA –1
(0.025 mM), UA (0.3 mM), BSA (70 g L ) and DA (50 nM) to the GsNc/GC electrode before the addition of 1mM H2O2. (c) Variation of current responses to 1 mM H2O2 versus storage time under saturated N2 and atmosphere at room temperature.
Fig. 5. (a) The quantity of immobilized ChOx on GsNc. (b) Current-time plot of ChOx/GsNc/GC electrode with successive addition of increasing [Choline] under saturated oxygen. Inset: Linear dependences of response currents –1
vs. [Choline]. (c) The interfering test of glucose (5 mM), UA (0.3 mM), AA (0.025 mM) and BSA (70 g L ) to the response of choline (1 mM) based on ChOx/GsNc/GC electrode.
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Table 1. Comparison of the performance of various H2O2 sensors. Electrode AuPd@GR/ITO AuNPs/N-GQDs/GC MNRS@NG/GS/GC Ag-AlOOH-rGO/GC His–CuNPs/RGO GsNc/GC
Linear range (mM) 0.005~11.5 0.00025~13.3 0.002~0.24 0.005~4.2 0.001~5.0 0.001~5.0
Sensitivity (µA mM–1cm–2) 186.9 186.2 707.9 115.4 424.4 1157.1
Response time (s) 3 5 5 2.7
Ref. [36] [37] [38] [39] [40] This work
Table 2. Comparison of the performance of various choline biosensors. Electrode CHOD/HRP/Fc-CRGO/GC PVA/Aunanorods/ChOx/Pt ChOx/IL/MWCNT/GC ChOx/MWCNT/ZrO2NP/GC PDDA/ChOx/ZnO/MWCNT/PG ChOx/GsNc/GC a
µA mM–1
Linear range (mM) 0.001~0.4 0.02~0.4 0.0069~0.67 0.00005~0.2 0.001~0.8 0.02~2.5
Sensitivity (µA mM–1cm–2) 15.7 2.59a 178 39.6
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Response time (s) 8 8 4 3.9
Ref. [45] [46] [47] [48] [49] This work
Scheme 1.
26
Fig. 1
27
Fig. 2
28
Fig. 3
29
Fig. 4
30
Fig. 5
31