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Sol-Gel Derived Nanostructured Metal Oxide Platform for Bacterial Detection Pratima R. Solanki,*a Manoj K. Patel,a Ajeet Kaushik,a M. K. Pandey,a R. K. Kotnala,a B. D. Malhotra *a, b a
Department of Science and Technology Centre on Biomolecular Electronics, Biomedical Instrumentation Section, Materials Physics and Engineering Division, National Physical Laboratory (CSIR), Dr. K. S. Krishnan Marg, New Delhi 110012, India tel.: + 91 11 45609152; fax: + 91 11 45609310 *e-mail:
[email protected] b Centre for NanoBioengineering & SpinTronics, Chungnam National University 220, Gung-Dong, Yuseong-GU, Daejeon, 305-764, Korea *e-mail:
[email protected] Received: July 4, 2011;& Accepted: August 15, 2011 Abstract O1 gene based 24-mer single stranded deoxyribonucleic acid probe (ssDNA) has been immobilized onto sol-gel derived nanostructured zirconium oxide (NanoZrO2) film fabricated onto indium-tin-oxide (ITO) coated glass plate to detect Vibrio cholerae. The X-ray diffraction and Atomic Force Microscopy techniques have been used to characterize the nanostructured ZrO2 (particle size of ~ 30–40 nm) and the ssDNA/ZrO2 bioelectrode. The hybridization of ssDNA/ZrO2 bioelectrode with the complementary and genomic DNA has been investigated using differential pulse voltammetry. The results of electrochemical studies suggest that electro-active and cationic NanoZrO2 provides an effective surface to bind with the phosphate group of DNA resulting in enhanced electron transport. The ssDNA/NanoZrO2 bioelectrode shows a detection range from 1 108 to 10 nM of complementary DNA of V. cholerae within 60 s of hybridization time at 25 8C using methylene blue as an electroactive indicator. This O1 gene based metal oxide (ZrO2) sensor exhibits sensitivity for ssDNA/NanoZrO2/ITO bioelectrode as 0.48 mA/nM cm2 for complementary DNA and 2.34 mA/nM cm2 for genomic DNA with regression coefficients (R) of 0.991 and 0.995, respectively. This DNA bioelectrode is stable for about 15 weeks when stored at 4 8C. Keywords: O1 gene, Nanostructured ZrO2, Sol-gel, DNA Biosensor, Vibrio cholerae
DOI: 10.1002/elan.201100351 Supporting Information for this article is available on the WWW under http://dx.doi.org/10.1002/elan.201100351.
1 Introduction There is increased interest for the development of rapid and sensitive analytical tools for estimation of bacterial toxins in desired fluids. Many biomolecules such as gangliosides, antibodies, cholera toxin (CT) and somatic antigens (O1 and O139) have been utilized for V. cholerae detection. A total of 26 strains of Vibrio cholerae, including members of the O1, O139, and non-O1, non-O139 serogroups from both clinical and environmental sources, have been found to be responsible for the presence of genes encoding cholera toxin (ctxA). Among these, O1 and O139 antigens have been reported to be associated with the cholera outbreak [1–2]. As compared to O139 antigen, O1 antigen is found in majority of the patients affected by V. cholerae and main sources are the contaminated food especially seafood products etc [3]. Thus, we have concentrated on virulent (O1) gene. Keeping this in view, we have conducted investigations on gram negative and curved-rod shaped bacterium Vibrio cholerae, that causes diarrheal disease via infection of the intestine. Electroanalysis 2011, 23, No. 11, 2699 – 2708
The conventional methods for V. cholerae detection include enzyme linked immunosorbent assay/immunological detection kits, southern hybridization and polymerase chain reaction [4–6]. A chemiluminescence biosensor based on supported lipid layer incorporated with ganglioside GM1 has been developed for detection of cholera in the range from 1 pg/mL to 1 ng/mL [7]. Carter et al. have fabricated a piezoelectric quartz crystal microbalance based immunosensor for rapid detection of V. cholerae serotype 0139 by immobilization of the antibody onto a gold transducer surface of a 10 MHz AT cut PZ crystal [8]. A disposable immunosensor has been found to detect eight colonies of V. cholerae bacterium in hand-pump and seawater, and 80 CFU/mL in sewer water and tap water [9]. A cantilever based cholera sensor has been developed by immobilization of antibody of V. cholerae O1 onto gold-coated microcantilever surface and the resonance frequency shift due to bacteria binding has been measured by dynamic force microscopy as a function of V. cholerae concentration ranging from 1 103 to 1 107 CFU/mL [10]. Anti-CT antibody has been adsorbed onto gold nanoparticles dispersed onto a polytyramine-modi-
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fied gold electrode that can detect CT upto 9 1020 M by potential-step capacitance [11]. However, the fabrication of an immunosensor requires many steps including immobilization of antibodies that are expensive. In spite of these interesting developments, there is a considerable scope for the development of fast, sensitive, reliable, and cost-effective biosensor for detection of V. cholerae. The deoxyribonucleic acid (DNA) based hybridization biosensors have recently been reported for monitoring sequence-specific hybridization events directly or by DNA intercalates (metal coordination complexes, antibiotics, etc.) that form complexes with nitrogenous bases of DNA [12]. The electrochemical sensors can be used to directly detect desired nucleic acid targets via hybridization, so that sensitivity and specificity problems associated with nucleic acid amplification in the presence of biological inhibitors are minimized. To fabricate a desired genosensor, the immobilization of DNA probe onto a suitable matrix is a crucial step. Nanostructured metal oxides (NMOs) including zirconium oxide (ZrO2), cerium oxide, iron oxide, zinc oxide, titanium oxide and tin oxide have recently been used as immobilizing supports for biosensing applications [13]. The NMOs provide good biocompatibility, high adsorption capability, high active surface area for loading of desired biomolecules for enhanced electron transfer, high sensitivity and good mechanical/thermal/chemical stability. Among the various NMOs, nanostructured ZrO2(nano ZrO2) has recently attracted much interest due to easy preparation, non-toxicity, biocompatibility, surface modification, redox properties and affinity to oxygen atom of biomolecules (especially to phosphate group of DNA). The high isoelectric point (~ 9.5) of nanoZrO2 helps in increased adsorption of a desired biomolecule of low isoelectric point (IEP, ~ 4–5) via electrostatic interactions resulting in improved sensing properties. Solanki et al. have fabricated a DNA biosensor by immobilization of 17 bases terminal of single stranded oligonucleotide probe (ssDNA) of 16s rRNA coding region of Escherichia coli onto ZrO2 film for E. coli detection [14]. Electrochemically deposited ZrO2 film onto gold surface has been used to immobilize ssDNA specific to Mycobacterium tuberculosis by utilizing affinity between the oxygen atom of phosphate group and zirconium to fabricate DNA biosensor [15, 16]. The electrochemically deposited nanoZrO2 film and ZrO2/carbon paste electrode have also been recently used for development of DNA biosensors [17]. We report results of the studies relating to the fabrication of a genosensor by utilizing sol-gel derived nanoZrO2 film for the immobilization 24-mer ssDNA based on O1 gene for V. cholerae detection using hybridization technique for complementary, noncomplementary and genomic DNA. The sol-gel technique is known to have many advantages such as simplicity, ability to control the size and morphology of nanostrutuctured film. Besides this, it results in the formation of a homogeneous film with a large area, and relatively low cost, low tempera2700
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ture processing, better thermal stability and flexible deposition parameters etc [18, 19].
2 Experimental 2.1 Chemicals and Reagents All chemicals including Tris (hydroxymethyl) amino methane, ethylene diamine tetra acetic acid (EDTA), sodium dihydrogen ortho-phosphate and di-sodium hydrogen ortho-phosphate were procured from Sigma-Aldrich, USA. Indium-tin oxide (ITO) coated glass plate (Balzers Ltd., UK) (sheet resistance: 15 W cm1) was used as a substrate for ZrO2 electrode fabrication. And Ag/AgCl reference was made available from Autolab, Netherlands. HPLC purified 24-mer based oligomeric probe (ssDNA) 5’-AGC AAGAGC ATT GTT GTTCCT ACC-3’ procured from Genxbio, India were used as a probe. All solutions were prepared using de-ionized water (Milli Q 10 TS) and these solutions were autoclaved prior to being used. DNA solutions were prepared in Tris EDTA buffer (TE, 10 Mm Tris, 1 mM EDTA, pH 8.0). All PCR chemicals were used from Banglore Genei, India. The genomic DNA of V. cholerae was collected from National Institute of Communicable Diseases (NICD), New Delhi, India. 2.2 Fabrication of ZrO2 Electrode Zirconium(IV) n-propoxide 70 wt.% was diluted in propanol and the resulting solution was hydrolyzed by drop wise addition of hydrochloric acid under continuous stirring by a magnetic stirrer in an anhydrous nitrogen atmosphere to avoid hydroxide precipitation steps. After completion of hydrolysis, the sol was continuously stirred for about an hour at ambient temperature (25 8C) to polymerize the gel (Scheme 1 Steps 1–3). Thus obtained sol gel solution was used to fabricate film onto ITO coated glass plate using the dip coating method in air and was dried at 100 8C for 3 h and was further annealed at 450 8C for 4 h to obtain smooth surface (Scheme 1 Steps 4–5) [14]. 2.3 Fabrication of ssDNA/ZrO2/ITO Bioelectrode The 24 bases ssDNA probe [100 nM/mL; (1.06 1011 pg/ mL)] was immobilized onto ZrO2/ITO electrode (0.25 cm2) and was kept in a humid chamber for 3 h at 25 8C. ssDNA/NanoZrO2/ITO bioelectrode was washed with TE buffer several times to remove the unbound probe from the surface. This ssDNA/ZrO2/ITO bioelectrode was hybridized with the complementary and genomic ssDNA (after denaturation at 95 8C) for 5 min. After hybridization the electrode was washed thrice with buffer to remove any unhybridized DNA. The electrochemical response of ssDNA/ZrO2/ITO bioelectrode was monitored using differential pulse voltammetric (DPV) studies in phosphate buffer saline (PBS, 0.05 M, pH 7.0,
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Scheme 1. Preparation of ssDNA/NanoZrO2/ITO bioelectrode using sol-gel method.
0.9 % NaCl) containing 20 mM methylene blue (MB) for given concentration of genomic DNA. The triplet sets were used for the desired studies. 2.4 Polymerase Chain Reaction (PCR) Amplification PCR assay was performed for the specific O1 gene target of V. cholerae in a single step in 25 mL reaction volume was obtained, final concentration containing 1X PCR buffer (1.5 mM MgCl2 ; 0.1 M TrisHCl (pH 8.3), 0.5 M KCl; 0.1 % gelatin), 1.5 mM MgCl2, 1 mM dNTP Mix, specific primers for O1 gene of V. cholerae, 10 pmol each (forward: 5’-TGC AAA TGG AAC ACC TCA AA-3’ and reverse: 5’-GCA ATC CTC AGG GTA TCC TTC3’), 0.5 U of Taq polymerase (Banglore Genei, India) and 50 ng/mL of DNA template of V. cholerae. The PCR studies were performed on a master cycler gradient (Eppendorf) with the following parameters: the first cycle of denaturation at 95 8C for 5 min, annealing at 58 8C for 40 s and polymerization at 72 8C for 1 min, followed by 30 cycles of 95 8C for 30 s, annealing at 55 8C for 40 s and polymerization at 72 8C for 1 min and final cycle of polymerization at 72 8C for 7 min. The amplicons (amplified PCR product) have been analyzed by electrophoresis on a standard 2 % agarose gel. Electroanalysis 2011, 23, No. 11, 2699 – 2708
The PCR amplicons of O1 gene at 232 bp regions have been separated by electrophoresis in 2 % agrose gel stained by ethidium bromide and visualized by UV florescence. The Gel Documentation system (Bio-Rad) has been used for obtaining the gel picture. The Figure A (Supporting Information) shows amplified PCR product (232 bp) with 100 bp DNA ladder (M). The genomic DNA products purified from DNA purification kit (Banglore Genei) have been used for hybridization with ssDNA-ZrO2/ITO bioelectrode.
2.5 Characterization Techniques Atomic force microscopy (AFM; Vicco440), an X-ray diffraction (XRD, Cu Ka radiation (Rigaku) and Fourier transform infrared (FTIR, Perkin-Elmer, Model 2000) studies have been used for characterization of the ZrO2/ ITO electrode and ssDNA/ZrO2/ITO bioelectrode. Electrochemical studies have been carried out with a Potentiostat/Galvanostat (Model 273 A, Princeton Applied Research, USA) using a three-electrode system containing ITO glass plate as working, Ag/AgCl as reference and platinum as counter electrode.
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3 Results and Discussion 3.1 Optical Characterization The fabrication and phase purity of NanoZrO2 film have been confirmed using XRD pattern. The observed reflection planes obtained at 2q = 30 and 32 reveal growth of the crystalline ZrO2 (JCPDS 79-1771) and coincide with pure ZrO2 (Figure 1A). The particle size of the ZrO2 nanoparticles estimated using Scherer formula [D = Kl/b cos q] has been found to be as ~ 40 nm and also correlates with the results obtained using AFM studies (~ 30– 40 nm). Figure 1 B shows AFM image of the NanoZrO2 surface recorded in tapping mode with the resonance frequency of 235–295 kHz and DNA/NanoZrO2/ITO bioelectrode under physiological conditions. The AFM image of NanoZrO2/ITO electrode (Image i) shows well-distributed granular rough morphology wherein the particle size varies from 30 to 100 nm. However, at some point the particles agglomerate due to high charge density of material and surface charged particle – particle interaction. The remarkable change of NanoZrO2/ITO electrode is observed after the immobilization of ssDNA, wherein the rough nanoporous morphology changes into another wellarranged morphology (Image ii). This reveals that ssDNA is successfully absorbed onto the ZrO2 surface via strong interactions arising due to negatively charged phosphate group of the backbone of the DNA with the oxygen atom of zirconium species (Scheme 1). Phosphates and phosphonates are known to exhibit high-affinity interactions with the metal oxides, and it appears that DNA is strongly associated with ZrO2via multiple interactions with the phosphate backbone. It is observed that the particle of NanoZrO2 on ssDNA immobilized electrode increases and varies from 20 to 150 nm whereas the roughness of bioelectrode decreases from 20.6 nm to 18.6 nm, 15.6 nm. These results show that fabricated ZrO2 electrode has highly nanoporous morphology that undergoes changes after immobilization of ssDNA. As compared to the NanoZrO2 film, the average peak height (Rpm) increases after immobilization of ssDNA (247 nm) and hybridization (dsDNA; 243 nm) onto ZrO2 surface (128 nm). It appears that ssDNA molecules are immobilized in standing form and the observed surface exhibits significant change in morphology (Image iii) confirming hybridization with the complementary DNA molecules onto ssDNA/NanoZrO2/ITO bioelectrode that has average roughness of ~ 18 nm. The variation of the peak-to-valley height (Rmax), average roughness (Ra) measured as 170 nm for ZrO2/ ITO electrode increases to 257 nm for ssDNA/NanoZrO2/ ITO bioelectrode, then decreases to146 nm for the dsDNA/NanoZrO2/ITO bioelectrode. These changes are assigned to the adsorbed phosphate group of DNA on ZrO2 film (Supporting Information Figure B). The energy dispersive X-ray spectroscopy analysis of NanoZrO2/ITO electrode (i) and ssDNA/ZrO2/ITO bioelectrode (ii) reveals the presence of oxygen atom and zir2702
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conium, oxygen atoms, respectively (Figure 1C). In case of the ssDNA/NanoZrO2/ITO electrode, the oxygen atom peak intensity is enhanced and the observed new peak of the phosphate atom reveals immobilization of the DNA molecules onto NanoZrO2/ITO electrode. In the FTIR studies (Supporting Information Figure C), NanoZrO2/ITO film (curve a) shows peak at 613 cm1 arising due to the binding of zirconia with oxygen ZrO2. The FTIR bands appear around 3273 and 1572 cm1 as stretching and bending frequencies, respectively, indicating presence of the adsorbed water molecule at the ZrO2 surface. On the other hand, ssDNA/NanoZrO2/ITO bioelectrode (curve b) exhibits a peak at about 972 cm1 assigned to the PO stretching (asymmetric), PO stretching (symmetric) for the PO2 group of phosphor diester bond between the phosphate group of DNA and deoxyribose sugar, respectively. The peaks seen in the region 1549–1640 cm1 correspond to C=O, C=N, C=C stretching and -NH2 bending vibrations of the DNA bases. It is observed that the 613 cm1 peak shifts to lower wavelength at about 509 cm1 due to electrostatic interaction between ZrO2 and DNA. 3.2 Electrochemical Characterization Electrochemical impedance spectroscopic (EIS) technique provides information on the step-wise changes of NanoZrO2 film after immobilization of ssDNA and hybridized with the genomic DNA measured in PBS containing 5 mM [Fe(CN)6]3/4 in the frequency range of 0.01–105 Hz. In the EIS spectra (Figure 2A), the semicircle diameter of the Nyquist diagram equals surface charge transfer resistance (Rct) of the electrode, the increase in diameter of the semicircle reflects increase in the interfacial Rct. Figure 2A shows the Nyquist plots obtained for the bare ITO electrode (curve a), NanoZrO2/ITO electrode (curve b), ssDNA/NanoZrO2/ITO bioelectrode (curve c) and hybridized dsDNA/NanoZrO2/ITO bioelectrode (curve d). The inset shows the experimentally obtained data modeled using a modified Randles equivalent circuit, wherein Rs is the electrolyte solution resistance, Rct is the electron transfer resistance, Zw is the Warburg impedance resulting from the diffusion of ions and Cdl is the double layer capacitance. The Rct for bare ITO electrode (curve a) has been found to be 2.062 103 W that is higher than that of the ZrO2 electrode (1.116 103 W, curve b). It indicates that presence of the ZrO2 molecules increases the electroactive surface area resulting in enhanced diffusion of redox species onto substrate that help to improve electron transfer from the medium to electrode. After immobilization of probe (ssDNA) onto ZrO2/ITO electrode, the value of Rct further decreases to 6.031 102 W due to penetration of electrons generated by the redox species via interfacial layer, resulting in improved electron transport. Moreover, the negative charge on the DNA molecules results in increased interaction with oxygen mole-
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Fig. 1. (A): XRD pattern of NanoZrO2/ITO film. (B) AFM images of NanoZrO2/ITO film (i), ssDNA/NanoZrO2/ITO (ii), dsDNA/ NanoZrO2/ITO bioelectrode (iii), (C) EDX of NanoZrO2/ITO electrode (i) and ssDNA/NanoZrO2/ITO bioelectrode (ii).
cules of zirconium oxide resulting in decrease of net negative charge on the ssDNA molecules (curve c) leading to decrease in Rct value. These results reveal that the ZrO2 surface provides an effective platform for the immobilization of DNA probe. Subsequently, as the genomic DNA Electroanalysis 2011, 23, No. 11, 2699 – 2708
is hybridized with ssDNA/NanoZrO2/ITO electrode, the Rct value further increases (1.011 103 W) due to presence of a large number of DNA molecules linked with the probe. The increased Rct value indicates that hybridization results in evolution of the negatively charged inter-
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Fig. 2. (A) Impedance spectra of (a) bare ITO, (b) NanoZrO2/ ITO electrode and (c) ssDNA/NanoZrO2/ITO bioelectrode and (d) dsDNA/NanoZrO2/ITO bioelectrode in PBS containing 5 mM [Fe(CN)6]3/4. (B) CV of ITO (a), NanoZrO2/ITO electrode (b) and (c) ssDNA/NanoZrO2/ITO bioelectrode,
face that electrostatically repels multicharged negative redox probe [Fe(CN)6]3/4. This repulsive force perhaps inhibits the interfacial electron transfer leading to enhanced Rct value. However, the value of Cdl is found to be decreased (4.64 106) after hybridization (curve d) compared to that of ssDNA (1.03 105 ; curve c) due to permittivity of the DNA species. These results reveal the hybridization of the DNA probe. This Rct can be translated into exchange current under the equilibrium (Io) and then mono electron-transfer rate constant (Kct) is determined [19]. The value of Kct for ssDNA/NanoZrO2/ITO bioelectrode is higher (3.54 103 cm/s) than that of the dsDNA/ NanoZrO2/ITO (2.11 103 cm/s) and ZrO2/ITO electrode (1.91 103 cm/ s). These studies bring out that the ssDNA/NanoZrO2/ 2704
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ITO bioelectrode contains enhanced redox centers that enable faster charge transfer. These redox sites perhaps get blocked resulting in reduced charge transfer. It has been observed that the charge transfer rate of ZrO2 electrode exhibits semiconducting behavior. The CV studies of the bare ITO electrode (a), ZrO2/ ITO electrode (b) and ssDNA/NanoZrO2/ITO bioelectrode (c) have been conducted in PBS containing 5 mM [Fe(CN)6]3/4 at scan rate of 30 mV/s (Figure 2B). It is observed that magnitude of the electrochemical response current obtained for the NanoZrO2/ITO electrode increases to about 1.5 fold in comparison to that of the ITO surface. This is attributed to faster charge transfer between the electrode and ZrO2 surface. The observed enhanced current indicates that the nanoparticles of ZrO2 exhibit higher surface area-to-volume ratio than that of their bulk counterpart. Moreover, the cationic nanoZrO2 also increases the adsorption of redox moieties resulting in fast electron transfer between medium and substrate. This perhaps results in modification of the electrochemical interface resulting in enhanced electro catalytic activity. Further, after the immobilization of ssDNA onto NanoZrO2/ITO electrode, the magnitude of current is found to be enhanced with slight shift towards the negative side, assigned to the oxidation of nitrogenous bases (guanine) immobilized DNA onto NanoZrO2/ITO surface. Moreover, NanoZrO2/ITO electrode provides a favorable environment for the immobilization of DNA. The CVs of NanoZrO2/ITO electrode and ssDNA/ NanoZrO2/ITO bioelectrode have been recorded at different scan rates (10–100 mV s1) (Supporting Information Figure D). It can be seen that the peak potential shifts towards positive side as the scan rate increases. And anodic peak potential shifts more towards the positive side and cathodic peak potential shifts in the reverse direction. Besides this, anodic and cathodic peak currents (Ipa and Ipc) show linear behavior with square root of scan rate (v1/2) (Supporting Information Figure D), revealing that the electron-transfer process is diffusion controlled. According to Laviron theory the slope value (RT/anF) for NanoZrO2/ITO and ssDNA/NanoZrO2/ITO has been calculated as 0.083 and 0.078. The surface concentration of the redox species (G) for NanoZrO2/ITO and ssDNA/NanoZrO2/ITO bioelectrode has been found to be as 5.52 109 mol/cm2 and 6.01 109 mol/cm2, respectively, using the Lavirons equation [20]. These results reveal that the more redox species are present onto ssDNA/NanoZrO2/ITO bioelectrode as compared to that of NanoZrO2/ITO electrode resulting in stronger interaction between the oxygen molecules of zirconium species and phosphate group of DNA backbone. Figure 3 shows results of the DPV studies of the modified electrode and the corresponding change in magnitude of the current recorded in PBS containing MB as an intercalator. MB being an organic dye belongs to the phenothiazine family and is used as a redox indicator widely employed in electrochemical DNA hybridization sensors. It produces different electrochemical signal in the pres-
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bility of bases compared to that of ssDNA bases. There is no significant difference in the value of current obtained for the ssDNA/NanoZrO2/ITO bioelectrode when it hybridizes with the noncomplementary sequence (curve v), revealing high selectivity of the DNA probe. The magnitude of current increases after it is hybridized with the genomic DNA due to long chain of DNA molecules. 3.3 Electrochemical Response Studies
Fig. 3. DPV of ITO (i), NanoZrO2/ITO electrode (ii), ssDNA/ NanoZrO2/ITO bioelectrode (iii), dsDNA/NanoZrO2/ITO bioelectrode with complementary (iv), noncomplementary (v) genomic dsDNA/NanoZrO2/ITO bioelectrode (vi).
ence of ssDNA and dsDNA, at low oxidation potential (generally 0.26 V vs. Ag/AgCl) thus allowing minimization of potential electrochemical interferences. The detection of hybridization using MB, wherein reversible electroactivity and strong association with immobilized ssDNA probe leads to significantly enhanced voltammetric signal. The magnitude of current is found to increase after ZrO2 film formation onto ITO glass plate indicating that the ZrO2 nanoparticles exhibit higher surface area-tovolume ratio than that of their bulk counterpart. Moreover, the cationic nanoZrO2 also increases the adsorption of redox moieties resulting in fast electron transfer between medium and substrate. This perhaps results in modification of the electrochemical interface resulting in enhanced electrocatalytic activity. Further, magnitude of the current increases after immobilization of the ssDNA onto NanoZrO2 film, due to binding of the MB molecules with purine bases of the DNA molecules. MB intercalates with the DNA base stack and participates in electron transfer mediated by stacked bases. It appears that at low concentration of salt (NaCl) into the medium, the guanine oxidation rate is independent of DNA molecules. Thus all immobilized guanine bases on NanoZrO2/ITO electrode present in DNA probe contribute to the observed catalytic current. On interaction of ssDNA/NanoZrO2/ITO bioelectrode with the complementary sequence, the magnitude of peak current decreases significantly. This decrease clearly reveals that the ssDNA/NanoZrO2/ITO bioelectrode is successfully hybridized with its complementary sequence, indicating reduced amount of MB onto bioelectrode due to the steric inhibition of MB packing. Moreover, there is decrease in the electrochemical response current after hybridization with genomic DNA due to decreased availaElectroanalysis 2011, 23, No. 11, 2699 – 2708
The effect of complementary DNA and genomic DNA concentration has been monitored using DPV in PBS containing MB solution as shown in Figure 4 and 5, respectively. It can be seen that magnitude of the anodic peak current of MB decreases with increased complementary DNA concentration varying from 1 108 to 10 nM (Figure 4). The inset in Figure 4 shows the plot obtained between the log values of complementary concentration vs current response. The increased interaction between DNA and intercalated MB makes it more energetically favorable to intercalate or to remain bound rather than diffuse to the electrode. It can be seen that MB peak height decreases with increase in the complementary DNA concentration indicating enhanced number of dsDNA molecules at the electrode surface. This decrease in magnitude of current shows that lesser number of MB molecules is associated with dsDNA indicating that lesser charge perhaps passes through dsDNA and upon intercalation the reduction potential perhaps becomes less cathodic. The decrease in MB peak with respect to complementary DNA concentration follows Equation 1. I dsDNAcomp ¼ 9:867 106 5:68 107 log ½DNAcomp ð1Þ The value of binding constant (Ka) for the genomic DNA hybridization has been calculated using the Langmuir-like adsorption curve following relation. ½T=I ¼ ½T=I sat þ 1=Ka I sat where [T] is the target concentration, I is the current density, Isat is the saturated current density, and Ka is the binding constant. The linearized Langmuir plot of the isotherm yields the term Ka = [T]/I 5 102 [T] – 0.1129, and hence, the saturated current density of complementary target is 5.92 105 A/cm2. The value of Ka has been estimated as 1.497 105 nM1. The higher value of Ka and its increased sensitivity (0.48 mA) results in detectable concentration of complementary DNA as 1 amol. The sensitivity of ssDNA/NanoZrO2/ITO bioelectrode estimated from the slope of the linear regression curve has been found as 0.48 mA/nM/cm2 with regression coefficient as 0.991. Figure 5 shows that magnitude of the peak current due to oxidized MB molecules increases with increased concentration of genomic DNA after hybridization with the
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Fig. 4. DPV response of the dsDNA/NanoZrO2/ITO bioelectrode after hybridization with complementary concentration. Inset: Linear graph log complementary concentrations vs. current variation.
Fig. 5. DPV response of the dsDNA/NanoZrO2/ITO bioelectrode after hybridization with genomic DNA concentration. Inset: Linear graph between genomic DNA concentration vs. current variation.
probe DNA. The inset in Figure 5 shows the variation of magnitude of current response vs. genomic DNA concentration. It appears that positively charged MB molecules are electrostatically attached to the negatively charged 2706
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phosphate backbone of double stranded DNA or these are intercalated after hybridization. Moreover, the magnitude of MB peak current increases with increased concentration of genomic DNA due to availability of increased
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Sol-Gel Derived Nanostructured Metal Oxide Platform for Bacterial Detection Table 1. The characteristics of ssDNA/NanoZrO2/ITO bioelectrode for cholera detection along with those reported in the literature. No.
Surface/matrix
Transducer
Detection limit
Linearity
Reference
1 2
Waveguide Gold transducer surface
0.5 ng/mL 105 cells/ mL
– 10 MHz AT
[6] [7]
3
Planar supported lipid membrane Gold-coated AFM microcantilevers Polytyramine-modified gold electrode ZrO2/ITO electrode
Array biosensor Quartz crystal microbalance Ultrasensitive chemiluminescence biosensor Microcantilever-based biosensor Immunosensor
800 ng/mL
1 pg/mL–1 ng/ mL
[9]
1 103 CFU/mL
1 103 to 1 107 CFU/mL
[10]
9 1011 nM
0.1 aM and 10 pM
[11]
DNA biosensor
1 108 nM
1 108–10 nM
Present work
4 5 6
number of free guanine bases (nonhybridized bases) of genomic DNA. This maximum decrease in MB oxidation current indicates the presence of DNA duplex formed at the electrode surface. The increase in the MB peak current with respect to genomic DNA follows Equation 2. I dsDNAGenomic ¼ 2:01 105 þ8:28 107 log ½DNAGenomic
ð2Þ
The sensitivity of this genosensor has been determined from the slope of the linear calibration curve as 2.34 mA/ nM/cm2 with value of the regression coefficient as 0.995. The storage stability of ssDNA/NanoZrO2/ITO bioelectrode has been determined using DPV technique in PBS containing MB solution by monitoring magnitude of current of the oxidized MB that binds with the free guanine bases at regular interval of about two weeks for about four months. It has been found that this geno-electrode exhibits 90 % of response up to 15 weeks after which the magnitude of the current decreases and reaches 75 % after about 15 weeks. Table 1 shows characteristics of the ssDNA/NanoZrO2/ITO bioelectrode for cholera detection along with those reported in the literature.
4 Conclusions The NanoZrO2 film based electrochemical DNA biosensor has been fabricated by immobilizing 24-mer ssDNA for detection of V. cholerae with complementary and genomic DNA using hybridization technique. It has been found that the ssDNA/NanoZrO2/ITO bioelectrode shows higher sensitivity for genomic DNA as compared to that of complementary DNA. The hybridization time has been obtained as about 5 min both for the complementary and genomic DNA and the response time of this sensor is 60 s. This NanoZrO2 based electrochemical DNA sensor is highly specific for detection of V. cholerae in real samples. And it should be interesting to utilize this NanoZrO2 electrode for immobilization of other biomolecules like O139, antibodies and ganglioside for cholera detection. Electroanalysis 2011, 23, No. 11, 2699 – 2708
Acknowledgements Authors thank the Director, NPL, New Delhi, India for the facilities. PRS is grateful to the Department of Science and Technology (DST), India for the Award of Young Scientist. We thank Mr. Saurabh Srivastava for valuable discussion and Mr. S. B. Samanta and Mr. Sandeep Thakur for AFM studies. We are also thankful to NICD, New Delhi for supply of genomic DNA. BDM thanks the National Research Foundation of Korea, the Ministry of Education, Science and Technology for giving the opportunity to visit the Centre for NanoBioengineering & SpinTronics under the WCU (World Class University) Program (R32-20026) during August 2011.
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