Polyaniline Langmuir−Blodgett Film Based Cholesterol Biosensor

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Polyaniline Langmuir-Blodgett Film Based Cholesterol Biosensor Zimple Matharu,†,‡ G. Sumana,† Sunil K. Arya,† S. P. Singh,† Vinay Gupta,‡ and B. D. Malhotra*,† Biomolecular Electronics and Conducting Polymer Research Group, National Physical Laboratory, Dr. K. S. Krishnan Marg, New Delhi-110012, India, and Department of Physics and Astrophysics, UniVersity of Delhi, Delhi-110007, India ReceiVed July 16, 2007 Cholesterol oxidase (ChOx) has been covalently linked to Langmuir-Blodgett (LB) monolayers of polyaniline (PANI)-stearic acid (SA) prepared onto indium-tin-oxide (ITO) coated glass plates via glutaraldehyde (Glu) chemistry. These ChOx/Glu/PANI-SA LB film/ITO electrodes have been characterized by FT-IR, cyclic voltammetry, and scanning electron microscopy, respectively. The results of response measurements carried out on these bioelectrodes using linear sweep voltammetry (LSV) reveal linearity from 25 to 400 mg/dL of cholesterol concentration with sensitivity of 88.9 nA mg-1 dL. The linear regression analysis of bioelectrode reveals standard deviation and correlation coefficient of 0.737 µA and 0.9988, respectively. The low value of the Michaelis-Menten constant of these bioelectrodes obtained as 1.21 mM for the immobilized enzyme indicates increased interaction between ChOx and cholesterol in the PANI-SA LB film.

Introduction Rapid growth in the development of new materials and improvements in sensing techniques have led to the evolution of advanced biosensors.1,2 To obtain faster response and increased binding, various immobilization matrices such as LangmuirBlodgett(LB) films,3,4 conducting polymers,5,6 nanomaterials,7-9 sol-gel films,10,11 etc. have been used. Besides this, immobilization methods of enzymes play a crucial role in the performance of a biosensor. LB films are considered to be more suitable for the fabrication of electrodes for biosensing applications, as the ordered arrangement of molecules and thickness in nanoscale are likely to result in a highly sensitive sensor with ultrafast response.3 These ultrathin films with ordered arrangement and nanorange thickness on desired substrates could be used as molecular wires between conducting surface and enzyme, which in turn significantly alter the electron-transfer process. The use of the LB technique provides an opportunity to fabricate biosensors at the molecular level.12 Conducting polymer-based LB films have recently aroused much interest because of their interesting features of biocom* To whom correspondence should be addressed. Phone: +91-1125734273. Fax: 25726938. E-mail: [email protected]. † National Physical Laboratory. ‡ University of Delhi. (1) Dong, S.; Chen, Xu. Mol. Biotechnol. 2002, 82, 303-323. (2) Jiang, T.; Minunni, M.; Wilson, P.; Zhang, J.; Turner, A. P. F.; Mascini, M. Biosen. Bioelectron. 2005, 20, 1939-1945. (3) Ohnuki, H.; Saiki, T.; Kusakari, A.; Endo, H. I.; Chihara, M.; Izumi, M. Langmuir 2007, 23, 4675-4681. (4) Zhu, D. G.; Petty, M. C.; Ancelin, H.; Yarwood, J. Thin Solid Films 1989, 176, 151-156. (5) Borole, D. D.; Kapadi, U. R.; Mahulikar, P. P.; Hundiwali, D. G. Poly. AdV. Tech. 2004, 15, 306-312. (6) Vidal, J. C.; Espuelas, G. R.; Castillo, J. R. Anal. Lett. 2002, 35(5), 837853. (7) Pandey, P.; Singh, S. P.;Arya, S. K.; Gupta, V.; Datta, M.; Singh, S.; Malhotra, B. D. Langmuir 2007, 23, 3333-3337. (8) Kouassi, G. K.; Irudayaraj, J.; McCarty, G. J. Nanobiotechnology 2005, 3, 1. (9) Shumyantseva, V.; Carrara, S.; Bavastrello, V.; Riley, D.J.; Bulko, T.V.; Skryabin, K. G.; Archakov, A. I.; Nicolini, C. Biosen. Bioelectron. 2005, 21, 217-222. (10) Wang, J. Anal. Chim. Acta 1999, 399, 21-27. (11) Li, J.; Peng, T.; Peng, Y. Electroanalysis 2003, 15, 1031-1037. (12) Petty, M. C. Ende. New Series 1983, 7(2), 65-69.

patibility, the possibility of miniaturization, etc.13 Attempts have recently been made to fabricate LB films of organic materials including conducting polymers for biosensor applications. In this context, Ohnuki et al. have incorporated glucose oxidase onto LB films of octadecyltrimethylammonium and nanostructured Prussian blue (PB) clusters.3 They have reported that presence of PB cluster helps in the detection of glucose at low potential by enhancing the electron-transfer rate. Zhang et al. have prepared mixed LB films of urease and octadecylamine onto ion-selective-field-effect transistor for urea biosensing and have found linearity of urea in the range 0-20 mM.14 Ramanathan et al. have utilized LB films of polyaniline for a glucose biosensor.15 Singhal et al. have used poly(3-dodecyl thiophene) LB films for fabrication of a glucose biosensor.16 Further, these authors have used LB films of poly(N-vinyl carbazole) for development of urea biosensor and have reported that these urea biosensing electrodes are stable over a wide range of pH and temperature.17 Sharma et al. have reported that P3HT LB film based galactose biosensor has a shelflife of more than 90 days and is stable up to 45 °C.18 In all these attempts, enzyme was physically adsorbed on the LB matrix or entrapped in the LB film by dissolving it in chloroform. Covalent immobilization of enzymes is known to have various requisites such as reproducibility, durability, and stability of the bioelectrode against pH, chemical nature of microenvironment, etc. Efforts have not yet been made to covalently immobilize enzyme on to LB films of polyaniline (PANI) for the development of a cholesterol biosensor. Recently, monitoring of cholesterol in human beings has been considered important since its elevated level results in heart disease and neurological disorders.19,20 In the present manuscript, we report the results of our studies relating to covalent immobilization (13) Petty, M. C. J. Biomed. Eng. 1991, 13, 209-214. (14) Zhang, A.; Renault, N. J.; Wan, J.; Hou, Y.; Chvelon, J. Mater. Sci. Eng. C 2002, 21, 91-96. (15) Ramanathan, K.; Ram, M. K.; Malhotra, B. D.; Murthy, A. S. N. Mater. Sci. Eng. C 1995, 3, 159-163. (16) Singhal, R.; Takashima; Kaneto, K.; Samanta, S. B.; Annapoorni, S.; Malhotra, B. D. Sens. Actuators, B 2002, 86, 42-48. (17) Singhal, R.; Gambhir, A.; Pandey, M. K.; Annapoorni, S.; Malhotra, B. D. Biosen. Bioelectron. 2002, 17, 697-703. (18) Sharma, S. K.; Singhal, R.; Malhotra, B. D.; Sehgal, N.; Kumar, A. Electrochim. Acta 2004, 49, 2479-2485.

10.1021/la702123a CCC: $37.00 © 2007 American Chemical Society Published on Web 11/15/2007

Polyaniline LB-Film-Based Cholesterol Biosensor

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Scheme 1. Tentative Mechanism of Covalent Immobilization of ChOx on PANI-SA LB Film

of cholesterol oxidase (ChOx) to LB films of PANI for application to a cholesterol biosensor. Experimental Section Chemicals and Reagents. ChOx (E.C. 1.1.3.6, from Pseudomonas fluorescens) with specific activity of 24 U/mg, horseradish peroxidase (HRP, E.C.1.11.1.7) with specific activity of 200 U/mg solid, o-dianisidine, 1-methyl-2-pyrrolidone, stearic acid, cadmium chloride, and glutaraldehyde were procured from Sigma-Aldrich. All other chemicals used are of analytical grade and have been used as received. Polyaniline was synthesized by chemical oxidative polymerization using ammonium persulphate as oxidant.21 LB Film Deposition. For LB monolayer fabrication,22,23 1-methyl2-pyrrolidone solution of 0.08 mg/mL polyaniline and 0.08 mg/mL stearic acid were mixed and ultrasonicated for about 2 h. Monolayer deposition was carried out in the LB trough (NIMA, UK). CdCl2 solution (0.2 mM) in deionized water (18.2 MΩ/cm) was used as subphase in the LB trough. The pressure-area isotherm of the monolayer was obtained by spreading 100 µL of this solution on the subphase and increasing the pressure by closing the barrier. The stability of the monolayer was monitored, holding the monolayer at the target pressure and observing the change in mean molecular area with time. At the optimized pressure and temperature, 15 monolayers were transferred onto precleaned ITO-coated glass substrates by vertically dipping at a speed of 5 mm/min. The target pressure was maintained by compressing the monolayer on the subphase at a speed of 40 cm2/min. (19) Fredrikson, D.S.; Levy, R.I. In The metabolic basis of inherited disease; Wyngarden, J. B, Fredrickson, D. D., Eds.; McGraw-Hill: New York, 1972; p 545. (20) Forster, R.; Cassidy, J.; Donoghue, E. O. Electroanalysis 2000, 12, 716. (21) Leon, M. J.D. Proceeding of The National Conference On Undergraduate Research; Lexington, KY, 2001. (22) Dhanabalan, A.; Dabke, R. B.; Kumar, N. P.; Talwar, S. S.; Major, S. S.; Contractor, A. Q. Langmuir 1997, 13, 4395-4400. (23) Dhanabalan, A.; Dabke, R. B.; Datta, S. N.; Kumar, N. P.; Major, S. S.; Talwar, S. S.; Contractor, A. Q. Thin Solid Films 1997, 295, 255-259.

The various isotherms recorded as a function of pH and temperature reveal that the ideal isotherm is obtained at pH 2.0 and 20 °C (see Supporting Information, Supplement 1a). The value of the pressure at which the quasi-solid behavior of PANI-SA monolayer appears, is found to be 16 mN/m. Further increase in the pressure (>32 mN/m) results in the collapse of the monolayer losing its monomolecular form. The observed limiting mean molecular area of ∼32 Å2 indicates the formation of uniform mixed monolayer. It may be noted that this monolayer is stable at the air/water interface for more than 2 h at a barrier speed of 40 cm2/min and a pressure of 16 mN/m (see Supporting Information, Supplement 1b). Immobilization of Cholesterol Oxidase. ChOx was covalently immobilized onto the PANI-SA LB film using glutaraldehyde24,25 (Scheme 1). For this purpose, these LB films were first incubated in a 1% solution of glutaraldehyde for about 6 h followed by washing with deionized water. For ChOx immobilization, the optimum amount of 20 µL of ChOx in phosphate buffer saline (PBS) solution (50 mM, pH 7.0, 0.9% NaCl) was spread onto a glutaraldehyde-modified surface and was kept overnight. The electrode was then washed with PBS buffer containing 0.05% Tween 20 and was stored at 4 °C when not in use. The incubation of ChOx solution on Glu/PANI-SA/ITO results in the covalent bond formation between the amine groups of lysine or hydroxylysine present in the ChOx molecules and aldehyde group of glutaraldehyde. These ChOx/Glu/PANI-SA LB film/ITO bioelectrodes were characterized by scanning electron microscopy (Leo 440), Fourier transform infrared (FT-IR) spectroscopy (PerkinElmer instrument, model spectrum BX using ATR accessory), and cyclic voltammetry (CV) (Autolab Potentiostat/Galvanostat, Eco Chemie, Netherlands), respectively. Enzyme activity measurements were carried out by UV-vis spectrophotometer (UV-160 A, Shimadzu) and linear sweep voltammetry (LSV). (24) Sai, V. V. R.; Mahajan, S.; Contractor, A. Q.; Mukherji, S. Anal. Chem. 2006, 78, 8368-8373. (25) Coelho, R. A. L.; Santos, G. M. P.; Azevedo, P. H. S.; Azevedo, W. M.; Carvalho, L. B., Jr. J. Biomed. Res. 2001, 56, 257.

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Figure 1. SEM textures of (i) PANI-SA LB film/ITO and (ii) ChOx/ Glu/PANI-SA LB film/ITO electrodes.

Figure 3. Cyclic voltammetric response of (i) PANI-SA LB film/ ITO, (ii) Glu/PANI-SA LB film/ITO, and (iii) ChOx/Glu/PANI-SA LB film/ITO electrodes.

Figure 2. FT-IR spectra of (i) PANI-SA LB film/ITO and (ii) ChOx/Glu/PANI-SA LB film/ITO electrodes.

Results and Discussion Scanning Electron Microscopic Studies. Figure 1 shows the SEM pictures obtained for PANI-SA LB film/ITO and ChOx/ Glu/PANI-SA LB film/ITO electrodes, respectively. The uniform morphology obtained in PANI-SA [Figure 1(i)] indicates a wellpacked, homogeneous LB film on the surface of ITO. The presence of uniformly distributed globular structures [Figure 1(ii)] can be attributed to the covalently bound ChOx enzyme molecules, as most proteins and enzymes possess globular structures. FT-IR Studies. The FT-IR spectrum [Figure 2(i)] of PANISA LB film shows quinoid and benzenoid ring stretching bands (CdC) present at 1593 and 1470 cm-1. The 1295 cm-1 peak is attributed to the C-N vibration of polyaniline. The characteristic peaks of polyaniline backbone are observed at 840 and 700 cm-1. The peak around 1720 cm-1 indicates the carbonyl stretching of carboxylic group present in stearic acid. The presence of all these peaks confirms the presence of PANI and stearic acid on the ITO electrode. The FT-IR spectrum of ChOx/Glu/PANI-SA LB film electrode [Figure 2(ii)] exhibits additional peaks at 1640 cm-1 and 1558 cm-1 attributed to the carbonyl stretch (amide I band) and NH bending (amide II band), respectively confirming ChOx binding. Also, a broad band seen between 3300 and 3100 cm-1 can be assigned to amide A and amide B of the enzyme. Electrochemical Studies. Cyclic Voltammetric (CV) and Linear Sweep Voltammetric (LSV) Studies. CV and LSV studies have been carried out using a three-electrode cell with platinum foil as counter and Ag/AgCl as reference electrode in acetate buffer of pH 5.6, 50 mM on an Autolab Potentiostat/ Galvanostat (Eco Chemie, Netherlands). CV Studies. Figure 3 shows the cyclic voltammograms for PANI-SA LB film/ITO, Glu/PANI-SA LB film/ITO, ChOx/Glu/ PANI-SA LB film/ITO electrodes in the potential range of -0.7 to 1.0 V at a scan rate of 50 mV/s.

The decrease in the oxidation current obtained for the Glu/ PANI-SA LB film/ITO electrode [Figure 3(ii)] compared to that of the PANI-SA LB film/ITO electrode [Figure 3(i)] reveals the glutaraldehyde binding since glutaraldehyde forms an insulating layer on the PANI-SA surface. Further, increase in the current obtained for the ChOx/Glu/PANI-SA LB film/ITO electrode [Figure 3(iii)] attributed to the presence of redox moieties at active sites of enzyme on the electrode surface suggests ChOx immobilization onto the Glu/PANI-SA LB film/ITO electrode surface. Response Studies ChOx/Glu/PANI-SA LB Film/ITO Bioelectrode by LSV. The LSV studies have been used to study the activity of the ChOx/Glu/PANI-SA LB film/ITO bioelectrode in acetate buffer (50 mM) of pH 5.6 in the voltage range of -0.5 and 0.9 V for different cholesterol concentrations. Figure 4a shows the linear sweep voltammogram of ChOx/ Glu/PANI-SA LB film/ITO bioelectrode obtained as a function of cholesterol concentration wherein the oxidation peak seen around 0.2 V corresponds to the oxidation of PANI. It is interesting to note that no peak related to the generation of H2O2 is observed in the LSV measurements (Figure 4a). This could perhaps be attributed to the well-aligned, uniformly packed polymer chains (in the PANI-SA LB film) that act as a molecular wire resulting in better electron-accepting behavior over molecular oxygen from the reduced enzyme. During the reoxidation of ChOx after enzymatic reaction, the well-aligned PANI chains in the LB film accept electrons from the reduced enzyme, thereby causing an increase in the oxidation current of PANI during LSV measurements26 (Figure 4a). The mechanism for enzymatic reaction and catalytic action of PANI has been shown in Scheme 2. It can be seen from the linear regression curve of the ChOx/Glu/PANISA LB film/ITO bioelectrode (Figure 4b) that the ChOx/Glu/ PANI-SA LB film/ITO bioelectrode can be used to estimate cholesterol from 25 to 400 mg/dL. The sensitivity of the ChOx/ Glu/PANI-SA LB film/ITO bioelectrode calculated from the slope of curve has been found to be 88.9 nA mg-1 dL. The standard deviation and correlation coefficient from the linear regression analysis for the bioelectrode have been found to be 0.737 µA and 0.9988, respectively. The results of triplicate sets (data not shown) reveal reproducibility within negligible error. (26) Chaubey, A.; Pande, K. K.; Singh, V. S.; Malhotra, B. D. Anal. Chim. Acta 2000, 407, 97-103.

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Figure 4. (a) LSV curve of ChOx/Glu/PANI-SA LB film/ITO as a function of cholesterol concentration (i) 0, (ii) 25, (iii) 50, (iv) 100, (v) 200, (vi) 300, and (vii) 400 mg/dL. (b) Linear regression curve of the ChOx/Glu/PANI-SA LB film/ITO bioelectrode.

Figure 6. Photometric response of the ChOx/Glu/PANI-SA LB film/ITO electrode as a function of temperature. Figure 5. Photometric response of the ChOx/Glu/PANI-SA LB film/ITO electrode as a function of cholesterol concentration. Scheme 2. Biochemical Reaction at the ChOx/Glu/PANI-SA LB Film/ITO Electrode

Photometric Studies of the ChOx/Glu/PANI-SA LB Film/ ITO Bioelectrode. UV-visible experiments have been carried out to estimate the enzyme activity, stability and shelflife of ChOx/Glu/PANI-SA LB film/ITO electrodes. The electrode is dipped in the 3 mL PBS solution containing 20 µL of HRP (1 mg/mL in PBS), 20 µL of o-dianisidine dye (1% in deionized water), and 100 µL of analyte (cholesterol solution) and is kept for about 6 min. Effect of Cholesterol Concentration. Figure 5 shows the difference between the initial and final absorbance value at 500 nm after 6 min of incubation of substrate. It can be seen (Figure 6) that the value of absorbance increases linearly with cholesterol concentration in the range of 25-400 mg/dL. The experiments carried out in triplicate sets were found to be reproducible within 5% error.

The apparent enzyme activity (U/cm2) has been calculated 2 using aenz app (U/cm ) ) AV/ts where A is the difference in absorbance before and after incubation, V is the total volume (3.00 cm3),  is the millimolar extinction coefficient (7.5 for o-dianisidine at 500 nm), t is the reaction time (min), and s is the surface area (cm2) of the electrode.27 The apparent enzyme activity has been found to be 1.33 × 10-3 U/cm2. Calculation of the Michaelis-Menten Constant (Km). The Michaelis-Menten kinetic parameters of enzymatic reaction that determine the affinity of enzyme have been estimated using Lineweaver-Burke plot. The Km value for the immobilized enzyme, 46.83 mg/dL (1.21 mM), has been found to be smaller than the corresponding value obtained for free ChOx (3.71 mM) under similar experimental conditions. The observed lower (27) Kumar, A.; Pandey, R. R.; Brantley, B. Talanta 2006, 69, 700-705. (28) Wang, H.; Mu, S. Sens. Actuators, B 1999, 56, 22-30. (29) Bongiovanni, C.; Ferri, T.; Poscia, A.; Varalli, M.; Santucci, R.; Desideri, A. Bioelectrochemistry 2001, 54, 17-22. (30) Shen, J.; Liu, C. C.; Sens. Actuators, B 2007, 120, 417-425. (31) Singh, S.; Solanki, P. R.; Pandey, M. K.; Malhotra, B. D. Sens. Actuators, B 2006, 115, 534-541. (32) Arya, S. K.; Solanki, P. R.; Singh, R. P.; Pandey, M. K.; Datta, M.; Malhotra, B. D. Talanta 2006, 69, 918-926. (33) Singh, S.; Chaubey, A.; Malhotra, B. D. Anal. Chim. Acta 2004, 502, 229-234. (34) Kumar, A.; Rajesh Chaubey, A.; Grover, S. K.; Malhotra, B. D. J. Appl. Polym. Sci. 2001, 82, 3486-3491.

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Table 1. Characteristics of the ChOx/Glu/PANI-SA LB Film/ITO Bioelectrode, along with Those Reported in Literature no.

immobilization matrix

sensing element

1

polyaniline

ChOx

2

polymeric film

ChOx/HRP

3

3-mercaptopropionic acid polyaniline

ChOx

4 5

method of immobilization electrochemical doping physical entrapment covalent

ChOx/ChEt/ HRP ChOx/HRP

covalent

ChOx

covalent electrochemical entrapment physical adsorption covalent

6

tetraethylorthosilicate (sol-gel) modified ODT

7

polypyrrole

ChOx/ChEt

8

DBS-doped PPy

9

polyaniline

ChOx/ K3Fe(CN)6 ChOx

covalent

linearity

transducer used

Km

0.05-0.5 mM 0.07-0.27mM 50-200 mg/dL (1.29-5.17mM) up to 500 mg/dL (7.68 mM) 2-12 mM

2.27 and 3.26 mM 6 mM

amperometry (flow injection) amperometry spectrophotometry

75 mg/dL 1.94 mM 21.2 mM

amperometry

50-500 mg/dL (1.29-12.92 mM) 1-8 mM

amperometry

2-8 mM

amperometry

25-400 mg/dL (0.65-10.34 mM)

electrochemical and spectrophotometry

value of Km for bound enzyme than that for free enzyme shows enhanced affinity of the immobilized cholesterol oxidase with cholesterol in the PANI-SA LB film. This result can be assigned to the uniform distribution of ChOx molecules on to the PANISA LB film surface. Effect of Temperature on ChOx/Glu/PANI-SA LB Film/ ITO Electrodes. To study the effect of temperature, the reaction of enzyme (ChOx) in the ChOx/Glu/PANI-SA LB film/ITO with the analyte (cholesterol) has been carried out at temperature varying from 20 to 45 °C of phosphate buffer saline solution pH 7.0 (Figure 6). The buffer solution containing 100 µL of 200 mg/dL cholesterol, 20 µL of HRP, and 20 µL of o-dianisidine has been used for these studies. The reaction rate is found to increase continuously with temperature up to 35 °C, whereafter it shows a sharp decrease, indicating that ChOx gets denatured at temperatures >35 °C. The value of the activation energy calculated using the Arrhenious plot, i.e., plot of ln(absorbance) with inverse of absolute temperature (data not shown), has been found to be 32.27 kJ/mol in the lower temperature range (e35 °C). Effect of Interferents on the ChOx/Glu/PANI-SA LB Film/ ITO Electrode and Shelf Life. The effect of interferents [uric acid (0.1 mM), glucose (5 mM), lactic acid (0.5 mM), ascorbic acid (0.05 mM), and urea (1 mM)] on cholesterol measurement has been studied by taking a solution containing a 1:1 ratio of cholesterol (200 mg/dL or 5.17 mM) and the interferent (data not shown). Excluding urea and glucose that have been found to exhibit interference of 7-9%, all other compounds (lactic acid, ascorbic acid, uric acid) show negligible interference. The results of experiments carried out with ChOx/Glu/PANI-SA LB film/ITO electrode for serum samples reveal a free cholesterol level that is about 25-30% of the value, obtained using an auto analyzer of the same samples for total cholesterol. The value is in good agreement, as the serum contains only 30% free cholesterol. The electrode stability has been studied by carrying out UVvisible experiments at regular interval of 1 week. The ChOx/ Glu/PANI-SA LB film/ITO electrodes were stored at 4 °C prior to being used. The electrodes were found to be stable up to 10

optical 9.8 mM

1.21 mM (bound enzyme) 3.71 mM (free enzyme)

shelf life

ref

11 days

28

7 days

29

4 days

30

6 weeks

31

8 weeks

27

2 months

32

4 weeks

33

∼3 months

34

10 weeks

present work

weeks, and less than 10% decrease in the value of absorbance was found even after 10 weeks. Table 1 shows the characteristics of the cholesterol biosensor based on the ChOx/Glu/PANI-SA LB film/ITO electrode along with those reported in literature. It can be seen from Table 1 that the ChOx/Glu/PANI-SA LB film/ITO electrode shows linearity in the broad range of 0.65-10.34 mM and has a high affinity (low Km) for its analyte. The high affinity for the electrode can be attributed to the availability of uniformly distributed functional groups on the surface of the LB film arising due to the ordered arrangement of PANI molecules that facilitate uniform distribution of ChOx on the LB surface.

Conclusions Mixed monolayers of PANI-SA prepared on ITO-coated glass using the LB technique have been successfully utilized for fabrication of cholesterol biosensor. These monolayers are stable for more than 2 h on the air/water interface. The ChOx/Glu/ PANI-SA LB film/ITO bioelectrodes exhibit linearity over a concentration range of 25-400 mg/dL, improved stability (10 weeks) and low Km(1.21 mM). These ChOx/Glu/PANI-SA LB film/ITO based biosensor shows sensitivity of about 88.9 nA mg-1 dL and correlation coefficient of 0.9988. The relatively low value of Km (1.21 mM) compared to that of free enzyme (3.71 mM) indicates a distinct advantage of PANI-SA LB film as a potential electrode for development of efficient cholesterol sensor. Acknowledgment. We are grateful to Dr. Vikram Kumar, Director, National Physical Laboratory, New Delhi, India for his interest in this work. Zimple Matharu is thankful to the Council of Scientific and Industrial Research (CSIR), India, for the award of a research fellowship. The authors thank Dr. K. N. Sood (NPL, New Delhi, India) for SEM measurements and all members of the BECPRG for interesting discussions. Supporting Information Available: Isotherm of polyanilinestearic acid monolayer and stability of the monolayer. This material is available free of charge via the Internet at http://pubs.acs.org. LA702123A