7398

Langmuir 2007, 23, 7398-7403

Cholesterol Biosensor Based on Amino-Undecanethiol Self-Assembled Monolayer Using Surface Plasmon Resonance Technique
Pratima R. Solanki, Sunil K. Arya, Y. Nishimura, M. Iwamoto, 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 Physical Electronics, Tokyo Institute of Technology, 2-12-1-S3-33, O-okayama, Maguro, Tokyo 152-8552, Japan ReceiVed February 7, 2007. In Final Form: March 21, 2007
Cholesterol oxidase has been covalently immobilized onto 11-amino-1-undecanethiol hydrochloride (AUT) selfassembled monolayer (SAM) fabricated on gold (Au) substrates using glutaraldehyde as a cross-linker. These ChOx/ AUT/Au bioelectrodes characterized using contact angle (CA) measurements; electrochemical technique and atomic force microscopy (AFM) have been utilized for the estimation of cholesterol in solution using the surface plasmon resonance (SPR) technique. These biosensing electrodes exhibiting linearity from 50 to 500 mg/dL of cholesterol in solution and sensitivity of 1.23 m0/(mg dL), can be used more than 20 times and have a shelf life of about 10 weeks when stored at 4 C.

Introduction
In the past two decades, biosensors have received much attention due to fast response, small size, reliable and reproducible results for estimation of various blood analytes.1-7 In order to obtain higher accuracy, precision, and reliability, the surface plasmon resonance (SPR) technique has received much interest. SPR, a surface-sensitive technique based on the detection of change in refractive index (RI), can be used to monitor changes occurring at an interface.8-14 This interesting technique can be utilized to quantify biomolecular interactions in real time in a label-free environment wherein one of the interactants (e.g., enzymes and antibodies) is immobilized onto a sensor surface and an analyte is passed over the sensor surface. The rate of change of an SPR signal can be analyzed to estimate the apparent rate constants of the association and dissociation phases of the biochemical reactions. Recently, SPR techniques have been used for the detection of biochemical reactions between immobilized
* E-mail: bansi.malhotra@gmail.com. Phone: 91 11-25734273. Fax: 9111-25726938. National Physical Laboratory, New Delhi. Tokyo Institute of Technology, Japan.
(1) Hoccoun, J.; Piro, B.; Noel, V.; Pham, M. C. Bioelectrochemistry 2006, 68, 218. (2) Rajesh; Takashima, W.; Kaneto, K. Biomaterials 2005, 26, 3683. (3) Lucarelli, F.; Marrazza, G.; Turner, A. P. F.; Mascini, M. Biosens. Bioelectron. 2004, 19, 515. (4) Gerard, M.; Ramanathan, K.; Chaubey, A.; Malhotra, B. D. Electroanalysis 1999, 6, 450. (5) Ramanathan, K.; Pandey, S. S.; Kumar, R.; Malhotra, B. D.; Murthy, A. S. N. J. Appl. Polym. Sci. 2000, 78, 662. (6) Pandey, S. S.; Ram, M. K.; Srivastava, V. K.; Malhotra, B. D. J. Appl. Polym. Sci. 1997, 65, 2745. (7) Dobay, R.; Harsanyi, G.; Visy, C. Anal. Chim. Acta 1999, 385, 187. (8) Green, R. J.; Frazier, R. A.; Shakesheff, K. M.; Davies, M. C.; Roberts, C. J.; Tendler, S. J. B. Biomaterials 2000, 21, 1823. (9) Liu, X.; Sun, Y.; Song, D.; Zhang, Q.; Tian, Y.; Zhang, H. Talanta 2006, 68, 1026. (10) Rich, R. L.; Day, Y. S. N.; Morton, T. A.; Myszka, D. G. Anal. Biochem. 2001, 296, 197. (11) Mullett, W. M.; Lai, E. P. C.; Yeung, J. M. Methods 2000, 22, 77. (12) Nishimura, S.; Yoshidome, T.; Tokuda, T.; Mitsushio, M.; Higo, M. Anal. Sci. 2002, 18, 261. (13) Shen, B.; Shimmon, S.; Smith, M. M.; Ghosh, P. J. Pharm. Biomed. Anal. 2003, 31, 83. (14) Torrance, L.; Ziegler, A.; Pittaman, H.; Paterson, M.; Toth, R.; Eggleston, I. J. Virol. Methods 2006, 134, 164.

enzymes (glucose oxidase,15cholesterol oxidase,16 urease17) and substrate in solutions. Gaus and Hall (1999) have carried out studies relating to the measurement of change in the SPR angle for estimation of LDL and HDL cholesterol using heparin.18 Immobilization of biomolecules (enzymes, DNA) onto various matrices such as conducting polymers,19 Langmuir-Blodgett (LB) films,20,21 and sol-gels22 using different techniques such as physical adsorption,23 covalent bonding,24 and entrapment25 have been utilized for development of clinical biosensors. However, these biosensors have a number of disadvantages such as denaturation of enzymes during entrapment or at the time of polymer synthesis or due to harsh experimental conditions.26,27 Moreover, reaction kinetics of the immobilized biomolecules in a thick polymer film may get hampered by the sluggish transport of charge carriers within a polymer matrix.28 To overcome these problems, homogeneous and well-defined substrates that can control the properties of the desired solid surface, resulting in improved interaction between the substrate and biomolecules, have been used. Self-assembled monolayers (SAMs) have recently been used as substrates for the immobilization of proteins,29-31 as these are
(15) Hsieh, H. V.; Pfeiffer, Z. A.; Amiss, T. J.; Sherman, D. B.; Pitner, J. B. Biosens. Bioelectron. 2004, 19, 653. (16) Arya, S. K.; Solanki, P. R.; Singh, R. P.; Pandey, M. K.; Datta, M.; Malhotra, B. D. Talanta 2006, 69, 918. (17) May, L. M.; Russell, D. A. Anal. Chim. Acta 2003, 500, 119. (18) Gaus, K.; Hall, E. A. H. J. Colloid Interface Sci. 1999, 217, 111. (19) Malhotra, B. D.; Chaubey, A.; Singh, S. P. Anal. Chim. Acta 2006, 578, 59. (20) Wan, K.; Chovelon, J. M.; Jaffrezic-Renault, N. Talanta 2000, 52, 663. (21) Hou, Y.; Jaffrezic-Renault, N.; Zhang, A.; Wan, J.; Errachid, A.; Chovelon, J. M. Sens. Actuator, B 2002, 86, 143. (22) Li, J.; Peng, T.; Peng, Y. Electroanalysis 2003, 15, 1031. (23) Bean, L. S.; Heng, L. Y.; Yamin, B. M.; Ahmad, M. Thin Solid Films 2005, 477, 104. (24) Campuzano, S.; Galvez, R.; Pedrero, M.; Manuel, de Villena, F. J.; Pingarron, J. M. J. Electroanal. Chem. 2002, 526, 92. (25) Singh, S.; Chaubey, A.; Malhotra, B. D. Anal. Chim. Acta 2004, 502, 229. (26) Hou, S. F.; Yang, K. S.; Fang, H. Q.; Chen, H. Y. Talanta 1998, 47, 561. (27) Iijima, S.; Mizutani, F.; Yabuki, S.; Tanaka, Y.; Aasi, M.; Katsura, T. Anal. Chim. Acta 1993, 281, 483. (28) Dong, X. D.; Lu, J.; Cha, C. J. Electroanal. Chem. 1995, 381, 195. (29) Martins, M. C. L.; Fonseca, C.; Barbosa, M. A.; Ratner, B. D. Biomaterials 2003, 24, 3697. (30) Barrias, C. C.; Martins, M. C. L.; Miranda, M. C. S.; Barbosa, M. A. Biomaterials 2005, 26, 2695.

10.1021/la700350x CCC: $37.00 2007 American Chemical Society Published on Web 05/25/2007

Cholesterol Biosensor Based on AUT SAM

Langmuir, Vol. 23, No. 13, 2007 7399

Figure 1. (A) Blank gold. (B) AUT/Au. (C) ChOx/AUT/Au bioelectrode. Scheme 1 . Schematic of Covalent Immobilization of ChOx on AUT/Au SAM Using Glutaraldehyde as Linking Reagent

likely to result in shorter response time, reduce non-Faradaic background current, and show good reproducibility.32 Also, SAMs are well-ordered, homogeneous, simple, and versatile systems for immobilization of the desired biomolecules. In this context, the terminal group of the alkanethiol SAM can be chemically modified, enabling the control of the surface structure and thus increasing the versatility of the SAM.33,34 Cholesterol is an important component of the membranes of cells, providing stability over a higher temperature interval. It is a major precursor for the synthesis of vitamin D, various steroid hormones (cortisol, cortisone, and aldosterone in the adrenal glands) and sex hormones like progesteron, estrogen, and testosterone.35 Further, recent research has shown that cholesterol plays an important role for the brain synapses, as well as in the immune system including protection against cancer. However, a high level of blood cholesterol is said to promote atherosclerosis or heart attacks and coronary heart disease.36,37 Thus, estimation of cholesterol in blood is very important for the treatment of heart disease. Cholesterol oxidase is a common and important enzyme used for the estimation of cholesterol concentration. Most cholesterol biosensors developed to date are based on either amperometric or colorimetric techniques.38-41 It has been found that electrochemical determination of cholesterol suffers
(31) Onoe, H.; Matsumoto, K.; Shimoyama, I. J. Microelectromech. Syst. 2004, 13, 6003. (32) Yi, X.; Huang-Xian, Ju.; Hong-Yuan, C. Anal. Chem. 2000, 278, 22. (33) Mirsky, V. M.; Riepl, M.; Wolfbeis, O. S. Biosens. Bioelectron. 1997, 12, 977. (34) Lin, J.; Chaung, W. J. Biomed. Mater. Res. 2000, 51, 413. (35) Myant, N. B. The biology of cholesterol and related steroids; Willium Heinemann: London, 1981. (36) 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. (37) Forster, R.; Cassidy, J.; Donoghue, E. O. Electroanalysis 2000, 12, 716. (38) Singh, S.; Solanki, P. R.; Pandey, M. K.; Malhotra, B. D. Anal. Chim. Acta 2006, 568, 126. (39) Singh, S.; Solanki, P. R.; Pandey, M. K.; Malhotra, B. D. Sens. Actuator, B 2006, 115, 534. (40) Tan, X.; Li, M.; Cai, P.; Luo, L.; Zou, X. Anal. Biochem. 2005, 337, 111. (41) Li, G.; Liao, J. M.; Hu, G. Q.; Ma, N. Z.; Wu, P. J. Biosens. Bioelectron. 2005, 20, 2140.

from interference of some compounds such as ascorbic acid, glucose, and so forth, which are commonly present in blood and serum samples. To minimize the effect of the interferents, many different strategies including the use of various mediators and modification of electrode surfaces have been utilized. Among these, the self-assembly technique is of particular interest because its chemistry can be easily manipulated via changing functional groups that become compatible for the immobilization of biomolecules and biosensor fabrication, and so forth.42-50 In the present manuscript, we report results of the studies carried out on covalently immobilized cholesterol oxidase onto 11-amino-undecanethiol hydrochloride (AUT) SAM using glutaraldehyde as a cross-linker using the SPR technique for application to the cholesterol biosensor. These ChOx/AUT/Au biosensing electrodes characterized using contact angle (CA) measurements, electrochemical technique, and atomic force microscopy (AFM) have been used for estimation of cholesterol concentration in solution using the SPR technique. Experimental Section
Materials. 11-Amino-1-undecanethiol hydrochloride (product number A423) was procurred from Dojindo, Japan. Glutaraldehyde (product number 079-00533) was from Wako, Tokyo. K3Fe(CN) 6, KCl, and ethanol (99.5%) were procured from Koso Chemical Co., Ltd., Tokyo, Japan. Cholesterol oxidase {(ChOx; EC 1.1.3.6 CHOD, from Streptomyces species (strain SA-COO) having length
(42) Dong, S.; Li, J. Bioelectrochem. Bioenerg. 1997, 42, 7. (43) Subramanian, A.; Irudayaraj, J.; Ryan, T. Sens. Actuator, B.2006, 114, 192. (44) Mauriz, E.; Calle, A.; Lechuga, L. M.; Quintana, J.; Montoya, A.; Manclus, J. J. Anal. Chim. Acta 2006, 561, 40. (45) Yang, M.; Yang. Y.; Yang, H.; Shen, G.; Yu, R. Biomaterials 2006, 27, 246. (46) Vidal, J. C.; Espuelas, J.; Ruiz, E. G.; Castillo, J. R. Talanta 2004, 64, 655. (47) Vidal, J. C.; Espuelas, J.; Castillo, J. R. Anal. Biochem. 2004, 333, 88. (48) Gobi, K. V.; Mizutani, F. Sens. Actuators, B 2001, 80, 272. (49) Bokoch, M. P.; Devadoss, A.; Palencsar, M. S.; Burgess, J. D. Anal. Chim. Acta 2004, 519, 47. (50) Chou, L. C. S.; Liu, C. C. Sens. Actuator, B 2005, 110, 204.

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Figure 4. Cyclic voltammogram (CV) of Au, AUT/Au SAM, and ChOx/AUT/Au in 5 mM K3Fe(CN6) in 1 M KCl. Figure 2. Impedance spectra of (i) AUT/Au. (ii) Blank gold. (iii) ChOx/AUT/Au. ethanol solution of 11-amino-1-undecanethiol hydrochloride for 24 h at room temperature. The SAM-modified gold plates were rinsed with ethanol and water several times and dried under a stream of nitrogen. Immobilization of Cholesterol Oxidase (ChOx) on AUT/Au Electrode. The AUT/Au SAM was modified with glutaraldehyde by dipping SAM plates in 0.1% of glutaraldehyde for about 2 h, after which these plates were washed with water a number of times. Cholesterol oxidase (ChOx) was immobilized onto glutaraldehydemodified SAM plates by dispensing cholesterol oxidase (10 L) and keeping these for about 12 h at room temperature. The resulting ChOx/AUT/Au electrodes were washed with phosphate buffer (50 mM, pH 7.0) to remove any unbound enzyme and were stored at 4 C when not in use.

Results and Discussion

Mechanisms of Covalent Immobilization of ChOx onto AUT/Au Surface. For binding of cholesterol oxidase onto 11amino-1-undecanethiol hydrochloride/Au (AUT/Au) having NH2 group at outer surface, the condensation reaction between amino and aldehyde was exploited.52,53 Since glutaraldehyde is a biofunctional compound, the aldehyde group at one end of the glutaraldehyde binds with the NH2 group of the SAM, and the other group binds with the amino group of enzymes. The mechanism of covalent immobilization of ChOx on the NH2 terminal of AUT SAM, using glutaraldehyde as a cross-linker, is given in Scheme 1. Contact Angle Studies. Contact angle technique based on the sessile drop method was used to assess the hydrophobic/ hydrophilic nature of the surface,54 as the quality of a monolayer can be estimated from the wetting measurements; the shape of the liquid drop is affected by the free energy of this surface. The contact angle value obtained was found to vary from 50 for blank gold (Au), 61 for AUT/Au, and 45 for ChOx/AUT/Au (Figure 1). The change in the contact angle value from 50 for blank gold (Au) and 61 for the AUT/Au indicates the formation of SAM with a hydrophilic amino group on the AUT surface. Further change in the contact angle value after ChOx immobilization confirms the binding of ChOx. Electrochemical Impedance Spectroscopy. Impedance spectroscopy (IS) is a versatile technique for investigating the electrical properties of a variety of different materials that may be ionic, semiconducting, or even insulating. It gives information about
(52) Delvaux, M.; Demoustier-Champagne, S. Biosens. Bioelectron. 2003, 18, 943. (53) Hsu, H.-P.; Shih, J.-S. J. Chin. Chem. Soc. 2001, 48, 167. (54) Abdelghani, A.; Chovelon, J. M.; Krafft, J. M.; Renault, N. J.; Trouillet, A.; Veillas, C.; Trioli, C. R.; Gagnaire, H. Thin Solid Films 1996, 157, 284-285.

Figure 3. AFM pictures of (a) AUT/Au and (b) ChOx/AUT/Au bioelectrode. 546 and molecular weight 58 994 Da} with specific activity of 24 units per milligram (U/mg) was procurred from Sigma-Aldrich (U.S.A.). Preparation of Gold Substrate and NH2 Terminal (AUT) SAM. Gold (Au) coated glass plates were prepared using glass substrates (1.3 cm 3.9 cm) by the vacuum evaporation technique.51 SAM of 11-amino-1-undecanethiol hydrochloride/SAM (AUT SAM) was formed by immersion of the precleaned Au substrate into a 1 mM
(51) King, D. E. J. Vac. Sci. Technol., A 1995, 13, 1247.

Cholesterol Biosensor Based on AUT SAM

Langmuir, Vol. 23, No. 13, 2007 7401

Figure 5. Cyclic voltammogram of ChOx/AUT/Au bioelectrode in PBS (50 mM, pH 7.0, 0.9% NaCl) containing 5 mM FeCN63-/4- in the range of -0.5 to 0.8 V for cholesterol concentrations (i) 0 mg/dL, (ii) 50 mg/dL, (iii) 100 mg/dl, (iv) 200 mg/dL, (v) 300 mg/dL, (vi) 400 mg/dL, and (vii) 500 mg/dL; inset shows amperometric response as a function of cholesterol concentration (50-500 mg/dL).

Figure 6. A typical SPR biosensing experiment showing the change in angle vs time. The ChOx/AUT/Au bioelectrode with different stages of SPR angle change: (a) baseline; (b) association phase; (c) dissociation phase; (d,e) regeneration phase in phosphate buffer (50 mM, pH 7.0).

Figure 7. Variation in SPR signal as a function of cholesterol concentration [(i) PBS, (ii) 50 mg/dL, (iii) 100 mg/dL, (iv) 200 mg/dL, (v) 300 mg/dL, (vi) 400 mg/dL, and (vii) 500 mg/dL] in another freshly prepared ChOx/AUT/Au bioelectrode.

the materials bulk phase (e.g., conductivity, dielectric constant) and their inner and outer interfaces55 (e.g., capacitance of the interfacial region and derived quantities). Figure 2 shows the Faradic impedance spectra in the frequency range 0.01-105 Hz, presented by the Nyquist plots of the AUT/Au, blank gold, and ChOx/AUT/Au electrodes, respectively, using Autolab potentiostat/galvanostat (Eco Chemie, The Netherlands) in phosphate buffered saline (50 mM, pH 7.0, 0.9% NaCl) containing 5 mM FeCN63-/4- as the redox probe. The value of Rct, charge-transfer resistance, obtained as 589.5 (curve i) for AUT/Au SAM is smaller than that of the bare gold electrode (Rct 792.0 (curve ii)).56 This result may be attributed to the presence of redox couples present in the solution. Moreover, the AUT/Au surface
(55) Chen, H.; Heng, C. K.; Puiu, P. D.; Zhou, X. D.; Lee, A. C.; Lim, T. M.; Tan, S. N. Anal. Chim. Acta 2005, 554, 52. (56) Ge, C.; Liao, J.; Yu, W.; Gu, N. Biosens. Bioelcctron. 2003, 18, 53.

shows smaller charge-transfer resistance due to the polarization of terminal NH2 groups, resulting in a net positive charge on the surface attracting the negative charge of FeCN63-/4- enhancing charge transfer from solution to electrode surface. Takehara et al.57 have reported similar results for SAMs of different head groups for FeCN63-/4-. Curve iii (Figure 2) is the impedance spectrum obtained after the immobilization of cholesterol oxidase onto AUT/Au SAM, and the value of Rct is obtained as 3235.5 . The increased value of Rct obtained for ChOx/AUT/Au electrode indicates hindrance to the electron transfer, confirming the successful covalent binding of the NH2 group of cholesterol oxidase with the carbonyl group of glutaraldehyde (Scheme 1). Atomic Force Microscopic (AFM) Studies of AUT/Au, ChOx/AUT/Au Bioelectrode. AFM has been used for the characterization of the AUT/Au, ChOx/AUT/Au electrode, and
(57) Takehara, K.; Takemura, H.; Ide, Y. Electrochim. Acta 1994, 39, 817.

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present work 10 amino-undecane-thiol self-assembled monolayer covalently 50-500 mg/dL 50 mg/dl optical 10 weeks 9 drop cast 0.01-0.07 mM 0.01mM amperometry 20 times 1.23 m0/ (mg dL)

ref

[60]

[48]

[22]

[46]

[16]

[49]

[50]

1.04 m0/ (mg dL) amperometric amperometry cyclic voltammetry amperometry 10-1000 M 0.2-3.0 mM 66-700 nM layer by layer vesicle fuion entrapment 0-2 mM covalently template 1-hexadecanethiol 4 5 6 7 PSS/PEI/ChOx thiolipid SAM 3 8 covalently 50-500 mg/dL 50 mg/dL 8 M 1 M optical 10 weeks 25 days 60 days 7 months <15

[61]

Table 1. Characteristics of Various Cholesterol Biosensors

images obtained in air using a NanoScope IIIa (Digital Instruments) in tapping mode with standard silicon cantilevers having a normal spring constant of 28 N m-1 are shown in the Figure 3. The morphology of the AUT/Au shows a smooth surface. However, after immobilization of cholesterol oxidase, the presence of a uniformly distributed structure indicates the immobilization of cholesterol oxidase onto the AUT/Au electrode.58 Cyclic Voltammetric Response Studies. Figure 4 shows cyclic voltammograms obtained for the bare Au, AUT/Au, and ChOx/AUT/Au in 5 mM K3Fe(CN)6 in 1 M KCl. The cyclic voltammogram of the bare gold exhibits oxidation and reduction peaks at 0.3 and at 0.27 V, respectively, indicating a reversible diffusion-controlled profile. In the case of the AUT/Au electrode, the redox potential between 0.35 and 0.22 V points to the reversible diffusion-controlled system.57 The similar magnitude of the current obtained for the bare gold (Au) and with AUT/Au, resulting due to electron transfer between the AUT SAM and Au substrate, reveals the similar nature of the NH2 terminal SAM and Au bare gold. However, the redox potential of the ChOx/ AUT/Au electrode does not show a reversible diffusion profile as compared to the AUT/Au substrate, indicating hindrances of the electron transfer to the AUT/Au surface. The results obtained by impedance spectroscopic studies support the data obtained using cyclic voltammetric measurements. Figure 5 exhibits various cyclic voltammograms obtained for the ChOx/AUT/Au bioelectrode as a function of the cholesterol concentration (50-500 mg/dL) prepared in Triton X100 as a surfactant.39 The current increases with the increase in cholesterol concentration, indicating an increase in the H2O2 concentration produced due to the interaction of cholesterol oxidase with cholesterol. The inset shows the amperometric response as a function of cholesterol concentration, when plotted as a function of change in the oxidation current. Surface Plasmon Resonance Studies. SPR studies were conducted on an Autolab SPR procured from Eco Chemie (The Netherlands). The SPR instrument has a vibrating mirror to modulate the angle of incidence of the p-polarized light beam (670 nm), and intensity of the reflected light is measured over a range of 4000 millidegrees (m). Figure 6 shows the change in SPR angle measured as a function of cholesterol concentration. During SPR experiments, first 100 s were given for the baseline correction with phosphate buffer (50 mM, pH 7.0), whereafter the cholesterol solution containing concentrations varying from
(58) Cabrita, J. F.; Abrantes, L. M.; Viana, A. S. Electrochim. Acta 2005, 50, 2117.

2 months

<15

Figure 8. Variation in the SPR angle change as a function of cholesterol concentration (50-500 mg/dL) (Figure 7).

reusability

optical

linearity

immobilization technique

immobilization matrix

S no.

modified ODT

silicic solgel/ Prussian blue/carbon Pt/SAM/Prussian blue(PB) alkanethiol

modified poly(3-hexythiophene) screen-printed rhodium-graphite

cholesterol oxidase cholesterol oxidase cholesterol oxidase cholestrol oxidase and microperoxidase cholesterol oxidase molecular imprinted layer cholesterol oxidase cholesterol oxidase cytochrome p450scc on gold nanoparticles cholesterol oxidase

sensing element

covalently

entrapment

50-500 mg/dL 1 10-6 to 8 10-5 0.35 mM

50 mg/dL

1.2 10-7

detection limit

amperometric

transducer used

35 days

shelf life

0.329 A M

0.13 A M-1

sensitivity

[62]

Cholesterol Biosensor Based on AUT SAM

Langmuir, Vol. 23, No. 13, 2007 7403

Figure 9. Variation in SPR signal: (i) PBS; (ii) 1% Triton; (iii) palmitate (200 mg/dL); (iv) oleate (200 mg/dL); (v) 10% Triton.

50 to 500 mg/dL was added, allowing for the interaction of the ChOx/AUT/Au bioelectrode with substrate (cholesterol) in the next 300 s (association phase). After completion of the association phase (300 s), the ChOx/AUT/Au bioelectrode was washed to remove any extra cholesterol solution, and the phosphate buffer was introduced into the system (dissociation phase), resulting in similar conditions before and after the association phase. The change in the SPR angle corresponds to the amount of binding, since all other parameters are constant. After the completion of dissociation phase, the AUT/Au surface is fully regenerated by rigorous washing in the regeneration phase (Figure 6). The increase in the SPR angle (Figure 7) with the increase of cholesterol concentration indicates interaction of ChOx with cholesterol, whereas in the control experiment (i.e., with no cholesterol), no increase in the SPR angle is observed (curve i, Figure 7). It may be noted that similar results with error of around 4% are obtained during repeated experiments. For quantification, the shift in the SPR angle change is plotted as a function of cholesterol concentration. The straight line obtained (Figure 8) shows linearity from 50 to 500 mg/dL obtained for the estimation of cholesterol using the ChOx/AUT/Au bioelectrode. The association (Ka) and dissociation (Kd) constants of reaction on the ChOx/AUT/Au bioelectrode with cholesterol concentration (50-500 mg/dL) have been calculated as 4.222 919 (ms) and 1.279 416 10-2 1/s, respectively, using the ESPRIT kinetic evaluation software.59 The sensitivity of the ChOx/AUT/Au bioelectrode has been measured as 1.23 m (mg/dL)-1. Attempts have been made to investigate the effect of interferents on the response of the ChOx/AUT/Au bioelectrode as the metabolites such as urea, uric acid, ascorbic acid, and glucose present in the serum may interfere in the estimation of cholesterol concentration. It has been observed that the addition of a solution containing a 1:1 ratio of cholesterol (200 mg/dL) and the interferents like ascorbic acid (0.05 mM), uric acid (0.2 mM), glucose (5 mM), and urea (2 mM) does not produce any change in the SPR signal (data not shown), showing the specificity and advantage of SPR over other techniques. The effect of cholesterol esters (cholesterol palmitate and cholesterol oleate) and surfactant (Triton X100) has been investigated on the response of the ChOx/AUT/Au bioelectrode
(59) http://www.brinkmann.com/products/electrochem_SPR_sw.asp.

using the SPR technique. It can be seen (Figure 9) that no significant change in SPR angle occurs as a consequence of exposure of the ChOx/AUT/Au bioelectrode to cholesterol esters (cholesterol palmitate and cholesterol oleate). Similar results are obtained upon exposure of the ChOx/AUT/Au bioelectrode to Triton X100 concentration (1% and 10%). The activity of the ChOx/AUT/Au bioelectrode stored at 4 C has been measured at different time intervals (0, 7, 15, 30, 45, 60, and 75 days) using UV-visible spectrophtometry. It has been observed that the activity of cholesterol oxidase immobilized onto the AUT/Au surface decreases to 75-70% after 45 days and to 50% after 75 days compared to the original activity measured on 0 day (100%). Table 1 shows the characteristics of the cholesterol biosensor based on cholesterol oxidase covalently immobilized onto the modified 11-amino-undecanethiol SAM on gold plates along with those values reported in literature.

Conclusions
It has been shown that cholesterol oxidase can be covalently immobilized onto AUT/Au SAM. The ChOx/AUT/Au bioelectrode can be utilized to estimate cholesterol (50-500 mg/dL) using SPR and electrochemical techniques. The presence of interferents such as glucose, urea, and uric acid does not appear to affect the observed SPR response of the ChOx/AUT/Au bioelectrode. Attempts should be made to immobilize cholesterol oxidase, cholesterol esterase, and peroxidase onto the AUT/Au electrode surface for estimation of LDL and HDL cholesterol in serum samples. Acknowledgment. We are grateful to Dr. Vikram Kumar, Director, National Physical Laboratory, New Delhi, India for providing facilities. P.R.S. thanks the Council of Scientific & Industrial Research, India, for the award of the Senior Research Associateship.
LA700350X
(60) Ram, M. K.; Bertoncello, P.; Ding, H.; Paddeu, S.; Nicolini, C. Biosens. Bioelectron. 2001, 16, 849. (61) Arya, S. K.; Solanki, P. R.; Singh, S. P.; Kaneto, K.; Pandey, M. K.; Datta, M.; Malhotra, B. D. Biosens. Bioelectron. 2007, 22, 2516. (62) Shumyantseva, V. V.; Carrara, S.; Bavastrello, V.: Riley, D. J.; Bulko, T. V.; Skryabin, K. G.; Archakov, A. I.; Nicolini, C. Biosens. Bioelectron. 2005, 21, 217.