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Electrochemical characterization of enzymatic organo-metallic coating of TiO2 nanoparticles
Sensors and Actuators B 196 (2014) 589–595
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Electrochemical characterization of enzymatic organo-metallic coating of TiO2 nanoparticles Kyung Hee Park a , Ravi Ranjan Pandey b , Chang Kook Hong c , Krishan Kumar Saini b , Marshal Dhayal d,∗ a
The Research Institute for Advanced Engineering Technology, Chosun University, Gwangju 501-759, South Korea CSIR- National Physical Laboratory, New Delhi 110 012, India c School of Applied Chemical Engineering, Chonnam National University, Gwangju 500-757, South Korea d Clinical Research Facility, CSIR- Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500007, India b
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Article history: Received 18 November 2013 Received in revised form 7 February 2014 Accepted 10 February 2014 Available online 19 February 2014 Keywords: Nano structures Biosensor TiO2 Soybean oil base binder Thin films.
a b s t r a c t Triglyceride molecules in acrylated epoxidized soybean oil (AESO) and maleinized acrylated epoxidized soybean oil (MAESO) base binders were used to achieve binding of TiO2 nanoparticles on conducting glass surfaces. These surfaces were coated with glucose oxidase enzyme after annealing at 450 ◦ C. The annealed film surface was examined by SEM and AFM observations confirm enhancement of surface porosity and morphology, before and after enzyme attachment. Electrochemical techniques were used to study the glucose sensing properties of these substrates prepared with these natural plant oils binders. It has been observed that substrates prepared by using AESO and MAESO fatty acid binders have higher enzyme adsorption capability and exhibit better charge transport properties. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Electrochemical studies of metal base nanomaterials and surfaces functionalized with different biomolecules have shown rapid growth over the last two decades [1–3]. The surface physicochemical properties of these metals and metal oxides are changed by doping [4–7] and using different types of surface treatments methods [8,9] which led to distinctive structural and functional changes for different applications. Other approaches for surface modifications include developing complexes with polymeric materials [10]. Despite several advantages of metal and metal oxide based nanomaterials, it is still a challenging task to make stable thin film from these materials and to apply them for quantification of electrochemical processes at the surfaces of these nanoparticles. Polyethylene glycol (PEG) is a commonly used binder for preparing TiO2 nano-particles films by screen printing methods. However the metal oxide nanoparticle films prepared with PEG base binders can resist the electron transport at the surface [11]. Thus, there is a need to search for binder materials, to attach nanoparticles
∗ Corresponding author. Tel.: +91 040 271 92520; fax: +91 040 271 60591. E-mail addresses: [email protected], [email protected] (M. Dhayal). http://dx.doi.org/10.1016/j.snb.2014.02.040 0925-4005/© 2014 Elsevier B.V. All rights reserved.
on conducting surfaces, which can provide enhanced charge transport properties and at the same time does not alter the surface property and structural properties of TiO2 nano-materials. Previously only TiO2 and TiO2 with different polymer matrixes have been used as platforms for the immobilization of different enzymes [12,13] and antibodies for detection of toxic compounds [14,15]. Stability and usefulness of these TiO2 surfaces for immobilization of biomolecules for biosensor have been extensively studied [16–18]. Use of chitosan for preparation of TiO2 nanoparticles thin films for developing immunosensor has also been reported [13]. The objective of this study is to investigate the effects of chemically functionalized acrylated epoxidized soybean oil (AESO) and maleinized acrylated epoxidized soybean oil (MAESO) on binding of TiO2 nanoparticle on conducting surfaces and functionality of the emerged structure. Binding of nanoparticles is expected from natural plant oils, such as AESO and MAESO, due to the presence of triglyceride molecules where three fatty acids join at glycerol junction [19]. Here we report our investigations on the functional and electrochemical properties of the TiO2 electrodes prepared using AESO, and MAESO binders. Electrodes with PEG binders are fabricated for comparison purposes. Usefulness of these platforms was demonstrated by immobilizing glucose oxidase (GOx ) enzyme which is a well studied model system for electrochemical biosensors [18]. Relative amount of glucose in the electrolyte
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was estimated on these GOx immobilized surfaces. Experiments were conducted with the use of all three binders PEG, AESO and MAESO for preparation of TiO2 coating on FTO substrate conducting surfaces. 2. Experimental Acrylated epoxidized soybean oil, maleic anhydride, benzyldimethylamine (BDMA) and hydroquinone were obtained from Sigma–Aldrich. Anatase crystallite phase TiO2 nanoparticles having diameter ∼25 nm were obtained from Nippon Aerosil (P25). Analytical grade solvents and substrates were procured from local vendor. MAESO has been synthesized by mixing AESO and hydroquinone in ratio 1000:1. Mixture was stirred for 15–20 min and the temperature was raised to 70 ◦ C. Then 16.3 wt.% of maleic acid & 1.7 wt.% of BDMA catalysts were added to the above mixture and temperature was further raised to 80 ◦ C and kept at this temperature for six hours. TiO2 paste was prepared by adding TiO2 nanoparticles, deionized water, plant oil binder (AESO or MAESO, ethanol, acetylacetone and nitric acid into the above mixture followed by vigorous stirring, as per the procedure described elsewhere [20]. For control experiments 0.4 g of polyethylene glycol (PEG) and 0.1 g of Triton X-100 as a dispersing agent were used to prepare TiO2 paste in place of AESO or MAESO whereas other ingredients remain the same. The similar amount of TiO2 paste was casted on the pre-cleaned fluorine-doped tin dioxide (FTO, Pilkington TEC glass, 8 cm−2 ) glass substrates by screen printing process for all three conditions. In this way TiO2 nanoparticle films with three types of binders viz; (a) PEG, (b) AESO and (c) MAESO, were prepared which were subsequently annealed at 450 ◦ C for 30 min under open atmospheric conditions. Glucose oxidase (GOx ) enzyme was adsorbed onto the above electrodes by soaking them in 0.1 M enzyme solution, prepared in standard phosphate buffer (PBS) of pH 7.4, for 30 min. After that the electrodes were rinsed with buffer solution and stored under refrigerated conditions (4 ◦ C) before further measurements. FTIR spectra of the samples have been recorded in reflection mode with glancing angle of 35◦ from 500 to 5000 cm−1 range with Perkin Elmer Model Spectrum BX-100 spectrometer. Surface morphological investigations are carried with Hitachi model S-4700 scanning electron microscope. Atomic Force Microscope model AFM CP-II, Veeco has been employed for surface roughness studies, to determine root mean square (RMS) value of surface roughness; whereas Ecochemie Model 302N (Netherlands) electrochemical workstation is used for electrochemical measurements. These measurements are carried out in three electrode configuration with Pt wire as counter electrode, Ag/AgCl reference electrode and enzyme loaded TiO2 nano particles film on conducting glass substrates, being the working electrode and PBS (pH 7.4) electrolyte as described in our previous studies [21]. 3. Results and discussion Fig. 1 shows XRD pattern of (a) TiO2 nanoparticles, (b) FTO substrate and (c) representative XRD pattern of P25 TiO2 nanoparticles thin film coated on FTO substrate. Prominent diffraction peaks in the XRD pattern of TiO2 nanoparticles (Fig. 1a) appears at 2 values of 25.16, 27.46, 36.4, 37.76, 48.2, 53.9, 55.15, 62.71 and 68.7 degrees which are assigned (hkl) values A101, R110, A103, A004, A112, A200, A105, A211, A204 and A116 respectively which confirms the formation of mainly anatase phase of TiO2 [22]. FTO substrate shows several strong peaks of Sn which were assigned (hkl) values as (1 1 0), (0 0 2), (1 1 2), (2 0 2) and (1 1 3) planes in XRD spectra (Fig. 1b) as measured previously [23]. XRD spectra (Fig. 1c)
Fig. 1. XRD spectra of (a) TiO2 nanoparticles, (b) FTO substrates and (c) TiO2 nanoparticle film coatings on FTO substrate.
of TiO2 films on FTO substrate which is similar for all coating conditions shows prominent diffraction peaks at 2 values of 25.16 and small peaks at 27.46, 36.4, 37.76, 48.2, 53.9, 55.15, 62.71 and 68.7 degrees which are assigned (hkl) values A101, R110, A103, A004, A112, A200, A105, A211, A204 and A116 respectively. The XRD pattern also shows strong diffraction peaks associated with FTO substrates and above results confirms the anatase phase of TiO2 in films deposited on these substrates. SEM images of TiO2 nanoparticle films surfaces, prepared with three different binders, are shown in Fig. 2A. Surface morphologies of the three samples and individual TiO2 particles are clearly visible in high resolution images. There appears to be no aggregation of TiO2 nanoparticles/crystallites and the formation of larger size nanostructures may be due to the different nature of binders. An increase in surface porosity and microstructure of TiO2 film with AESO and MAESO binders are observed under AFM analysis (Fig. 2B). FTIR spectra of three different types of TiO2 nano-particle film surfaces are shown in Fig. 3. Spectral peak in the wavenumber ranging from 3100 cm−1 to 3600 cm−1 has been assigned to hydroxyl groups present at the film surface. A complex peak pattern in the range 500–900 cm−1 mainly corresponds to metal ion and oxygen interactions associated with Ti–O–Ti bands. Peak at 1110 cm−1 is associated to Si–O–Si as part of silicon and oxygen atoms interaction in conducting glass substrate. The presence of the small peak at ∼853 cm−1 can be assigned to Ti–O–Si functional group contributed by the binding of titanium atoms with glass substrate. FTIR peak at ∼1640 cm−1 is attributed to the C=C bond. There is a significant enhancement in the intensity of this peak in TiO2 film samples with AESO and MAESO binder. Tow peaks at 1087 cm−1 and 1139 cm−1 were observed which seem spiting of Si–O–Si peak observed at 1110 cm−1 . In addition to these there were several other weak peaks between 2700 and 3100 cm−1 and these were assigned to C–H vibrations. The existence of C–H is attributed to the presence of the organic residue at surface, which has not been removed during the calcination process adopted here. Higher intensity of peak in the range 2700–3100 cm−1 of FTIR spectra of MAESO added TiO2 nano-particles film surface confirms the stability of reactive sites associated with fatty acid molecule present in the plant oil base binders used in this study. Fig. 4 shows FTIR spectra of GOx coated TiO2 nano-particles film surfaces in which carbonyl absorption peak of amide around 1650 cm−1 was observed. The C–H vibrations at 2876 and 2994 cm−1 disappears after enzyme (GOx ) immobilization on the
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Fig. 2. (A) SEM and (B) AFM images of TiO2 nano particles films prepared with (a) PEG binder, (b) AESO binder and (c) MAESO binder.
film surface. Significant difference in the NH band strength between 1600 and 1640 cm−1 wavenumber has been observed after GOx enzyme coating on the TiO2 film surfaces prepared with PEG, and plant oil binders such as AESO and MAESO. The disappearance of C–H vibrations at 2876 and 2994 cm−1 suggests that the enzyme binds with these moieties at the surfaces of these films. Higher FTIR peak intensity in the range of 2800–3100 cm−1 of MAESO added TiO2 nano particles film surface confirms the stability of
reactive sites associated with fatty acid molecule presence in the plant oil base binders used. Presence of carbonyl absorption peak of amide around 1650 cm−1 in FTIR spectra of GOx coated surfaces confirms enzyme functionality at the surface. The peak intensity at ∼853 cm−1 was increased for AESO and MAESO which was assigned to Ti-O-Si functional group contributed by the binding of titanium atoms with glass substrate. Hence this increase can indicates a
Fig. 3. FT-IR spectra of TiO2 nano particles film prepared with (a) PEG binder, (b) AESO binder & (c) MAESO binder.
Fig. 4. FT-IR spectra of glucose oxidase enzyme immobilised TiO2 nano particles film prepared with (a) PEG binder, (b) AESO binder and (c) MAESO binder.
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Fig. 5. Cyclic voltameteric response of TiO2 nano particles films prepared with PEG binder. Small letters assigned in the figure represents CV response (a) of TiO2 nano particles films in PBS buffer, (a’) of GOx enzyme immobilized TiO2 nano particles films in PBS buffer, (b) of TiO2 nano particles films after addition of ∼12 mg/mL glucose in PBS buffer, and (b’) of GOx enzyme immobilized TiO2 nano particles films after addition of ∼12 mg/mL glucose in PBS buffer.
stronger bonding of TiO2 on substrates maintained after enzyme immobilization. Fig. 5 shows CV response of TiO2 nanoparticles films electrode surface, prepared with PEG binder, before and after GOx enzyme loading is recorded at a scan-rate of 30 mV s−1 from −0.4 V to +1.0 V as previously optimized with different kind of biomolecules [13]. During the measurements we had observed a small difference in CV response for the first and follow up scans; therefore to avoid any discrepancy in the measurements we discard the first scan and used follow up scans and keep the scan cycle constants in all the measurements. Results show decrease in both oxidation and reduction current after enzyme loading on the electrode surface. This decrease in current after enzyme loading is due to the interaction of enzyme molecules with TiO2 nanoparticle in such a manner which reduces surface charge transportation. To further test the specificity of glucose detection, ∼12 mg/mL glucose was added in PBS buffer and CV response of TiO2 nanoparticles films, prepared with PEG binder, with and without enzyme immobilized surfaces was observed. It was expected that the GOx enzyme,
present on the electrode surface, catalyzes the oxidation of -dglucose to d-glucono-␦-lactone which can induce relative change in CV response. The reaction kinetics can be well described by the Michaelis–Menten kinetics [24]. Therefore CV peak dictates the reaction as well as the ability of the substrate to transfer charge carriers to the electrode. Relative variation in the CV response of the electrode without GOx immobilization, after addition of glucose in PBS, was considered as non-specific electrochemical response. There was an increase in oxidation peak current after addition of glucose in PBS for both with and without enzyme loaded TiO2 nanoparticles films electrode surface prepared with PEG binder. However, an increase in CV response was expected only on enzyme immobilized electrode surfaces but a similar increase in the current has been observed on the TiO2 nanoparticles films electrode surface, prepared with PEG binder, without immobilization of enzyme. This observation indicates the non-specific electrochemical responses of glucose with TiO2 electrode surfaces prepared with PEG binder. The phenomenon for this non-specific interaction is not well understood for us but it may be due to the interaction of PEG residue molecules, used for binding of TiO2 nanoparticles for film preparation, with the glucose molecules. To assess the usefulness of natural binders, AESO and MAESO, CV response of the electrodes prepared with these binders, was recorded under similar conditions and are shown in Fig. 6. An increase in oxidation and reduction current has been observed after GOx attachment on the TiO2 working electrode, for both AESO and MAESO binders. This suggests increased charge transportation on these surfaces, which hints at chemical attachment of GOx enzyme on the film surface prepared with these binders. This type of attachment is possible through the functional moieties of fatty acid molecules of AESO and MAESO binders which result in better charge communication between TiO2 –Gox and TiO2 –TiO2 nanoparticles. These moieties may also further support better charge transfer between TiO2 nanoparticle and electrode. CV response of all the above electrodes was also measured in PBS electrolyte containing 12 mg/mL glucose to assess the glucose oxidation kinetics on their surface. The CV response of the electrode without GOx immobilization on AESO binder used TiO2 nanoparticles film electrode was considered as non-specific electrochemical response. MAESO binder used TiO2 nanoparticles film electrode did not show any non-specific interactions. Therefore specific electrochemical response of the electrode increases significantly for the use of MAESO binders.
Fig. 6. Cyclic voltameteric response of TiO2 nano particles films prepared with (A) AESO binder and (B) MAESO binder. Small letters assigned in the figure represents CV response (a) of TiO2 nano particles films in PBS buffer, (a’) of GOx enzyme immobilized TiO2 nano particles films in PBS buffer, (b) of TiO2 nano particles films after addition of ∼12 mg/mL glucose in PBS buffer, and (b’) of GOx enzyme immobilized TiO2 nano particles films after addition of ∼12 mg/mL glucose in PBS buffer.
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Fig. 7. Relative change in the peak current observed from cyclic voltameteric responses of TiO2 nano particles films prepared with PEG binder (TiO2 ), AESO binder (TiO2 –AESO) and MAESO binder (TiO2 –MAESO). (a) The peak current difference before and after adding glucose in electrolyte on TiO2 nano particles films electrodes surfaces and (b) the peak current difference before and after adding glucose in electrolyte on GOx enzyme immobilized TiO2 nano particles films.
To evaluate the relative specific versus non-specific performance of different TiO2 nanoparticles film substrates prepared with different binder materials, described here, we plot the peak current value (I = IG − I0 ) in Fig. 7 which was obtained from the CV response, shown in Figs. 5 and 6, at glucose concentration of 12 mg/mL in the PBS. The observed difference in I values was normalized separately for all the binders with the maximum peak current of CV responses for fresh and GOx enzyme incorporated TiO2 film electrodes. Working electrodes having TiO2 film coatings with PEG and AESO binders has shown large non-specificity in electrochemical response as compared to the MAESO added TiO2 electrode which has shown very minimal non-specific response. The study indicates that MAESO added TiO2 electrode surface is
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superior for glucose determination as compared to PEG or AESO added substrates. To assess the glucose level monitoring abilities of these electrodes, chronoamperometeric (CA) studies were carried out with both GOx immobilized and without GOx immobilized electrodes for glucose concentration between 0 and 12 mg/ml in the PBS electrolyte. CA response can account for both capacitive and Faradaic current at electrode surfaces. Capacitive current (IC ) at electrode surfaces decays exponentially (IC = e-kt ) for a constant applied potential whereas Faradaic current (IF ) at electrode surfaces is associated with oxidation of molecules at electrode surface which can only be replenished by diffusion process. At longer times the ratio IF /IC is larger because capacitive current decreases more rapidly than Faradaic current. In this study the potential of the working electrode was stepped at 0.7 V and the resulting current from electrode was monitored as a function of time as shown in Fig. 8. These time dependent electron transfer events at surfaces can be described by the Cottrell equation in CA response [25]. √ nFAC D i = √ (1) t where, i = current at electrode surface; n = number of electrons associated to reduction/oxidization of one molecule of analyte; F = Faraday constant; A = area of the planar electrode surface; C = concentration of the reducible analyte; D = diffusion coefficient and t = time at which the current observed. Cottrell equation shows liner dependence of current with analyte concentration (i ␣ n) at a fix time, hence the CA response was used to estimate linear response limit of glucose in the PBS electrolyte on electrode surfaces before and after coating of GOx . Peak current difference (IP = Ip,X − IP,0 ) has been calculated from chronoamperometric data at a contact time; where IP,0 is the peak current without adding glucose in the electrolyte and Ip,X the peak current at ‘X’ concentration of glucose in the electrolyte as shown in Fig. 9. Linear response for glucose concentration has been for electrodes prepared with PEG and AESO binders. This matches
Fig. 8. Chronoamperometry response at various concentration of glucose in electrolyte on TiO2 nanoparticles film electrode prepared with (A) PEG binder, (B) AESO binder and (C) MAESO binder. Chronoamperometry response at various concentration of glucose in electrolyte on GOx enzyme immobilized TiO2 nano particles film electrode prepared with (D) PEG binder, (E) AESO binder and (F) MAESO binder.
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Fig. 9. Glucose concentration vs peak current response curve before and after GOx enzyme immobilization on TiO2 nanoparticles films electrodes prepared with (A) PEG binder, (B) AESO binder and (C) MAESO binder. Ip,0 is the peak current of enzyme immobilized electrode without glucose in electrolyte and Ip,x is peak current of enzyme immobilized electrode at ‘x’ concentration of glucose added in electrolyte.
with Michaelis–Menten kinetics but there is large non-specificity for these binders. On the other hand for MAESO added TiO2 electrode, there are deviation from the standard equation; but this electrode does not exhibit non-specific interaction. Also detection sensitivity of this electrode is comparatively poor. Literature survey shows several reports [26,27] which are extensively focused on describing the effects of nano-structured materials in enhancing detection sensitivity of biomolecules [28,29]. These reports have stressed on the role of surface microstructure and available active centers for charge sharing for the sensitivity and selectivity of enzymatic sensor. In this study we have explored the performance of natural binders to support nano-particle film formation and surface microstructure for biological applications. These materials improve chemical binding of nano-materials at substrate surfaces and at the same time provide active functional support for immobilization of bio-molecules. But some anomalous observations are also there which stresses for further detailed investigation of these materials for specific property.
4. Conclusion In this study we have demonstrated the possible use of natural binders to support the nano-particles film formation for biological applications. The use of such binders enhances chemical binding of nano-materials at substrate surfaces as well as provides active functional support for immobilization of bio-molecules. The reactive sites associated with fatty acid molecule present in the plant oil base binders are stable and these natural plant oils base binders increases surface porosity and lead novel microstructure which improves the enzyme adsorption capability and improved charge transport property of these surfaces. The MAESO binder suppresses the non-specific interaction but at the same time shows deviation from standard equations. On the other hand AESO and PEG binders show non-specific interactions but confirm to the standard equations for enzymatic interactions. Better charge sharing capabilities of the electrodes with these binders suggest chemical binding of GOx enzyme on the electrode surface prepared with these binders.
Acknowledgment The work was supported by Council for Scientific and Industrial Research (CSIR) and Department of Biotechnology, Ministry of Science and Technology, Govt. of India. KHP is thankful for the support by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (NRF-2012R1A1A3010655).
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Biographies Kyung Hee Park received her PhD degree in 2000 from Chonnam National University, South Korea in major of electrochemistry. She got a grant to work at Chonnam National University as post-doctoral fellow. She is currently a research professor in Chosun University, South Korea. Her research interest is focusing in preparing films based applied chemistry for nanostructured organic–inorganic hybrid materials, biomaterials and polymers.
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Ravi Ranjan Pandey received his M.Phil and Ph.D from University of Delhi in 2000 and 2009, respectively and MSc from Bhim Rao Ambedkar Bihar University, Muzaffarpur, India in 1998. Currently he is working as Post-doctoral research fellow at University of British Columbia, Vancouver, Canada. He worked as a post-doctoral fellow at Centre for Cellular and Molecular Biology, Hyderabad, India in 2010. He had obtained various prestigious fellowship/award GATE (2004), SRF-CSIR (2005) and young scientist award (2010) in chemistry from Department of Science and Technology Govt. of India. His research interests include in multidiscipline area of chemistry, biology and materials. Chang Kook Hong received his MS degree from Chonnam National University in 1993 and his PhD degree in 2001 from University of Akron, Ohio, USA. He is currently professor in the School of Applied Chemical Engineering at Chonnam National University. He has years of experience in the field of chemo/biosensing and electroanalytical chemistry, including the QCM, electro-chemical devices. His research interests focus on functional polymer for electrochemical and bioanalytical applications. K. K. Saini received his BSc (1975), MSc (1980), M. Phil (1981) from Kurukshetra University, Kurukshetra and Ph. D (physics) from Delhi University in 1994. He joined National Physical Laboratory in 1983 as Scientist. He has been elevated to various positions during this tenure and at present a chief scientist at National Physical Laboratory, New Delhi. He is also heading Center for Calibration and Testing at National Physical Laboratory, New Delhi. He is member of American Chemical Society, American Vacuum Society, Materials Research Society of India, Electron Microscopy society, Indian Meterological society, VLSI society of India. He has developed technology for the manufacture of self-cleaning architectural glass with an economic process. Marshal Dhayal graduated with a M.Sc. degree from University of Rajasthan and PhD from UMIST, Manchester in 1999 and 2004. He had worked at Biology Research Center for Industrial Accelerators, Dongshing University, South Korea as a Research Professor and held Scientist Fellow position at National Physical Laboratory, Delhi. Before joined as a Scientist at CSIR-Center for Cellular and Molecular Biology in 2009, he was a Senior Fellow in Department of Bioengineering, University of Washington, Seattle. He is currently Editor-in-Chief, Advanced Electrochemistry published by American Scientific Publishers and also committee member of MEMS/NEMS of technical group of American Vacuum Society, USA.
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Electrochemical characterization of enzymatic organo-metallic coating of TiO2 nanoparticles Kyung Hee Park a , Ravi Ranjan Pandey b , Chang Kook Hong c , Krishan Kumar Saini b , Marshal Dhayal d,∗ a
The Research Institute for Advanced Engineering Technology, Chosun University, Gwangju 501-759, South Korea CSIR- National Physical Laboratory, New Delhi 110 012, India c School of Applied Chemical Engineering, Chonnam National University, Gwangju 500-757, South Korea d Clinical Research Facility, CSIR- Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500007, India b
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Article history: Received 18 November 2013 Received in revised form 7 February 2014 Accepted 10 February 2014 Available online 19 February 2014 Keywords: Nano structures Biosensor TiO2 Soybean oil base binder Thin films.
a b s t r a c t Triglyceride molecules in acrylated epoxidized soybean oil (AESO) and maleinized acrylated epoxidized soybean oil (MAESO) base binders were used to achieve binding of TiO2 nanoparticles on conducting glass surfaces. These surfaces were coated with glucose oxidase enzyme after annealing at 450 ◦ C. The annealed film surface was examined by SEM and AFM observations confirm enhancement of surface porosity and morphology, before and after enzyme attachment. Electrochemical techniques were used to study the glucose sensing properties of these substrates prepared with these natural plant oils binders. It has been observed that substrates prepared by using AESO and MAESO fatty acid binders have higher enzyme adsorption capability and exhibit better charge transport properties. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Electrochemical studies of metal base nanomaterials and surfaces functionalized with different biomolecules have shown rapid growth over the last two decades [1–3]. The surface physicochemical properties of these metals and metal oxides are changed by doping [4–7] and using different types of surface treatments methods [8,9] which led to distinctive structural and functional changes for different applications. Other approaches for surface modifications include developing complexes with polymeric materials [10]. Despite several advantages of metal and metal oxide based nanomaterials, it is still a challenging task to make stable thin film from these materials and to apply them for quantification of electrochemical processes at the surfaces of these nanoparticles. Polyethylene glycol (PEG) is a commonly used binder for preparing TiO2 nano-particles films by screen printing methods. However the metal oxide nanoparticle films prepared with PEG base binders can resist the electron transport at the surface [11]. Thus, there is a need to search for binder materials, to attach nanoparticles
∗ Corresponding author. Tel.: +91 040 271 92520; fax: +91 040 271 60591. E-mail addresses: [email protected], [email protected] (M. Dhayal). http://dx.doi.org/10.1016/j.snb.2014.02.040 0925-4005/© 2014 Elsevier B.V. All rights reserved.
on conducting surfaces, which can provide enhanced charge transport properties and at the same time does not alter the surface property and structural properties of TiO2 nano-materials. Previously only TiO2 and TiO2 with different polymer matrixes have been used as platforms for the immobilization of different enzymes [12,13] and antibodies for detection of toxic compounds [14,15]. Stability and usefulness of these TiO2 surfaces for immobilization of biomolecules for biosensor have been extensively studied [16–18]. Use of chitosan for preparation of TiO2 nanoparticles thin films for developing immunosensor has also been reported [13]. The objective of this study is to investigate the effects of chemically functionalized acrylated epoxidized soybean oil (AESO) and maleinized acrylated epoxidized soybean oil (MAESO) on binding of TiO2 nanoparticle on conducting surfaces and functionality of the emerged structure. Binding of nanoparticles is expected from natural plant oils, such as AESO and MAESO, due to the presence of triglyceride molecules where three fatty acids join at glycerol junction [19]. Here we report our investigations on the functional and electrochemical properties of the TiO2 electrodes prepared using AESO, and MAESO binders. Electrodes with PEG binders are fabricated for comparison purposes. Usefulness of these platforms was demonstrated by immobilizing glucose oxidase (GOx ) enzyme which is a well studied model system for electrochemical biosensors [18]. Relative amount of glucose in the electrolyte
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was estimated on these GOx immobilized surfaces. Experiments were conducted with the use of all three binders PEG, AESO and MAESO for preparation of TiO2 coating on FTO substrate conducting surfaces. 2. Experimental Acrylated epoxidized soybean oil, maleic anhydride, benzyldimethylamine (BDMA) and hydroquinone were obtained from Sigma–Aldrich. Anatase crystallite phase TiO2 nanoparticles having diameter ∼25 nm were obtained from Nippon Aerosil (P25). Analytical grade solvents and substrates were procured from local vendor. MAESO has been synthesized by mixing AESO and hydroquinone in ratio 1000:1. Mixture was stirred for 15–20 min and the temperature was raised to 70 ◦ C. Then 16.3 wt.% of maleic acid & 1.7 wt.% of BDMA catalysts were added to the above mixture and temperature was further raised to 80 ◦ C and kept at this temperature for six hours. TiO2 paste was prepared by adding TiO2 nanoparticles, deionized water, plant oil binder (AESO or MAESO, ethanol, acetylacetone and nitric acid into the above mixture followed by vigorous stirring, as per the procedure described elsewhere [20]. For control experiments 0.4 g of polyethylene glycol (PEG) and 0.1 g of Triton X-100 as a dispersing agent were used to prepare TiO2 paste in place of AESO or MAESO whereas other ingredients remain the same. The similar amount of TiO2 paste was casted on the pre-cleaned fluorine-doped tin dioxide (FTO, Pilkington TEC glass, 8 cm−2 ) glass substrates by screen printing process for all three conditions. In this way TiO2 nanoparticle films with three types of binders viz; (a) PEG, (b) AESO and (c) MAESO, were prepared which were subsequently annealed at 450 ◦ C for 30 min under open atmospheric conditions. Glucose oxidase (GOx ) enzyme was adsorbed onto the above electrodes by soaking them in 0.1 M enzyme solution, prepared in standard phosphate buffer (PBS) of pH 7.4, for 30 min. After that the electrodes were rinsed with buffer solution and stored under refrigerated conditions (4 ◦ C) before further measurements. FTIR spectra of the samples have been recorded in reflection mode with glancing angle of 35◦ from 500 to 5000 cm−1 range with Perkin Elmer Model Spectrum BX-100 spectrometer. Surface morphological investigations are carried with Hitachi model S-4700 scanning electron microscope. Atomic Force Microscope model AFM CP-II, Veeco has been employed for surface roughness studies, to determine root mean square (RMS) value of surface roughness; whereas Ecochemie Model 302N (Netherlands) electrochemical workstation is used for electrochemical measurements. These measurements are carried out in three electrode configuration with Pt wire as counter electrode, Ag/AgCl reference electrode and enzyme loaded TiO2 nano particles film on conducting glass substrates, being the working electrode and PBS (pH 7.4) electrolyte as described in our previous studies [21]. 3. Results and discussion Fig. 1 shows XRD pattern of (a) TiO2 nanoparticles, (b) FTO substrate and (c) representative XRD pattern of P25 TiO2 nanoparticles thin film coated on FTO substrate. Prominent diffraction peaks in the XRD pattern of TiO2 nanoparticles (Fig. 1a) appears at 2 values of 25.16, 27.46, 36.4, 37.76, 48.2, 53.9, 55.15, 62.71 and 68.7 degrees which are assigned (hkl) values A101, R110, A103, A004, A112, A200, A105, A211, A204 and A116 respectively which confirms the formation of mainly anatase phase of TiO2 [22]. FTO substrate shows several strong peaks of Sn which were assigned (hkl) values as (1 1 0), (0 0 2), (1 1 2), (2 0 2) and (1 1 3) planes in XRD spectra (Fig. 1b) as measured previously [23]. XRD spectra (Fig. 1c)
Fig. 1. XRD spectra of (a) TiO2 nanoparticles, (b) FTO substrates and (c) TiO2 nanoparticle film coatings on FTO substrate.
of TiO2 films on FTO substrate which is similar for all coating conditions shows prominent diffraction peaks at 2 values of 25.16 and small peaks at 27.46, 36.4, 37.76, 48.2, 53.9, 55.15, 62.71 and 68.7 degrees which are assigned (hkl) values A101, R110, A103, A004, A112, A200, A105, A211, A204 and A116 respectively. The XRD pattern also shows strong diffraction peaks associated with FTO substrates and above results confirms the anatase phase of TiO2 in films deposited on these substrates. SEM images of TiO2 nanoparticle films surfaces, prepared with three different binders, are shown in Fig. 2A. Surface morphologies of the three samples and individual TiO2 particles are clearly visible in high resolution images. There appears to be no aggregation of TiO2 nanoparticles/crystallites and the formation of larger size nanostructures may be due to the different nature of binders. An increase in surface porosity and microstructure of TiO2 film with AESO and MAESO binders are observed under AFM analysis (Fig. 2B). FTIR spectra of three different types of TiO2 nano-particle film surfaces are shown in Fig. 3. Spectral peak in the wavenumber ranging from 3100 cm−1 to 3600 cm−1 has been assigned to hydroxyl groups present at the film surface. A complex peak pattern in the range 500–900 cm−1 mainly corresponds to metal ion and oxygen interactions associated with Ti–O–Ti bands. Peak at 1110 cm−1 is associated to Si–O–Si as part of silicon and oxygen atoms interaction in conducting glass substrate. The presence of the small peak at ∼853 cm−1 can be assigned to Ti–O–Si functional group contributed by the binding of titanium atoms with glass substrate. FTIR peak at ∼1640 cm−1 is attributed to the C=C bond. There is a significant enhancement in the intensity of this peak in TiO2 film samples with AESO and MAESO binder. Tow peaks at 1087 cm−1 and 1139 cm−1 were observed which seem spiting of Si–O–Si peak observed at 1110 cm−1 . In addition to these there were several other weak peaks between 2700 and 3100 cm−1 and these were assigned to C–H vibrations. The existence of C–H is attributed to the presence of the organic residue at surface, which has not been removed during the calcination process adopted here. Higher intensity of peak in the range 2700–3100 cm−1 of FTIR spectra of MAESO added TiO2 nano-particles film surface confirms the stability of reactive sites associated with fatty acid molecule present in the plant oil base binders used in this study. Fig. 4 shows FTIR spectra of GOx coated TiO2 nano-particles film surfaces in which carbonyl absorption peak of amide around 1650 cm−1 was observed. The C–H vibrations at 2876 and 2994 cm−1 disappears after enzyme (GOx ) immobilization on the
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Fig. 2. (A) SEM and (B) AFM images of TiO2 nano particles films prepared with (a) PEG binder, (b) AESO binder and (c) MAESO binder.
film surface. Significant difference in the NH band strength between 1600 and 1640 cm−1 wavenumber has been observed after GOx enzyme coating on the TiO2 film surfaces prepared with PEG, and plant oil binders such as AESO and MAESO. The disappearance of C–H vibrations at 2876 and 2994 cm−1 suggests that the enzyme binds with these moieties at the surfaces of these films. Higher FTIR peak intensity in the range of 2800–3100 cm−1 of MAESO added TiO2 nano particles film surface confirms the stability of
reactive sites associated with fatty acid molecule presence in the plant oil base binders used. Presence of carbonyl absorption peak of amide around 1650 cm−1 in FTIR spectra of GOx coated surfaces confirms enzyme functionality at the surface. The peak intensity at ∼853 cm−1 was increased for AESO and MAESO which was assigned to Ti-O-Si functional group contributed by the binding of titanium atoms with glass substrate. Hence this increase can indicates a
Fig. 3. FT-IR spectra of TiO2 nano particles film prepared with (a) PEG binder, (b) AESO binder & (c) MAESO binder.
Fig. 4. FT-IR spectra of glucose oxidase enzyme immobilised TiO2 nano particles film prepared with (a) PEG binder, (b) AESO binder and (c) MAESO binder.
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Fig. 5. Cyclic voltameteric response of TiO2 nano particles films prepared with PEG binder. Small letters assigned in the figure represents CV response (a) of TiO2 nano particles films in PBS buffer, (a’) of GOx enzyme immobilized TiO2 nano particles films in PBS buffer, (b) of TiO2 nano particles films after addition of ∼12 mg/mL glucose in PBS buffer, and (b’) of GOx enzyme immobilized TiO2 nano particles films after addition of ∼12 mg/mL glucose in PBS buffer.
stronger bonding of TiO2 on substrates maintained after enzyme immobilization. Fig. 5 shows CV response of TiO2 nanoparticles films electrode surface, prepared with PEG binder, before and after GOx enzyme loading is recorded at a scan-rate of 30 mV s−1 from −0.4 V to +1.0 V as previously optimized with different kind of biomolecules [13]. During the measurements we had observed a small difference in CV response for the first and follow up scans; therefore to avoid any discrepancy in the measurements we discard the first scan and used follow up scans and keep the scan cycle constants in all the measurements. Results show decrease in both oxidation and reduction current after enzyme loading on the electrode surface. This decrease in current after enzyme loading is due to the interaction of enzyme molecules with TiO2 nanoparticle in such a manner which reduces surface charge transportation. To further test the specificity of glucose detection, ∼12 mg/mL glucose was added in PBS buffer and CV response of TiO2 nanoparticles films, prepared with PEG binder, with and without enzyme immobilized surfaces was observed. It was expected that the GOx enzyme,
present on the electrode surface, catalyzes the oxidation of -dglucose to d-glucono-␦-lactone which can induce relative change in CV response. The reaction kinetics can be well described by the Michaelis–Menten kinetics [24]. Therefore CV peak dictates the reaction as well as the ability of the substrate to transfer charge carriers to the electrode. Relative variation in the CV response of the electrode without GOx immobilization, after addition of glucose in PBS, was considered as non-specific electrochemical response. There was an increase in oxidation peak current after addition of glucose in PBS for both with and without enzyme loaded TiO2 nanoparticles films electrode surface prepared with PEG binder. However, an increase in CV response was expected only on enzyme immobilized electrode surfaces but a similar increase in the current has been observed on the TiO2 nanoparticles films electrode surface, prepared with PEG binder, without immobilization of enzyme. This observation indicates the non-specific electrochemical responses of glucose with TiO2 electrode surfaces prepared with PEG binder. The phenomenon for this non-specific interaction is not well understood for us but it may be due to the interaction of PEG residue molecules, used for binding of TiO2 nanoparticles for film preparation, with the glucose molecules. To assess the usefulness of natural binders, AESO and MAESO, CV response of the electrodes prepared with these binders, was recorded under similar conditions and are shown in Fig. 6. An increase in oxidation and reduction current has been observed after GOx attachment on the TiO2 working electrode, for both AESO and MAESO binders. This suggests increased charge transportation on these surfaces, which hints at chemical attachment of GOx enzyme on the film surface prepared with these binders. This type of attachment is possible through the functional moieties of fatty acid molecules of AESO and MAESO binders which result in better charge communication between TiO2 –Gox and TiO2 –TiO2 nanoparticles. These moieties may also further support better charge transfer between TiO2 nanoparticle and electrode. CV response of all the above electrodes was also measured in PBS electrolyte containing 12 mg/mL glucose to assess the glucose oxidation kinetics on their surface. The CV response of the electrode without GOx immobilization on AESO binder used TiO2 nanoparticles film electrode was considered as non-specific electrochemical response. MAESO binder used TiO2 nanoparticles film electrode did not show any non-specific interactions. Therefore specific electrochemical response of the electrode increases significantly for the use of MAESO binders.
Fig. 6. Cyclic voltameteric response of TiO2 nano particles films prepared with (A) AESO binder and (B) MAESO binder. Small letters assigned in the figure represents CV response (a) of TiO2 nano particles films in PBS buffer, (a’) of GOx enzyme immobilized TiO2 nano particles films in PBS buffer, (b) of TiO2 nano particles films after addition of ∼12 mg/mL glucose in PBS buffer, and (b’) of GOx enzyme immobilized TiO2 nano particles films after addition of ∼12 mg/mL glucose in PBS buffer.
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Fig. 7. Relative change in the peak current observed from cyclic voltameteric responses of TiO2 nano particles films prepared with PEG binder (TiO2 ), AESO binder (TiO2 –AESO) and MAESO binder (TiO2 –MAESO). (a) The peak current difference before and after adding glucose in electrolyte on TiO2 nano particles films electrodes surfaces and (b) the peak current difference before and after adding glucose in electrolyte on GOx enzyme immobilized TiO2 nano particles films.
To evaluate the relative specific versus non-specific performance of different TiO2 nanoparticles film substrates prepared with different binder materials, described here, we plot the peak current value (I = IG − I0 ) in Fig. 7 which was obtained from the CV response, shown in Figs. 5 and 6, at glucose concentration of 12 mg/mL in the PBS. The observed difference in I values was normalized separately for all the binders with the maximum peak current of CV responses for fresh and GOx enzyme incorporated TiO2 film electrodes. Working electrodes having TiO2 film coatings with PEG and AESO binders has shown large non-specificity in electrochemical response as compared to the MAESO added TiO2 electrode which has shown very minimal non-specific response. The study indicates that MAESO added TiO2 electrode surface is
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superior for glucose determination as compared to PEG or AESO added substrates. To assess the glucose level monitoring abilities of these electrodes, chronoamperometeric (CA) studies were carried out with both GOx immobilized and without GOx immobilized electrodes for glucose concentration between 0 and 12 mg/ml in the PBS electrolyte. CA response can account for both capacitive and Faradaic current at electrode surfaces. Capacitive current (IC ) at electrode surfaces decays exponentially (IC = e-kt ) for a constant applied potential whereas Faradaic current (IF ) at electrode surfaces is associated with oxidation of molecules at electrode surface which can only be replenished by diffusion process. At longer times the ratio IF /IC is larger because capacitive current decreases more rapidly than Faradaic current. In this study the potential of the working electrode was stepped at 0.7 V and the resulting current from electrode was monitored as a function of time as shown in Fig. 8. These time dependent electron transfer events at surfaces can be described by the Cottrell equation in CA response [25]. √ nFAC D i = √ (1) t where, i = current at electrode surface; n = number of electrons associated to reduction/oxidization of one molecule of analyte; F = Faraday constant; A = area of the planar electrode surface; C = concentration of the reducible analyte; D = diffusion coefficient and t = time at which the current observed. Cottrell equation shows liner dependence of current with analyte concentration (i ␣ n) at a fix time, hence the CA response was used to estimate linear response limit of glucose in the PBS electrolyte on electrode surfaces before and after coating of GOx . Peak current difference (IP = Ip,X − IP,0 ) has been calculated from chronoamperometric data at a contact time; where IP,0 is the peak current without adding glucose in the electrolyte and Ip,X the peak current at ‘X’ concentration of glucose in the electrolyte as shown in Fig. 9. Linear response for glucose concentration has been for electrodes prepared with PEG and AESO binders. This matches
Fig. 8. Chronoamperometry response at various concentration of glucose in electrolyte on TiO2 nanoparticles film electrode prepared with (A) PEG binder, (B) AESO binder and (C) MAESO binder. Chronoamperometry response at various concentration of glucose in electrolyte on GOx enzyme immobilized TiO2 nano particles film electrode prepared with (D) PEG binder, (E) AESO binder and (F) MAESO binder.
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Fig. 9. Glucose concentration vs peak current response curve before and after GOx enzyme immobilization on TiO2 nanoparticles films electrodes prepared with (A) PEG binder, (B) AESO binder and (C) MAESO binder. Ip,0 is the peak current of enzyme immobilized electrode without glucose in electrolyte and Ip,x is peak current of enzyme immobilized electrode at ‘x’ concentration of glucose added in electrolyte.
with Michaelis–Menten kinetics but there is large non-specificity for these binders. On the other hand for MAESO added TiO2 electrode, there are deviation from the standard equation; but this electrode does not exhibit non-specific interaction. Also detection sensitivity of this electrode is comparatively poor. Literature survey shows several reports [26,27] which are extensively focused on describing the effects of nano-structured materials in enhancing detection sensitivity of biomolecules [28,29]. These reports have stressed on the role of surface microstructure and available active centers for charge sharing for the sensitivity and selectivity of enzymatic sensor. In this study we have explored the performance of natural binders to support nano-particle film formation and surface microstructure for biological applications. These materials improve chemical binding of nano-materials at substrate surfaces and at the same time provide active functional support for immobilization of bio-molecules. But some anomalous observations are also there which stresses for further detailed investigation of these materials for specific property.
4. Conclusion In this study we have demonstrated the possible use of natural binders to support the nano-particles film formation for biological applications. The use of such binders enhances chemical binding of nano-materials at substrate surfaces as well as provides active functional support for immobilization of bio-molecules. The reactive sites associated with fatty acid molecule present in the plant oil base binders are stable and these natural plant oils base binders increases surface porosity and lead novel microstructure which improves the enzyme adsorption capability and improved charge transport property of these surfaces. The MAESO binder suppresses the non-specific interaction but at the same time shows deviation from standard equations. On the other hand AESO and PEG binders show non-specific interactions but confirm to the standard equations for enzymatic interactions. Better charge sharing capabilities of the electrodes with these binders suggest chemical binding of GOx enzyme on the electrode surface prepared with these binders.
Acknowledgment The work was supported by Council for Scientific and Industrial Research (CSIR) and Department of Biotechnology, Ministry of Science and Technology, Govt. of India. KHP is thankful for the support by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (NRF-2012R1A1A3010655).
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Biographies Kyung Hee Park received her PhD degree in 2000 from Chonnam National University, South Korea in major of electrochemistry. She got a grant to work at Chonnam National University as post-doctoral fellow. She is currently a research professor in Chosun University, South Korea. Her research interest is focusing in preparing films based applied chemistry for nanostructured organic–inorganic hybrid materials, biomaterials and polymers.
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Ravi Ranjan Pandey received his M.Phil and Ph.D from University of Delhi in 2000 and 2009, respectively and MSc from Bhim Rao Ambedkar Bihar University, Muzaffarpur, India in 1998. Currently he is working as Post-doctoral research fellow at University of British Columbia, Vancouver, Canada. He worked as a post-doctoral fellow at Centre for Cellular and Molecular Biology, Hyderabad, India in 2010. He had obtained various prestigious fellowship/award GATE (2004), SRF-CSIR (2005) and young scientist award (2010) in chemistry from Department of Science and Technology Govt. of India. His research interests include in multidiscipline area of chemistry, biology and materials. Chang Kook Hong received his MS degree from Chonnam National University in 1993 and his PhD degree in 2001 from University of Akron, Ohio, USA. He is currently professor in the School of Applied Chemical Engineering at Chonnam National University. He has years of experience in the field of chemo/biosensing and electroanalytical chemistry, including the QCM, electro-chemical devices. His research interests focus on functional polymer for electrochemical and bioanalytical applications. K. K. Saini received his BSc (1975), MSc (1980), M. Phil (1981) from Kurukshetra University, Kurukshetra and Ph. D (physics) from Delhi University in 1994. He joined National Physical Laboratory in 1983 as Scientist. He has been elevated to various positions during this tenure and at present a chief scientist at National Physical Laboratory, New Delhi. He is also heading Center for Calibration and Testing at National Physical Laboratory, New Delhi. He is member of American Chemical Society, American Vacuum Society, Materials Research Society of India, Electron Microscopy society, Indian Meterological society, VLSI society of India. He has developed technology for the manufacture of self-cleaning architectural glass with an economic process. Marshal Dhayal graduated with a M.Sc. degree from University of Rajasthan and PhD from UMIST, Manchester in 1999 and 2004. He had worked at Biology Research Center for Industrial Accelerators, Dongshing University, South Korea as a Research Professor and held Scientist Fellow position at National Physical Laboratory, Delhi. Before joined as a Scientist at CSIR-Center for Cellular and Molecular Biology in 2009, he was a Senior Fellow in Department of Bioengineering, University of Washington, Seattle. He is currently Editor-in-Chief, Advanced Electrochemistry published by American Scientific Publishers and also committee member of MEMS/NEMS of technical group of American Vacuum Society, USA.