Hydrogen peroxide ± sensitive enzyme sensorbased on phthalocyanine thin ®lm

T.A. Sergeyevaa,*, N.V. Lavrikb, A.E. Rachkova,Z.I. Kazantsevab, S.A. Piletskya, A.V. El'skayaa

aInstitute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine, 150 Zabolotnogo Str., 252143 Kiev, UkrainebInstitute of Semiconductors Physics, National Academy of Sciences of Ukraine, 45 Prospekt Nauki Str., 252650 Kiev, Ukraine

Received 30 September 1998; received in revised form 10 February 1999; accepted 21 February 1999

Abstract

An enzyme biosensor speci®c for hydrogen peroxide was developed using a new conductometric transducer based on tetra-

tert-butyl copper phthalocyanine (ttb-CuPc) thin ®lms and horseradish peroxidase as sensitive element. This analytical system

is based on detection of molecular iodine produced as a result of the oxidation of the iodide ions by hydrogen peroxide in the

presence of horseradish peroxidase. For the detection of the peroxidase-initiated reaction the ability of the ttb-CuPc thin ®lm

to change its conductivity in response to the appearance of molecular iodine is used. To minimise the interfering effect of the

aqueous electrolyte on the conductometric response of the ttb-CuPc thin ®lm itself, gold interdigitated electrodes bearing ttb-

CuPc layer were covered with a hydrophobic gas-permeable membrane. Thermally evaporated calixarene or plasma

polymerised hexamethyldisiloxane was used as a gas-permeable membrane material. In order to assess the optimum sensor

technology as well as the operating regime, impedance spectroscopy data were analysed. For biosensor creation horseradish

peroxidase was deposited on the sensitive part of the electrodes in a cross-linked bovine serum albumin matrix. The possibility

of hydrogen peroxide detection with the biosensor proposed in the range 5±300 mM was demonstrated. The operational

stability of biosensor was at least 7 h and the relative standard deviation did not exceed 10%. When stored at �48C the sensor

response was stable for more than 90 days. The dependencies of the sensor response on pH, buffer and NaCl concentrations

were investigated. # 1999 Elsevier Science B.V. All rights reserved.

Keywords: Horseradish peroxidase; Biosensor; Hydrogen peroxide detection; Interdigitated planar electrodes; Tetra-tert-butyl copper

phthalocyanine; Langmuir±Blodgett ®lms

1. Introduction

Development of reliable methods of hydrogen per-

oxide determination is of great importance in both

biological and industrial ®elds. Detection of hydrogen

peroxide is important from several points of view:

1. it is widely used in food industry as a sterilising

agent and may be present in ®nal products which

can lead to the loss of nutritional value and

appearance of toxic compounds such as hydro-

peroxides, epoxides and aldehydes [1];

2. hydrogen and organic peroxides can be released

in the environment from industrial processes

and during the ozonation of drinking water

[2,3];

Analytica Chimica Acta 391 (1999) 289±297

*Corresponding author. Tel.: +380-044-266-07-49; fax: +380-

044-266-07-59; e-mail: sergeeva@biosensor.kiev.ua

0003-2670/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved.

PII: S 0 0 0 3 - 2 6 7 0 ( 9 9 ) 0 0 2 0 3 - 2

3. measurements of hydrogen peroxide produced dur-

ing the reaction of many important chemicals in the

presence of oxidases forms the basis for several

biological and medical tests [4±6];

4. hydrogen peroxide is frequently used in pharma-

ceutical and cosmetic formulations [7];

5. horseradish peroxidase is widely used as label in

enzyme-linked immunosorbent assays (ELISA).

Conventional methods for the determination of

hydrogen peroxide including spectrophotometry [8],

colorimetry [9] and chemiluminescence [10] often

involve complicated methods and expensive equip-

ment. A commonly used electrochemical method for

the detection of hydrogen peroxide is its oxidation at

high overpotentials at a wide variety of electrodes [11±

17]. The main disadvantage of this approach is its poor

speci®city because the high voltage applied to the

working electrode can lead to unacceptable interfer-

ences. To reduce the interfering effect caused by

substances which can be oxidised at used potentials,

alternative systems which can operate at much

reduced voltages are being developed for hydrogen

peroxide detection. A number of amperometric

enzyme sensors combining the speci®city of enzy-

matic reaction with the high sensitivity of electroche-

mical transducer have been proposed for this purpose

[18±22]. An amperometric sensor based on horserad-

ish peroxidase (HRP) modi®ed platinised carbon par-

ticles which are capable of reducing hydrogen

peroxide at 0.0 V (vs. SCE) has been developed by

Cardosi [23]. A new scheme for fabricating bulk-

modi®ed amperometric biosensors based on gra-

phite±PTFE electrodes which give a possibility of

hydrogen peroxide detection at 0.0 V (vs. SCE) with

a detection limit of 2.5 mM, and a linear dynamic

range of 2.5±150 mM has been reported [24]. A

potentiometric sensor for this purpose has been pro-

posed by Zul®car et al. [25]. The potentiometric

response of this sensor in the ¯ow system was linear

in the concentration range 0.75±50 mM.

As one can conclude considerable progress and

practical achievements in the ®eld of hydrogen per-

oxide determination have been obtained by using

amperometric and potentiometric biosensors. Until

now no conductometric biosensors for this purpose

have been reported. The main reason for this is that

because of the relatively low impedance of aqueous

medium signi®cant dif®culty is created in proper

operation of the conductometric biosensors. Never-

theless, conductometric biosensors can be more easily

integrated since they do not need a reference electrode

and the conductometric transducers can be manufac-

tured using simple thin ®lm technology [26±28].

In the present study, the possibility of hydrogen

peroxide monitoring was investigated with a perox-

idase enzyme sensor based on a conductometric trans-

ducer having tetra-tert-butyl Cu-phthalocyanine thin

®lms as a sensitive element. The construction of the

transducer is simple, of low cost and has good pos-

sibilities for mass production.

2. Experimental

2.1. Materials

Tetra-tert-butyl copper phthalocyanine (ttb-CuPc)

of high purity was purchased from the Research

Institute of Organic Dyes (Moscow, Russia). Chloro-

form and benzene of a reagent grade were obtained

from Sigma and were distilled prior to use. Hexa-tert-

butyl-calix[6]arene was synthesised at the Institute of

Organic Chemistry (Kiev, Ukraine) in Dr. Kalchenko's

laboratory as described elsewhere [29]. Hexamethyl-

disiloxane was obtained from the microelectronics

plant `̀ Kvazar'' (Ukraine). Glutaraldehyde and bovine

serum albumin (BSA) were purchased from Sigma.

HRP (EC 1.11.1.7) with an activity 257 U/mg (guaia-

col as a reducing substrate) was purchased from

Biozyme. Hydrogen peroxide standardised by iodi-

metric titration was freshly prepared in deionised

water. Iodine (I2) was purchased from Sigma as

crystals of 99% purity. I2 crystals were dissolved in

96% (v/v) ethanol to prepare a 0.2 M stock iodine

solution for further experiments. Potassium iodide

(KI) was obtained from Sigma and a 0.5 M stock

solution of KI was prepared by dissolving in

0.02 M K-phosphate buffer, pH 6.0 immediately

before use.

2.2. Iodine-sensitive transducer and measurements

of the I2 concentration

Interdigitated microelectrodes were purchased from

Emokon (Kiev, Ukraine). The ceramic chips of

290 T.A. Sergeyeva et al. / Analytica Chimica Acta 391 (1999) 289±297

5 mm�30 mm size and 0.2 mm thickness contained

two identical pairs of gold interdigitated electrodes.

The electrodes were deposited onto a ceramic sub-

strate by using conventional thin-®lm technology

which includes the following main stages:

1. vacuum deposition of 0.1 mm chromium adhesive

layers onto ceramic substrates;

2. vacuum deposition of 1 mm gold on the top of the

chromium layers;

3. patterning of the interdigitated microstructures by

photolithography.

Each finger in the electrode arrays was 20 mm wide

and approximately 1 mm long. The separation

between the adjacent fingers was 20 mm. The total

sensitive area of each electrode array was about

1.5 mm2.

Iodine-sensitive transducers were prepared by

deposition of thin ttb-CuPc ®lm onto the active area

of the interdigitated electrodes by one of the following

methods: casting from chloroform/benzene solutions,

Langmuir±Blodgett (LB) deposition, and thermal eva-

poration in vacuum. The details of the thermal eva-

poration and LB deposition procedures are given

elsewhere [30,31]. Hydrophobic gas-permeable mem-

branes (HGPMs) deposited on the ttb-CuPc ®lms were

formed by either thermal evaporation of hexa-tert-

butyl-calix[6]arene (HGPM-1) or plasma polymerisa-

tion of hexamethyldisiloxane (HGPM-2) as described

elsewhere [32]. The cross-section of the resulting

structures is schematically shown in Fig. 1. The thick-

ness of each of the deposited organic layers was

de®ned by ellipsometric measurements using Si-

monocrystal satellites.

To measure impedance spectra and conductometric

responses of the sensor, the differential pair of the

interdigitated electrodes with the ttb-CuPc/HGPM

layer was connected to the electronic units as

described earlier [33]. The output voltage was

recorded with a chart recorder and the values corre-

sponding to the steady-state response were taken as a

sensor response. These values were also used for

impedance analysis. To plot impedance spectra, the

absolute values of the complex impedance |Z| of the

sensor were calculated using the following equation:

jZj � Ro�Ug ÿ Ux�=Ux; (1)

where Ro is the resistance across the a.c. nanovolt-

meter input, Ug the a.c. generator output voltage

(60 mV) and Ux is the voltage measured with the

a.c. nanovoltmeter.

2.3. Enzyme immobilisation and measurements of

H2O2 concentration

Horseradish peroxidase was immobilised onto the

transducer surface in a cross-linked BSA matrix

according to the following procedure. Peroxidase

and BSA were dissolved separately in 0.1 M K-phos-

phate buffer solutions, pH 8.0. The resulting protein

concentrations were 100 mg/ml in both solutions.

Equal aliquots of these solutions were mixed with

inositol (Sigma, St. Louis, USA) and ZnSO4 so that

the concentrations of the inositol and ZnSO4 in the

resulting mixture were 5% (w/v) and 0.9 mM, respec-

tively. Approximately 1 ml of this mixture was depos-

ited on a sensitive area of the electrodes. To complete

the polymerisation of the membrane, the sensor chip

was exposed to glutaraldehyde vapours for 30 min and

then dried at room temperature for 15 min.

For H2O2 determination, the prepared enzyme sen-

sor was placed in a 2.5 ml glass cell with gently stirred

0.02 M K-phosphate buffer (pH 6.0) containing

0.01 M KI and 0.15 M NaCl. The measurements were

carried out at room temperature. The sensor responses

were recorded after successive additions of the stock

H2O2 solution into the buffer.

3. Results and discussion

The conductometric method of H2O2 detection is

based on the reaction of iodide ions oxidation by H2O2

Fig. 1. The scheme of a conductometric horseradish peroxidase-

based enzyme sensor with a chemoresistive layer separated from

the region where the enzymatic reaction occurs.

T.A. Sergeyeva et al. / Analytica Chimica Acta 391 (1999) 289±297 291

in the presence of HRP:

H2O2 � 2Iÿ � 2H� !peroxidase2H2O� I2 (2)

Therefore, the amount of hydrogen peroxide used in

this reaction can be monitored by measurement of

either iodide consumption or free iodine release.

Iodide-sensitive potentiometric electrodes have been

used previously in order to measure the iodide con-

centration [34,35], but integration of potentiometric

biosensors implies quite sophisticated technology. On

the other hand, the reaction (2) can be monitored by

measurement of the free iodine released. In particular,

we propose to monitor the iodine concentration using

a conductometric transducer with a phthalocyanine

thin ®lm as a sensitive element. To detect the mole-

cular iodine concentration, the ability of ttb-CuPc ®lm

to change its conductivity in response to appearance of

I2 is used in the present study. Chemoresistors includ-

ing phthalocyanins are widely recognised as one of the

most attractive basis for chemical and biosensors due

to their direct electrical responses and good compat-

ibility with electronics [36]. However, high electrical

conductivity of aqueous electrolyte prevents realisa-

tion of biosensors on this basis, therefore properties of

chemoresistors were mainly studied in gaseous envir-

onments only [30,31]. A speci®c feature of the con-

ductometric transducer designed is that ttb-CuPc ®lm

was covered by a hydrophobic gas-permeable mem-

brane (HGPM) to suppress interfering effect of an

aqueous electrolyte (Fig. 1).

To de®ne the optimum technological procedures

and operating frequency, impedance spectra of a set of

the prepared structures were measured in air (Fig. 2,

curve 1) and in various electrolytes, with and without

molecular iodine (Fig. 2, curves 2±4). As can be seen,

at the lowest frequency, the increase of the electrolyte

conductivity has no signi®cant effect on the impe-

dance (Fig. 2, curves 2 and 3), especially in the case of

LB-deposited ttb-CuPc ®lms. At the same time, low-

frequency impedance decreases when 20 mM of free

iodine is present in the electrolyte (Fig. 2, curve 4). It

was shown that the shift of the impedance spectra due

to the presence of iodine is much lower in the case of

the casted ®lms. Therefore, samples with the latter

type of ttb-CuPc ®lms were eliminated from further

experiments. The structures with the LB-deposited

and thermally evaporated ®lms exhibited quite similar

characteristics. Thermal evaporation of the ttb-CuPc

®lms provides all-dry processing of the transducers up

to biomaterial immobilisation. On the other hand, the

LB deposition uses more effectively the material to be

deposited. To minimise the interfering effect of the

electrolyte, the working frequency of 1 Hz was

selected for monitoring the sensor impedance in all

subsequent experiments. The chemoresistors with a

LB-deposited and thermally evaporated ttb-CuPc

layer demonstrated similar sensitivity to the appear-

ance of free iodine in solution. Similar results were

obtained in the case of the thermally evaporated active

layers. The response was rather slow and at least

10 min was needed to reach the steady-state response.

As noted before [29,30] such a slow kinetics is typical

for conductivity changes in phthalocyanine ®lms at

room temperature.

Similar responses on iodine were obtained for both

types of hydrophobic gas permeable membranes,

HGPM-1 and HGPM-2, of equal thickness

(100 nm). However, HGPM-2 was chosen for the

further experiments because of its better adhesion

and mechanical stability.

HRP was immobilised on a surface of the designed

iodine-sensitive transducer in a cross-linked BSA

matrix. To compensate non-speci®c reactions of the

biosensor, an enzyme-free BSA matrix was deposited

on a reference transducer. All measurements were

carried out in a differential pair mode. The calibration

curve of the biosensor obtained in 20 mM potassium-

phosphate buffer, pH 6.0 was found to be linear within

the range of the hydrogen peroxide concentration from

5 to 300 mM (Fig. 3), each point represents the aver-

age of three measurements and the sensor standard

deviation did not exceed 10%. However, no sensor

response was observed when H2O2 was added to the

buffer solution in the absence of KI. Addition of the

iodide in the absence of the substrate, H2O2, did not

cause any noticeable response as well.

The dependence of the enzyme sensor response on

the pH of the sample solution was investigated. The

greatest response takes place at a pH between 5.0 and

6.5 (Fig. 4). This corresponds approximately to the

region where the peroxidase activity has a maximum.

One can therefore conclude that the observed

responses are indeed controlled by the rate of the

enzymatic reaction (2).

The generally recognised main drawback of con-

ductometric biosensors is their poor speci®city exhib-

292 T.A. Sergeyeva et al. / Analytica Chimica Acta 391 (1999) 289±297

ited particularly in measurements in buffers with a

high capacity and ionic strength. The dependencies of

the buffer capacity and ionic strength on the magni-

tude of the peroxidase enzyme sensor response have

been investigated. It has been shown that neither ionic

strength nor buffer capacity affected the magnitude of

the sensor responses signi®cantly (Figs. 5 and 6). This

fact seems to be of great importance because it gives a

Fig. 2. Impedance spectra of iodine-sensitive conductometric transducer with casted (a) and Langmuir±Blodgett deposited (b) tetra-tert-butyl

copper phthalocyanine films. The impedance spectra were measured for: 1 ± dry electrodes, 2 ± electrodes immersed in distilled water,

3 ± 0.15 M NaCl solution, 4 ± 0.15 M NaCl solution containing 20 mM I2.

T.A. Sergeyeva et al. / Analytica Chimica Acta 391 (1999) 289±297 293

Fig. 3. Dependence of horseradish peroxidase-based enzyme sensor response on substrate concentration. Measurements were carried out in

0.02 M potassium phosphate buffer, pH 6.0, containing 0.15 M NaCl and 0.01 M KI.

Fig. 4. The pH dependence of the activity of horseradish peroxidase immobilised on the surface of the iodine-sensitive transducer.

Measurements were carried out in 0.02 M potassium phosphate buffer containing 0.15 M NaCl, 50 mM H2O2 and 0.01 M KI.

294 T.A. Sergeyeva et al. / Analytica Chimica Acta 391 (1999) 289±297

Fig. 5. Dependence of horseradish peroxidase-based enzyme sensor response on NaCl concentration in working solution. Measurements were

carried out in 0.02 M potassium phosphate buffer containing 50 mM H2O2 and 0.01 M KI.

Fig. 6. Dependence of horseradish peroxidase-based enzyme sensor response on buffer concentration of working solution. Measurements

were carried out in potassium phosphate buffer containing 0.15 M NaCl, 50 mM H2O2 and 0.01 M KI.

T.A. Sergeyeva et al. / Analytica Chimica Acta 391 (1999) 289±297 295

possibility to avoid the main drawback of the con-

ductometric detection method. The results obtained

prove that the proposed transducers have high and

speci®c sensitivity to the iodine generated by the

enzymatic reaction. Thus, despite some slight effect

of the electrolyte on the measured sensor impedance,

the hydrophobic membrane is thick enough to provide

stable operating of the sensor in the range of physio-

logical pH and ionic strength.

Stability of a biosensor system is a very important

characteristic for their commercial application. The

operational stability test demonstrated that the steady-

state response was not decreased for at least 7 h

(approximately 30 measurements). When stored at

�48C, the biosensor response was stable for at least

three months.

The main characteristics of the horseradish perox-

idase-based biosensor are summarised in Table 1.

Both the response and recovery times could be sig-

ni®cantly reduced if the initial rate of the conductivity

increases was measured instead of steady state

responses. However, this operation mode would also

lead to an increased error due to a much lower signal/

noise ratio in the beginning of the response kinetics.

The range of the detectable concentrations can be

extended to the upper end by adding a diluting system

based, for instance, on a micro¯uidic device. At the

same time, the threshold sensitivity of the proposed

sensor has to be still improved to make it useful for

biodiagnostic analysis of hydrogen peroxide. One of

the possible ways to achieve this is optimisation of

diffusion properties of the matrix for the enzyme

immobilisation. The stability of a biosensor over a

long period of time is one of the most critical char-

acteristics that de®nes the potential for commercial

applications. Although 90 day shelf-lifetime requires

refrigerating at�48C, this can be considered as appro-

priate for most of the practical applications.

4. Conclusions

A chemoresistor based on the organic semiconduc-

tor material, phthalocyanine, has been used in aqueous

medium as a transducer for biosensors. Both the

thermal evaporation and Langmuir±Blodgett deposi-

tion of the chemoresistive material provided transdu-

cers with satisfactory and quite reproducible

parameters. Due to the presence of hydrophobic

gas-permeable membrane covering the iodine-sensi-

tive ®lm, neither ionic strength nor buffer capacity has

a signi®cant in¯uence on the measured responses. The

separation of the chemoresistive material and the

aqueous medium effectively reduces electrolyte inter-

ference, and thus, practically eliminates the main

disadvantage of the conductometric method as applied

to biosensors. It has been shown that free iodine

generated due to the peroxidase-initiated reaction

can be effectively monitored with the designed trans-

ducers.

Acknowledgements

Financial support from Ministry Ukraine for

Science and Technology (Grant 05.41.07/005-92) is

gratefully acknowledged. The authors thank Tammy

Calvert for linguistic advice.

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Table 1

Characteristics of HRP-based biosensor

Operation mode Steady state

Optimum pH 6.0

Linear dynamic range 5±300 mM

Baseline recovery time 15 min

Relative standard deviation 10%

Storage stability >90 days

Operational stability 7 h

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