Appl Biochem Biotechnol DOI 10.1007/s12010-014-0986-z
Sensitive and Reliable Ascorbic Acid Sensing by Lanthanum Oxide/Reduced Graphene Oxide Nanocomposite Navin Kumar Mogha & Vikrant Sahu & Meenakshi Sharma & Raj Kishore Sharma & Dhanraj T. Masram
Received: 15 January 2014 / Accepted: 19 May 2014 # Springer Science+Business Media New York 2014
Abstract A simple strategy for the detection and estimation of ascorbic acid (AA), using lanthanum oxide–reduced graphene oxide nanocomposite (LO/RGO) on indium tin oxide (ITO) substrate, is reported. LO/RGO displays high catalytic activity toward the oxidation of AA, and the synergism between lanthanum oxide and reduced graphene oxide was attributed to the successful and efficient detection. Detection mechanism and sensing efficacy of LO/ RGO nanocomposite are investigated by electrochemical techniques. Chronoamperometric results under optimal conditions show a linear response range from 14 to 100 μM for AA detection. Commercially available vitamin C tablets were also analyzed using the proposed LO/RGO sensor, and the remarkable recovery percentage (97.64–99.7) shows the potential application in AA detection. Keywords Lanthanum oxide . Reduced graphene nanocomposite . Ascorbic acid . Electrochemical sensor
Introduction Ascorbic acid (AA, vitamin C) is a very important constituent of our food, and plays a key role in our biological metabolism. It plays essential role in healing of injuries, cell development, collagen synthesis, blood vessel formation, and prevention of common cold, treatment of infertility, mental illness, and cancer [1–3]. It is also widely used as an antioxidant in food and beverages. Although AA is found in both plants and animal tissues, however, our bodies do not to synthesize it; hence, in daily diet, a normal human require about 70–90 mg of AA intake. Inadequate intake may cause scurvy, gingival bleeding, and on the other hand, excessive intake leads to urinary stones, stomachache, etc. [4]. It is therefore desirable to N. K. Mogha : V. Sahu : R. K. Sharma (*) : D. T. Masram (*) Department of Chemistry, University of Delhi, Delhi 110007, India e-mail: [email protected] e-mail: [email protected] M. Sharma Dr. B.R. Ambedkar Center for Biomedical Research, University of Delhi, Delhi 110007, India
Appl Biochem Biotechnol
determine the AA concentration with high selectivity and sensitivity for food safety issue. AA determination has been reported by various methods such as electrophoresis [5], fluorescence [6], chemiluminescence [7], liquid chromatography [8], and electrochemical methods [9–13]. Among these, electrochemical method has shown a viable, simple, and cost-effective detection with high sensitivity. Despite several advantages, high overpotential and electrode fouling are a challenge in the electrochemical detection of AA [14, 15]. Furthermore, the electrochemical oxidation of uric acid and dopamine gives overlapping peaks with AA, leading to ambiguous selectivity and reproducibility. Unlike the bulk or thin films, the application of nanomaterials leads to improved performance in sensing materials. In designing and development of electrochemical sensors, bare electrodes have been modified with nanomaterials and are used as the platform for electrochemical reactions. Thus, the synthesis of novel nanomaterials for efficient sensing is most necessary, challenging as well attractive. Nanocomposites consisting of nanocarbons and metal nanoparticles have shown interesting catalytic, electronic, and optical properties [16]. Since its discovery [17], graphene has attracted great attention as an attractive material with strong mechanical strength, high thermal conductivity, excellent electric conductivity, and very large theoretical surface area, 2,603 m2g−1 [18, 19]. Unique properties, like high electro catalysis, have been observed by modifying the graphene oxide or graphene surface with metal nanoparticles. Lanthanum, a rare earth metal, has shown extraordinary catalytic properties, with applications in environmental chemistry and other catalytic reactions [20, 21]. In addition, the lanthanide ion has tendency to bind with oxygen containing functional groups [22]. These characteristics prompt us to synthesize lanthanum oxide–reduced graphene nanocomposite and use it as the platform for electrochemical sensing of AA as represent in Fig. 1.
Materials and Methods Materials and Reagents Graphite powder (~200 mesh, 99.9 %) was purchased from Alfa Aesar, India; La(NO3)3·6H2O was obtained from Merck Millipore, India; poly(ethylene glycol), MW~35,000, was purchased from Sigma-Aldrich, USA; ascorbic acid, extra pure, was purchased from Sisco Research Laboratories (SRL), India, All other materials used were of high quality.
Fig. 1 Schematic representation of electrocatalytic oxidation of ascorbic acid
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Deionized water was used throughout this study. Phosphate buffered solutions (PBS) (pH 6.0) were prepared using 0.1 M Na2HPO4, 0.1 M NaH2PO4, and 0.1 M KCl. X-ray diffraction pattern of LO/RGO nanocomposite was recorded using X-ray diffractometer (model no. D8 DISCOVER). Morphological characterization was carried out using Zeies Ultra 55 field emission scanning electron microscope (FESEM). Electrochemical sensing tests of the LO/ RGO electrodes were carried out using CH electrochemical workstation model no. 604D and 660D. Fourier transform infrared spectroscopy results were obtained using PerkinElmer spectrum BX model. Preparation of Graphene Oxide Graphene oxide (GO) was prepared by the modified Hummers method [23]. Briefly, 9:1 mixture of concentrated H2SO4/H3PO4 was added to a mixture of graphite flakes and KMnO4. The reaction mixture was then heated to 50 °C and stirred for 12 h. Then, the reaction mixture was cooled to room temperature and poured onto ice with 30 % H2O2. Mixture was then sifted through a metal (300 μm) sieve and filtered through polyester fiber. Filtrate was centrifuged (4,000 rpm for 4 h), and the supernatant was decanted. The remaining solid material was then washed in succession of water followed by 30 % HCl and ethanol. Mixture was sifted through the US Standard Testing Sieve and then filtered through polyester fiber during each wash. The filtrate was centrifuged (4,000 rpm for 4 h) and the supernatant decanted. The obtained material coagulated with diethyl ether, and the resulting suspension was filtered through a PTFE membrane (0.45 μm), and vacuum-dried overnight. Preparation of LO/RG Nanocomposite Lanthanum oxide–graphene nanocomposite (LO/RGO) was synthesized by the following method; in the first step, about 20 mg of La(NO3)3·6H2O and 0.2 g of poly(ethylene glycol) were mixed in 20 ml DI water and ultrasonicated for about 1 h, and to this, 200 mg graphene oxide was added and further ultrasonicated for 30 min; formed suspension was loaded into a 50 mL Teflonlined autoclave. Finally, the autoclave was sealed and maintained at 170 °C for 24 h. The autoclave was then allowed to cool down to room temperature naturally. The precipitate was filtered off, washed with absolute ethanol and DI water several times, and then dried in air at 80 °C for 1 hr followed by the annealing at 250 °C for 3 h, resulting in desired product. Fabrication of Electrode Indium tin oxide (ITO)-coated glass was used as the substrate for electrode preparation; firstly, it was cleaned by a hydrolyzing solution consisting of deionized water, ammonia solution, and hydrogen peroxide (6:1:1 ratio), by dipping it in for 40 min at 80 °C, and left for drying in a vacuum. Afterwards, about 20 mg of LO/RGO nanocomposite was dispersed in isopropanol with 5 wt% nafion binder and spray deposited on ITO glass (1 cm2 area), and left for drying at 60 °C for 4 h. Electrochemical Measurements Electrochemical characterizations were performed using CHI 604D and CHI 660D electrochemical workstation. Conventional three-electrode system, comprising LO/RGO nanocomposite over ITO as a working electrode, Pt wire as an auxiliary electrode, and saturated Ag/AgCl as a reference, were used. All the measurements were done at room
Appl Biochem Biotechnol Fig. 2 UV–visible spectra of (a) GO, (b) LO/RGO in ethanol
temperature in a three-electrode system assembly, with 0.1 M PBS solution at pH 6.0. Potential range in cyclic voltammetric analysis was optimized from −0.2 to 1.1 V. Amperometric study was carried out at potential of 0.27 V, and the ascorbic acid was added in different steps from 10 to 300 μM.
Result and Discussion Characterization of LO/RGO Nanocomposite UV–Vis spectra of GO and LO/RGO are shown in Fig. 2, exhibiting a maximum absorption peak at about 240 nm, corresponding to π–π* transition of aromatic C–C bonds. In LO/RGO, spectrum peak at 245 nm corresponds to absorption by ionic oxide of lanthanum [24], La2O3. The shoulder at 270 nm is red shifted due to the reduction of GO to RGO. Figure 3 shows the FT-IR spectra for graphene oxide (GO) and LO/RGO nanocomposites. Compared to GO, the peak position and intensity of LO/RGO changed considerably. The bands at 1,631 and 1,385 cm−1 attributed to carboxy asymmetric and symmetric telescopic vibration of GO shifted to 1,564 and 1,452 cm−1 after coordination with La3+. In addition, another peak at 1,243 cm−1 due to C–O stretch of epoxide is seen shifted to 1,157 cm−1. These Fig. 3 FTIR spectra of (a) LO/RGO nanocomposites and (b) GO
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Fig. 4 Raman spectra of LO/RGO and GO
results indicate the La3+ bonding with carboxyl group of graphene oxide to form LO/RGO is complex. Raman spectra of GO and LO/RGO in Fig. 4 show the D and G bands of carbon. The Dband is attributed to the disordered structures or defects in the curved graphene nanosheets, and the G-band is related to the phonons propagating along the graphitic structures [25]. Their intensity ratio is used to predict the defect in graphene samples. Reduction of GO to RGO introduces more disorderness in RGO nanosheets, which is associated with the increase in intensity ratio of D- to G-band. This increase in intensity ratio can be seen in the spectra of LO/ RGO which also indicates a good reduction of GO to form LO/RGO.
Fig. 5 FESEM images of (a) GO and (b) LO/RGO showing small spherical lanthanum oxide nanoparticles embedded over RGO
Appl Biochem Biotechnol Fig. 6 XRD spectra of LO/RGO and GO
The field emission scanning electron micrograph of pure GO and LO/RGO clearly distinguishes the LO/RGO nanocomposite with well-dispersed decorated RGO with La2O3 nodular grains (40–50 nm) (Fig. 5). These La2O3 nanospheres are anchored over RGO surface through oxygen surface groups, and therefore, overrule the possibility of relative movement among the grains and forming clusters affecting performance in redox cycling. LO/RGO X-ray diffraction pattern in Fig. 6 shows characteristic peak at 2θ=30° of 101 plane of La2O3. The peaks at 2θ=26 and 42° are characteristic peaks of reduced graphene oxide (RGO). Also, the characteristic peaks at 2θ=10.2 and 42o are also seen in the diffraction peak pattern of GO. All these peaks confirm the simultaneous formation of LO/RGO composite. Electrocatalytic Oxidation of AA The oxidation of AA at LO/RGO/ITO electrode was studied by cyclic voltammetry (CV) in 0.1 M PBS solution containing 100 μM AA, and was compared with the bare RGO/ITO
Fig. 7 CVof (a) LO/RGO/ITO electrode and bare RGO/ITO electrode, containing 100 μM ascorbic acid, (b) (a) LO/RGO/ITO without ascorbic acid, (b) with 200 μM ascorbic acid, in 0.1 M PBS at pH 6.0
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Fig. 8 a Scan rate dependence of LO/RGO/ITO, in 0.1 M PBS containing 100 μM AA. (b) Peak current variation with scan rate
electrode under same conditions, shown in Fig. 7. As seen, LO/RGO/ITO electrode exhibited an electrocatalytical oxidation response toward AA, in the form of an anodic peak at about 0.2 V. CV plots of LO/RGO/ITO electrode with and without ascorbic Acid suggest that peak current increase many folds in the presence of ascorbic acid. It is known that AA oxidation proceeds via covalent linkage and the electron transfer kinetics is sensitive to the electrode surface characteristics [26]. In this report, catalytic activity of the lanthanum oxide nanoparticles is responsible for such oxidation. In addition, the high density of edge plane-like offers some favorable sites for transferring the electron to biomolecules, which would facilitate/accelerate the electron transfer between electrode surface and electroactive species in solution [27]. Effect of Scan Rate Figure 8a shows the CVs of LO/RGO/ITO electrode in the presence of 100 μM AA at various scan rates. Obviously, both the oxidation peak current and oxidation peak potential were increased with scan rate. This suggests a kinetic limitation in the reaction between redox sites of the LO/RGO/ITO electrode and AA. Meanwhile, the anodic peak current is proportional to the scan rate (Fig. 8b), indicating a typical behavior for a mass transfer controlled reaction, following the regression equation of y=163.464+8.389x, with R2 being 0.994.
Fig. 9 a CV showing response of LO/RGO/ITO electrodes at different concentrations of AA, ranging from 0 to 100 μM, in 0.1 M PBS solution at pH 6.0. b Peak current variations w.r.t. concentrations of AA
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Fig. 10 a Amperometric response of LO/RGO/ITO electrode to the successive addition of AA, in 0.1 M PBS solution at pH 6.0. b Plot of electrocatalytic current of AA vs. its concentrations
Determination of Ascorbic Acid Figure 9a depicts the cyclic voltammetry (CV) responses of LO/RGO-modified ITO electrode toward different concentrations of AA. The LO/RGO/ITO voltammogram exhibits clear peaks of AA oxidation at 0.27 V. With the increase in AA concentration, oxidation increases and thus the peak current due to AA increases. Peak currents increased linearly (up to 50 μM only) on increasing the concentration, with regression equation y=114.1661+10.679x, and correlation coefficient R2 =0.994 as shown in Fig. 9b. The current deviates from linearity at higher AA concentrations, i.e., more than 50 μM concentration, most possibly due to the saturation of active sites at the surface of electrode and the passivation of the electrode and/or the formation of 2,3-diketogluconic acid, as this is known to occur in alkaline media [28]. Amperometric Study of Ascorbic Acid The amperometric response of the LO/RGO/ITO electrode to successive additions of AA was further evaluated under optimized conditions. Figure 10 shows the amperometric current–time response of AA at 0.2 V. As illustrated, upon addition of an aliquot of AA to the stirred PBS, the oxidation current increased steeply and reached a steady-state current. The amperometric signal showed a good linear correlation to AA concentration in the range from 14.4 μM to Fig. 11 Amperometric response of LO/RGO/ITO electrode to the successive addition of AA, glucose (Glu), NaCl, L-cysteine (Cys) in 0.1 M PBS solution at pH 6.0
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Table 1 Determination of AA in vitamin c tablets
S. No.
Content (μM)
Added (μM)
Recovered (μM)
Standard deviation
Recovery %
1
11.4
50
61.26
0.305
99.7
2
22.8
50
72.16
0.208
99.12
3
32.4
50
80.46
0.568
97.64
0.1 mM. The linear regression equation was expressed y=1.7515+0.173x with a correlation coefficient of R2 =0.981. Interference Studies In order to investigate the selectivity of the LO/RGO/ITO electrode, several compounds from common co-existing substances were investigated by detecting the current response of the modified electrode to AA, using chronoamperometry technique. Figure 11 indicated that the change in current due to interference species (glucose (200 μM), NaCl (500 μM), and Lcysteine (200 μM)) is negligible. Real Sample Analysis Commercially available vitamin C tablets were analyzed to study the applicability of the LO/ RGO/ITO electrode. First, vitamin C tablets were dissolved in PBS (pH 6.0). In the analysis, the standard addition method was applied, by which a known amount of AA in PBS (pH 6.0)
Fig. 12 a, b shows the×100 images of culture media of control and LO/RGO nanocomposite. c, d shows the high magnification (×400 image) of control and LO/RGO nanocomposite
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was added into the test solution. The recovery for the determination of AA was found in the range of 97.64–99.7 % for three samples, as shown in Table 1. Each experiment was performed in three replicates and then mean was calculated to determine the recovered percentage. Results indicate that the LO/RGO/ITO electrode is very much useful for the practical determination of AA in real samples. Bio-compatibility Assay To check the bio-compatibility of LO/RGO nanocomposite, 1 mg of LO/RGO nanocomposite was dispersed in 1 ml of incomplete Dulbecco’s modified eagle medium to achieve a stock concentration of 1 mg/ml. The final concentration assayed was 100–200 μg/ml. Briefly, the SiHa cells were seeded in T.25 culture flask and incubated at 37 °C with 5 % CO2. The cells were treated after 24 h of incubation with LO/RGO nanocomposite and appropriate control for another 48 h to observe the cell morphology and growth of culture. The treated and control flasks were photographed at×100 and×400 magnification under a Nikon inverted microscope. The treated and control SiHa cultures observed under×100 magnification showed no significant change in growth percentage. The confluency of cells after treatment was similar to the control flask (i.e., 70–80 % confluent culture). However, to check the morphology of cells in treated culture, the flasks were observed under×400 magnification (Fig. 12). Both cultures showed similar morphology, size, and adherent property, suggesting that LO/RGO nanocomposite is bio-compatible and is not leading to any abrupt change in morphology of cells or leading to any cell death. Further, MTT assay was also performed to check the cell viability and score any cell death. The result showed no significant cell death in treated cultures as compared to control culture.
Conclusion We have demonstrated LO/RGO/ITO as an efficient, selective, and sensitive electrode for ascorbic acid sensing. High catalytic activity of LO/RGO toward the oxidation of AA, and the synergistic effects between lanthanum oxide and reduced graphene oxide were the main attributes of the successful detection of ascorbic acid. Moreover, the proposed method was applied to the determination of AA in real samples with remarkable efficient results. Thus, it is believed that this proposed nanocomposite, LO/RGO, can provide a novel platform for the construction of sensitive electrochemical sensors for ascorbic acid. Acknowledgments We greatly acknowledge Department of Science and Technology (SERB), India for funding through the project no-SR/FT/CS-123/2010 dated 08/02/2012. We are also Thankful to the CSIR (Council of Scientific & Industrial Research) FOR Senior research fellowship to VS.
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Sensitive and Reliable Ascorbic Acid Sensing by Lanthanum Oxide/Reduced Graphene Oxide Nanocomposite Navin Kumar Mogha & Vikrant Sahu & Meenakshi Sharma & Raj Kishore Sharma & Dhanraj T. Masram
Received: 15 January 2014 / Accepted: 19 May 2014 # Springer Science+Business Media New York 2014
Abstract A simple strategy for the detection and estimation of ascorbic acid (AA), using lanthanum oxide–reduced graphene oxide nanocomposite (LO/RGO) on indium tin oxide (ITO) substrate, is reported. LO/RGO displays high catalytic activity toward the oxidation of AA, and the synergism between lanthanum oxide and reduced graphene oxide was attributed to the successful and efficient detection. Detection mechanism and sensing efficacy of LO/ RGO nanocomposite are investigated by electrochemical techniques. Chronoamperometric results under optimal conditions show a linear response range from 14 to 100 μM for AA detection. Commercially available vitamin C tablets were also analyzed using the proposed LO/RGO sensor, and the remarkable recovery percentage (97.64–99.7) shows the potential application in AA detection. Keywords Lanthanum oxide . Reduced graphene nanocomposite . Ascorbic acid . Electrochemical sensor
Introduction Ascorbic acid (AA, vitamin C) is a very important constituent of our food, and plays a key role in our biological metabolism. It plays essential role in healing of injuries, cell development, collagen synthesis, blood vessel formation, and prevention of common cold, treatment of infertility, mental illness, and cancer [1–3]. It is also widely used as an antioxidant in food and beverages. Although AA is found in both plants and animal tissues, however, our bodies do not to synthesize it; hence, in daily diet, a normal human require about 70–90 mg of AA intake. Inadequate intake may cause scurvy, gingival bleeding, and on the other hand, excessive intake leads to urinary stones, stomachache, etc. [4]. It is therefore desirable to N. K. Mogha : V. Sahu : R. K. Sharma (*) : D. T. Masram (*) Department of Chemistry, University of Delhi, Delhi 110007, India e-mail: [email protected] e-mail: [email protected] M. Sharma Dr. B.R. Ambedkar Center for Biomedical Research, University of Delhi, Delhi 110007, India
Appl Biochem Biotechnol
determine the AA concentration with high selectivity and sensitivity for food safety issue. AA determination has been reported by various methods such as electrophoresis [5], fluorescence [6], chemiluminescence [7], liquid chromatography [8], and electrochemical methods [9–13]. Among these, electrochemical method has shown a viable, simple, and cost-effective detection with high sensitivity. Despite several advantages, high overpotential and electrode fouling are a challenge in the electrochemical detection of AA [14, 15]. Furthermore, the electrochemical oxidation of uric acid and dopamine gives overlapping peaks with AA, leading to ambiguous selectivity and reproducibility. Unlike the bulk or thin films, the application of nanomaterials leads to improved performance in sensing materials. In designing and development of electrochemical sensors, bare electrodes have been modified with nanomaterials and are used as the platform for electrochemical reactions. Thus, the synthesis of novel nanomaterials for efficient sensing is most necessary, challenging as well attractive. Nanocomposites consisting of nanocarbons and metal nanoparticles have shown interesting catalytic, electronic, and optical properties [16]. Since its discovery [17], graphene has attracted great attention as an attractive material with strong mechanical strength, high thermal conductivity, excellent electric conductivity, and very large theoretical surface area, 2,603 m2g−1 [18, 19]. Unique properties, like high electro catalysis, have been observed by modifying the graphene oxide or graphene surface with metal nanoparticles. Lanthanum, a rare earth metal, has shown extraordinary catalytic properties, with applications in environmental chemistry and other catalytic reactions [20, 21]. In addition, the lanthanide ion has tendency to bind with oxygen containing functional groups [22]. These characteristics prompt us to synthesize lanthanum oxide–reduced graphene nanocomposite and use it as the platform for electrochemical sensing of AA as represent in Fig. 1.
Materials and Methods Materials and Reagents Graphite powder (~200 mesh, 99.9 %) was purchased from Alfa Aesar, India; La(NO3)3·6H2O was obtained from Merck Millipore, India; poly(ethylene glycol), MW~35,000, was purchased from Sigma-Aldrich, USA; ascorbic acid, extra pure, was purchased from Sisco Research Laboratories (SRL), India, All other materials used were of high quality.
Fig. 1 Schematic representation of electrocatalytic oxidation of ascorbic acid
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Deionized water was used throughout this study. Phosphate buffered solutions (PBS) (pH 6.0) were prepared using 0.1 M Na2HPO4, 0.1 M NaH2PO4, and 0.1 M KCl. X-ray diffraction pattern of LO/RGO nanocomposite was recorded using X-ray diffractometer (model no. D8 DISCOVER). Morphological characterization was carried out using Zeies Ultra 55 field emission scanning electron microscope (FESEM). Electrochemical sensing tests of the LO/ RGO electrodes were carried out using CH electrochemical workstation model no. 604D and 660D. Fourier transform infrared spectroscopy results were obtained using PerkinElmer spectrum BX model. Preparation of Graphene Oxide Graphene oxide (GO) was prepared by the modified Hummers method [23]. Briefly, 9:1 mixture of concentrated H2SO4/H3PO4 was added to a mixture of graphite flakes and KMnO4. The reaction mixture was then heated to 50 °C and stirred for 12 h. Then, the reaction mixture was cooled to room temperature and poured onto ice with 30 % H2O2. Mixture was then sifted through a metal (300 μm) sieve and filtered through polyester fiber. Filtrate was centrifuged (4,000 rpm for 4 h), and the supernatant was decanted. The remaining solid material was then washed in succession of water followed by 30 % HCl and ethanol. Mixture was sifted through the US Standard Testing Sieve and then filtered through polyester fiber during each wash. The filtrate was centrifuged (4,000 rpm for 4 h) and the supernatant decanted. The obtained material coagulated with diethyl ether, and the resulting suspension was filtered through a PTFE membrane (0.45 μm), and vacuum-dried overnight. Preparation of LO/RG Nanocomposite Lanthanum oxide–graphene nanocomposite (LO/RGO) was synthesized by the following method; in the first step, about 20 mg of La(NO3)3·6H2O and 0.2 g of poly(ethylene glycol) were mixed in 20 ml DI water and ultrasonicated for about 1 h, and to this, 200 mg graphene oxide was added and further ultrasonicated for 30 min; formed suspension was loaded into a 50 mL Teflonlined autoclave. Finally, the autoclave was sealed and maintained at 170 °C for 24 h. The autoclave was then allowed to cool down to room temperature naturally. The precipitate was filtered off, washed with absolute ethanol and DI water several times, and then dried in air at 80 °C for 1 hr followed by the annealing at 250 °C for 3 h, resulting in desired product. Fabrication of Electrode Indium tin oxide (ITO)-coated glass was used as the substrate for electrode preparation; firstly, it was cleaned by a hydrolyzing solution consisting of deionized water, ammonia solution, and hydrogen peroxide (6:1:1 ratio), by dipping it in for 40 min at 80 °C, and left for drying in a vacuum. Afterwards, about 20 mg of LO/RGO nanocomposite was dispersed in isopropanol with 5 wt% nafion binder and spray deposited on ITO glass (1 cm2 area), and left for drying at 60 °C for 4 h. Electrochemical Measurements Electrochemical characterizations were performed using CHI 604D and CHI 660D electrochemical workstation. Conventional three-electrode system, comprising LO/RGO nanocomposite over ITO as a working electrode, Pt wire as an auxiliary electrode, and saturated Ag/AgCl as a reference, were used. All the measurements were done at room
Appl Biochem Biotechnol Fig. 2 UV–visible spectra of (a) GO, (b) LO/RGO in ethanol
temperature in a three-electrode system assembly, with 0.1 M PBS solution at pH 6.0. Potential range in cyclic voltammetric analysis was optimized from −0.2 to 1.1 V. Amperometric study was carried out at potential of 0.27 V, and the ascorbic acid was added in different steps from 10 to 300 μM.
Result and Discussion Characterization of LO/RGO Nanocomposite UV–Vis spectra of GO and LO/RGO are shown in Fig. 2, exhibiting a maximum absorption peak at about 240 nm, corresponding to π–π* transition of aromatic C–C bonds. In LO/RGO, spectrum peak at 245 nm corresponds to absorption by ionic oxide of lanthanum [24], La2O3. The shoulder at 270 nm is red shifted due to the reduction of GO to RGO. Figure 3 shows the FT-IR spectra for graphene oxide (GO) and LO/RGO nanocomposites. Compared to GO, the peak position and intensity of LO/RGO changed considerably. The bands at 1,631 and 1,385 cm−1 attributed to carboxy asymmetric and symmetric telescopic vibration of GO shifted to 1,564 and 1,452 cm−1 after coordination with La3+. In addition, another peak at 1,243 cm−1 due to C–O stretch of epoxide is seen shifted to 1,157 cm−1. These Fig. 3 FTIR spectra of (a) LO/RGO nanocomposites and (b) GO
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Fig. 4 Raman spectra of LO/RGO and GO
results indicate the La3+ bonding with carboxyl group of graphene oxide to form LO/RGO is complex. Raman spectra of GO and LO/RGO in Fig. 4 show the D and G bands of carbon. The Dband is attributed to the disordered structures or defects in the curved graphene nanosheets, and the G-band is related to the phonons propagating along the graphitic structures [25]. Their intensity ratio is used to predict the defect in graphene samples. Reduction of GO to RGO introduces more disorderness in RGO nanosheets, which is associated with the increase in intensity ratio of D- to G-band. This increase in intensity ratio can be seen in the spectra of LO/ RGO which also indicates a good reduction of GO to form LO/RGO.
Fig. 5 FESEM images of (a) GO and (b) LO/RGO showing small spherical lanthanum oxide nanoparticles embedded over RGO
Appl Biochem Biotechnol Fig. 6 XRD spectra of LO/RGO and GO
The field emission scanning electron micrograph of pure GO and LO/RGO clearly distinguishes the LO/RGO nanocomposite with well-dispersed decorated RGO with La2O3 nodular grains (40–50 nm) (Fig. 5). These La2O3 nanospheres are anchored over RGO surface through oxygen surface groups, and therefore, overrule the possibility of relative movement among the grains and forming clusters affecting performance in redox cycling. LO/RGO X-ray diffraction pattern in Fig. 6 shows characteristic peak at 2θ=30° of 101 plane of La2O3. The peaks at 2θ=26 and 42° are characteristic peaks of reduced graphene oxide (RGO). Also, the characteristic peaks at 2θ=10.2 and 42o are also seen in the diffraction peak pattern of GO. All these peaks confirm the simultaneous formation of LO/RGO composite. Electrocatalytic Oxidation of AA The oxidation of AA at LO/RGO/ITO electrode was studied by cyclic voltammetry (CV) in 0.1 M PBS solution containing 100 μM AA, and was compared with the bare RGO/ITO
Fig. 7 CVof (a) LO/RGO/ITO electrode and bare RGO/ITO electrode, containing 100 μM ascorbic acid, (b) (a) LO/RGO/ITO without ascorbic acid, (b) with 200 μM ascorbic acid, in 0.1 M PBS at pH 6.0
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Fig. 8 a Scan rate dependence of LO/RGO/ITO, in 0.1 M PBS containing 100 μM AA. (b) Peak current variation with scan rate
electrode under same conditions, shown in Fig. 7. As seen, LO/RGO/ITO electrode exhibited an electrocatalytical oxidation response toward AA, in the form of an anodic peak at about 0.2 V. CV plots of LO/RGO/ITO electrode with and without ascorbic Acid suggest that peak current increase many folds in the presence of ascorbic acid. It is known that AA oxidation proceeds via covalent linkage and the electron transfer kinetics is sensitive to the electrode surface characteristics [26]. In this report, catalytic activity of the lanthanum oxide nanoparticles is responsible for such oxidation. In addition, the high density of edge plane-like offers some favorable sites for transferring the electron to biomolecules, which would facilitate/accelerate the electron transfer between electrode surface and electroactive species in solution [27]. Effect of Scan Rate Figure 8a shows the CVs of LO/RGO/ITO electrode in the presence of 100 μM AA at various scan rates. Obviously, both the oxidation peak current and oxidation peak potential were increased with scan rate. This suggests a kinetic limitation in the reaction between redox sites of the LO/RGO/ITO electrode and AA. Meanwhile, the anodic peak current is proportional to the scan rate (Fig. 8b), indicating a typical behavior for a mass transfer controlled reaction, following the regression equation of y=163.464+8.389x, with R2 being 0.994.
Fig. 9 a CV showing response of LO/RGO/ITO electrodes at different concentrations of AA, ranging from 0 to 100 μM, in 0.1 M PBS solution at pH 6.0. b Peak current variations w.r.t. concentrations of AA
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Fig. 10 a Amperometric response of LO/RGO/ITO electrode to the successive addition of AA, in 0.1 M PBS solution at pH 6.0. b Plot of electrocatalytic current of AA vs. its concentrations
Determination of Ascorbic Acid Figure 9a depicts the cyclic voltammetry (CV) responses of LO/RGO-modified ITO electrode toward different concentrations of AA. The LO/RGO/ITO voltammogram exhibits clear peaks of AA oxidation at 0.27 V. With the increase in AA concentration, oxidation increases and thus the peak current due to AA increases. Peak currents increased linearly (up to 50 μM only) on increasing the concentration, with regression equation y=114.1661+10.679x, and correlation coefficient R2 =0.994 as shown in Fig. 9b. The current deviates from linearity at higher AA concentrations, i.e., more than 50 μM concentration, most possibly due to the saturation of active sites at the surface of electrode and the passivation of the electrode and/or the formation of 2,3-diketogluconic acid, as this is known to occur in alkaline media [28]. Amperometric Study of Ascorbic Acid The amperometric response of the LO/RGO/ITO electrode to successive additions of AA was further evaluated under optimized conditions. Figure 10 shows the amperometric current–time response of AA at 0.2 V. As illustrated, upon addition of an aliquot of AA to the stirred PBS, the oxidation current increased steeply and reached a steady-state current. The amperometric signal showed a good linear correlation to AA concentration in the range from 14.4 μM to Fig. 11 Amperometric response of LO/RGO/ITO electrode to the successive addition of AA, glucose (Glu), NaCl, L-cysteine (Cys) in 0.1 M PBS solution at pH 6.0
Appl Biochem Biotechnol
Table 1 Determination of AA in vitamin c tablets
S. No.
Content (μM)
Added (μM)
Recovered (μM)
Standard deviation
Recovery %
1
11.4
50
61.26
0.305
99.7
2
22.8
50
72.16
0.208
99.12
3
32.4
50
80.46
0.568
97.64
0.1 mM. The linear regression equation was expressed y=1.7515+0.173x with a correlation coefficient of R2 =0.981. Interference Studies In order to investigate the selectivity of the LO/RGO/ITO electrode, several compounds from common co-existing substances were investigated by detecting the current response of the modified electrode to AA, using chronoamperometry technique. Figure 11 indicated that the change in current due to interference species (glucose (200 μM), NaCl (500 μM), and Lcysteine (200 μM)) is negligible. Real Sample Analysis Commercially available vitamin C tablets were analyzed to study the applicability of the LO/ RGO/ITO electrode. First, vitamin C tablets were dissolved in PBS (pH 6.0). In the analysis, the standard addition method was applied, by which a known amount of AA in PBS (pH 6.0)
Fig. 12 a, b shows the×100 images of culture media of control and LO/RGO nanocomposite. c, d shows the high magnification (×400 image) of control and LO/RGO nanocomposite
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was added into the test solution. The recovery for the determination of AA was found in the range of 97.64–99.7 % for three samples, as shown in Table 1. Each experiment was performed in three replicates and then mean was calculated to determine the recovered percentage. Results indicate that the LO/RGO/ITO electrode is very much useful for the practical determination of AA in real samples. Bio-compatibility Assay To check the bio-compatibility of LO/RGO nanocomposite, 1 mg of LO/RGO nanocomposite was dispersed in 1 ml of incomplete Dulbecco’s modified eagle medium to achieve a stock concentration of 1 mg/ml. The final concentration assayed was 100–200 μg/ml. Briefly, the SiHa cells were seeded in T.25 culture flask and incubated at 37 °C with 5 % CO2. The cells were treated after 24 h of incubation with LO/RGO nanocomposite and appropriate control for another 48 h to observe the cell morphology and growth of culture. The treated and control flasks were photographed at×100 and×400 magnification under a Nikon inverted microscope. The treated and control SiHa cultures observed under×100 magnification showed no significant change in growth percentage. The confluency of cells after treatment was similar to the control flask (i.e., 70–80 % confluent culture). However, to check the morphology of cells in treated culture, the flasks were observed under×400 magnification (Fig. 12). Both cultures showed similar morphology, size, and adherent property, suggesting that LO/RGO nanocomposite is bio-compatible and is not leading to any abrupt change in morphology of cells or leading to any cell death. Further, MTT assay was also performed to check the cell viability and score any cell death. The result showed no significant cell death in treated cultures as compared to control culture.
Conclusion We have demonstrated LO/RGO/ITO as an efficient, selective, and sensitive electrode for ascorbic acid sensing. High catalytic activity of LO/RGO toward the oxidation of AA, and the synergistic effects between lanthanum oxide and reduced graphene oxide were the main attributes of the successful detection of ascorbic acid. Moreover, the proposed method was applied to the determination of AA in real samples with remarkable efficient results. Thus, it is believed that this proposed nanocomposite, LO/RGO, can provide a novel platform for the construction of sensitive electrochemical sensors for ascorbic acid. Acknowledgments We greatly acknowledge Department of Science and Technology (SERB), India for funding through the project no-SR/FT/CS-123/2010 dated 08/02/2012. We are also Thankful to the CSIR (Council of Scientific & Industrial Research) FOR Senior research fellowship to VS.
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