Immobilization of urease on poly(N-vinyl carbazole)/stearic acidLangmuir�/Blodgett films for application to urea biosensor

Rahul Singhal a,b, Anamika Gambhir a, M.K. Pandey a, S. Annapoorni b,B.D. Malhotra a,*

a Biomolecular Electronics and Conducting Polymer Research Group, National Physical Laboratory, Dr. K.S. Krishnan Road, New Delhi 110012, Indiab Department of Physics and Astrophysics, University of Delhi, Delhi 110007, India

Received 30 January 2001; received in revised form 26 November 2001; accepted 11 January 2002

Abstract

Urease was immobilized in mixed monolayers of poly(N -vinyl carbazole) (PNVK) and stearic acid (SA) formed at an air�/water

interface. The monolayers were transferred onto indium-tin-oxide (ITO) coated glass plates using Langmuir�/Blodgett (LB) film

deposition technique. Urease immobilized on PNVK/SA LB films, characterized using FTIR and UV�/visible spectroscopy, was

found to exhibit increased stability over a wide pH (6.5�/8.5) and temperature (25�/50 8C) range. Potentiometric measurements on

these urease electrodes were carried out using an ammonium ion analyzer. Two values for K mapp were obtained at lower and higher

concentrations of substrate urea. # 2002 Published by Elsevier Science B.V.

Keywords: Langmuir�/Blodgett films; Enzyme; Urease; Immobilization; Urea biosensor

1. Introduction

The metabolic function of kidney is reflected in the

concentration of organic compounds such as urea in

blood or urine. Therefore, the estimation of urea is

frequently performed in the medical field. The use of

urease as a biocatalyst for the development of ureabiosensor has attracted continued interest from bio-

chemical and clinical analysts. The general principle for

fabricating a urea biosensor is based on immobilization

of urease onto a membrane or support in which urea is

catalytically converted into ammonium and bicarbonate

ions:

(NH2)2CO�3H2O 0UREASE

2NH�4 �OH��HCO�

3 (1)

For monitoring the enzymatic products, various

techniques such as spectrophotometry, potentiometry

(Senillou et al., 1999; Koncki et al., 2000), flow injection

technique (Milardovic et al., 1999), coulometry (Loba-nov et al., 1995) and amperometry (Malitesta et al.,

1990) have been proposed. Table 1 lists the character-

istics of some urea biosensors. Potentiometric and

amperometric methods of determination are however

frequently used. Enzymatic urea electrodes are the most

studied potentiometric biosensors (Mascini et al., 1983).

Some of the potentiometric transducers used to con-

struct urea biosensors are ion-selective membrane elec-

trodes (H� or NH4�) or gas potentiometric electrodes

(NH3 or CO2), which maintain the combination of these

potentiometric transducers with enzyme-based mem-

branes.

More recently, enzyme-catalyzed polymer transfor-

mation with electrochemical ac-impedance detection

(Ho et al., 1999) has been employed for the measure-

ment of urea. An essential prerequisite for the develop-

ment of a biosensor is the immobilization of a

biomolecule by a method wherein the enzyme remains

active. Immobilization of enzyme/protein has been

attempted in conducting polymeric matrices which are

currently drawing much attention. Conducting polymers

possess the ability to bind oppositely charged complex

entities in their oxidized conducting states and release

them in their neutral insulating states (Hailin and

Wallace, 1989). Immobilization of a biomolecule in

conducting polymers can be achieved by several techni-

* Corresponding author. Tel.: �91-11-582-4620; fax: �91-11-585-

2678.

E-mail address: bansi@csnpl.ren.nic.in (B.D. Malhotra).

Biosensors and Bioelectronics 17 (2002) 697�/703

www.elsevier.com/locate/bios

0956-5663/02/$ - see front matter # 2002 Published by Elsevier Science B.V.

PII: S 0 9 5 6 - 5 6 6 3 ( 0 2 ) 0 0 0 2 0 - 9

ques. These include physical adsorption, electrochemical

entrapment, chemical cross-linking and covalent cou-

pling etc. For optimum activity of an immobilized

enzyme, it is important that its active sites are oriented

towards the free surface of the immobilizing matrix.

Morphological studies have revealed that the surface of

a conducting polymer is critically dependent on the

method of preparation (Diaz and Bargon, 1986) and

plays an important role in the effective immobilization

of a desired enzyme (Ramanathan et al., 1994, 1995a,b).

Fig. 1 shows a schematic for the biosensor configura-

tion.

Langmuir�/Blodgett technique for monolayer deposi-

tion is known to facilitate the desired orientation of a

biomolecule (Fig. 2). Only a few reports have appeared

on this subject. Formation of LB films of lipid�/enzyme

mixtures by using a conventional constant-perimeter

barrier trough has been described (Sriyudthsak et al.,

1988; Wan et al., 2000; Choi et al., 1998; Barmin et al.,

1994; Zhu et al., 1989; Dubrovsky et al., 1994; Girard-

Egrot et al., 1997; Paddeu et al., 1995). Immobilization

of Glucose oxidase (GOD) on a phospholipid analogous

vinyl polymer has been recently reported by Yasuzawa

et al. (2000). LB films of polyemeraldine base of

polyaniline with GOD entrapped between the layers

was deposited on indium-tin-oxide (ITO) glass plate by

Ramanathan et al. (1995b). Guiomar et al. (1997) found

that glucose oxidase can be immobilized on LB films of

cellulose acetate propionate deposited on a self-as-

sembled coated substrate.

In the present paper we report the studies on technical

development of urea biosensor based on the immobili-

zation of urease within an LB film of poly(N -vinyl

carbazole)/stearic acid (PNVK/SA). When the immobi-

lized urease is placed in a solution containing urea, urea

diffuses into the PNVK/SA LB film of the immobilized

enzyme. The enzyme urease then catalyzes the decom-position of urea into ammonium ions. The potentio-

metric response as a result of hydrolysis of urea has been

measured by an ammonium ion analyzer.

2. Materials and methods

Poly(N -vinyl carbazole) (Aldrich, USA) was used asreceived. Stearic acid was recrystallized three times using

acetone prior to being used. Monolayers of PNVK and

stearic acid dissolved in chloroform were spread onto

the aqueous subphase containing 2�/10�4 M CdCl2.

The resulting solution was sonicated for about 30 min

prior to being dispensed onto the subphase of the

Joyce�/Loebl LB (Model Trough 4) trough. Deionized

water from a Millipore water purification system wasused as the subphase. The pressure�/area (p �/A ) iso-

therms of PNVK/SA system were obtained at a com-

pression rate of 0.5 cm/min. LB film deposition was

carried out at a surface pressure of 30 mN/m onto the

ITO coated glass plates at pH 7. The temperature of the

subphase was maintained at 27 8C using a refrigerated

water circulator (BioRad E4870). The speed of the

dipping head was 5 mm/min. For entrapment about7.35 mg of urease was mixed with 0.5 ml solution of

stearic acid and PNVK in choloroform (1 IU/ml) just

before LB deposition. The solution was carefully loaded

onto the surface of deionized water (Millipore 10 RTS)

in the LB trough with a microliter syringe.

For physical adsorption a stock solution of urease

was made using 1.46 mg/100 ml of phosphate buffer to

give a concentration of 1 IU/ml. Different aliquots fromthe stock were applied to the LB films of PNVK

deposited on ITO glass plates. The activity of urease

in the adsorbed and entrapped states was measured in a

measuring cell (Fig. 3) containing phosphate buffer

(0.01 M, pH 7.0) by an ammonium ion analyzer (AR

25, Fisher Scientific). The distance between ammonium

ion sensitive electrode and PNVK/SA/UREASE elec-

trode was 1 cm. Different concentrations of substrate(urea) were added in the presence of immobilized urease

on ITO glass. The concentration of NH4� ion produced

was measured in parts per million (ppm) and the

Table 1

Characteristics of some urea biosensors

Matrix Method Linearity range (mM) Response time (min) Shelf-life (days) Reference

PVC ammonium electrode Amperometry 15�/80 1 12�/14 Campanella et al. (1990)

Screen printed electrode Conductometry 5�/25 1 �/ Ho et al. (1999)

Porous PVC Potentiometry 0.16�/12.5 10 120 Hirose et al. (1983)

Polypyrrole FIA 0.01�/30 4 �/ Osaka et al. (1999)

PNVK/SA/urease LB film Potentiometry 0.5�/68 2 35 Present work

Fig. 1. Schematic of a biosensor.

R. Singhal et al. / Biosensors and Bioelectronics 17 (2002) 697�/703698

resulting change in potential was measured in millivolts

(mV).The thermal stability of urease immobilized on

PNVK/SA LB films was investigated by measuring the

urease activity as a function of temperature by holding

the film in a 1 ml phosphate buffer at varying

temperature between 25 and 60 8C maintained in a

hot water bath for about 10 min. The films were then

tested for urease activity by the method described

earlier.Leaching of urease from adsorbed and entrapped

states on PNVK/SA LB films was monitored by placing

the film in a 2 ml phosphate buffer solution and

periodically withdrawing 100 ml of buffer for testing

the presence of urease. Response studies of the PNVK/

SA/urease LB films were conducted as a function of pH

and concentration of urea.

3. Results and discussion

3.1. Characteristics of LB films of PNVK/stearic acid

Fig. 4 shows the pressure�/area isotherm of PNVK/

SA mixed monolayer obtained at 30 8C. It can be seen

that the gas�/liquid phase transition occurs at about a

surface pressure of 2.6 mN/m at a molecular area of 41.3

A2. The liquid�/solid phase transition occurs at a surface

pressure of 26 mN/m at a molecular area of 31 A2. Thecompressibility of gas to liquid phase transition changes

from 0.03 to 0.02 m/mN and for liquid to solid phase

transition, it changes from 0.008 to 0.005 m/mN.

3.2. FTIR spectra of urease immobilized PNVK/SA LB

films

FTIR studies conducted on urease entrapped PNVK/

SA LB films show (Fig. 5) bands at 795 and 840 cm�1

ascribed to C�/H bending of 1,2,4-trisubstitution indi-

cating that carbazole ring is intact in the polymer. The

bands at 693 and 745 cm�1 have been ascribed to

C�/H bending of 1,2-disubstituted rings in PNVK. The

peak at 1230 cm�1 has been attributed to C�/H

stretching. The absorbance at 722 cm�1 is attributed

to the intrachain dication transition. These results

are in agreement with the values reported in literature

(Verghese, 1997).

The peptide groups of urease (protein) backbone

cause five vibrations in the CONH plane and five out-

of-plane (CONH) vibrations. A strong band observed at

about 3300 cm�1 and a less stronger band at about 3100

cm�1 have been assigned to amide A and amide B

bands, respectively. The amide A band is caused by the

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 the

amide A vibration. At about 1650 cm�1 the amide I

band has been understood to arise due to C�/O

stretching vibrations (Belanger et al., 1989). Suppressed

C�/N stretching and NH bending vibrations at 1540

cm�1 can be assigned to partial shielding effect of

deposited monolayers of PNVK and stearic acid. The

presence of vibrations corresponding to a-helix and b-

pleated sheet structure indicates that urease immobilized

Fig. 2. Schematic of monolayers of PNVK/SA/UREASE obtained on a water surface: (a) expanded; (b) partially compressed; (c) closed packed

molecules.

Fig. 3. Schematic of the measuring cell used for the estimation of urea

concentration.

Fig. 4. Pressure�/area isotherm of mixed monolayers of PNVK and

SA at a subphase temperature of 30 8C and pH 7.0.

R. Singhal et al. / Biosensors and Bioelectronics 17 (2002) 697�/703 699

on LB films of PNVK/SA has not lost its secondary

structure.

3.3. Response characteristics

3.3.1. Enzyme loading

Fig. 6 shows the results of experiments carried out for

estimating the minimum enzyme units required for

activity. The experiments for assessing minimum en-zyme units required for activity was carried out using 68

mM of urea substrate. It is clear from figure that no

detectable response was obtained below 5 IU of urease

activity. However, as the concentration approaches 10

IU the enzyme remains unsaturated even after 15 min of

the reaction. Thus this concentration was used for

further experiments. The increased requirement for

enzyme can be attributed to the fact that urease wasdissolved in chloroform for entrapment in LB films of

PNVK/SA.

3.3.2. pH profile of urease immobilized PNVK/SA LB

films

Fig. 7 shows optimum pH requirement of free and

immobilized urease at 30 8C at a substrate concentra-

tion of 10 mM. The pH optimum of immobilized urease

is comparable to that for free urease which shows a pH

optimum of 7.2. It can be noted that the fall in activity

in the immobilized state is more gradual on both sides of

optimum pH as compared to free urease. A broadening

of profile towards both acidic and alkaline range was

observed implying that the enzyme becomes less sensi-

tive to pH changes when it is immobilized. Such an

Fig. 5. FTIR spectra of urease entrapped in LB film of PNVK and SA: (a) active urease (inset shows the expended region 600�/800 cm�1); (b)

denatured urease.

Fig. 6. Effect of enzyme concentration immobilized per cm2 of PNVK/

SA LB films on activity at a urea concentration of 93 mM in phosphate

buffer at pH 7.0.

Fig. 7. Effect of pH on the activity of urease (10 IU/cm2) in (")

solution and in (j) immobilized PNVK/SA LB films.

R. Singhal et al. / Biosensors and Bioelectronics 17 (2002) 697�/703700

effect is anticipated when neither proton partitioning

nor diffusion limitation is present. It appears that the

effect is due to substrate diffusion limitation alone. An

immobilized enzyme preparation that has a high enzymeloading (large quantity of enzyme activity per unit

polymer) may be subject to substrate diffusion limita-

tion. Since in LB films the enzyme is deposited

symmetrically in monolayers along with the polymer,

the enzyme present in the first few PNVK/SA mono-

layers is likely to come into contact with the substrate. If

the enzyme has high intrinsic specific activity, which is

the case when the enzyme is immobilized in the PNVK/SA LB monolayers, the substrate concentration gradient

through the particle will be steep and consequently the

substrate may not penetrate to the center of the

immobilized enzyme (urease) molecules. If some con-

straint like a change in pH is subsequently applied

leading to the reduced enzyme activity, the substrate

concentration gradient will become less steep, thus

allowing the substrate to penetrate further into theimmobilized enzyme molecules. Effectively the enzyme

concentration increases resulting in the availability of

increased number of urease molecules to the substrate.

These two antagonistically factors tend to moderate the

effect of pH change (Trevan, 1980). The fact that the

immobilized enzyme is more stable in varying pH has

also been reported by other researchers (Ramanathan et

al., 1997; Gambhir et al., in press, Carr and Bowers,1980).

3.3.3. Determination of enzyme activity and substrate

kinetics

A good linear correlation between potential sensed by

an ammonium ion selective electrode and urea concen-

tration was obtained in the range from 0.5 to 93 mM

when this electrode was used. Two linear ranges were

obtained viz., 0.5�/10 and 10�/68 mM (Fig. 8a and b).Lineweaver�/Burke plots (Fig. 9) for the immobilized

urease gave two values of Kmapp. Sensitivity was higher in

the 10�/68 mM range and Kmapp was 9 mM. The value of

Kmapp for the lower range was justifiably higher (30 mM).

Various reports available on the effect of immobilization

on enzyme kinetic parameters investigating the relation-

ship between substrate concentration (S ) and activity

(V ) over a narrow range of substrate concentrationindicate the reciprocal plots (Lineweaver�/Burke) as

linear and a single Kmapp is obtained by extrapolating

the plot. The two Kmapp values obtained in this report

suggest that Kmapp is perhaps dependent upon the

substrate concentration used. The higher Km at low

substrate concentration can be attributed to the higher

substrate diffusion limitation at lower substrate concen-

trations. Besides this, partitioning effects will be greaterat lower substrate concentrations. Both these factors are

likely to influence the apparent ease with which enzyme

and substrate can associate and will thus affect Kmapp.

3.3.4. Sensitivity, repeatability and detection limit

The response time of the urease/PNVK/SA LB

electrode was about 2 min. The detection limit and the

sensitivity for this urease electrode have been experi-

mentally determined as 5 mM and 10 mV/mM, respec-

tively. This urease electrode could be used about 10times.

3.3.5. Storage and stability

The observed leaching of about 5% and shelf-life of

about 5 weeks has been attributed to very thin andstable architecture of PNVK/SA LB films. It is impor-

tant to point out here that urease immobilized on 60�/70

monolayers of PNVK/SA obtained by the physical

Fig. 8. (a) Response curve of PNVK/SA/urease LB films as a function

of urea concentration (0�/10 mM). (b) Response curve of PNVK/SA/

urease LB films as a function of urea concentration (0�/93 mM).

Fig. 9. Lineweaver�/Burke plots for immobilized urease on PNVK/SA

LB films: (a) 1 for 0�/10 mM urea; (b) 10�/68 mM urea concentration.

R. Singhal et al. / Biosensors and Bioelectronics 17 (2002) 697�/703 701

adsorption technique shows high leaching (75%) and a

short shelf-life (2 days). A very thin and smooth surface

of LB film is perhaps unable to retain the physically

adsorbed enzyme on the surface.

A higher temperature (41 8C) optimum (Fig. 10) was

obtained for PNVK/SA/urease electrode, which could

perhaps be due to higher intermolecular interactions.

About 75% activity was recorded at 45 8C after which a

steady decrease was observed which is likely to be due to

the denaturation of protein at higher temperatures. This

decrease in activity was reversible until 49 8C indicating

that the enzyme perhaps attains its near original

conformation after returning to normal temperature.

The value of activation energy before and after critical

temperature calculated from the Arrhenius plot was

found to be 4027 and 7595 cal, respectively.

4. Conclusions

It can be seen that stable PNVK/SA monolayers can

be formed at the air�/water interface. Further, urease

can be immobilized onto these PNVK/SA LB mono-

layers. These PNVK/SA/UREASE monolayers trans-

ferred onto ITO coated glass plates can be utilized for

urea sensing. The detection limit and sensitivity of these

electrodes was found to be 5 mM and 10 mV/mM,

respectively. The shelf-life of these electrodes was found

to be 5 weeks at 4 8C.

Acknowledgements

We are grateful to Dr K. Lal, Director, NPL for his

interest in this work. The financial support received

under the DST funded project (SP/S2/M-52/96) and the

Indo-Polish project (INT/POL/P015/2000) are gratefully

acknowledged. A.G. and R.S. are grateful to CSIR for

award of Research Associateship (RA) and Senior

Research Fellowship (SRF), respectively.

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