Langmuir–Blodgett films of poly(3-dodecyl thiophene) for application to glucose biosensor

Sensors and Actuators B 86 (2002) 42–48

Langmuir–Blodgett films of poly(3-dodecyl thiophene) for application to glucose biosensor Rahul Singhala, W. Takashimac, K. Kanetoc, S.B. Samantad, S. Annapoornib, B.D. Malhotraa,* a

Biomolecular Electronics and Conducting Polymer Research Group, National Physical Laboratory, Dr. K.S. Krishnan Road, New Delhi 110012, India b Department of Physics and Astrophysics, University of Delhi, Delhi 110007, India c Department of Biological Functions and Engineering, Graduate School of Life Sciences and System Engineering, Kyushu Institute of Technology, Iizuka, Fukuoka 820-8502, Japan d Superconductivity Division, National Physical Laboratory, Dr. K.S. Krishnan Road, New Delhi 110012, India Received 28 August 2001; received in revised form 13 March 2002; accepted 22 March 2002

Abstract Monolayers of poly(3-dodecyl thiophene) (P3DT) have been obtained on indium–tin-oxide (ITO) coated glass plates by dispensing mixed solution of P3DT and stearic acid (SA) prepared in chloroform onto water subphase by a microsyringe. The pressure–area isotherms of these P3DT–SA monolayers were studied as a function of temperature and pH. The monolayer stability onto the water subphase has been experimentally studied at different temperatures, pH and surface pressure. These P3DT–SA monolayers fabricated onto the ITO-coated glass plates were characterized using FTIR and cyclic voltammetry studies. The desired enzyme monolayers were fabricated by dispensing glucose oxidase mixed with P3DT/SA in chloroform and were transferred onto desired ITO-coated glass. An attempt has been made to utilize these P3DT/SA/GOX LB films for fabrication of a glucose biosensor. Published by Elsevier Science B.V. Keywords: Poly(3-dodecyl thiophene); Glucose biosensor; Langmuir–Blodgett film; Conducting polymer; Shelf-life

1. Introduction Conducting organic polymers, in recent years have generated wide-spread interest as potential materials for a variety of applications in different areas including EMI shielding, anti-static and electronic junction devices, electro-chromic displays, opto-electronic systems, semiconductor protection, information storage, capacitors, gas sensors and biosensors. Agbor et al. [1] have reported the effect of various gases (NO2, H2S, CO, SO2, N2 and CH4) on surface plasmon resonance of Langmuir–Blodgett (LB) films of polyaniline. Stella et al. [2] have characterized olive oil using an electronic nose based on polypyrrole. Li et al. [3] fabricated polyaniline composite ultrathin films with isopolymolybdic acid and demonstrated that the conductivity of the films is sensitive to humidity, NO2 and NH3. Besides this, the use of conducting polymer films for the development of biosensor has attracted significant attention. Umana and Waller [4] have electrochemically polymerized pyrrole * Corresponding author. Tel.: þ91-11-5734273; fax: þ91-11-5852678. E-mail address: [email protected] (B.D. Malhotra).

0925-4005/02/$ – see front matter. Published by Elsevier Science B.V. PII: S 0 9 2 5 - 4 0 0 5 ( 0 2 ) 0 0 1 4 5 - 4

in the presence of enzyme glucose oxidase (GOX) and showed that these enzyme electrodes can be used as glucose biosensors. Chaubey et al. [5] recently demonstrated that LDH immobilized PPY–PVS films can be used to estimate lactate concentration from 12 to 24 mM. A biosensor is an analytical device incorporating a biological or biologically derived material, either intimately associated or integrated within a physico-chemical transducer. The aim is to produce an electronic response that is proportional to the concentration of analyte. Specificity of the desired molecule can be achieved by immobilizing the appropriate enzyme into the polymer matrix. However, the usefulness of immobilized enzyme electrodes depends on factors such as the immobilization method, the chemical and physical conditions (pH, temperature and contaminants), the thickness and stability of the membrane used to couple the enzyme. Immobilization of enzyme in several matrices has been used for the fabrication of biosensors for estimation of glucose [6,7], urea [8,9], cholesterol [10], etc. A number of papers relating to development of conducting polymer sensors (chemical and electrochemical) self-assembled monolayer assembly, LB deposition, etc. have recently appeared

R. Singhal et al. / Sensors and Actuators B 86 (2002) 42–48

in literature [11–15]. Among these methods LB deposition has currently drawn much attention [15–17]. The LB film deposition technique is known to be capable of preparing highly ordered monolayer films with a densely packed structure and precisely controlled thickness. The immobilization of monolayers, or submonolayers, of enzymes on electrode surfaces forms the basis of molecular level fabrication of enzyme biosensors. Monolayer enzyme electrodes have been fabricated via covalent or electrostatic binding of the recognition molecule onto electrodes modified with LB films [18,19]. Owaku et al. [20] fabricated protein A molecular membrane on a quartz surface by LB method. Protein A has specific efficiency to the fc part of anti-human IgG antibody. The antibodies were selfassembled onto a protein A LB film. These films can be used for optical immunosensing. Chen et al. [21] found that chemically synthesized poly (o-anisidine) is able to form stable monolayer on the water surface. These monolayers transferred onto quartz crystal microbalance (QCM) can be used as organic vapor sensor. The advantage of using LB films lies in that this technique provides well-defined surface for protein immobilization [22]. The prevalence of diabetes in industrialized countries amounts to approximately 4%. Therefore the selective determination of blood glucose is of utmost importance for screening and treatment of diabetes. The normal concentration of glucose in blood serum ranges between 80 and 140 mg/dl. Among organic polymers, that have been used as the sensing materials, poly(3-dodecyl thiophene) (P3DT) is rated as one of the most technologically promising electrically conducting polymer due to its ease of synthesis, low cost, versatile processability and relatively stable electrical conductivity. Present paper deals with the systematic studies on the immobilization and characterization of GOX onto P3DT LB films for application to glucose biosensor. The response of these P3DT/SA/GOX LB films was measured as a function of glucose concentration using amperometric technique.

43

2.2. Equipments Monolayers were formed onto water subphase of the Langmuir–Trough (Joyce-Loebl, Model 4). The pH of the subphase was determined with a pH meter (Kent EIL 7045/ 46). FTIR spectra were obtained using Fourier transform infra-red spectrometer (Nicolet 510P). Cyclic voltammetry was carried out using an electrochemical interface (Schlumberger Model SI 1286) and the output was recorded directly on a Hewlett-Packard (Model HP 7440A) colorpro plotter. Amperometric measurements were conducted with a Keithley Electrometer (Model 617). 2.3. Preparation of LB film The monolayers of P3DT/SA were fabricated by dispensing a solution (1:1) of P3DT (1 mM) and SA (2 mM) in chloroform onto water subphase containing CdCl2 (2  104 M), using a Joyce-Loebl LB trough (Model 4). The pressure–area isotherms of the P3DT/SA monolayers were obtained at the barrier compression rate of 4 mm/min at different temperatures and pH. The monolayer stability onto the water subphase was measured as a function of surface pressure, temperature and subphase pH, respectively. Such P3DT/SA monolayers were transferred onto the ITO-coated glass plates at a surface pressure 30 mN/m and 30 8C by vertical dipping method. The dipping speed during upstroke and downstroke was maintained at 5 mm/min. 2.4. Immobilization of GOX The 5 mg (200 units/mg) of enzyme GOX mixed in a solution of P3DT/SA in chloroform was spread onto air– water interface of the LB trough. These P3DT/SA/GOX monolayers were later transferred onto desired indium–tinoxide (ITO) coated glass plates. 2.5. Activity of GOX/P3DT/SA films

2. Experimental 2.1. Materials All reagents: cadmium chloride, stearic acid (SA), chloroform, b-D-glucose (all from Merck), GOX type X-S (EC1.1.3.4 from Aspergillus niger, Sigma), horse-radish peroxidase (HRP) (Sisco Research Laboratories); K2HPO4, KH2PO42H2O (Merck) and ITO-coated glass plates (Balzers) were used as-received without further purification. Regioregular P3DT prepared by Rieke method [23] were a gift from Prof. M. Rikukawa of the Sophia University, Tokyo, Japan. Number average molecular weight (Mn) and weight average molecular weight (Mw) of P3DT was found to be 29,000 and 41,000, respectively. The aqueous solution was prepared in doubly distilled-deionized water (Millipore RO 10TS-water purification system).

In order to determine the presence of GOX in LB film the activity measurements of the P3DT/SA/GOX LB films were carried out using o-dianisidine method. The 2.5 ml of o-dianisidine in the presence of peroxidase (0.1 ml) and glucose (0.5 ml) results in brown colored dye, which absorbs at 540 nm. The activity of the immobilized GOX was experimentally determined by amperometric method. Different concentrations of glucose were prepared for the estimation of glucose concentration. The b-D-glucose was allowed to mutarotate for about 24 h at 30 8C before use. 3. Results and discussion 3.1. Pressure–area isotherm of P3DT/SA LB films Fig. 1 shows pressure–area isotherm of P3DT/SA mixed monolayer spread onto the water subphase containing

44

R. Singhal et al. / Sensors and Actuators B 86 (2002) 42–48

3.2. FTIR studies

Fig. 1. Pressure–area isotherm of mixed monolayer of P3DT and SA at: (a) 10 8C; (b) 15 8C; (c) 20 8C; (d) 25 8C; (e) 30 8C; (f) 35 8C; (g) 40 8C and pH 7.0.

2  104 M CdCl2 at different subphase temperatures. It can be seen that monolayer of P3DT/SA remains stable onto air– water interface during the temperature range 5–40 8C. The nature of each curve is almost the same and the best isotherm was, however, obtained at 30 8C and pH 7. It was found (Fig. 2) that gas–liquid transition occurs at a surface pressure ˚ 2. The liquid–solid of 4.12 mN/m and area/molecule 36.6 A phase transition can be seen at a surface pressure of 30 mN/m ˚ 2 (area/molecule). The compressibility for gas– and 33 A liquid phase transition varies from 0.006 to 0.003 and for the liquid–solid phase transition it changes from 0.01 to 0.008. It has been found that monolayers remain stable onto water subphase at a temperature of 30 8C, pH 7 and a surface pressure of 30 mN/m. These monolayers were transferred onto ITO-coated glass plates at a surface pressure of 30 mN/ m and 30 8C, respectively. These monolayers obtained at dipping head speed of 3 mm/min were utilized for the fabrication of the desired glucose biosensing device.

FTIR spectra of P3DT/SA films with and without enzyme (GOX) are shown in Fig. 3(a) and (b). Curve a is the FTIR spectra obtained for P3DT/SA LB films. The peaks at 1350 and 1450 cm1 have been attributed to symmetric and asymmetric vibrations of COO group and the peak for symmetric and asymmetric vibrations of CH2 group arise at 2820 and 2918 cm1, respectively. Curve b is the FTIR spectra obtained for P3DT/SA/GOX LB film. The peak at 1080 and 1650 cm1 can be attributed to C–O stretching vibration and C=O stretching vibration, respectively. A strong band observed at about 3300 cm1 and a less stronger band at about 3100 cm1 have been assigned to amide A and amide B bands, respectively. The amide A band arises due to NH stretching vibration, whereas the amide B band arises due to the first overtone of the amide II vibration that becomes intensified by Fermi resonance with amide A vibration. 3.3. Cyclic voltammetric studies In order to study the electroactivity of P3DT/SA LB films, cyclic voltammetry experiments were conducted at a scan rate of 50 mV/s. Tetrabutylammonium perchlorate in acetonitrile (0.1 M) was used as electrolyte. LB film of P3DT/SA on ITO-coated glass plate, Pt, standard calomel electrode (SCE) were utilized as working electrode, counter electrode and reference electrode, respectively. Typical cyclic voltammogram (Fig. 4) obtained shows a broad oxidation peak in region 0.8–1.1 V for P3DT/SA, in agreement with the results reported in literature [24]. It is clear that these P3DT/SA LB films remain electroactive and can be used for the immobilization of enzyme (GOX). 3.4. Activity measurements and response time The results of activity measurements are given in Fig. 5. GOX immobilized P3DT/SA LB films yields brown color dye with o-dianisidine in the presence of peroxidase and glucose. All measurements were taken at room temperature and at glucose concentration of 100 mg/dl. It can be seen (Fig. 5) that absorbance at 540 nm increases up to 120 s, after which it becomes stable. The response time of the GOX/PNVK/SA LB electrode was found to be about 2 min. The observed longer response time has been attributed to the slower diffusion of glucose molecules to the active sites of GOX entrapped in P3DT/SA LB films for desired biochemical reaction. Such a P3DT/SA/GOX electrode could be used six times for glucose estimation. 3.5. Amperometric response measurements

Fig. 2. Pressure–area isotherm of mixed monolayer of P3DT and SA at a subphase temperature of 30 8C and pH 7.0.

The results of amperometric response determined for P3DT/SA/GOX LB films are shown in Fig. 6. All measurements were performed at room temperature using P3DT/SA LB film immobilized with enzyme (GOX) as working

R. Singhal et al. / Sensors and Actuators B 86 (2002) 42–48

45

Fig. 3. FTIR spectra of P3DT/SA films: (a) in the absence of GOX; (b) in the presence of GOX immobilized onto the films.

electrode polarized at 0.4 V and Pt as counter electrode. The working solution comprised of phosphate buffer (pH 7.0) and glucose. The overall reaction involves the catalytic oxidation of glucose by GOX at the P3DT/SA/GOX LB electrode GOX

b-glucose þ O2 ! b-gluconic acid þ H2 O2

(1)

followed by the amperometric determination of H2O2 by electrochemical oxidation at 0.4 V vs. SCE 2H2 O2 ! O2 þ 2Hþ þ 2e

(2)

P3DT/SA/GOX LB electrode shows linearity from 100 to 400 mg/dl after which a limiting value of current was obtained.

46

R. Singhal et al. / Sensors and Actuators B 86 (2002) 42–48

Fig. 4. Cyclic voltammogram of P3DT/SA LB film in the solution of TBAP in acetonitrile (0.1 M) and at a scan rate 50 mV/s.

Fig. 7. Effect of temperature on the response current for P3DT/SA/GOX electrodes in the presence of glucose (100 mg/dl) in phosphate buffer (pH 7).

3.6. Effect of temperature The thermal stability of GOX immobilized P3DT/SA LB films was investigated using amperometric measurements. Fig. 7 shows the results of enzyme (GOX) activity measurements obtained as a function of temperature by holding the P3DT/SA/GOX film in a solution of phosphate buffer (pH 7) and glucose solution (100 mg/dl). It can be seen that GOX activity increases up to about 40 8C where after it decreases drastically.

Fig. 5. Activity measurement of GOX immobilized P3DT/SA LB films in the presence of o-dianisidine at 540 nm and at a glucose concentration of 100 mg/dl.

Fig. 6. Amperometric response of P3DT/SA/GOX LB film in phosphate buffer (pH 7.0) at 0.4 V (bias voltage).

Fig. 8. Response of P3DT/SA/GOX electrodes as a function of storage time in the presence of glucose (100 mg/dl) in phosphate buffer (pH 7).

R. Singhal et al. / Sensors and Actuators B 86 (2002) 42–48

47

Table 1 Characteristics of some conducting polymer based glucose biosensors Matrix

Method of enzyme immobilization

Response limit (mM)

Stability (day)

Effect of interferents

Response time

References

ISFET Polypyrrole Polypyrrole Polypyrrole Polyaniline N-substituted polypyrrole

Cross-linking via GA Physical adsorption Electropolymerization Covalent bonding Physical adsorption Cross-linking

12 10 10 18 1 5

– 10 7 – – 40

– 1 min – – – –

[25] [29] [4] [13] [30] [26]

ISFET Modified Pt electrode Polyaniline P3DT

Physical adsorption Physical adsorption LB technique LB technique

5 40 30 25

– – – 40

– – – – – Uric acid: 3.4%, ascorbic acid: 26.5% – – – Uric acid: 3.8%, ascorbic acid: 4.5%

– 20 s – 2 min

[27] [28] [17] Present work

3.7. Shelf-life

Acknowledgements

P3DT/SA/GOX LB films were tested for stability under similar operating conditions as those for the response measurements. Fig. 8 shows the amperometric response of the P3DT/SA/GOX LB films to 100 mg/dl glucose solution in phosphate buffer (pH 7). It can be seen that the response of these electrodes remains almost same for about 10 days after which there is a gradual decrease in its value up to about 40 days after which it becomes stable. The half-life of these P3DT/SA/GOX LB films has been determined as about 25 days.

We are grateful to Dr. Krishan Lal, Director, NPL, for providing the facilities to carry out the experiments. RS is grateful to CSIR for the award of Senior Research Fellowship. Financial support received under the DST sponsored project (SP/S2/M-52/96), Indo-Polish (INT/POL/ P.015/2000) and Indo-Japan project (INT/IJJC/I-17/97) is gratefully acknowledged.

3.8. Effect of interferents The effect of interferents in the presence of their physiological maximum levels with glucose was studied under the same conditions as those for response measurements. The response current for 5.5 mM glucose in the presence of ascorbic acid (0.1 mM) or uric acid (0.5 mM) were within the range of relative error of 5% from the actual value without addition. Table 1 gives the results of the experiments using P3DT/ SA/GOX LB films along with those reported in literature.

4. Conclusions It has been shown that GOX can be immobilized in P3DT/ SA LB films. These GOX/P3DT/SA LB films found to be stable up to 40 8C. However, the shelf-life of these electrodes is about 40 days at 4 8C. Compared to the GOX physisorbed P3DT/SA LB films, which are known to have slower response time, GOX entrapped P3DT/SA LB films have negligible leaching. These GOX immobilized P3DT/ SA films can be used for the estimation of glucose from 100 to 400 mg/dl. The experiments are currently in progress to improve the response time and shelf-life of GOX/P3DT/SA LB films.

References [1] N.E. Agbor, J.P. Cresswell, M.C. Petty, A.P. Monkman, An optical gas sensor based on polyaniline Langmuir–Blodgett films, Sensors and Actuators B 41 (1997) 137–141. [2] R. Stella, J.N. Barisci, G. Serra, G.G. Wallace, D. De Rossi, Characterization of olive oil by an electronic nose based on conducting polymer sensor, Sensors and Actuators B 63 (2000) 1–9. [3] D. Li, Y. Jiang, Z. Wu, X. Chen, Y. Li, Self-assembly of polyaniline ultrathin films based on doping-induced deposition effect and applications for chemical sensors, Sensors and Actuators B 66 (2000) 125–127. [4] M. Umana, J. Waller, Protein modified electrodes. The glucose oxidase/polypyrrole system, Anal. Chem. 58 (1986) 2979–2983. [5] A. Chaubey, K.K. Pande, V.S. Singh, B.D. Malhotra, Immobilization of lactate dehydrogenase on electrochemically prepared polypyrrole– polyvinylsulphonate composite films for application to lactate biosensors, Anal. Chim. Acta 407 (2000) 97–103. [6] U. Narang, P.N. Prasad, F.V. Bright, K. Ramanathan, N.D. Kumar, B.D. Malhotra, M.N. Kamalasanan, S. Chandra, Glucose biosensor based on sol–gel derived platform, Anal. Chem. 66 (1994) 3139– 3144. [7] Y. Mishima, J. Motonaka, I. Maruyama, I. Nakabayashi, S. Ikeda, Glucose sensor based on titanium dioxide electrode modified with potassium hexacyanoferrate(III), Sensors and Actuators B 65 (2000) 343–345. [8] P.C. Pandey, A.P. Mitra, Conducting polymer-coated enzyme microsensor for urea, Analyst 113 (1988) 329–331. [9] W.O. Ho, S. Krause, C.J. McNeil, J.A. Pritchard, R.D. Armstrong, D. Athey, K. Rawson, Electrochemical sensor for measurement of urea and creatinine in serum based on ac impedance measurement of enzyme catalyzed polymer transformation, Anal. Chem. 71 (1999) 1940–1946.

48

R. Singhal et al. / Sensors and Actuators B 86 (2002) 42–48

[10] A. Kumar, Rajesh, B.D. Malhotra, S.K. Grover, Co-immobilization of cholesterol oxidase and horse radish peroxidase in a sol–gel film, Anal. Chim. Acta 414 (2000) 43–50. [11] G. Bidan, New sensitive matrices to build up chemical or electrochemical sensors, Sensors and Actuators B 6 (1992) 45–56. [12] A.V. Barmin, A.V. Eremenko, I.N. Kurochkin, A.A. Sokolovsky, Cyclic voltammetry of ferrocenecarboxylic acid monomolecular films and their reaction with glucose-oxidase, Electroanalysis 6 (1994) 107. [13] W. Schuhmann, C. Kranz, J. Huber, H. Wohlschla¨ ger, Conducting polymer based amperometric enzyme electrodes. Towards the development of miniaturized reagentless biosensor, Synth. Met. 61 (1993) 31–35. [14] J.J. Gooding, D.B. Hibbert, The application of alkanethiol selfassembled monolayers of enzyme electrodes, TrAC 18 (1999) 525–533. [15] H. Tsuji, K. Mitsubayashi, An amperometric glucose sensor with modified Langmuir–Blodgett films, Electroanalysis 9 (1997) 161– 164. [16] A. Ulman, An Introduction to Ultra-thin Organic Films from Langmuir–Blodgett to Self-assembly, Academic Press, London, 1991. [17] K. Ramanathan, M.K. Ram, B.D. Malhotra, A.S.N. Murthy, Application of polyaniline Langmuir–Blodgett films as a glucose biosensor, Mater. Sci. Eng. C 3 (1995) 159–163. [18] M. Sriyudthsak, H. Yamagishi, T. Moriizumi, Enzyme immobilized Langmuir–Blodgett films for biosensor, Thin Solid Films 160 (1988) 463–469. [19] D.G. Zhu, M.C. Petty, H. Ancelin, J. Yarwood, On the formation of Langmuir–Blodgett films containing enzymes, Thin Solid Films 176 (1989) 151–156. [20] K. Owaku, M. Goto, Y. Iikariyama, M. Aizawa, Protein A Langmuir–Blodgett film for antibody immobilization and its use in optical immunosensing, Anal. Chem. 67 (1995) 1613–1616. [21] Z.-K. Chen, S.-C. Ng, S.F.Y. Li, L. Zhang, L. Yu, H.S.O. Chan, The fabrication and evaluation of a vapour sensor based on quartz crystal

[22]

[23]

[24] [25]

[26]

[27]

[28]

[29]

[30]

microbalance coated with poly(o-anisidine) Langmuir–Blodgett layers, Synth. Met. 87 (1997) 201–204. A.P. Girard-Egort, R.M. Morelis, P.R. Coulet, Bioactive nanostructure with glutamate dehydrogenase associated with LB films: protecting role of the enzyme molecules on the structural lipidic organization, Thin Solid Films 292 (1997) 282–289. T.-A. Chen, X. Wu, R.D. Rieke, Regiocontrolled synthesis of poly(3-alkylthiophenes) mediated by Rieke zinc: their characterization and solid state properties, J. Am. Chem. Soc. 117 (1995) 233–244. R.D. McCullough, P. Ewbank, Handbook of Conducting Polymer 1998, Marcel Dekker, New York, pp. 225–258 (Chapter 9). C.-H. Lee, H.-I. Seo, Y.-C. Lee, B.-W. Cho, H. Jeong, B.-K. Sohn, All solid type ISFET glucose sensor with fast response and high sensitivity characteristics, Sensors and Actuators B 64 (2000) 37–41. M. Yasuzawa, T. Nieda, T. Hirano, A. Kunugi, Properties of glucose sensor based on the immobilization of glucose oxidase in N-substituted polypyrrole film, Sensors and Actuators B 66 (2000) 77–79. H.-I. Seo, C.-S. Kim, B.-K. Sohn, T. Yeow, M.-T. Son, M. Haskard, ISFET glucose sensor based on a new principle using the electrolysis of hydrogen peroxide, Sensors and Actuators B 40 (1997) 1–5. E. Mann-Buxbaum, F. Pittner, T. Schalkhammer, A. Jachimowicz, G. Jobst, F. Olcaytug, G. Urban, New microminiaturized glucose sensor using covalent immobilization techniques, Sensors and Actuators B 1 (1990) 518–522. E. Tamiya, I. Karube, S. Hattori, M. Suzuki, K. Yokoyama, Micro-glucose sensor using electron mediators immobilized an a polypyrrole modified electrode, Sensors and Actuators B 18 (1989) 297–307. V. Lucachova, A. Karyakin, E.E. Karyakina, L. Gorton, The improvement of polyaniline glucose biosensor stability using enzyme immobilization from water–organic mixtures with a high content of organic solvent, Sensors and Actuators B 44 (1997) 356–360.