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Nanostructured zinc oxide platform for mycotoxin detection
Bioelectrochemistry 77 (2010) 75–81
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Bioelectrochemistry j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b i o e l e c h e m
Nanostructured zinc oxide platform for mycotoxin detection Anees A. Ansari ⁎,1, Ajeet Kaushik, Pratima R. Solanki, B.D. Malhotra ⁎ Department of Science & Technology Centre on Biomolecular Electronics, National Physical Laboratory, Dr. K. S. Krishnan Marg, New Delhi-110012, India
a r t i c l e
i n f o
Article history: Received 7 April 2009 Received in revised form 23 June 2009 Accepted 25 June 2009 Available online 8 July 2009 Keywords: Sol–gel Zinc oxide Impedimetric immunosensor Mycotoxin Ochratoxin-A
a b s t r a c t Nanostructured zinc oxide (Nano-ZnO) film has been deposited onto indium–tin–oxide (ITO) glass plate for co-immobilization of rabbit-immunoglubin antibodies (r-IgGs) and bovine serum albumin (BSA) for ochratoxin-A (OTA) detection. The results of X-ray diffraction (XRD) studies reveal the formation of Nano-ZnO with average particle size as ~5.0 nm. Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), electrochemical impedance spectroscopy (EIS) techniques have been used to characterize Nano-ZnO/ITO electrode and BSA/r-IgGs/Nano-ZnO/ITO immunoelectrode. Electrochemical impedimetric response of BSA/r-IgGs/Nano-ZnO/ITO immunoelectrode obtained as a function of OTA concentration exhibits linearity as 0.006–0.01 nM/dm3, detection limit of 0.006 nM/dm3, response time as 25 s and sensitivity of 189 Ω/nM/dm3cm− 2 with a regression coefficient of 0.997. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Electrochemical immunosensors have recently aroused much interest for detection of desired proteins, biomarkers, biological toxins and bio-warfare agents in critical situations, food, environment, pharmaceutical chemistry and clinical diagnostics [1–4]. To obtain a sensitive, compact and stable immunosensor platform, the desired antibodies should be directed in a given orientation on the electrode. The selection of a matrix for the immobilization of antibodies (Abs) is extremely important to obtain desired characteristics of an immunoelectrode [1,2]. Nanostructured metal oxides have recently attracted much attention due to their interesting electrocatalytic, piezoelectric, photonic and tunable size dependent surface properties that make them attractive for biosensing applications [5–7]. In this context, zirconium oxide [8], titanium oxide [9,10], tin oxide [11], cerium oxide [12] and zinc oxide (ZnO) [13–16] have been utilized for immobilization of proteins, enzymes and antigens for accelerated electron transfer between desired immobilized biomolecules and electrode. These nanomaterials exhibit interesting properties such as large surface-tovolume ratio, high surface reaction activity, high catalytic efficiency, and strong adsorption ability that make them potential candidate materials to play a catalytic role in the fabrication of an immunosensor. The large surface area of a nanostuctured metal oxide is likely
⁎ Corresponding authors. Ansari is to be contacted at Tel.: +91 11 45609152; fax: +91 11 45609310. Malhotra, Tel.: +91 11 45609151; fax: +91 11 45609310. E-mail addresses: [email protected] (A.A. Ansari), [email protected] (B.D. Malhotra). 1 Presently at the King Abdullah Institute for Nanotechnology, King Saud University, Riyadh-Saudi Arabia. 1567-5394/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.bioelechem.2009.06.014
to lead to increased Abs loading per unit mass of particles. Moreover, multipoint attachment of enzyme molecules to nanomaterial surfaces reduces protein unfolding resulting in enhanced stability of the biomolecule attached to nanoparticles surface. In this context, the enzyme-attached nanoparticles facilitate enzymes to act as free enzymes in solution and in turn improve enzyme–substrate interaction by minimizing potential aggregation of the free enzyme. Among the various metal oxides, nanostructured ZnO has been used for immunosensor applications [6,7,13–16]. Its unique properties such as high isoelectric point (IEP ~9.5) and biocompatibility facilitate immobilization of an enzyme and protein having low IEP via electrostatic interactions. Besides this, the positively charged ZnO nanoparticles (IEP ~ 9.5) not only provide a friendly microenvironment for immobilizing negatively charged rabbit antibodies (r-IgGs; IEP ~ 5.5) and retain its bioactivity but also accelerate electron transfer communication between protein and the electrode to a large extent [6,7]. Moreover, non-toxicity, high chemical stability and high electron transfer capability make Nano-ZnO a promising material for immobilization of desired biomolecules for fabrication of an immunosensor. The sol–gel derived Nano-ZnO has recently emerged as an attractive material due to its ease of preparation under ambient conditions, tunable porosity, high thermal stability, chemical inertness and negligible swelling in aqueous and non-aqueous solutions for immobilization of desired biomolecules (enzymes, proteins and antibodies) [9,12]. Liu et al. have prepared stable nano-sized flower like ZnO film by hydrothermal method for H2O2 sensor [16]. ZnO nanocomb has been fabricated in bulk quantity by vapor phase transport method for glucose detection [7]. Wei et al have grown ZnO nanorods on Au electrode for development of enzymatic glucose biosensor [6]. However, sol–gel derived Nano-ZnO has not yet been utilized for development of immunosensor for detection of ochratoxin-A (OTA).
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Ochratoxin-A [7-(L-β-phenylalanylcarbonyl)-carboxyl-5-chloro8-hydroxy-3,4-dihydro-3R-methylisocumarin, OTA] is one of the most abundant food contaminating mycotoxins [17–20]. OTA is found in tissues and organs of animals including human blood and breast milk and is known to produce nephrotoxic, tetratogenic, carcinogenic and immune toxic activity in several animal species. OTA contamination has been reported in cereals, coffee, wines, dried fruits and animal feeds, as well as in tissues and blood of animals and human beings [18–20]. It affects humans mainly through consumption of improperly stored food products and causes carcinogenicity. The International Agency of Research on Cancer (IARC) has classified OTA as a possible carcinogenic compound (Group 2B, possibly by induction of oxidative DNA damage) for humans (World Health Organization, 1996) since it causes immuno suppression and immuno toxicity [19,20]. Ochratoxin can be detected using various techniques such as thinlayer chromatography (TLC), high performance liquid chromatography (HPLC), enzyme linked immune sorbent assay (ELISA) and electrochemical techniques [1–4,21–23]. Among them, electrochemical immunosensors based on electrochemical impedance spectroscopy (EIS) have recently attracted considerable interest for antigen detection [17,24]. In EIS a nondestructive means for the adsorption/ desorption of biomolecules on conductive supports can be assayed by monitoring interfacial electron transfer features at the electrode surface. In this manuscript, we report results of studies relating to the preparation and characterization of sol–gel derived Nano-ZnO film deposited onto ITO for application as impedimetric sensor for OTA detection. 2. Materials and methods 2.1. Reagents and materials Ochratoxin-A (Aspergillus ochraceus), rabbit immunoglobulin antibodies (r-IgGs), bovine albumin serum (BSA; 98% purity), zinc acetate dehydrate and Triton X-100 have been procured from Sigma Aldrich (USA). NH4OH and HNO3 reagents have been procured from Merck India Ltd, Mumbai, India. All these chemicals are of analytical grade and have been used without further purification. Indium–tin– oxide (ITO) coated glass plates have been obtained from Balzers, UK.
The deionized water obtained from Millipore water purification system (Milli Q 10 TS) has been used for preparation of the solutions and buffers. 2.2. Fabrication of sol–gel derived nanostructured ZnO film/electrode Firstly, 1 g of zinc acetate dihydrate [Zn (CH3COO)2 2H2O] is dissolved in 10 ml ethanol. Then 3 ml (1 M) solution of ammonium hydroxide (NH4OH) is added drop wise to this solution with constant stirring at room temperature to maintain pH = 9–10. A white milky precipitate of Zn(OH)2 thus obtained is centrifuged and is followed by washing with deionized water until neutral pH is achieved. Subsequently, this precipitate is dispersed in distilled water and dilute HNO3 (1 M) in ambient conditions for peptization. A transparent viscous solution is obtained for film fabrication on ITO coated glass plate via dip-coating technique. To achieve uniform coating onto a desired ITO electrode, 2 wt.% TritonX-100 is added to the resulting solution. These films are then allowed to dry at 400 °C for about 1 h [25]. 2.3. Fabrication of immunoelectrode A solution of rabbit immunoglobulin antibodies (r-IgGs) is prepared in phosphate buffer (PB, 50 mM, pH = 7.0). Bovine serum albumin (BSA, 98%) dissolved in PB solution has been used as the blocking agent for non-specific binding sites. Both the solutions containing 0.15 M NaN3 as a preservative are aliquoated and are stored at −20 °C. Immobilization of r-IgGs has been carried out by spreading 10 μL solution onto the sol–gel derived Nano-ZnO/ITO electrode. Both r-IgGs and BSA are immobilized onto the Nano-ZnO/ITO electrode under similar conditions to delineate the role of Nano-ZnO film and synergy between the various components. Scheme 1 shows stepwise fabrication of the BSA/r-IgGs/Nano-ZnO/ITO immunosensor along with the biochemical reaction of r-IgGs with OTA on the Nano-ZnO surface. Ochratoxin-A (A. ochraceus; OTA) solution is prepared in phosphate buffer (50 mM, pH = 7.0) with 10% methanol. 2.4. Characterization X-ray diffraction (XRD, Cu Ka radiation (Rigaku)) studies have been performed to identify the structure of Nano-ZnO. Scanning
Scheme 1. Schematic of fabrication of BSA/r-IgGs/Nano-ZnO/ITO immunosensor along with the biochemical reaction between OTA and immunosensor.
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with preferred (002) orientation with average grain size of ~ 5 nm (estimated using Scherrer's formula). Interestingly, the observed sharp and intensified reflection plane (002) in XRD pattern reveals that Nano-ZnO film grows along c-axis direction. The lattice constant (a) for the Nano-ZnO film calculated from the peak position is found to be (a) = 0.544 Å, which is slightly higher than that of the bulk ZnO (0.520 Å). The higher value of the lattice constant may be attributed to the fact that unit cell is slightly elongated along the direction of growth. Moreover, high value of the lattice constant reveals lattice
Fig. 1. X-ray diffraction pattern of sol–gel derived ZnO film; inset shows absorption spectrum of sol–gel derived Nano-ZnO film.
electron microscopy (SEM, LEO-440) studies have been conducted to examine its surface morphology. UV–visible absorption spectrum (Model 160A, Shimadzu spectrophotometer) has been recorded for the characterization of Nano-ZnO. FTIR spectra of sol–gel derived Nano-ZnO films have been recorded using FTIR (Perkin-Elmer) spectrophotometer to investigate binding of r-IgGs and BSA onto ZnO/ITO film. Electrochemical measurements have been conducted on an Autolab Potentiostat/Galvanostat (Eco Chemie, Netherlands) using a three-electrode cell containing ITO as working electrode, Ag/AgCl as reference electrode and platinum (Pt) wire as counter electrode in phosphate buffer saline (PBS, 50 mM, pH = 7.0, 0.9% NaCl) containing 5 mM [Fe (CN)6]3−/4−. 3. Results and discussion 3.1. Characterization of Nano-ZnO film Fig. 1 shows X-ray diffraction (XRD) pattern of sol–gel derived Nano-ZnO film that exhibits reflection planes (100, 002, 101, 102, 110 and 103) in agreement with the literature (JCPDS #751526)[5,6,13]. Nano-ZnO film exhibits a polycrystalline hexagonal wurtzite structure
Fig. 2. FTIR spectra of Nano-ZnO electrode (a), r-IgGs/Nano-ZnO/ITO electrode (b) and BSA/r-IgGs/Nano-ZnO/ITO electrode (c).
Fig. 3. SEM image of (a) Nano-ZnO/ITO film (b) r-IgGs/Nano-ZnO/ITO electrode and (c) BSA/r-IgGs/Nano-ZnO/ITO electrode.
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expansion effect resulting from increased oxygen vacancies and Zn2+ ions with decreased particle size. The inset in Fig. 1 shows UV–visible absorption spectrum of the Nano-ZnO film on glass substrate. The absorption maxima seen at 371 nm for the precursor ZnO film in absorption spectrum is in agreement with the literature suggesting that precursor species are ZnO molecules [5]. 3.2. FTIR spectroscopic studies FTIR spectra of Nano-ZnO/ITO (curve a), r-IgGs/Nano-ZnO/ITO immunoelectrode (curve b), and BSA/r-IgGs/Nano-ZnO/ITO immunoelectrode (curve c) are shown in Fig. 2. Nano-ZnO/ITO film exhibits bands at 3441, 1591, 1111 cm− 1 corresponding to O–H stretching and bending vibrations of physically adsorbed water molecules on the electrode surface. The band at 506 cm− 1 is attributed to Zn–O stretching vibration modes revealing preparation of the Nano-ZnO film. The 1643 cm− 1 peak observed in the spectrum of r-IgGs/NanoZnO/ITO immunoelectrode (curve b), corresponds to amide II band of
r-IgGs (β-sheet, main secondary structure element of IgG), indicating presence of IgG immobilized onto Nano-ZnO/ITO electrode. The new bands appearing around 1365, 1053, 960 and 849 cm− 1 are attributed to the N–H frequency (amide group), indicating existence of r-IgGs on the electrode surface [24]. A diffused band seen at 3441 cm− 1 (O–H stretching vibration) is shifted towards 3214 cm− 1 (-CONH stretching vibration) due to the presence of amide groups of r-IgGs macromolecules associated with the Zn–O nanoparticles. Hence, the Zn–O– Zn inorganic network is bonded with r-IgGs macromolecules by hydrogen bonding as well as electrostatic interactions in r-IgGs and ZnO nanoparticles. However, presence of 1647 cm− 1 peak in the spectrum of BSA/r-IgGs/Nano-ZnO/ITO bioelectrode (curve c) corresponds to the amide II band of BSA indicating immobilization of BSA on the immunoelectrode [26]. 3.3. Scanning electron microscopy studies Surface morphology of Nano-ZnO/ITO electrode (Fig. 3, image a), r-IgGs/Nano-ZnO/ITO immunoelectrode (Fig. 3, image b) and
Fig. 4. A) Cyclic voltammogram of bare ITO (a) sol–gel derived Nano-ZnO/ITO electrode (b) r-IgGs/Nano-ZnO/ITO electrode (c) and BSA/r-IgGs/Nano-ZnO/ITO electrode (d), B) DPV studies of sol–gel derived Nano-ZnO/ITO electrode (a), r-IgGs/Nano-ZnO/ITO electrode (b) and BSA/r-IgGs/Nano-ZnO/ITO electrode (c) in PBS solution (50 mM, pH = 7.0, 0.9% NaCl) containing 5 mM of [Fe(CN|6]3− and 5 mM of [Fe(CN|6]4−. C) CV of BSA/r-IgGs/Nano-ZnO/ITO electrode as a function of scan rate (10–100 mV/s− 1) in ascending order in PBS solution (50 mM, pH = 7.0, 0.9% NaCl) containing 5 mM of [Fe(CN|6]3− and 5 mM of [Fe(CN|6]4−, D) Magnitude of cathodic and anodic response current as a function of scan rate (10–100 mV/s).
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BSA/r-IgGs/Nano-ZnO/ITO immunoelectrode (Fig. 3, image c) has been studied using scanning electron microscopy (SEM). The SEM image of Nano-ZnO/ITO reveals the formation of smooth, uniform fine granular morphology. After the immobilization of r-IgGs onto the Nano-ZnO/ITO film, granular morphology of Nano-ZnO film changes into spherical flower like morphology with the nano-edges (image b and inset). The nano-edges are inter-connected and exhibit free volume favoring the immobilization of BSA. After the immobilization of BSA, the morphology of the r-IgGs/Nano-ZnO/ ITO immunoelectrode further changes into densely well-arranged flower like morphology (image c) revealing the immobilization of BSA. It is known that adsorbed r-IgG molecules are favorably oriented when Fc (carboxyl terminated group) part is attached to the electrode and its Fab (amino terminated site) part binds with a given antigen with high level of specificity. Scheme 1 shows various steps relating to fabrication of the immunosensor and the biochemical reaction of r-IgGs with OTA at the nanobiocomposite surface. In this Scheme, positively charged Nano-ZnO (IEP ~9.5) binds to the carboxyl groups of r-IgG via electrostatic interactions and free amino terminal sites of r-IgG preferably bind with the carboxylic group of OTA molecules. 3.4. Cyclic voltammetry studies Fig. 4A shows cyclic voltammograms of bare ITO (curve a), NanoZnO/ITO electrode (curve b), r-IgGs/Nano-ZnO/ITO immunoelectrode (curve c) and BSA/r-IgGs/Nano-ZnO/ITO immunoelectrode (curve d) in PBS (50 mM, pH = 7.0, 0.9% NaCl) containing 5 mM [Fe(CN)6]3−/4− obtained at a scanning rate of 50 mVs− 1. It is observed that magnitude of the current response of Nano-ZnO/ ITO electrode (curve b) is higher than that of the bare ITO (curve a) revealing that Nano-ZnO has increased electroactive surface of the electrode resulting in enhanced electron transport between medium and the electrode. After the immobilization of r-IgGs onto Nano-ZnO/ITO electrode, significant increase in the magnitude of current response is observed (curve c). This suggests that Nano-ZnO film provides increased surface area for r-IgGs immobilization resulting in enhanced electron kinetics. Additionally, the presence of non-binding sites on r-IgGs increases the magnitude of current response. Moreover, sol–gel derived ZnO matrix provides a three-dimensional stage and some of the restricted orientations also favour direct and faster electron communication between r-IgGs and the electrode surface. The magnitude of
Fig. 5. Electrochemical Impedance spectra of sol–gel derived Nano-ZnO electrode (a), r-IgGs/Nano-ZnO/ITO electrode (b) and BSA/r-IgGs/Nano-ZnO/ITO electrode (c) in PBS solution (50 mM, pH = 7.0, 0.9% NaCl) containing 5 mM [Fe(CN|6]3−/4−, inset: diagram of electronic circuit.
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current decreases after the immobilization of BSA (curve d) revealing that blocking of non-binding sites of r-IgGs hinders diffusion of ferricyanide ions toward the electrode surface. These results indicate the immobilization of BSA onto r-IgGs/Nano-ZnO/ITO immunoelectrode. Similar results have been obtained using differential pulse voltammetry (DPV) studies (Fig. 4B) of Nano-ZnO/ITO electrode (curve a), r-IgGs/ Nano-ZnO/ITO immunoelectrode (curve b) and BSA/ r-IgGs/Nano-ZnO/ITO immunoelectrode (curve c). It can be seen that magnitude of the current response of r-IgGs/ Nano-ZnO/ITO immunoelectrode (curve b) is higher than that of the Nano-ZnO/ITO electrode (curve a). And the magnitude of current response decreases after the immobilization of BSA onto r-IgGs/ Nano-ZnO/ITO immunoelectrode (curve c). Fig. 4C exhibits results of the CV studies of BSA/r-IgGs/Nano-ZnO/ ITO immunoelectrode as a function of scan rate (10–100 mV/s) carried out in PBS (50 mM, pH = 7.0, 0.9% NaCl). It is observed that cathodic and anodic peak current response of BSA/r-IgGs/Nano-ZnO/ ITO immunoelectrode increases (Fig. 4D) suggesting that electrochemical reaction is a diffusion-controlled process. Moreover, cathodic and anodic peak potentials separation also increases indicating facile charge transfer kinetics in the 10–100 mV/s range of scan rates. These results reveal that sol–gel derived Nano-ZnO/ITO electrode provides a congenial microenvironment similar to that of redox protein in a native system and allows protein molecules more freedom for orientation, thus facilitating close approach of the proteins (IgGs) to the sensing surface due to reduced insulating behaviour of the protein shell for direct electron transfer. This could be thought of as “electron antennae” that perhaps accelerates electrons between electrode and immobilized r-IgGs indicating availability of the immobilized r-IgGs molecules. The activity of the BSA/r-IgGs/Nano-ZnO/ITO immunoelectrode has been estimated as a function of pH varying from 6.0 to 8.0 at room temperature (25 °C). The high magnitude of current obtained at pH = 7.0 (data not shown) indicates that BSA/r-IgGs/Nano-ZnO/ITO immunoelectrode is more active at pH = 7.0 at which r-IgGs and BSA retain natural structure and do not denature. Thus all the experiments have been conducted at a pH = 7.0. 3.5. Electrochemical impedance spectroscopy studies Electrochemical impedance spectroscopy (EIS) is an effective method to investigate biochemical processes at the modified electrode surfaces to investigate the deposition of a ZnO film onto ITO
Fig. 6. Linear response curve of BSA/r-IgGs/Nano-ZnO/ITO immunosensor obtained between OTA concentration and RCT value.
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electrode and the immobilization of r-IgGs and BSA. The semicircle diameter of EIS equals electron transfer resistance (RCT) that controls electron transfer kinetics of the redox-probe at the electrode interface. RCT value varies when different substances are adsorbed onto the electrode surface. RCT and electrochemical reaction time constant (τ) can be calculated as t = 1=2π fmax = Rp ⋅Cdl
ð1Þ
where fmax is the frequency at which maximum Z″ is obtained, Rp is polarization resistance and Cdl is double layer capacitance. The experimentally obtained EIS data is fitted to the electrical equivalent circuit (Randles and Ershler Model 1) as shown in the inset Fig. 5. RCT for r-IgGs/Nano-ZnO/ITO electrode (curve b, Fig. 5) is estimated to be 2.12 k Ω, that is lower as compared to that of the Nano-ZnO/ITO electrode (RCT, 4.4 kΩ; curve a). It implies that a conductive layer (rIgGs) is assembled on the electrode surface resulting in accelerated electron communication between r-IgGs and the Nano-ZnO/ITO electrode (IEP of IgGs, ~5.5; IEP of ZnO, 9.5). After immobilization of BSA onto the r-IgGs/Nano-ZnO/ITO electrode, significant enhancement in RCT (3.34 k Ω, curve c) is observed. This may be due to the insulating BSA layer that perhaps hinders diffusion of ferricyanide ions toward the electrode surface resulting in increased value of RCT. The observed change in RCT after BSA immobilization can be assigned to the dielectric and insulating features at the electrode–electrolyte interface that is associated with successive immobilization of biomolecules. EIS analysis of Nano-ZnO/ITO electrode, r-IgGs/Nano-ZnO/ITO immunoelectrode and BSA/r-IgGs/Nano-ZnO/BSA immunoelectrode have been repeated several times (~15 times) on the same film surface in the electrolyte and different films have been prepared from the same stock solution .The results have been found to be reproducible (within 4% error). 3.6. Electrochemical impedimetric response studies Dependence of the electron transfer resistance, RCT, on the OTA concentration for the BSA/r-IgCs/Nano-ZnO/ITO immunosensor in 10 mL phosphate buffer (50 mM, pH = 7.0 an 0.9% NaCl) containing [Fe(CN)6]3−/4− and subsequent addition of OTA concentration (1–6 ng/dL) is stirred for 30 s using EIS technique in triplet set. The interaction of OTA with IgGs on the electrode surface results in significant changes in impedimetric parameters (RCT, Cdl, Rs and Zw). Fig. 6 shows the linear curve between the RCT values obtained during EIS response of the BSA/r-IgGs/Nano-ZnO/ITO immunoelectrode as a function of OTA (mycotoxin) concentration. The value of RCT increases on addition of OTA concentration. This may be attributed to the increased number of OTA molecules bound to the immobilized antibodies that perhaps provide a kinetic barrier for the electron transfer. BSA/r-IgGs/Nano-ZnO/ITO immunoelectrode exhibits improved sensing characteristics such as linearity as 0.006–0.01 nM/ dm3, detection limit as 0.006 nM/dm3, short response time of 25 s, and long term stability (45 days), sensitivity of 189 Ω/nM/dm3 cm2, reproducibility and the regression coefficient of 0.997. It may be noted that conformational changes are known to affect a biological reaction that in turn may be influenced by the nature of immobilization matrix and its surface morphology. The increased activity of r-IgGs due to favourable conformational changes results in enhanced interaction between the Nano-ZnO/ITO electrode and the active site of r-IgGs as indicated by the observed high value of stability constant (Ka, 7.6 × 1011 L/m) revealing high affinity of r-IgGs towards OTA due to prevalent electrostatic interactions. This may be attributed to favourable conformation of r-IgGs and increased loading of r-IgGs provided by the microenvironment of sol–gel derived Nano-ZnO film.
4. Conclusions It has been demonstrated that sol–gel derived Nano-ZnO film can be used for the immobilization of r-IgGs and BSA for blocking nonspecific binding sites of r-IgGs to detect OTA. The results show that Nano-ZnO electrode results in amplified electrochemical signal due to strong r-IgGs-OTA interaction occurring at the immunoelectrode surface. A good linear relationship between the electron transfer resistance and OTA concentration in the range, 0.006–0.01 nM/dm3 with improved sensitivity and detection limit etc has been obtained. It should be interesting to utilize the BSA/r-IgGs/Nano-ZnO/ITO platform for detection of other mycotoxins such as alphatoxin, ochratoxin B, citrinin, ergot akaloids, fumonisins, patulin, trichothecenes, and zearalenone, etc. And the findings of these studies have implications in clinical diagnostics, antibody screening and proteomics research. Acknowledgements We thank Dr. Vikram Kumar, Director, NPL, New Delhi, India for the facilities. Financial support received under the Department of Science & Technology (DST), Govt. of India the Department of Science and Technology (DST) projects [DST/TSG/ME/2008/18 and GAP- 070932], in-house project (OLP-070632D), India–Japan project (DST/INT/JAP/P21/07), and the Department of Biotechnology, Govt. of India (DBT/ GAP070832) is gratefully acknowledged. References [1] P.B. Luppa, L.J. Sokoll, D.W. Chan, Immunosensors—principles and applications to clinical chemistry, Clin. Chim. Acta 314 (2001) 1–26. [2] E. Katz, I. Willner, Probing biomolecular interactions at conductive and semiconductive surfaces by impedance spectroscopy: routes to impedimetric immunosensors, DNA-sensors, and enzyme biosensors, Electroanalysis 15 (2003) 913–947. [3] X.M. Li, X.Y. Yang, S.S. Zhang, Electrochemical enzyme immunoassay using model labels, Trends Anal. Chem. 27 (2008) 543–553. [4] M. Pumera, S. Sanchez, I. Ichinose, J. Tang, Electrochemical nanobiosensors, Sens. Actuators B 123 (2007) 1195–1205. [5] L. Irimpan, V.P.N. Nampoori, P. Radhakrishnan, A. Deepthy, B. Krishnan, Size dependent fluorescence spectroscopy of nanocolloids of ZnO, J. Appl. Phys. 102 (2007) 063524-5. [6] A. Wei, X.W. Suna, J.X. Wang, Y. Lei, X.P. Cai, C.M. Li, Z.L. Dong, W. Huang, Enzymatic glucose biosensor based on ZnO nanorod array grown by hydrothermal decomposition, Appl. Phys. Lett. 89 (2006) 123902-3. [7] J.F. Wang, X.W. Sun, A. Wei, Y. Lei, X.P. Cai, C.M. Li, Z.L. Dong, Zinc oxide nanocomb biosensor for glucose detection, Appl. Phys. Lett. 88 (2006) 233106-3. [8] S. Zong, Y. Cao, Y. Zhou, H. Ju, Zirconia nanoparticles enhanced grafted collagen tri helix scaffold for unmediated biosensing of hydrogen peroxide, Langmuir 22 (2006) 8915–8919. [9] J. Yu, H. Ju, Preparation of porous titania sol–gel matrix for immobilization of horseradish peroxidase by a vapor deposition method, Anal. Chem. 74 (2002) 3579–3583. [10] E. Topoglidis, C.J. Campbell, A.E.G. Cass, J.R. Durrant, Factors that affect protein adsorption on nanostructured titania films. A novel spectroelectrochemical application to sensing, Langmuir 17 (2001) 7899–7906. [11] E. Topoglidis, Y. Astuti, F. Duriaux, M. Gratzel, J.R. Durrant, Direct electrochemistry and nitric oxide interaction of heme proteins adsorbed on nanocrystalline tin oxide electrodes, Langmuir 19 (2003) 6894–6900. [12] A.A. Ansari, P.R. Solanki, B.D. Malhotra, Sol–gel derived nanostructured cerium oxide film for glucose sensor, Appl. Phys. Lett. 92 (2008) 263901-3. [13] A.A. Ansari, R. Singh, G. Sumana, B.D. Malhotra, Sol–gel derived nano-structured zinc oxide film for sexually transmitted disease sensor, Analyst 134 (2009) 997–1002. [14] P.R. Solanki, A. Kaushik, A.A. Ansari, G. Sumana, B.D. Malhotra, Zinc oxide–chitosan nanobiocomposite for urea sensor, Appl. Phys. Lett. 93 (2008) 163903-3. [15] E. Topoglidis, A.E.G. Cass, B. O'Regan, J.R. Durrant, Immobilization and bioelectrochemistry of proteins on nanoporous TiO2 and ZnO films, J. Electroanal. Chem. 517 (2001) 20–27. [16] Y.L. Liu, Y.H. Yang, F.H. Yang, M.Z. Liu, L.G. Shen, Q.R. Yu, Nanosized flower-like ZnO synthesized by a simple hydrothermal method and applied as matrix for horseradish peroxidase immobilization for electro-biosensing, J. Inorg. Biochem. 99 (2005) 2046–2053. [17] A.E. Radi, X.M. Berbel, V. Lates, J.L. Marty, Label-free impedimetric immunosensor for sensitive detection of ochratoxin A, Biosens. Bioelectron. 24 (2009) 1888–1892. [18] L.A. Pfohl, B.T. Petkova, I.N. Chernozemsky, M. Castegnaro, Balkan endemic nephropathy and the associated urinary tract tumors: review on etiological cause, potential role of mycotoxines, Food Addit. Contam. 19 (2002) 282–302.
A.A. Ansari et al. / Bioelectrochemistry 77 (2010) 75–81 [19] T.K. Goodman, in: M. Miraglia, H. van Edmond, C. Brera, J. Gilbert (Eds.), Mycotoxins and phycotoxins—developments in chemistry, toxicology and food safety. alaken Inc., Fort Collins, USA, 1998, p. 25. [20] B. Zimmerli, R. Dick, Ochratoxin A in table wine and grape-juice: occurrence and risk assessment, Food Addit. Contam. 13 (1996) 655–668. [21] A. Pittet, Modern methods and trends in mycotoxin analysis, Mitt. Lebensm. Hyg. 96 (2005) 424–444. [22] P.A. Burdaspal, T.M. Legarda, Ochratoxin A in wines and grape products originated from Spain and other European countries, Alimentaria 299 (1999) 107–113. [23] A.C. Barton, F. Davis, S.P.J. Higson, Labeless immunosensor assay for prostate specific antigen with picogram per milliliter limits of detection based upon an Ac impedance protocol, Anal. Chem. 80 (2008) 6198–6205.
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[24] A. Kaushik, P.R. Solanki, A.A. Ansari, S. Ahmad, B.D. Malhotra, Chitosan–iron oxide nanobiocomposite based immunosensor for ochratoxin-A, Electrochem. Commun. 10 (2008) 1364–1368. [25] A.A. Ansari, A. Kaushik, P.R. Solanki, B.D. Malhotra, Sol–gel derived nanoporous cerium oxide film for application to cholesterol biosensor, Electrochem. Commun. 10 (2008) 1246–1268. [26] R.C. Triulzi, M. Micic, J. Orbulescu, S. Giordani, B. Muellerd, R.M. Leblanc, Antibody– gold quantum dot–PAMAM dendrimer complex as an immunoglobulin immunoassay, Analyst 133 (2008) 667–672.
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Bioelectrochemistry j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b i o e l e c h e m
Nanostructured zinc oxide platform for mycotoxin detection Anees A. Ansari ⁎,1, Ajeet Kaushik, Pratima R. Solanki, B.D. Malhotra ⁎ Department of Science & Technology Centre on Biomolecular Electronics, National Physical Laboratory, Dr. K. S. Krishnan Marg, New Delhi-110012, India
a r t i c l e
i n f o
Article history: Received 7 April 2009 Received in revised form 23 June 2009 Accepted 25 June 2009 Available online 8 July 2009 Keywords: Sol–gel Zinc oxide Impedimetric immunosensor Mycotoxin Ochratoxin-A
a b s t r a c t Nanostructured zinc oxide (Nano-ZnO) film has been deposited onto indium–tin–oxide (ITO) glass plate for co-immobilization of rabbit-immunoglubin antibodies (r-IgGs) and bovine serum albumin (BSA) for ochratoxin-A (OTA) detection. The results of X-ray diffraction (XRD) studies reveal the formation of Nano-ZnO with average particle size as ~5.0 nm. Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), electrochemical impedance spectroscopy (EIS) techniques have been used to characterize Nano-ZnO/ITO electrode and BSA/r-IgGs/Nano-ZnO/ITO immunoelectrode. Electrochemical impedimetric response of BSA/r-IgGs/Nano-ZnO/ITO immunoelectrode obtained as a function of OTA concentration exhibits linearity as 0.006–0.01 nM/dm3, detection limit of 0.006 nM/dm3, response time as 25 s and sensitivity of 189 Ω/nM/dm3cm− 2 with a regression coefficient of 0.997. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Electrochemical immunosensors have recently aroused much interest for detection of desired proteins, biomarkers, biological toxins and bio-warfare agents in critical situations, food, environment, pharmaceutical chemistry and clinical diagnostics [1–4]. To obtain a sensitive, compact and stable immunosensor platform, the desired antibodies should be directed in a given orientation on the electrode. The selection of a matrix for the immobilization of antibodies (Abs) is extremely important to obtain desired characteristics of an immunoelectrode [1,2]. Nanostructured metal oxides have recently attracted much attention due to their interesting electrocatalytic, piezoelectric, photonic and tunable size dependent surface properties that make them attractive for biosensing applications [5–7]. In this context, zirconium oxide [8], titanium oxide [9,10], tin oxide [11], cerium oxide [12] and zinc oxide (ZnO) [13–16] have been utilized for immobilization of proteins, enzymes and antigens for accelerated electron transfer between desired immobilized biomolecules and electrode. These nanomaterials exhibit interesting properties such as large surface-tovolume ratio, high surface reaction activity, high catalytic efficiency, and strong adsorption ability that make them potential candidate materials to play a catalytic role in the fabrication of an immunosensor. The large surface area of a nanostuctured metal oxide is likely
⁎ Corresponding authors. Ansari is to be contacted at Tel.: +91 11 45609152; fax: +91 11 45609310. Malhotra, Tel.: +91 11 45609151; fax: +91 11 45609310. E-mail addresses: [email protected] (A.A. Ansari), [email protected] (B.D. Malhotra). 1 Presently at the King Abdullah Institute for Nanotechnology, King Saud University, Riyadh-Saudi Arabia. 1567-5394/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.bioelechem.2009.06.014
to lead to increased Abs loading per unit mass of particles. Moreover, multipoint attachment of enzyme molecules to nanomaterial surfaces reduces protein unfolding resulting in enhanced stability of the biomolecule attached to nanoparticles surface. In this context, the enzyme-attached nanoparticles facilitate enzymes to act as free enzymes in solution and in turn improve enzyme–substrate interaction by minimizing potential aggregation of the free enzyme. Among the various metal oxides, nanostructured ZnO has been used for immunosensor applications [6,7,13–16]. Its unique properties such as high isoelectric point (IEP ~9.5) and biocompatibility facilitate immobilization of an enzyme and protein having low IEP via electrostatic interactions. Besides this, the positively charged ZnO nanoparticles (IEP ~ 9.5) not only provide a friendly microenvironment for immobilizing negatively charged rabbit antibodies (r-IgGs; IEP ~ 5.5) and retain its bioactivity but also accelerate electron transfer communication between protein and the electrode to a large extent [6,7]. Moreover, non-toxicity, high chemical stability and high electron transfer capability make Nano-ZnO a promising material for immobilization of desired biomolecules for fabrication of an immunosensor. The sol–gel derived Nano-ZnO has recently emerged as an attractive material due to its ease of preparation under ambient conditions, tunable porosity, high thermal stability, chemical inertness and negligible swelling in aqueous and non-aqueous solutions for immobilization of desired biomolecules (enzymes, proteins and antibodies) [9,12]. Liu et al. have prepared stable nano-sized flower like ZnO film by hydrothermal method for H2O2 sensor [16]. ZnO nanocomb has been fabricated in bulk quantity by vapor phase transport method for glucose detection [7]. Wei et al have grown ZnO nanorods on Au electrode for development of enzymatic glucose biosensor [6]. However, sol–gel derived Nano-ZnO has not yet been utilized for development of immunosensor for detection of ochratoxin-A (OTA).
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Ochratoxin-A [7-(L-β-phenylalanylcarbonyl)-carboxyl-5-chloro8-hydroxy-3,4-dihydro-3R-methylisocumarin, OTA] is one of the most abundant food contaminating mycotoxins [17–20]. OTA is found in tissues and organs of animals including human blood and breast milk and is known to produce nephrotoxic, tetratogenic, carcinogenic and immune toxic activity in several animal species. OTA contamination has been reported in cereals, coffee, wines, dried fruits and animal feeds, as well as in tissues and blood of animals and human beings [18–20]. It affects humans mainly through consumption of improperly stored food products and causes carcinogenicity. The International Agency of Research on Cancer (IARC) has classified OTA as a possible carcinogenic compound (Group 2B, possibly by induction of oxidative DNA damage) for humans (World Health Organization, 1996) since it causes immuno suppression and immuno toxicity [19,20]. Ochratoxin can be detected using various techniques such as thinlayer chromatography (TLC), high performance liquid chromatography (HPLC), enzyme linked immune sorbent assay (ELISA) and electrochemical techniques [1–4,21–23]. Among them, electrochemical immunosensors based on electrochemical impedance spectroscopy (EIS) have recently attracted considerable interest for antigen detection [17,24]. In EIS a nondestructive means for the adsorption/ desorption of biomolecules on conductive supports can be assayed by monitoring interfacial electron transfer features at the electrode surface. In this manuscript, we report results of studies relating to the preparation and characterization of sol–gel derived Nano-ZnO film deposited onto ITO for application as impedimetric sensor for OTA detection. 2. Materials and methods 2.1. Reagents and materials Ochratoxin-A (Aspergillus ochraceus), rabbit immunoglobulin antibodies (r-IgGs), bovine albumin serum (BSA; 98% purity), zinc acetate dehydrate and Triton X-100 have been procured from Sigma Aldrich (USA). NH4OH and HNO3 reagents have been procured from Merck India Ltd, Mumbai, India. All these chemicals are of analytical grade and have been used without further purification. Indium–tin– oxide (ITO) coated glass plates have been obtained from Balzers, UK.
The deionized water obtained from Millipore water purification system (Milli Q 10 TS) has been used for preparation of the solutions and buffers. 2.2. Fabrication of sol–gel derived nanostructured ZnO film/electrode Firstly, 1 g of zinc acetate dihydrate [Zn (CH3COO)2 2H2O] is dissolved in 10 ml ethanol. Then 3 ml (1 M) solution of ammonium hydroxide (NH4OH) is added drop wise to this solution with constant stirring at room temperature to maintain pH = 9–10. A white milky precipitate of Zn(OH)2 thus obtained is centrifuged and is followed by washing with deionized water until neutral pH is achieved. Subsequently, this precipitate is dispersed in distilled water and dilute HNO3 (1 M) in ambient conditions for peptization. A transparent viscous solution is obtained for film fabrication on ITO coated glass plate via dip-coating technique. To achieve uniform coating onto a desired ITO electrode, 2 wt.% TritonX-100 is added to the resulting solution. These films are then allowed to dry at 400 °C for about 1 h [25]. 2.3. Fabrication of immunoelectrode A solution of rabbit immunoglobulin antibodies (r-IgGs) is prepared in phosphate buffer (PB, 50 mM, pH = 7.0). Bovine serum albumin (BSA, 98%) dissolved in PB solution has been used as the blocking agent for non-specific binding sites. Both the solutions containing 0.15 M NaN3 as a preservative are aliquoated and are stored at −20 °C. Immobilization of r-IgGs has been carried out by spreading 10 μL solution onto the sol–gel derived Nano-ZnO/ITO electrode. Both r-IgGs and BSA are immobilized onto the Nano-ZnO/ITO electrode under similar conditions to delineate the role of Nano-ZnO film and synergy between the various components. Scheme 1 shows stepwise fabrication of the BSA/r-IgGs/Nano-ZnO/ITO immunosensor along with the biochemical reaction of r-IgGs with OTA on the Nano-ZnO surface. Ochratoxin-A (A. ochraceus; OTA) solution is prepared in phosphate buffer (50 mM, pH = 7.0) with 10% methanol. 2.4. Characterization X-ray diffraction (XRD, Cu Ka radiation (Rigaku)) studies have been performed to identify the structure of Nano-ZnO. Scanning
Scheme 1. Schematic of fabrication of BSA/r-IgGs/Nano-ZnO/ITO immunosensor along with the biochemical reaction between OTA and immunosensor.
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with preferred (002) orientation with average grain size of ~ 5 nm (estimated using Scherrer's formula). Interestingly, the observed sharp and intensified reflection plane (002) in XRD pattern reveals that Nano-ZnO film grows along c-axis direction. The lattice constant (a) for the Nano-ZnO film calculated from the peak position is found to be (a) = 0.544 Å, which is slightly higher than that of the bulk ZnO (0.520 Å). The higher value of the lattice constant may be attributed to the fact that unit cell is slightly elongated along the direction of growth. Moreover, high value of the lattice constant reveals lattice
Fig. 1. X-ray diffraction pattern of sol–gel derived ZnO film; inset shows absorption spectrum of sol–gel derived Nano-ZnO film.
electron microscopy (SEM, LEO-440) studies have been conducted to examine its surface morphology. UV–visible absorption spectrum (Model 160A, Shimadzu spectrophotometer) has been recorded for the characterization of Nano-ZnO. FTIR spectra of sol–gel derived Nano-ZnO films have been recorded using FTIR (Perkin-Elmer) spectrophotometer to investigate binding of r-IgGs and BSA onto ZnO/ITO film. Electrochemical measurements have been conducted on an Autolab Potentiostat/Galvanostat (Eco Chemie, Netherlands) using a three-electrode cell containing ITO as working electrode, Ag/AgCl as reference electrode and platinum (Pt) wire as counter electrode in phosphate buffer saline (PBS, 50 mM, pH = 7.0, 0.9% NaCl) containing 5 mM [Fe (CN)6]3−/4−. 3. Results and discussion 3.1. Characterization of Nano-ZnO film Fig. 1 shows X-ray diffraction (XRD) pattern of sol–gel derived Nano-ZnO film that exhibits reflection planes (100, 002, 101, 102, 110 and 103) in agreement with the literature (JCPDS #751526)[5,6,13]. Nano-ZnO film exhibits a polycrystalline hexagonal wurtzite structure
Fig. 2. FTIR spectra of Nano-ZnO electrode (a), r-IgGs/Nano-ZnO/ITO electrode (b) and BSA/r-IgGs/Nano-ZnO/ITO electrode (c).
Fig. 3. SEM image of (a) Nano-ZnO/ITO film (b) r-IgGs/Nano-ZnO/ITO electrode and (c) BSA/r-IgGs/Nano-ZnO/ITO electrode.
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expansion effect resulting from increased oxygen vacancies and Zn2+ ions with decreased particle size. The inset in Fig. 1 shows UV–visible absorption spectrum of the Nano-ZnO film on glass substrate. The absorption maxima seen at 371 nm for the precursor ZnO film in absorption spectrum is in agreement with the literature suggesting that precursor species are ZnO molecules [5]. 3.2. FTIR spectroscopic studies FTIR spectra of Nano-ZnO/ITO (curve a), r-IgGs/Nano-ZnO/ITO immunoelectrode (curve b), and BSA/r-IgGs/Nano-ZnO/ITO immunoelectrode (curve c) are shown in Fig. 2. Nano-ZnO/ITO film exhibits bands at 3441, 1591, 1111 cm− 1 corresponding to O–H stretching and bending vibrations of physically adsorbed water molecules on the electrode surface. The band at 506 cm− 1 is attributed to Zn–O stretching vibration modes revealing preparation of the Nano-ZnO film. The 1643 cm− 1 peak observed in the spectrum of r-IgGs/NanoZnO/ITO immunoelectrode (curve b), corresponds to amide II band of
r-IgGs (β-sheet, main secondary structure element of IgG), indicating presence of IgG immobilized onto Nano-ZnO/ITO electrode. The new bands appearing around 1365, 1053, 960 and 849 cm− 1 are attributed to the N–H frequency (amide group), indicating existence of r-IgGs on the electrode surface [24]. A diffused band seen at 3441 cm− 1 (O–H stretching vibration) is shifted towards 3214 cm− 1 (-CONH stretching vibration) due to the presence of amide groups of r-IgGs macromolecules associated with the Zn–O nanoparticles. Hence, the Zn–O– Zn inorganic network is bonded with r-IgGs macromolecules by hydrogen bonding as well as electrostatic interactions in r-IgGs and ZnO nanoparticles. However, presence of 1647 cm− 1 peak in the spectrum of BSA/r-IgGs/Nano-ZnO/ITO bioelectrode (curve c) corresponds to the amide II band of BSA indicating immobilization of BSA on the immunoelectrode [26]. 3.3. Scanning electron microscopy studies Surface morphology of Nano-ZnO/ITO electrode (Fig. 3, image a), r-IgGs/Nano-ZnO/ITO immunoelectrode (Fig. 3, image b) and
Fig. 4. A) Cyclic voltammogram of bare ITO (a) sol–gel derived Nano-ZnO/ITO electrode (b) r-IgGs/Nano-ZnO/ITO electrode (c) and BSA/r-IgGs/Nano-ZnO/ITO electrode (d), B) DPV studies of sol–gel derived Nano-ZnO/ITO electrode (a), r-IgGs/Nano-ZnO/ITO electrode (b) and BSA/r-IgGs/Nano-ZnO/ITO electrode (c) in PBS solution (50 mM, pH = 7.0, 0.9% NaCl) containing 5 mM of [Fe(CN|6]3− and 5 mM of [Fe(CN|6]4−. C) CV of BSA/r-IgGs/Nano-ZnO/ITO electrode as a function of scan rate (10–100 mV/s− 1) in ascending order in PBS solution (50 mM, pH = 7.0, 0.9% NaCl) containing 5 mM of [Fe(CN|6]3− and 5 mM of [Fe(CN|6]4−, D) Magnitude of cathodic and anodic response current as a function of scan rate (10–100 mV/s).
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BSA/r-IgGs/Nano-ZnO/ITO immunoelectrode (Fig. 3, image c) has been studied using scanning electron microscopy (SEM). The SEM image of Nano-ZnO/ITO reveals the formation of smooth, uniform fine granular morphology. After the immobilization of r-IgGs onto the Nano-ZnO/ITO film, granular morphology of Nano-ZnO film changes into spherical flower like morphology with the nano-edges (image b and inset). The nano-edges are inter-connected and exhibit free volume favoring the immobilization of BSA. After the immobilization of BSA, the morphology of the r-IgGs/Nano-ZnO/ ITO immunoelectrode further changes into densely well-arranged flower like morphology (image c) revealing the immobilization of BSA. It is known that adsorbed r-IgG molecules are favorably oriented when Fc (carboxyl terminated group) part is attached to the electrode and its Fab (amino terminated site) part binds with a given antigen with high level of specificity. Scheme 1 shows various steps relating to fabrication of the immunosensor and the biochemical reaction of r-IgGs with OTA at the nanobiocomposite surface. In this Scheme, positively charged Nano-ZnO (IEP ~9.5) binds to the carboxyl groups of r-IgG via electrostatic interactions and free amino terminal sites of r-IgG preferably bind with the carboxylic group of OTA molecules. 3.4. Cyclic voltammetry studies Fig. 4A shows cyclic voltammograms of bare ITO (curve a), NanoZnO/ITO electrode (curve b), r-IgGs/Nano-ZnO/ITO immunoelectrode (curve c) and BSA/r-IgGs/Nano-ZnO/ITO immunoelectrode (curve d) in PBS (50 mM, pH = 7.0, 0.9% NaCl) containing 5 mM [Fe(CN)6]3−/4− obtained at a scanning rate of 50 mVs− 1. It is observed that magnitude of the current response of Nano-ZnO/ ITO electrode (curve b) is higher than that of the bare ITO (curve a) revealing that Nano-ZnO has increased electroactive surface of the electrode resulting in enhanced electron transport between medium and the electrode. After the immobilization of r-IgGs onto Nano-ZnO/ITO electrode, significant increase in the magnitude of current response is observed (curve c). This suggests that Nano-ZnO film provides increased surface area for r-IgGs immobilization resulting in enhanced electron kinetics. Additionally, the presence of non-binding sites on r-IgGs increases the magnitude of current response. Moreover, sol–gel derived ZnO matrix provides a three-dimensional stage and some of the restricted orientations also favour direct and faster electron communication between r-IgGs and the electrode surface. The magnitude of
Fig. 5. Electrochemical Impedance spectra of sol–gel derived Nano-ZnO electrode (a), r-IgGs/Nano-ZnO/ITO electrode (b) and BSA/r-IgGs/Nano-ZnO/ITO electrode (c) in PBS solution (50 mM, pH = 7.0, 0.9% NaCl) containing 5 mM [Fe(CN|6]3−/4−, inset: diagram of electronic circuit.
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current decreases after the immobilization of BSA (curve d) revealing that blocking of non-binding sites of r-IgGs hinders diffusion of ferricyanide ions toward the electrode surface. These results indicate the immobilization of BSA onto r-IgGs/Nano-ZnO/ITO immunoelectrode. Similar results have been obtained using differential pulse voltammetry (DPV) studies (Fig. 4B) of Nano-ZnO/ITO electrode (curve a), r-IgGs/ Nano-ZnO/ITO immunoelectrode (curve b) and BSA/ r-IgGs/Nano-ZnO/ITO immunoelectrode (curve c). It can be seen that magnitude of the current response of r-IgGs/ Nano-ZnO/ITO immunoelectrode (curve b) is higher than that of the Nano-ZnO/ITO electrode (curve a). And the magnitude of current response decreases after the immobilization of BSA onto r-IgGs/ Nano-ZnO/ITO immunoelectrode (curve c). Fig. 4C exhibits results of the CV studies of BSA/r-IgGs/Nano-ZnO/ ITO immunoelectrode as a function of scan rate (10–100 mV/s) carried out in PBS (50 mM, pH = 7.0, 0.9% NaCl). It is observed that cathodic and anodic peak current response of BSA/r-IgGs/Nano-ZnO/ ITO immunoelectrode increases (Fig. 4D) suggesting that electrochemical reaction is a diffusion-controlled process. Moreover, cathodic and anodic peak potentials separation also increases indicating facile charge transfer kinetics in the 10–100 mV/s range of scan rates. These results reveal that sol–gel derived Nano-ZnO/ITO electrode provides a congenial microenvironment similar to that of redox protein in a native system and allows protein molecules more freedom for orientation, thus facilitating close approach of the proteins (IgGs) to the sensing surface due to reduced insulating behaviour of the protein shell for direct electron transfer. This could be thought of as “electron antennae” that perhaps accelerates electrons between electrode and immobilized r-IgGs indicating availability of the immobilized r-IgGs molecules. The activity of the BSA/r-IgGs/Nano-ZnO/ITO immunoelectrode has been estimated as a function of pH varying from 6.0 to 8.0 at room temperature (25 °C). The high magnitude of current obtained at pH = 7.0 (data not shown) indicates that BSA/r-IgGs/Nano-ZnO/ITO immunoelectrode is more active at pH = 7.0 at which r-IgGs and BSA retain natural structure and do not denature. Thus all the experiments have been conducted at a pH = 7.0. 3.5. Electrochemical impedance spectroscopy studies Electrochemical impedance spectroscopy (EIS) is an effective method to investigate biochemical processes at the modified electrode surfaces to investigate the deposition of a ZnO film onto ITO
Fig. 6. Linear response curve of BSA/r-IgGs/Nano-ZnO/ITO immunosensor obtained between OTA concentration and RCT value.
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electrode and the immobilization of r-IgGs and BSA. The semicircle diameter of EIS equals electron transfer resistance (RCT) that controls electron transfer kinetics of the redox-probe at the electrode interface. RCT value varies when different substances are adsorbed onto the electrode surface. RCT and electrochemical reaction time constant (τ) can be calculated as t = 1=2π fmax = Rp ⋅Cdl
ð1Þ
where fmax is the frequency at which maximum Z″ is obtained, Rp is polarization resistance and Cdl is double layer capacitance. The experimentally obtained EIS data is fitted to the electrical equivalent circuit (Randles and Ershler Model 1) as shown in the inset Fig. 5. RCT for r-IgGs/Nano-ZnO/ITO electrode (curve b, Fig. 5) is estimated to be 2.12 k Ω, that is lower as compared to that of the Nano-ZnO/ITO electrode (RCT, 4.4 kΩ; curve a). It implies that a conductive layer (rIgGs) is assembled on the electrode surface resulting in accelerated electron communication between r-IgGs and the Nano-ZnO/ITO electrode (IEP of IgGs, ~5.5; IEP of ZnO, 9.5). After immobilization of BSA onto the r-IgGs/Nano-ZnO/ITO electrode, significant enhancement in RCT (3.34 k Ω, curve c) is observed. This may be due to the insulating BSA layer that perhaps hinders diffusion of ferricyanide ions toward the electrode surface resulting in increased value of RCT. The observed change in RCT after BSA immobilization can be assigned to the dielectric and insulating features at the electrode–electrolyte interface that is associated with successive immobilization of biomolecules. EIS analysis of Nano-ZnO/ITO electrode, r-IgGs/Nano-ZnO/ITO immunoelectrode and BSA/r-IgGs/Nano-ZnO/BSA immunoelectrode have been repeated several times (~15 times) on the same film surface in the electrolyte and different films have been prepared from the same stock solution .The results have been found to be reproducible (within 4% error). 3.6. Electrochemical impedimetric response studies Dependence of the electron transfer resistance, RCT, on the OTA concentration for the BSA/r-IgCs/Nano-ZnO/ITO immunosensor in 10 mL phosphate buffer (50 mM, pH = 7.0 an 0.9% NaCl) containing [Fe(CN)6]3−/4− and subsequent addition of OTA concentration (1–6 ng/dL) is stirred for 30 s using EIS technique in triplet set. The interaction of OTA with IgGs on the electrode surface results in significant changes in impedimetric parameters (RCT, Cdl, Rs and Zw). Fig. 6 shows the linear curve between the RCT values obtained during EIS response of the BSA/r-IgGs/Nano-ZnO/ITO immunoelectrode as a function of OTA (mycotoxin) concentration. The value of RCT increases on addition of OTA concentration. This may be attributed to the increased number of OTA molecules bound to the immobilized antibodies that perhaps provide a kinetic barrier for the electron transfer. BSA/r-IgGs/Nano-ZnO/ITO immunoelectrode exhibits improved sensing characteristics such as linearity as 0.006–0.01 nM/ dm3, detection limit as 0.006 nM/dm3, short response time of 25 s, and long term stability (45 days), sensitivity of 189 Ω/nM/dm3 cm2, reproducibility and the regression coefficient of 0.997. It may be noted that conformational changes are known to affect a biological reaction that in turn may be influenced by the nature of immobilization matrix and its surface morphology. The increased activity of r-IgGs due to favourable conformational changes results in enhanced interaction between the Nano-ZnO/ITO electrode and the active site of r-IgGs as indicated by the observed high value of stability constant (Ka, 7.6 × 1011 L/m) revealing high affinity of r-IgGs towards OTA due to prevalent electrostatic interactions. This may be attributed to favourable conformation of r-IgGs and increased loading of r-IgGs provided by the microenvironment of sol–gel derived Nano-ZnO film.
4. Conclusions It has been demonstrated that sol–gel derived Nano-ZnO film can be used for the immobilization of r-IgGs and BSA for blocking nonspecific binding sites of r-IgGs to detect OTA. The results show that Nano-ZnO electrode results in amplified electrochemical signal due to strong r-IgGs-OTA interaction occurring at the immunoelectrode surface. A good linear relationship between the electron transfer resistance and OTA concentration in the range, 0.006–0.01 nM/dm3 with improved sensitivity and detection limit etc has been obtained. It should be interesting to utilize the BSA/r-IgGs/Nano-ZnO/ITO platform for detection of other mycotoxins such as alphatoxin, ochratoxin B, citrinin, ergot akaloids, fumonisins, patulin, trichothecenes, and zearalenone, etc. And the findings of these studies have implications in clinical diagnostics, antibody screening and proteomics research. Acknowledgements We thank Dr. Vikram Kumar, Director, NPL, New Delhi, India for the facilities. Financial support received under the Department of Science & Technology (DST), Govt. of India the Department of Science and Technology (DST) projects [DST/TSG/ME/2008/18 and GAP- 070932], in-house project (OLP-070632D), India–Japan project (DST/INT/JAP/P21/07), and the Department of Biotechnology, Govt. of India (DBT/ GAP070832) is gratefully acknowledged. References [1] P.B. Luppa, L.J. Sokoll, D.W. Chan, Immunosensors—principles and applications to clinical chemistry, Clin. Chim. Acta 314 (2001) 1–26. [2] E. Katz, I. Willner, Probing biomolecular interactions at conductive and semiconductive surfaces by impedance spectroscopy: routes to impedimetric immunosensors, DNA-sensors, and enzyme biosensors, Electroanalysis 15 (2003) 913–947. [3] X.M. Li, X.Y. Yang, S.S. Zhang, Electrochemical enzyme immunoassay using model labels, Trends Anal. Chem. 27 (2008) 543–553. [4] M. Pumera, S. Sanchez, I. Ichinose, J. Tang, Electrochemical nanobiosensors, Sens. Actuators B 123 (2007) 1195–1205. [5] L. Irimpan, V.P.N. Nampoori, P. Radhakrishnan, A. Deepthy, B. Krishnan, Size dependent fluorescence spectroscopy of nanocolloids of ZnO, J. Appl. Phys. 102 (2007) 063524-5. [6] A. Wei, X.W. Suna, J.X. Wang, Y. Lei, X.P. Cai, C.M. Li, Z.L. Dong, W. Huang, Enzymatic glucose biosensor based on ZnO nanorod array grown by hydrothermal decomposition, Appl. Phys. Lett. 89 (2006) 123902-3. [7] J.F. Wang, X.W. Sun, A. Wei, Y. Lei, X.P. Cai, C.M. Li, Z.L. Dong, Zinc oxide nanocomb biosensor for glucose detection, Appl. Phys. Lett. 88 (2006) 233106-3. [8] S. Zong, Y. Cao, Y. Zhou, H. Ju, Zirconia nanoparticles enhanced grafted collagen tri helix scaffold for unmediated biosensing of hydrogen peroxide, Langmuir 22 (2006) 8915–8919. [9] J. Yu, H. Ju, Preparation of porous titania sol–gel matrix for immobilization of horseradish peroxidase by a vapor deposition method, Anal. Chem. 74 (2002) 3579–3583. [10] E. Topoglidis, C.J. Campbell, A.E.G. Cass, J.R. Durrant, Factors that affect protein adsorption on nanostructured titania films. A novel spectroelectrochemical application to sensing, Langmuir 17 (2001) 7899–7906. [11] E. Topoglidis, Y. Astuti, F. Duriaux, M. Gratzel, J.R. Durrant, Direct electrochemistry and nitric oxide interaction of heme proteins adsorbed on nanocrystalline tin oxide electrodes, Langmuir 19 (2003) 6894–6900. [12] A.A. Ansari, P.R. Solanki, B.D. Malhotra, Sol–gel derived nanostructured cerium oxide film for glucose sensor, Appl. Phys. Lett. 92 (2008) 263901-3. [13] A.A. Ansari, R. Singh, G. Sumana, B.D. Malhotra, Sol–gel derived nano-structured zinc oxide film for sexually transmitted disease sensor, Analyst 134 (2009) 997–1002. [14] P.R. Solanki, A. Kaushik, A.A. Ansari, G. Sumana, B.D. Malhotra, Zinc oxide–chitosan nanobiocomposite for urea sensor, Appl. Phys. Lett. 93 (2008) 163903-3. [15] E. Topoglidis, A.E.G. Cass, B. O'Regan, J.R. Durrant, Immobilization and bioelectrochemistry of proteins on nanoporous TiO2 and ZnO films, J. Electroanal. Chem. 517 (2001) 20–27. [16] Y.L. Liu, Y.H. Yang, F.H. Yang, M.Z. Liu, L.G. Shen, Q.R. Yu, Nanosized flower-like ZnO synthesized by a simple hydrothermal method and applied as matrix for horseradish peroxidase immobilization for electro-biosensing, J. Inorg. Biochem. 99 (2005) 2046–2053. [17] A.E. Radi, X.M. Berbel, V. Lates, J.L. Marty, Label-free impedimetric immunosensor for sensitive detection of ochratoxin A, Biosens. Bioelectron. 24 (2009) 1888–1892. [18] L.A. Pfohl, B.T. Petkova, I.N. Chernozemsky, M. Castegnaro, Balkan endemic nephropathy and the associated urinary tract tumors: review on etiological cause, potential role of mycotoxines, Food Addit. Contam. 19 (2002) 282–302.
A.A. Ansari et al. / Bioelectrochemistry 77 (2010) 75–81 [19] T.K. Goodman, in: M. Miraglia, H. van Edmond, C. Brera, J. Gilbert (Eds.), Mycotoxins and phycotoxins—developments in chemistry, toxicology and food safety. alaken Inc., Fort Collins, USA, 1998, p. 25. [20] B. Zimmerli, R. Dick, Ochratoxin A in table wine and grape-juice: occurrence and risk assessment, Food Addit. Contam. 13 (1996) 655–668. [21] A. Pittet, Modern methods and trends in mycotoxin analysis, Mitt. Lebensm. Hyg. 96 (2005) 424–444. [22] P.A. Burdaspal, T.M. Legarda, Ochratoxin A in wines and grape products originated from Spain and other European countries, Alimentaria 299 (1999) 107–113. [23] A.C. Barton, F. Davis, S.P.J. Higson, Labeless immunosensor assay for prostate specific antigen with picogram per milliliter limits of detection based upon an Ac impedance protocol, Anal. Chem. 80 (2008) 6198–6205.
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[24] A. Kaushik, P.R. Solanki, A.A. Ansari, S. Ahmad, B.D. Malhotra, Chitosan–iron oxide nanobiocomposite based immunosensor for ochratoxin-A, Electrochem. Commun. 10 (2008) 1364–1368. [25] A.A. Ansari, A. Kaushik, P.R. Solanki, B.D. Malhotra, Sol–gel derived nanoporous cerium oxide film for application to cholesterol biosensor, Electrochem. Commun. 10 (2008) 1246–1268. [26] R.C. Triulzi, M. Micic, J. Orbulescu, S. Giordani, B. Muellerd, R.M. Leblanc, Antibody– gold quantum dot–PAMAM dendrimer complex as an immunoglobulin immunoassay, Analyst 133 (2008) 667–672.