Electrophoretic Fabrication of Chitosan−Zirconium-Oxide

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Electrophoretic Fabrication of Chitosan-Zirconium-Oxide Nanobiocomposite Platform for Nucleic Acid Detection Maumita Das,†,‡ Chetna Dhand,†,‡ Gajjala Sumana,† A. K. Srivastava,† R. Nagarajan,‡ Lata Nain,|| M. Iwamoto,§ Takaaki Manaka,§ and B. D. Malhotra*,† †

)

Department of Science & Technology Centre on Biomolecular Electronics, Biomedical Instrumentation Section, Materials Physics & Engineering Division, National Physical Laboratory, Dr. K. S. Krishnan Marg, New Delhi-110012, India ‡ Department of Chemistry, University of Delhi, Delhi-110007, India § Department of Physical Electronics, Tokyo Institute of Technology, Tokyo, Japan Division of Microbiology, Indian Agricultural Research Institute, New Delhi-110012, India ABSTRACT: The present work describes electrophoretic fabrication of nanostructured chitosan-zirconium-oxide composite (CHIT-NanoZrO2) film (180 nm) onto indium-tin-oxide (ITO)-coated glass plate. This nanobiocomposite film has been explored as immobilization platform for probe DNA specific to M. Tuberculosis as model biomolecule to investigate its sensing characteristics. It is revealed that pH-responsive behavior of CHIT and its cationic skeleton is responsible for the movement of CHIT-NanoZrO2 colloids toward cathode during electrophoretic deposition. The FT-IR, SEM, TEM, and EDX techniques have been employed for the structural, morphological, and composition analysis of the fabricated electrodes. The morphological studies clearly reveal uniform inter-linking and dispersion of hexagonal nanograins of ZrO2 (30-50 nm) into the chitosan matrix, resulting in homogeneous nanobiocomposite formation. Electrochemical response measurements of DNA/CHIT-NanoZrO2/ITO bioelectrode, carried out using cyclic voltammetry and differential pulse voltammetry, reveal that this bioelectrode can specifically detect complementary target DNA up to 0.00078 μM with sensitivity of 6.38  10-6 AμM-1.

’ INTRODUCTION Nanostructured metal oxides, owing to their small size, exhibit interesting chemical, physical, and electronic properties that are different from those of bulk materials and can be used to construct novel and improved sensing devices, in particular, electrochemical sensors and biosensors.1,2 They are known to have unique characteristics to promote faster electron transfer kinetics between the electrode and desired biomolecules. A large number of nanostructured metal oxides such as cerium oxide (CeO2), iron oxide (Fe3O4), nickel oxide (NiO), tin oxide (SnO2), zinc oxide (ZnO) and zirconium oxide (ZrO2) [42-50] have been explored for application in electrochemical biosensors.3-13 Among these, ZrO2 has recently attracted much attention because of its thermal stability, chemical inertness, biocompatibility, and affinity for the groups containing oxygen.14-16 However, the problems of cracking and aggregation have led to limited application of ZrO2 nanoparticles to biosensing. This problem can perhaps be addressed by modifying ZrO2 nanoparticles with chitosan (CHIT) to prepare a nanobiocomposite. CHIT has been found to be an interesting biopolymer for immobilization of biomolecules because of its excellent film-forming ability, high permeability, nontoxicity, biocompatibility, low cost, and so on. Moreover, chemical modification of amino groups of CHIT provides hydrophilic environment for the biomolecules.17-19 Feng et al. r 2011 American Chemical Society

have reported nanoporous CeO2/CHIT composite film as the immobilization matrix for fabrication of electrochemical DNA biosensor for colorectal cancer detection. This biocomposite matrix can be used for increased loading of probe DNA to obtain enhanced biosensor response.20 Kaushik et al. have developed a nucleic acid sensor based on Fe2O3/CHIT hybrid matrix for pyrethroid detection.21 They have observed that incorporation of Fe3O4 nanoparticles improves the electroactive surface area of CHIT for immobilization of DNA, resulting in accelerated electron transport. Tuberculosis is presently the world’s leading chronic pulmonary bacterial infectious disease, caused by Mycobacterium tuberculosis (M. Tuberculosis), killing over two million people annually. It is estimated that there are 1 billion persons infected with tuberculosis worldwide, with 8 million new cases and 3 million deaths per year. Available statistics indicate that there is a close association between the acquired immunodeficiency syndrome (AIDS) and tuberculosis. The number of cases of M. Tuberculosis infection with multiple drug resistance (MDR) is increasing, and this hampers both treatment and control programs worldwide. Received: November 2, 2010 Revised: December 10, 2010 Published: January 10, 2011 540

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Scheme 1. Schematic Showing the Fabrication of DNA/CHIT-NanoZrO2/ITO Bioelectrode

The resurgence of TB and increased risk for TB in HIV-infected persons has intensified the requirement for a rapid, costeffective, and accurate method for diagnosis.22-25 Conventional diagnostic methods such as polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) are timeconsuming and require trained expertise. There is, thus, an urgent need for sensitive, specific, stable, cost-effective, and reusable method for M. Tuberculosis detection. In this context, affinity biosensors have emerged as promising alternative for microbial detection using nucleic-acid-based detection of complementary target sequences.26,27 We report results of studies relating to the electrophoretically fabricated CHIT-NanoZrO2 film onto indium-tin-oxide (ITO)-coated glass plate and its application as an adhesion layer for the investigation of DNA hybridization specific to M. Tuberculosis.

Electrophoretic Deposition of CHIT-NanoZrO2/ITO Nanobiocomposite Film. Electrophoretic deposition (EPD) of CHITNanoZrO2/ITO composite film has been carried out with a DC battery (BioRad, model 200/2.0) using the setup shown in Scheme 1. For EPD, colloidal suspension of CHIT-NanoZrO2 is prepared by mixing 25 mg of CHIT and 5 mg of surface treated zirconia in 20 mL of acetonitrile. The surface modification of zirconia is accomplished by mixing 0.5 g of ZrOCl2 3 8H2O with 100 mL of 0.7% sodium salt of DBSA at pH 4 with continuous stirring for 6 h. A DC voltage of 200 V for 5 min is applied to obtain thin, uniform, and homogeneous films of CHIT-NanoZrO2 composite.

Fabrication of Nucleic-Acid-Fuctionalized CHIT-NanoZrO2 Bioelectrodes. Biotinylated single-stranded probe DNA (ssDNA;

10 μL of 21-mer), specific to M. Tuberculosis, is covalently immobilized onto the avidin-modified CHIT-NanoZrO2/ITO surface using avidin-biotin coupling. We have used 15 mM EDC and 30 mM NHS solution, prepared in deionized water, to activate 10 μL avidin solution (1 mg/mL) for 2 h of incubation at 25 °C. 2 μL of activated avidin is immobilized onto the CHIT-NanoZrO2 surface for 2 h. After washing with autoclaved deionized water, the avidin-CHIT-NanoZrO2 surface is immobilized with 21-mer biotinylated oligonucleotide specific to M. Tuberculosis for 5 min of incubation at 25 °C in a humid chamber. These fabricated ssDNA/CHIT-NanoZrO2/ITO bioelectrodes have been characterized using Fourier transform infrared spectroscopy (FT-IR, PerkinElmer, Spectrum BX II), scanning electron microscopy (SEM, LEO 40), and high-resolution transmission electron microscope (HR-TEM, Tecnaii-G2 F30 STWIN with field emission gun electron source). Electrochemical analysis has been conducted by Autolab Potentiostat/Galvanostat (Eco Chemie, AD Utrecht, The Netherlands) using a three-electrode system with ITO as working electrode, platinum wire as auxiliary electrode, and Ag/AgCl as reference electrode in PBS solution containing 5 mM [Fe(CN)6]3-/4- as redox probe. The ssDNA/CHIT-NanoZrO2/ITO bioelectrode is optimized for

’ EXPERIMENTAL SECTION Materials. Chitosan (Mw: 2.4  106), zirconium oxychloride (ZrOCl2 3 8H2O), dodecylbenzenesulfonic acid (sodium salt) (DBSA), N-hydroxysuccinimide (NHS), N-ethyl-N0 -(3-dimethylaminopropyl carbodiimide) (EDC), oligonucleotide probe sequence specific to M. Tuberculosis, complementary target, one-base mismatch, and noncomplementary DNA have been procured from Sigma-Aldrich, Milwaukee, WI. All solutions and glassware have been autoclaved prior to being used, and desired reagents (molecular biology grade) are prepared in deionized water (Milli Q 10 TS). Oligonucleotide sequences used in this work are: Biotinylated probe: biotin-50 -GGTCTTCGTGGCCGGCGTTCA-30 Complementary: 50 -TGAACGCCGGCCACGAAGACC-30 One-base mismatch: 50 -TGAACGCCGACCACGAAGACC-30 Noncomplementary: 50 -ATGTCTCAAGCCAGCTGCTG-30 541

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Figure 1. FT-IR spectra of (i) CHIT/ITO, (ii) NanoZrO2/ITO, (iii) CHIT-NanoZrO2/ITO, and (iv) DNA/CHIT-NanoZrO2/ITO electrodes.

symmetrical deformation of CH3 and CH2 group, and the peak at 1414 cm-1 corresponds to C-N axial deformation. The broad peak seen at 1149 cm-1 is assigned to β (1-4) glucosidic band in polysaccharide unit. Furthermore, the peaks seen at 1070 and 1028 cm-1 are attributed to stretching vibration mode of the hydroxyl group and C-O-C in glucosamine unit.29 The NanoZrO2/ITO electrode exhibits characteristic peaks at around 517 and 647 cm-1 arising due to symmetric stretching of ZrO-Zr species, indicating the presence of zirconia on the ITO surface. In the FT-IR spectra of CHIT-NanoZrO2/ITO composite film, the bands observed at 1460, 1561, and 1652 cm-1 are due to Zr-O-C vibrations. Moreover, the blue shift and broadening in IR bands corresponding to N-H stretching of chitosan and Zr-O stretching of ZrO2 indicate composite formation between these biocompatible components. The peaks seen at 1257, 1513, and 1738 cm-1 for DNA/CHIT-NanoZrO2/ITO bioelectrode are due to P-O stretching vibrations of the phosphate backbone and C-O stretching vibrations of purine and pirimidine rings of DNA, respectively. Furthermore, the broad peak seen at 3524 cm-1 is due to N-H stretching, and the band seen at 1657 cm-1 is attributed to amide stretching vibrations, indicating immobilization of DNA onto the CHIT-NanoZrO2 surface.

hybridization time and is subjected to incubation in desired concentration of complementary target solution for 60s at 25 °C. The ssDNA/CHITNanoZrO2/ITO bioelectrode is stored at 4 °C when not in use.

’ RESULTS AND DISCUSSION A key characteristic of chitosan used for preparation of CHITNanoZrO2 composite film fabrication is its unique response to applied electrical stimuli. When applied voltage is sufficient for protons to be reduced at the cathode surface, a localized pH gradient is generated, resulting in cathodic electrodeposition of thin CHIT-NanoZrO2 film (180 nm) with ZrO2 molecules entrapped within chitosan chains due to strong electrostatic interactions between these oppositely charged moieties.28 The proposed mechanism of cathodic electrodeposition of CHITNanoZrO2 onto ITO plate is shown in Scheme 1. Figure 1 shows FT-IR spectra of the pristine CHIT/ITO (curve i), NanoZrO2/ITO (curve ii), CHIT-NanoZrO2/ITO (curve iii), and DNA/CHIT-NanoZrO2/ITO (curve iv), respectively. For CHIT/ITO electrode, peaks seen at 1740, 1655, and 1564 cm-1 are assigned to the carbonyl (CdO) stretching of -COOH group, C-O stretching along with N-H deformation mode of amide-I group, and N-H deformation of NH2 group, respectively. The band seen at 1468 cm-1 is due to the 542

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Figure 2. (a) SEM images of CHIT/ITO, NanoZrO2/ITO, CHIT-NanoZrO2/ITO, and DNA/CHIT-NanoZrO2/ITO electrodes and (b) EDX analysis of CHIT/ITO, NanoZrO2/ITO, and CHIT-NanoZrO2/ITO electrodes, respectively.

Figure 2a shows SEM images of CHIT/ITO, NanoZrO2/ ITO, CHIT-NanoZrO2/ITO, and DNA/CHIT-NanoZrO2/ ITO electrodes, respectively. The SEM image of CHIT/ITO film reveals a dense, fibrous morphology with homogeneous distribution of chitosan. The NanoZrO2/ITO film shows the formation of well-dispersed, globular nanostructure of ZrO2 with porous topology. The observed regularity in CHIT-NanoZrO2 nanobiocomposite reveals uniform dispersion of ZrO2 nanoparticles in the CHIT matrix. This may be attributed to electrostatic interactions between the cationic chitosan and the surface-charged ZrO2 nanoparticles. The SEM picture of DNA/CHIT-NanoZrO2/ITO clearly shows uniform binding of DNA that result in flattening and smoothening of the CHIT-NanoZrO2 biocomposite surface due to reduced porosity. This suggests that this nanobiocomposite provides a favorable microenvironment for high loading of probe DNA. The electron diffraction X-ray (EDX) analysis (Figure 2b) of CHIT/ITO and ZrO2/ITO electrode reveals its composition comprising of carbon, nitrogen, oxygen atoms (CHIT) and zirconium, oxygen atoms (ZrO2), respectively. The presence of all these atoms in CHIT-NanoZrO2/ ITO electrode indicates the composite formation. An analytical HR-TEM has been employed to characterize bare chitosan and the CHIT-NanoZrO2 nanocomposite (Figure 3). The HR-TEM micrograph of chitosan exhibits uniform microstructure

with grain size of about 10-20 nm having poor contrast, delineating an amorphous phase of chitosan as the matrix material. The distribution of ZrO2 in the matrix of chitosan is in ultrafine nanoscale with individual grain size of about 30-50 nm. Interestingly, the nanograined ZrO2 is interlinked and is well-dispersed into the matrix. A local area chemical analysis of these grains, performed in situ by utilizing TEM-EDX, reveals the existence of zirconium and oxygen along with other elements pertaining to chitosan. The other elements evolved with EDX spectra are due to the presence of chitosan and carbon-coated Nigrid used as support for the TEM specimen. The micrograph of ZrO2 clearly shows faceted grains at their periphery and contours, which could be correlated with the crystallized structure of this secondary phase dispersed in an amorphous matrix. A hexagonal-shaped grain of ZrO2 with individual edges of about 20-30 nm in length clearly elucidates that crystallographic symmetry is preserved during growth. However, the presence of several other random-shaped faceted grains further presumes that during growth due to physical constraints, the crystallographic symmetry is perhaps not retained on the overall topography. However, crystallinity within the individual grains conceivably remains unaltered. The good quality of the composite material is expected because of the existence of a clear interface between the chitosan (amorphous) and ZrO2 (crystalline) in the 543

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Figure 3. TEM micrographs of (a) chitosan and (b) CHIT-NanoZrO2 nanobiocomposite wherein the dark regions marked by the white circles reveal the presence of crystalline zirconia within the amorphous chitosan environment. (c) TEM-EDX of CHIT-NanoZrO2 nanobiocomposite on nickel grid showing its elemental composition.

Table 1. Characteristics of DNA/CHIT-NanoZrO2/ITO biosensor along with those reported in literature M. Tuberculosis Detection matrix

biocompatibility

method of immobilization

ZrO2/Au

yes

physical adsorption

polyaniline

no

covalent binding by glutaraldehyde as cross-linker

Ppy-PVS

yes

pyrrole-2-carboxy aldehyde

CHIT NanoZrO2 /ITO

yes

covalent binding using avidin-biotin coupling

entire specimen, without any distortion at the interface between these two components. The biocompatibility of this nanocomposite has been investigated with both bacterial and plant systems. For the bacterial system, a culture of Gram negative bacteria, that is, Pseudomonas sp. is grown overnight on a nutrient broth medium at 28 ( 2 °C in a rotary shaker at 150 rpm. The culture (100 μL) is spread on a nutrient agar plate, and wells (2 mm dia) are made with the sterile core borer. The aqueous solution of the compound of 10 μL each (concentration: 0.001, 0.01, 0.1, and 1 mg mL-1) is added to the wells, and the plate is incubated at about 28 ( 2 °C. After ∼48 h of incubation, no zone of inhibition is seen in the plate, indicating biosafety of the compound (Figure 4i). For plant germination, wheat seed surface has been first strained with 0.1% HgCl2, followed by washing with sterilized water. Seeds of wheat have then been immersed in different concentrations of CHIT-NanoZrO2 solution (0.001, 0.1, and 1 mg dL-1) and placed on soft agar for germination. After incubation at 28 °C in the dark for 3 to 4 days, germination has been checked and compared with that of controlled seeds. The observations pertaining to the effect of

stability

reusability

16 weeks

10-12 times

4

6-7 times

31

8 times

32

10-12 times

present work

18 weeks

reference

CHIT-NanoZrO2 composite on seed germination have been given in Figure 4ii-v and reveal appropriate seed germination in all of the concentrations, again showing the biocompatible nature of this nanocomposite. Figure 5a exhibits results of the cyclic voltammetric (CV) studies carried out on CHIT-NanoZrO2/ITO (curve ii) and DNA/ CHIT-NanoZrO2/ITO (curve i) electrodes, respectively which show well-defined redox peaks at 0.31 and 0.25 V with significant decrease in oxidation peak current from (5.56 to 3.10)  10-5 A after DNA binding. This decrease may be attributed to repulsion between the negatively charged DNA, covalently immobilized on CHIT-NanoZrO2/ITO nanocomposite matrix, and [Fe(CN)6]3-/4- moieties present in the buffer solution, resulting in slow electron transfer kinetics. Figure 5b shows cyclic voltammograms of the DNA/CHIT-NanoZrO2/ITO bioelectrode recorded at different scan rates (50-250 mV s-1). It can be seen that with the increase in the scan rate, the anodic peak shifts toward more positive potential and vice versa for the cathodic peak. Besides this, the redox peak currents (Ipa and Ipc) show linear behavior with square root of scan rate (ν1/2) (inset in 544

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Figure 4. Snapshot of the agar plates containing (i) Pseudomonas sp. after 48 h of incubation, revealing no inhibition zone formation in the plate using CHIT-NanoZrO2 solution and (ii-v) wheat seeds after 4 days of incubation, revealing proper seed germination, showing the biocompatibility of CHIT-NanoZrO2 composite.

Figure 5b), suggesting a diffusion-controlled process occurring at the modified electrode.30 The respective linear regression equations for the observed anodic and cathodic current behavior with square root of scan rate are given below Ipa ðAÞ ¼ 1:63  10 - 5 þ 5:36  10 - 6 ν1=2 ðV s - 1 Þ; R ¼ 0:998; SD ¼ 1:04  10 - 6 Ipc ðAÞ ¼ - 1:05  10

-5

- 2:8  10

- 6 1=2

ν

R ¼ - 0:995; SD ¼ 1:006  10

ðV s -6

ð1Þ -1

Figure 5. Cyclic voltammograms of (a) (i) DNA/CHIT-NanoZrO2/ ITO and (ii) CHIT-NanoZrO2/ITO electrodes and (b) DNA/ CHIT-NanoZrO2/ITO bioelectrode as a function of scan rate (50 to 250 mV/s) (inset shows plot of current as a function of square root of scan rate). (c) Stability studies of DNA/CHIT-NanoZrO2/ITO bioelectrode at a regular interval of 3 weeks.

Þ; ð2Þ

where R is the regression coefficient and SD is the standard deviation. The surface concentration of ionic species has been found to be 5.96  10-4 moles cm-2. The stability of a biosensor is indicated by its shelf-life (storage stability) and reproducibility. Shelf-life is determined by the duration for which the sensor can be used or it retains maximum of its activity. The reproducibility is a measure of the scatter or

the drift in a series of observations or results performed over a period of time. Besides this, the nature of materials of the fabricated film and the method of biomolecule immobilization are some of the other factors that affect the stability of the biosensing electrode. To investigate the stability, several DNA/CHIT-NanoZrO2/ITO bioelectrodes (concentration of 545

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0.025 μM) have been prepared and stored under desiccated conditions at 25 °C. It is observed that DPVs of these bioelectrodes recorded at a regular interval of 3 weeks exhibit no loss of guanine oxidation current up to a maximum of 18 weeks, after which there is sudden decrease in the guanine oxidation current, indicating stability of this bioelectrode up to 18 weeks (Figure 5c). Furthermore, it has been found that this bioelectrode can be regenerated and utilized for about 10-12 times. The stability of this bioelectrode, based on electrophoretically deposited nanobiocomposite matrix, is assigned to high surface free energy, provided by uniform distribution of ultrafine, nanosized grains of ZrO2 (30-50 nm) in the polymeric chitosan matrix that helps to strengthen binding of the biomolecule. Covalent immobilization of DNA using avidin-biotin coupling further results in direct and strong binding of DNA, specific to M. Tuberculosis, with CHIT-NanoZrO2 matrix via amide bond formation, preventing leaching that may perhaps be responsible for decrease in the sensing response during prolonged measurements. Electrochemical response measurements have been carried out as a function of target DNA concentration by monitoring guanine oxidation of ssDNA/CHIT-NanoZrO2/ITO bioelectrode for 60 s incubated at 25 °C (Figure 6a). The insignificant change in guanine redox peak is observed at e0.05 μM concentration of ssDNA. Guanine peak height is found to increase linearly with concentration from 0.05 to 0.00078 μM (inset in Figure 6a), indicating that 0.05 μM is sufficient to saturate the ssDNA/CHIT-NanoZrO2/ITO bioelectrode surface. The inset exhibits percentage reduction in the oxidation peak current of the ssDNA/CHIT-NanoZrO2/ITO bioelectrode as a function of complementary DNA concentration. The sensitivity of ssDNA/CHIT-NanoZrO2/ITO bioelectrode, estimated from the slope of the linear regression curve, has been found to be 6.38  10-6 A μM-1 and follows equation 3 I ðAÞ ¼ 6:37  10

-6

½lnðtarget DNA concentration in μ MÞ þ 4:67  10 - 5

ð3Þ

with regression coefficient (R) as 0.98. Figure 6b shows results of the hybridization studies carried out on the ssDNA/CHIT-NanoZrO2/ITO bioelectrode surface using methylene blue (MB) as a redox hybridization indicator for various complementary target concentrations (0.05 to 0.00078 μM) by differential pulse voltammetry. MB is known to associate with the unpaired nitrogenous bases of single-stranded DNA as compared with the double-stranded DNA. It can be seen that upon increasing the concentration of the complementary target DNA from 0.00078 to 0.05 μM, MB peak height decreases up to 0.05 μM, indicating that all of the hybridization sites on the ssDNA/CHIT-NanoZrO2/ITO bioelectrode are covered. Therefore, it can be concluded that ssDNA/CHIT-NanoZrO2/ ITO bioelectrode can be used effectively for detection of M. Tuberculosis DNA in the linear range from 0.00078 to 0.05 μM with detection limit of 0.00078 μM using both guanine oxidation and MB indicator as hybridization detection methods. Figure 6c exhibits differential pulse voltammograms of ssDNA/ CHIT-NanoZrO2/ITO before and after 60 s of incubation with complementary target, noncDNA, and one-base mismatch using MB as redox indicator. The peak current is found to decrease after hybridization because of steric and conformational changes induced during the hybridization process with complementary

Figure 6. (a) Cyclic voltammograms and (b) differential pulse voltammograms of ssDNA/CHIT-NanoZrO2/ITO electrodes after hybridization with complementary target DNA (0.05 to 0.00078 μM) by monitoring guanine oxidation and after 20 μM MB pretreatment at þ0.1 V for 20 s, respectively, in phosphate buffer (0.05M, pH 7.0) at pulse height of 50 mV and pulse width of 70 ms (inset shows a linear plot of percentage peak current reduction as a function of concentration of complementary target DNA and anodic peak current as a function of ln concentration of complementary target). (c) Differential pulse voltammograms of ssDNA/CHIT-NanoZrO2/ITO electrode after hybridization with complementary, noncomplementary, and one-base mismatch sequence in phosphate buffer (0.05M, pH 7.0) using MB as redox indicator. 546

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target DNA. However, under similar conditions, minor change in the peak current is found after incubation with one-base mismatch target DNA, indicating specificity of this ssDNA/CHITNanoZrO2/ITO bioelectrode. Besides this, no significant change in the electrochemical peak current is observed on exposure to noncDNA, revealing selectivity of the bioelectrode for M. Tuberculosis DNA hybridization detection. Table 1 shows characteristics of DNA/CHIT-NanoZrO2/ ITO bioelectrode along with those reported in literature for M. Tuberculosis detection.

(8) Jia, N. Q.; Xu, J.; Sun, M. H.; Jiang, Z. Y. Anal. Lett. 2005, 38, 1237–1248. (9) Wang, J. X.; Sun, X. W.; Wei, A.; Lei, Y.; Cai, X. P.; Li, C. M.; Dong, Z. L. Appl. Phys. Lett. 2006, 88, 233106-1–233106-3. (10) Deng, Z.; Rui, Q.; Yin, X.; Liu, H.; Tian, Y. Anal. Chem. 2008, 80, 5839–5846. (11) Deng, Z.; Tian, Y.; Yin, X.; Rui, Q.; Liu, H.; Luo, Y. Electrochem. Commun. 2008, 10, 818–820. (12) Zong, S.; Cao, Y.; Zhou, Y.; Ju, H. Biosens. Bioelectron. 2007, 22, 1776–1782. (13) Zong, S.; Cao, Y.; Zhou, Y.; Ju, H. Langmuir 2006, 22, 8915– 8919. (14) Das, M.; Sumana, G.; Nagarajan, R.; Malhotra, B. D. Appl. Phys. Lett. 2010, 96, 133703. (15) Bellezza, F.; Cipiciani, A.; Quotadamo, M. Langmuir 2005, 21, 11099–11104. (16) Liu, S. Q.; Dai, Z. H.; Chen, H. Y.; Ju, H. X. Biosens. Bioelectron. 2004, 19, 963–969. (17) Solanki, P. R.; Kaushik, A.; Ansari, A. A.; Sumana, G.; Malhotra, B. D. Appl. Phys. Lett. 2008, 93, 163903. (18) Miao, Y.; Tan, S. N. Analyst 2000, 125, 1591–1594. (19) Liao, J. D.; Lin, S. P.; Wu, Y. T. Biomacromolecules 2005, 6, 392–399. (20) Feng, K. J.; Yang, Y. H.; Wang, Z. J.; Jiang, J. H.; Shen, G. L.; Yu, R. Q. Talanta 2006, 70, 561–565. (21) Kaushik, A.; Solanki, P. R.; Ansari, A. A.; Malhotra, B. D.; Ahmad, S. Biochem. Eng. J. 2009, 46, 132–140. (22) Chun, A. L. Nat. Nanotechnol. 2009, 4, 698–699. (23) Prabhakar, N.; Arora, K.; Arya, S. K.; Solanki, P. R.; Iwamoto, M.; Singh, H.; Malhotra, B. D. Analyst 2008, 133, 1587–1592. (24) Thanyani, S. T.; Roberts, V.; Siko, D. R. G.; Very, P.; Verschoor, J. A. J. Immunol. Methods 2008, 332, 61–72. (25) Das, M.; Sumana, G.; Nagarajan, R.; Malhotra, B. D. Thin Solid Films 2010, 519, 1196–1201. (26) Russell, D. G. Nat. Rev. Mol. Cell Biol. 2001, 2, 569–577. (27) Good, M. C.; Greenstein, A. E.; Young, T. A.; Ng, H. L.; Alber, T. J. Mol. Biol. 2004, 339, 459–469. (28) Yi, H.; Wu, L. Q.; Bentley, W. E.; Ghodssi, R.; Rubloff, G. W.; Culver, J. N.; Payne, G. F. Biomacromolecules 2005, 6, 2881–2894. (29) Khan, R.; Kaushik, A.; Solanki, P. R.; Ansari, A. A.; Pandey, M. K.; Malhotra, B. D. Anal. Chim. Acta 2008, 616, 207–213. (30) Yang, M.; Yang, Y.; Yang, H.; Shen, G.; Yu, R. Biomaterials 2006, 27, 246–255. (31) Gao, Y.; Masuda, Y.; Ohta, H.; Koumoto, K. Chem. Mater. 2004, 16, 2615–2622. (32) Shacham, R.; Mandler, D.; Avnir, D. Chemistry 2004, 10, 1936– 1943.

’ CONCLUSIONS We have successfully fabricated nanostructured ZrO2-chitosan thin film (180 nm) onto ITO-coated glass substrate by EPD technique. This electrodeposition from nanostructured ZrO2CHIT colloidal suspension can be attributed to the pH-responsive behavior of CHIT that helps in dragging the positively charged CHIT-ZrO2 composite colloid particles toward negatively biased electrode (cathode). The morphological studies carried out using SEM and TEM techniques clearly reveal incorporation of the hexagonal grains of ZrO2 (30-50 nm) in the amorphous network of chitosan. Interestingly, these nanograined ZrO2 have been shown to be inter-linked and welldispersed into the chitosan matrix, corroborating the homogeneous nanobiocomposite formation. This has been attributed to electrostatic interactions between cationic CHIT and surfacecharged ZrO2 nanoparticles. The DNA/CHIT-NanoZrO2/ITO bioelectrode, fabricated by covalent immobilization of biotinylated probe DNA specific to M. Tuberculosis onto this nanobiocomposite matrix, has been found to be selective and can detect complementary target up to 0.00078 μM with sensitivity of 6.38  10-6 A μM-1. This interesting platform should further be explored for application toward detection of other infectious diseases including microorganisms and pathogens.

’ AUTHOR INFORMATION Corresponding Author

*Tel: þ91 11 45609152. Fax: þ91 11 45609312. E-mail: [email protected].

’ ACKNOWLEDGMENT We thank Prof. R.C. Budhani, Director, National Physical Laboratory, India for the facilities. Maumita Das and Chetna Dhand are thankful to CSIR, India for the award of Senior Research Fellowships. The financial support received from Department of Science and Technology, India under the projects GAP 081132 and GAP 080232 is greatly acknowledged. ’ REFERENCES (1) Wanekaya, A. K.; Chen, W.; Myung, N. V.; Mulchandani, A. Electroanalysis 2006, 18, 533–550. (2) Luo, X.; Morrin, A.; Killard, A. J.; Smyth, M. R. Electroanalysis 2006, 18, 319–326. (3) Ansari, A. A.; Solanki, P. R.; Malhotra, B. D. Appl. Phys. Lett. 2008, 92, 263901-1–263901-3. (4) Kouassi, G. K.; Irudayaraj, J. J. Nanobiotechnol. 2005, 3, 1–9. (5) Cao, D. F.; He, P. L.; Hu, N. F. Analyst 2003, 128, 1268–1274. (6) Salimi, A.; Sharifi, E.; Noorbakhsh, A.; Soltanian, S. Biosens. Bioelectron. 2007, 22, 3146–3153. (7) Topoglidis, E.; Astuti, Y.; Duriaux, F.; Gratzel, M.; Durrant, J. R. Langmuir 2003, 19, 6894–6900. 547

dx.doi.org/10.1021/bm1013074 |Biomacromolecules 2011, 12, 540–547