6
Recent trends in biosensors Bansi D. Malhotra a, * , Rahul Singhal a , Asha Chaubey b , Sandeep K. Sharma c , Ashok Kumar c a Biomolecular Electronics & Conducting Polymer Research Group, National Physical Laboratory, Dr. K.S. Krishnan Marg, New Delhi 110 012, India b Regional Research Laboratory, Jammu, India c Institute of Genomics and Integrative Biology, Mall Road, Delhi 110 007, India Available online 17 July 2004 Abstract Rapid advances in biosensors have recently been reported. This has been possible due to rapid growth in the development of new biomaterials such as conducting polymers, copolymers and sol gels etc and the reported improvements in sensing techniques. Bio- sensors are miniaturized devices employing biochemical molecular recognition as the basis for a selective analysis. The response gen- erated as a result of biochemical reaction is detected by a transducer to give a signal (optical/electrical/thermal) that can be used with or without amplification for the estimation of the concentration of an analyte in a given test sample. Among the various biosensors, electrochemical sensors, especially amperometric biosensors presently hold a leading position. Due to specificity, portability, simplicity, high sensitivity, potential ability for real-time and on-site analysis coupled with the speed and low cost, biosensors have been projected to have applications in food analysis, environment control, clinical detection, drug and agriculture industries etc. Besides this, biosensors offer exciting opportunities for numerous decentralized clinical applica- tions, ranging from emergency room screening, home self testing and alternative site testing, continuous and real-time in vivo mon- itoring. New generation of biosensors combining new bioreceptors with the ever-growing number of transducers is emerging. The present paper highlights some of the recent advances in the area of biosensors contributed by our laboratory. Ó 2004 Elsevier B.V. All rights reserved. PACS: 68.18; 68.47.p; 72.80; 87.80 Keywords: Langmuir–Blodgett films; Electrical conductivity; Biological techniques; Biomedical engineering 1. Introduction In the recent past, there has been a tremendous de- mand of modern techniques that have great potential for industrial applications for a variety of analytes. In this context, biosensors have the potential to overcome most of the disadvantages of the conventional methods. Though literature on the biosensor technology is well documented [1–31], there is still a lack of proper utiliza- tion of the knowledge of biosensor technology for com- mercial applications. Biosensors are in general small devices based on direct spatial coupling between a bio- logically active compound and a signal transducer equipped with an electronic amplifier. Biosensors are analytical devices incorporating bio- logical materials such as enzymes, tissues, micro-organ- isms, antibodies, cell receptors or biologically derived materials or a biomimic component in intimate con- tact with a physico-chemical transducer or transducing microsystems. Transducers are the components that convert a biochemical signal into a quantifiable electri- cal signal. The transducing microsystem may be elec- trochemical, thermometric, optical, piezoelectric or magnetic. Biosensors have found immense applications in medical diagnostics, environmental monitoring and genetics, food processing industries and defense. Due 1567-1739/$ - see front matter Ó 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.cap.2004.06.021 * Corresponding author. E-mail address: [email protected] (B.D. Malhotra). www.elsevier.com/locate/cap Current Applied Physics 5 (2005) 92–97

Recent trends in biosensors

Embed Size (px)

Citation preview

Page 1: Recent trends in biosensors

www.elsevier.com/locate/cap

Current Applied Physics 5 (2005) 92–97

Recent trends in biosensors

Bansi D. Malhotra a,*, Rahul Singhal a, Asha Chaubey b,Sandeep K. Sharma c, Ashok Kumar c

a Biomolecular Electronics & Conducting Polymer Research Group, National Physical Laboratory, Dr. K.S. Krishnan Marg, New Delhi 110 012, Indiab Regional Research Laboratory, Jammu, India

c Institute of Genomics and Integrative Biology, Mall Road, Delhi 110 007, India

Available online 17 July 2004

Abstract

Rapid advances in biosensors have recently been reported. This has been possible due to rapid growth in the development of new

biomaterials such as conducting polymers, copolymers and sol gels etc and the reported improvements in sensing techniques. Bio-

sensors are miniaturized devices employing biochemical molecular recognition as the basis for a selective analysis. The response gen-

erated as a result of biochemical reaction is detected by a transducer to give a signal (optical/electrical/thermal) that can be used with

or without amplification for the estimation of the concentration of an analyte in a given test sample. Among the various biosensors,

electrochemical sensors, especially amperometric biosensors presently hold a leading position.

Due to specificity, portability, simplicity, high sensitivity, potential ability for real-time and on-site analysis coupled with the

speed and low cost, biosensors have been projected to have applications in food analysis, environment control, clinical detection,

drug and agriculture industries etc. Besides this, biosensors offer exciting opportunities for numerous decentralized clinical applica-

tions, ranging from emergency room screening, home self testing and alternative site testing, continuous and real-time in vivo mon-

itoring. New generation of biosensors combining new bioreceptors with the ever-growing number of transducers is emerging. The

present paper highlights some of the recent advances in the area of biosensors contributed by our laboratory.

� 2004 Elsevier B.V. All rights reserved.

PACS: 68.18; 68.47.p; 72.80; 87.80Keywords: Langmuir–Blodgett films; Electrical conductivity; Biological techniques; Biomedical engineering

1. Introduction

In the recent past, there has been a tremendous de-

mand of modern techniques that have great potential

for industrial applications for a variety of analytes. Inthis context, biosensors have the potential to overcome

most of the disadvantages of the conventional methods.

Though literature on the biosensor technology is well

documented [1–31], there is still a lack of proper utiliza-

tion of the knowledge of biosensor technology for com-

mercial applications. Biosensors are in general small

1567-1739/$ - see front matter � 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.cap.2004.06.021

* Corresponding author.

E-mail address: [email protected] (B.D. Malhotra).

devices based on direct spatial coupling between a bio-

logically active compound and a signal transducer

equipped with an electronic amplifier.

Biosensors are analytical devices incorporating bio-

logical materials such as enzymes, tissues, micro-organ-isms, antibodies, cell receptors or biologically derived

materials or a biomimic component in intimate con-

tact with a physico-chemical transducer or transducing

microsystems. Transducers are the components that

convert a biochemical signal into a quantifiable electri-

cal signal. The transducing microsystem may be elec-

trochemical, thermometric, optical, piezoelectric or

magnetic. Biosensors have found immense applicationsin medical diagnostics, environmental monitoring and

genetics, food processing industries and defense. Due

Page 2: Recent trends in biosensors

B.D. Malhotra et al. / Current Applied Physics 5 (2005) 92–97 93

to their simplicity, high sensitivity and potential ability

for real-time and onsite analysis, biosensors have been

widely applied in various fields including industrial proc-

ess, clinical detection, environmental control etc. [4–7].

The most important part of biosensor is the immobi-

lization of a desired enzyme. However, the usefulness ofimmobilized enzyme electrodes depends on factors such

as the immobilization method, the chemical and physi-

cal conditions (pH, temperature and contaminants),

thickness and stability of the membrane used to couple

the enzyme. Immobilization of enzyme in several matri-

ces has been used for the fabrication of biosensors for

estimation of glucose [8,9], urea [10,11], cholesterol

[12] etc. A number of papers relating to developmentof conducting polymer sensors (chemical and electro-

chemical), self-assembled monolayer assembly, Lang-

muir–Blodgett (LB) film deposition etc. have recently

appeared in literature [13,14].

Among the conducting polymers, poly-alkyl-thiophe-

nes (PATs) have rapidly become the subject of consider-

able interest. From theoretical point of view, PATs have

often been considered as a model for the study of chargetransport in conducting polymers with non-degenerate

ground states. The high environmental stability of both

its doped and undoped states together with its structural

versatility has led to various applications such as elec-

trode materials, organic semiconductors etc. Besides

this, polyaniline can also be used for the sensor applica-

tion because it exhibits two redox couples in the conven-

ient potential range to facilitate an enzyme-polymercharge transfer. Remarkable stability and solubility of

polyaniline in various solvents makes it an attractive

candidate for the technical development of a biosensor.

The present paper relates to some of the recent develop-

ments on application of Langmuir–Blodgett films to

biosensors carried out at our laboratories.

2. Langmuir–Blodgett films

Langmuir—Blodgett (LB) films are formed by first

dispensing a small quantity of an amphiphilic material

dissolved in a volatile organic solvent onto the surface

of purified water (sub-phase). As the solvent evaporates,

a monolayer is formed as dictated by the amphiphilic

nature of the molecules; the head group is immersedon the water surface and the tail groups remain outside.

The molecules in their closest packed arrangement

(solid phase) are removed from the surface of water by

suitably dipping and raising a desired substrate through

air/water interface.

Three deposition types viz X, Y, Z deposition are pos-

sible depending on the nature of substrate. If the sub-

strate is hydrophilic, the first monolayer is transferredas the substrate is raised through the sub-phase and

these molecules stack in a head-to-head and tail-to-tail

configuration. This deposition mode is referred to as

Y-type deposition. This results in an odd number of

monolayers being transferred onto the solid substrate.

However, if the solid substrate is hydrophobic, a mono-

layer will be deposited as it is first lowered into the sub-

phase, thus a Y-type film containing an even number ofmonolayers can be fabricated. If a monolayer is depos-

ited on the substrate when the solid substrate enters

the sub-phase this deposition is called X-type deposition.

On the other hand, if a monolayer is deposited on the

substrate when it is withdrawn from the sub-phase, it

is called Z-type deposition.

3. Biosensors based on Langmuir–Blodgett films

There are several methods for immobilization of en-

zymes. Some of these suffer from drawbacks. For exam-

ple, the physical adsorption method is prone to leaching

and shows instability whereas the covalent linking re-

sults in reduced activity of the biomolecule. Most of

these methods do not provide the control on the amountof enzyme to be immobilized. Langmuir–Blodgett tech-

nique can be used to obtain highly ordered and desired

orientation of the enzyme molecules, leading to faster

response [15,30]. LB technique is known to be an impor-

tant method for the immobilization of desired biomole-

cule. With a view to develop a desired biosensor, we

have used Langmui–Blodgett films of poly-3-hexylthio-

phene for the immobilization of developed galactoseoxidase (GaO), lactase (b-galactosidase, b-Gal) and glu-

cose oxidase (GOX), respectively.

3.1. Galactose biosensor

An enzymatic amperometric biosensor has been

developed for the estimation of galactose in milk and

blood serum. Galactose oxidase was immobilized withpoly(3-hexyl thiophene)/stearic acid (P3HT/SA) onto in-

dium tin-oxide (ITO) coated glass plates using Lang-

muir–Blodgett film deposition technique. The effect of

galactose concentration, pH, and stability of the immo-

bilized galactose oxidase in LB films were studied.

Fig. 1 shows the amperometric response of P3HT/SA/

GaO LB film at 25 �C with varying concentration of

galactose in phosphate buffer (pH 7.0). P3HT/SA/GaOLB electrode shows linearity for 1–4 g/dl galactose in

0.1 M phosphate buffer and soya milk, after which a lim-

iting value of current was obtained. It was also observed

that the same electrodes could be repeatedly used for

about 10 times. Thereafter, a severe drop in the current

was noticed. The repeated polarization of the electrode

at 0.4 V might have perhaps caused the denaturation

of GaO.Amperometric response of the P3HT/SA/GaO LB

electrodes was also taken at room temperature and at

Page 3: Recent trends in biosensors

0

150

300

450

600

750

900

0 1 2 3 4 5 6 7Galactose (g/dL)

Cur

rent

(nA)

Fig. 1. Amperometric response of P3HT/SA/GaO LB film electrode at

different concentrations of galactose in 0.1 M phosphate buffer, pH 7.0

(�) and in soya (lactose/galactose free) milk solution (n).

300

400

500

600

700

0 20 40 60 80 100No. of days

Cur

rent

(nA)

Fig. 3. Stability of P3HT/SA/GaO LB film electrode on storage at 4

�C. Stability was monitored at 10 days intervals amperometrically

using 3 g/dl galactose in 0.1 M phosphate buffer, pH 7.0.

94 B.D. Malhotra et al. / Current Applied Physics 5 (2005) 92–97

2 g/dl galactose solution using different pH of the solu-

tion. The current increases with the increase in pH.

The highest value of the current was obtained at pH

7.0 (Fig. 2), indicating that these P3HT/SA/GaO LB

electrodes can be used at pH 7.0 for the detection of

galactose.P3HT/SA/GaO LB films were tested for stability

under the same operating conditions as those for amper-

ometric response measurements. The response of the

P3HT/SA/GaO LB electrode was measured once in 7

days. The enzyme LB electrodes were stored at 4 �Cwhen not in use. The response was measured on a fresh

electrode. Fig. 3 shows the amperometric response of the

P3HT/SA/GaO LB film electrode in galactose solution(3 g/dl) in 0.1 M phosphate buffer (pH 7.0) as a function

of days. It can be seen that the response of these elec-

trodes is almost same for about 20 days after which

the electrodes show a gradual decrease in current re-

sponse. This may be due to partial decay in the enzyme

activity. The amperometric current decreases from 675

100

300

500

700

900

5.5 6.0 6.5 7.0 7.5 8.0 8.5pH

Cur

rent

(nA)

Fig. 2. Effect of pH on the amperometric response of P3HT/SA/GaO

LB film electrode at 3 g/dl galactose in 0.1 M phosphate buffers of

different pH at 0.4 V.

to 655 nA for 28 days and it decreases to 625 lA for

35 days. The half-life of these P3HT/SA/GaO electrodes

was determined to be 90 days.

Different concentrations of galactose were also pre-pared in serum samples (containing galactose <0.05

g l�1). P3HT/SA/GaO LB electrode showed linearity

from 0.05 to 0.5 g l�1 in blood serum after which a lim-

iting value of current was obtained (Fig. 4). Separate

electrodes were used for different concentration of galac-

tose.

Further, no significant effect of the interferents (as-

corbic acid, calcium chloride and uric acid) was ob-served at their physiological concentrations. Further, it

was found that P3HT/SA/GaO electrodes can be used

for galactose estimation in the temperature range 25–

40 �C.The stability of P3HT/SA/GaO LB films was tested

under the same operating conditions as those for amper-

ometric response measurements. The response of the

P3HT/SA/GaO electrodes was measured once in 10days. The response was measured on a fresh electrode.

The enzyme electrodes were stored at 4 �C, when not

in use. The amperometric current response was found

to decrease from 85 to 75 nA in 30 days and about

20

40

60

80

100

120

140

0 0.1 0.2 0.3 0.4 0.5Concentration (gl-1)

Cur

rent

(nA)

Fig. 4. Amperometric response of P3HT/SA/GaO LB films at different

concentrations of galactose in 0.1 M phosphate buffer, pH 7.4 in blood

serum.

Page 4: Recent trends in biosensors

55

65

75

85

0 20 40 60 80No. of Days

Cur

rent

(nA)

Fig. 5. Stability of P3HT/SA/GaO LB films on storage at 4 �C.Stability was monitored at 10 days interval amperometrically using 0.2

g l�1 galactose in 0.1 M phosphate buffer, pH 7.4.

80

90

100

B.D. Malhotra et al. / Current Applied Physics 5 (2005) 92–97 95

35% loss in amperometric response (Fig. 5) was ob-

served in three months.

0

10

20

30

40

50

60

70

2 4 6 8 10 12Lactose (g/dL)

Cur

rent

(nA)

Fig. 6. Amperometric response of P3HT/SA/b-Gal/GaO LB films at

different concentration of lactose in phosphate buffer, pH 7.0 (�) and

in milk (m). Different concentrations of lactose were prepared in

lactose and galactose free milk and change in current was observed.

0

50

100

150

200

250

10 20 30 40 50 60 70Temperature (°C)

Cur

rent

(nA)

Fig. 7. Effect of temperature on the amperometric response of P3HT/

SA/b-Gal/GaO LB films in the presence of 3 g/dl lactose in phosphate

buffer, pH 7.0 at 0.4 V (bias voltage).

4. Lactose biosensor

Lactose is the major carbohydrate present in the

milk. Most people, who suffer from the deficiency lac-

tase (b-galactosidase b-Gal), are not able to metabolize

lactose, present in most dairy foods. The absence or de-

crease of lactase activity in human leads to the clinical

syndrome ‘‘lactose intolerance’’. Lactose intolerance

causes various physical symptoms such as excessive

intestinal gas, nausea, cramps and diarrhea.We have developed an amperometric biosensor sensi-

tive to lactose as well as galactose, by immobilizing

b-galactosidase (b-Gal) and galactose oxidase (GaO)

in Langmuir–Blodgett (LB) films of poly-3-hexyl thio-

phene (P3HT) mixed with stearic acid (SA). The mono-

layers of P3HT/SA were fabricated by dispensing a

solution (1:1) of P3HT (1 mM) and SA (2 mM) in chlo-

roform onto water sub-phase containing CdCl2 (0.2mM), using Joyce-Loebl LB trough. Such P3HT/SA

monolayers were transferred onto the ITO-coated glass

plates at a surface pressure of 30 mN/m at 30 �C by ver-

tical dipping method. The dipping speed during up-

stroke and downstroke was maintained at 5 mm/min.

b-Galactosidase and galactose oxidase (2.5 mg each)

were mixed in a solution of P3HT/SA in chloroform

and this solution was spread onto air-water interfaceof the LB trough. Thirty monolayers of P3HT/SA/

b-Gal/GaO were then transferred onto indium-tin-oxide

(ITO) coated glass plates by vertical dipping method.

The b-Gal/GaO immobilized P3HT/SA LB films were

characterized using Fourier-Transform-Infra-Red spectr-

oscopy (FTIR) and scanning electron microscopy (SEM)

technique. Performance and characteristics of P3HT/

SA/b-Gal/GaO LB electrodes were studied with respect

to varying concentrations of lactose, temperature and

pH.

The results of the amperometric response determined

at room temperature for P3HT/SA/b-Gal/GaO LB films

are shown in Fig. 6. P3HT/SA/b-Gal/GaO LB electrode

shows linearity from 1 to 6 g/dl after which a limitingvalue of current was obtained. The thermal stability

and effect of pH on b-Gal/GaO immobilized P3HT/SA

LB film was investigated at 3 g/dl lactose concentra-

tion by amperometric measurements. Fig. 7 shows the

results of enzyme (b-Gal/GaO) response measurements

obtained as a function of temperature by holding the

P3HT/SA/b-Gal/GaO film in a lactose solution (3 g/dl

in phosphate buffer, pH 7) at different temperatures. Itshows that b-Gal/GaO activity increases up to 40 �Cand then it decreases drastically.

Page 5: Recent trends in biosensors

30

50

70

90

110

130

5.5 6 6.5 7 7.5 8 8.5pH

Cur

rent

(nA)

Fig. 8. Effect of pH on the amperometric response of P3HT/SA/b-Gal/

GaO LB films in the presence of 3 g/dl lactose in phosphate buffer of

different pH at 0.4V (bias voltage).

0

0.2

0.4

0.6

0.8

100 200 300 400 500 600Glucose Concentration (mg/dL)

Abso

rban

ce (O

.D.)

Fig. 9. Absorbance of P3HT/SA/GOX LB electrode at 540 nm as a

function of glucose concentration.

96 B.D. Malhotra et al. / Current Applied Physics 5 (2005) 92–97

Fig. 8 shows the results of P3HT/SA/b-Gal/GaO film

response measurements obtained as a function of varia-

tion of pH by holding the electrode in a lactose solution(3 g/dl) made in different pH buffers. It shows the max-

imum activity at pH range 7.0–7.2. It can be concluded

from these studies that the P3HT/SA/b-Gal/GaO

electrodes can be used for lactose estimation in the tem-

perature and pH range 25–40 �C and 7.0–7.2, respec-

tively.

5. Glucose biosensor

The prevalence of diabetes in industrialized countries

amounts to approximately 4% and hence the demand

and the necessity for the determination of blood glucose.

The normal concentration of glucose in blood serum

ranges between 4.2 and 5.2 mmol/l. The determination

of glucose is one of the most frequently performed rou-tine analyses in clinical chemistry as well as in the micro-

biological and food industries [29–31]. Keeping the

above in view, an attempt has been made towards the

preparation and characterization of LB films of poly-

3-hexyl thiophene mixed with stearic acid (SA). Further,

enzyme glucose oxidase (GOX) has been immobilized

onto the P3HT/SA LB films via LB technique. The

GOX immobilized P3HT/SA LB films have been sys-tematically investigated.

The activity of the glucose oxidase (GOX) immobi-

lized onto P3HT/SA LB films were performed by color-

imetric method using UV-visible spectrophotometer

(Schimatzu 160A). The following reaction occurs and

gives a brown color dye.

ð1Þ

ð2Þ

The dye produced adsorbs the light at 540 nm. Pho-

tometric response of glucose oxidase (GOX) immobi-

lized P3HT/SA LB film was also monitored with

varying concentration of glucose in phosphate buffer

(pH 7.0). The intensity of dye produced was found to

be directly proportional to the concentration of glucose

in the solution.

A plot between the absorbance at 540 nm with vary-ing concentration of glucose is plotted and is shown in

Fig. 9. It is clear from the fig. that the absorbance in-

creases linearly as glucose concentration increases form

100 to 500 mg/dl. These results suggest that these P3HT/

SA/GOX LB electrodes may be used for the estimation

of glucose from 100 to 500 mg/dl glucose solution [26].

6. Conclusions

An attempt has been made to present some of the re-

cent work in the area of biosensors carried out at our

laboratories. Further, it has been shown that conducting

polymer based Langmuir–Blodgett films of poly3-hexyl-

thiophene can be used for immobilization of galactose

oxidase (GaO), lactase (b-galactocidase, b-Gal) and glu-cose oxidase, respectively. These P3HT/SA/GaO, P3HT/

SA/b-Gal/GaO, P3HT/SA/GOX and P3DT/SA/GOX

LB electrodes can be used for the estimation of galac-

tose, lactose and glucose in desired test specimens,

respectively.

Acknowledgments

We are grateful to Dr. Vikram Kumar, Director,

NPL for his interest in this work. We are also thankful

Page 6: Recent trends in biosensors

B.D. Malhotra et al. / Current Applied Physics 5 (2005) 92–97 97

to Prof. Keiichi Kaneto, Kyushu Institute of Engineer-

ing and Technology, Iizuka, Fukuoka, Japan for his val-

uable suggestions.

References

[1] B.D. Malhotra, A.P.F. Turner (Eds.), Advances in Biosensors:

Perspectives in Biosensors, Jai Press, Elsevier, Netherlands, 2003.

[2] P.W. Stoecker, A.M. Yacynych, Selective Electrode Rev. 12

(1990) 137.

[3] A.O. Scott (Ed.), Biosensors for Food Analysis, The Royal Soc. of

Chem., Cambridge, UK, 1998.

[4] L.C. Clark Jr., Trans. Am. Artif. Intern. Organs 2 (1956) 41.

[5] L.C. Clark Jr., C. Lyons, Ann. NY Acad. Sci. 102 (1962) 29.

[6] S.J. Updike, G.P. Hicks, Nature 214 (1967) 986.

[7] G.G. Guiltbault, J. Montalvo, J. Am. Chem. Soc. 91 (1969) 2164.

[8] U. Narang, P.N. Prasad, F.V. Bright, K. Ramanathan, N.D.

Kumar, B.D. Malhotra, M.N. Kamalasanan, S. Chandra, Anal.

Chem. 66 (1994) 3139.

[9] Y. Mishima, J. Motonaka, I. Maruyama, I. Nakabayashi,

S. Ikeda, Sensors Actuators B 65 (2000) 343.

[10] P.C. Pandey, A.P. Mitra, Analyst 113 (1988) 329.

[11] W.O. Ho, S. Krause, C.J. McNeil, J.A. Pritchard, R.D. Arm-

strong, D. Athey, K. Rawson, Anal. Chem. 71 (1999) 1940.

[12] A. Kumar, Rajesh, B.D. Malhotra, S.K. Grover, Anal. Chim.

Acta 414 (2000) 43.

[13] V. Barmin, A.V. Eremenko, I.N. Kurochkin, A.A. Sokolovsky,

Electroanalysis 6 (1994) 107.

[14] W. Schuhmann, C. Kranz, J. Huaber, H. Wohlschlager, Synth.

Met. 1 (1993) 31.

[15] R. Singhal, Ph.D Thesis, University of Delhi, Delhi, India, 2003.

[16] A. Haouz, A. Geloso-Meyer, C. Burstein, Enzyme Microbiol.

Technol. 16 (1994) 292–297.

[17] G.G. Guilbault, Analytical Uses of Immobalizated Enzymes,

Marcel Dekker, New York, 1984.

[18] K. Hajizadeh, H.T. Tang, H.B. Halsall, W.R. Heinemann,

Electranalysis 8 (1991) 575–581.

[19] A. Guiseppi-Elie, G.G. Wallace, T. Matsue, in: T. Skotheim,

R. Elsenbaumer, J.R. Reynolds (Eds.), Handbook of Conducting

Polymers, second ed., Marcel Dekker, New York, 1997, p. 963.

[20] M. Gerard, A. Chaubey, B.D. Malhotra, Biosens. Bioelectron. 17

(2002) 345–359.

[21] C.H. Lee, H. Seo, Y.C. Lee, B.W. Cho, H. Jeong, B.K. Sohn,

Sensors Actuators B 64 (2000) 37.

[22] K.Y. Park, S.B. Choi, M. Lee, B.K. Sohn, S.Y. Choi, Sensors

Actuators B 83 (2002) 90.

[23] G.G. Guilbault, B. Danielsson, C.F. Mendenlus, K. Mosbach,

Anal. Chem. 55 (1983) 1582–1585.

[24] R.-J. Pei, J.-M. Hu, Y. Hu, Y. Zeng, J. Chem. Technol.

Biotechnol. 73 (1998) 59–63.

[25] S.L.R. Barker, H.A. Clark, S.F. Swallen, R. Kopelman, A.W.

Tsang, J.A. Swanson, Anal. Chem. 71 (1999) 1767.

[26] R. Singhal, A. Chaubey, K. Kaneto, W. Takashima, M.D.

Malhotra, Biotechnol. Bioeng. 85 (2004) 277–282.

[27] S.F. Cheng, L.K. Chau, Anal. Chem. 75 (2000) 16.

[28] R.J. Green, R.A. Frazier, K.M. Shakesheff, M.C. Davies, C.J.

Roberts, S.J.B. Tendler, Biomaterials 21 (2000) 1823–1835.

[29] D.G. Myszka, J. Mol. Recognit. 12 (1999) 390–408.

[30] R.L. Rich, D.G. Myszka, J. Mol. Recognit. 13 (1999) 388–

407.

[31] M. Sriyudthsak, H. Vamagishi, T. Moriizumi, Thin Solid Films

160 (1988) 463.