NOVEL COMBINED TEMPLATE FOR AMPEROMETRIC

BIOSENSORS WITH CHANGEABLE SELECTIVITY

Julija Razumiene

, Vidute Gureviciene

, Jurgis Barkauskas

Virginijus Bukauskas

and Arunas Setkus

Institute of Biochemistry, Department of Bioanalysis, Mokslininku 12, 08662 Vilnius, Lithuania

Vilnius University, Department of General and Inorganic Chemistry, Naugarduko 24, 03225 Vilnius, Lithuania

Semiconductor Physics Institute, Sensors Laboratory, A. Gostauto 11, LT01108 Vilnius, Lithuania

Keywords: Biosensors, Enzymes, Carbon Nnotubes, Medical Application.

Abstract: Present paper describes innovative approach in design of amperometric biosensors useful in various appli-

cations. Original template of the electrodes has been prepared on a base of carbon nanotube support layer

deposited on the polycarbonate membrane. Novel template and changeable enzyme layer give rise to crea-

tion of new family of biosensors acceptable for detection of wide range of carbohydrates. The morphology

and electric properties of the constituent parts of the template electrode are characterized by scanning probe

microscopy. The sensitivity, selectivity and stability are described for typical types of the biosensors.

1 INTRODUCTION

Electrochemical biosensors are the most common

class of biosensors in various practical applications

(A. Chaubey and B. D. Malhotra, 2002). In these

sensors, bio-interaction at specific sites of enzymes

results in extra electric charge. The extra charge can

be transferred from enzyme to electrode and de-

tected by an external electric circuit. Technology of

the electrodes and enzyme immobilization is crucial

for conversion of biochemical interaction into the

response signal and, still, is under intensive studies.

This type of sensors has well known advantages

such as acceptability for functioning in turbid media,

comparable instrumental sensitivity and amenability

to miniaturization, and etc. In this report, we present

an original approach in biosensor technology based

on immobilization of enzymes within special matrix

attachable to carbon nanotube electrode.

2 METHODS

AND EXPERIMENTS

We developed and tested an original technology

acceptable for production of a family of biosensors.

Selectivity of two component biosensors can be

changed simply by replacing special matrix contain-

ing enzymes attached to the single wall carbon na-

notube (SWCNT) based electrode. Therefore the

biosensors can be adjusted for detection of monosa-

charydes and disacharydes such as glucose, lactose,

galactose, maltose and et cetera.

Nanotube support layers on the polycarbonate

membranes were prepared from the industrial

SWCNT (Cheap Tubes Inc., USA). The main para-

meters of these nanotubes ware as follows: diameter

is 2 nm, 5.0 – 30.0 μm length, specific surface area

400 m2/g, electrical conductivity 10-2 S/cm. The

SWCNT were functionalized with carboxyl groups

(2.73 wt%).

In this study we introduced an original protocol

for coating of flexible support by SWCNT layer

acceptable for biosensor electrode. The protocol

includes special filtration of aqueous suspension

448

Razumiene J., Gureviciene V., Barkauskas J., Bukauskas V. and Setkus A. (2009).

NOVEL COMBINED TEMPLATE FOR AMPEROMETRIC BIOSENSORS WITH CHANGEABLE SELECTIVITY.

In Proceedings of the International Conference on Biomedical Electronics and Devices, pages 448-452

DOI: 10.5220/0001827604480452

 SciTePress

through the isopore polycarbonate membrane and is

crucial for electrochemical properties of biosensors.

The prototype biosensors were based on three

types of enzymes, namely glucose oxidase from

Aspergillus niger and pyrroloquinoline quinone

(PQQ) dependent glucose dehydrogenases and

aldose sugar dehydrogenase. The soluble glucose

dehydrogenase (s-PQQ-GDH) from Acinetobacter

calcoacetics L.M.D. 79.41 was purified by the me-

thod reported in (A. J. A. Olsthoorn and J. J. Duine,

1996). The membrane-bound enzyme (m-PQQ-

GDH) was purified from Erwinia sp. 34-1

(Marcinkevičienė et al., 1999). The water-soluble

aldose sugar dehydrogenase (s-PQQ-ADH) was

purified from Escherichia coli (Southall et al.,

2006). Each of the enzyme types was immobilized

on individual flexible support of polivinylalcohol

coated terylene. Adsorption and cross linking to the

support were the methods for immobilisation of

enzymes.

Our prototype amperiometric biosensors con-

sisted of SWCNT based electric charge drain and

changeable biosensitive detector. The sensor con-

struction is illustrated by a sketch in Fig. 1.

Surfaces of the sensor components, namely, elec-

trode support, SWNT coatings and matrix without

and with immobilized enzymes, were analyzed by

scanning probe microscope (SPM) D3100 / Nanos-

cope IVa (Veeco Instruments Inc.). Standard AFM

methods such as contact and tapping mode surface

scanning were used for visualization of the surface

morphology. The surface electrical characteristics

were evaluated from measurements of tunneling

current obtained in contact mode. Conductive probe

of the SPM was firmly pressed to the surface so that

it was not damaged. Special module SPM D3100

TUNA (Veeco Instruments Inc.) was used for these

experiments. The maps of the current and local point

volt-amperic characteristics (VACh) were obtained

for the components of the biosensor electrodes in

these experiments. The data and SPM mages were

processed by the NanoScope Software 6.14 (Veeco

Instruments Inc.).

Electrochemical experiments were performed us-

ing a conventional three-electrode system containing

a screen-printed carbon electrode as a working elec-

trode, a platinum wire as a counter electrode and an

Ag/AgCl in saturated KCl as a reference electrode

(all potential values presented in the text are vs. this

reference electrode). 0.05 M acetate buffer (pH 6.0)

containing 1 mM of Ca

and 0.2 mM N-

methylphenazonium methyl sulphate was used as a

default buffer. Steady state currents of the biosen-

sors were recorded at 0.4 V using a polarographic

analyzer “PARSTAT 2273” (Princeton Applied

Research, USA).

Figure 1: General side and top views (A) and the compo-

nents (B) of the biosensor: 1 – insulating film, 2 – enzyme

immobilized on terylene film, 3 – contact zone, 4 –

SWCNT-polycarbonate membrane, 5 – insulating film.

3 RESULTS AND DISCUSSIONS

Characteristics of the biosensor family were ob-

tained only for four types of the biosensors based on

the original prototype structure in present study. The

SWCNT layer on polycarbonate membrane and

changeable enzyme based detector are the most

important results of the sensor technology in present

study. It was proved by experiments with the proto-

type biosensors that SWCNT based structure is

acceptable for the sensor electrode and immobiliza-

tion of enzymes. The attachable enzyme detectors

were reproducible and stable for comparatively long

time.

3.1 Surface Properties of the

Electrodes

The morphology and electric properties were de-

scribed for separate components of the template

electrodes by the SPM experiments. The results

were obtained for the components at intermediate

stages of the technology.

Typical structure of the SWCNT coating is illu-

strated by a SPM image in Fig. 2. It is seen in Fig. 2

the SWCNT were found in vertical and horizontal

positions on the membrane. Since the membrane

contained the pores deep valleys were found in the

nanotube layer. It was revealed by high aspect ratio

SPM tests that SWCNT are in vertical position in

the areas corresponding to the pores in the mem-

brane. On the flat surfaces of the membrane there

were no preferable orientations of the SWCNT with

respect to the membrane surface. The SWCNT layer

NOVEL COMBINED TEMPLATE FOR AMPEROMETRIC BIOSENSORS WITH CHANGEABLE SELECTIVITY

449

was comparatively thick and at least several layers

of horizontal nanotubes were detectable.

It was proved by measurements of tunneling cur-

rent that electric conductivity highly depends on the

structure of the SWNT layer. The areas with vertical

nanotubes were more conductive that that with hori-

zontal SWCNT. Electrical properties of individual

areas of the SWCNT layer are compared in Fig. 3 by

typical voltamperic characteristics (VACh) that were

measured by special SPM module TUNA in contact

mode. The tunneling current was measured by the

SPM conductive cantilever tip diameter of which is

about 20 nm.

Figure 2: The SPM image of the surface of the SWCNT

coating on polycarbonate membrane. The surface area of

the image is 5x5 μm

and the maximum height is 400 nm.

In Fig. 3, the surface areas with the lowest and

the highest conductivity are represented by the

VACh measured at the tip-points of individual sur-

face area. The lowest conductivity was obtained for

the tip attached to the horizontal SWCNT (2 in

Fig. 3). The highest conductivity of the SWCNT

layer was found in the areas corresponding to the

pores in the membrane (1 in Fig. 3).

Detailed distribution of the electrical conductivi-

ty over the surface of the SWCNT layer was visua-

lized by scanning of the surfaces with the SPM

TUNA. It was found that the spots of high conduc-

tivity are measured over the flat surface of the mem-

brane if vertical SWCNT are detected in this area.

Comparatively large areas were characterized by

intermediate electric conductivity. It was supposed

that only part of the SWCNT are connected so that

produce conductive mesh of the electrode. The ma-

jor part of the vertical SWCNT is only partly con-

nected to this mesh and, therefore, limits electric

charge transfer from the enzymes to the measure-

ment circuit. This electric limitation reduces the

effectiveness of the biosensor electrode.

Figure 3: Tunneling current versus dc-potential between

the SPM cantilever and the sample.

3.2 Biosensor Characteristics

Since the main advantage of PQQ-dependent dehy-

drogenases is functional independence of oxygen

these enzymes are highly attractive for development

of biosensors (Razumiene et al., 2005; Razumiene et

al., 2006). All these enzymes were chosen also due

to different ability to oxidise a number of carbohy-

drates. Thus, integration of these biosensors in

whole sensing system allows detecting broad range

of sugars, encompassing clinically important such as

lactose, galactose, maltose and et cetera that is

usually not detectable in body fluids although are

associated with several diseases. In spite of numer-

ous modifications of these enzymes that can be

acceptable for detection of various important com-

pounds we probed only a few types in this study.

Typical calibration curve for glucose obtained

using s-PQQ-GDH based biosensor is shown in

Fig. 4.

In Fig. 4, the current generated at the electrode

during electrocatalytic oxidation of glucose by the

enzymes was measured as a function of glucose

concentration in the solution. Similar dependences

were measured for all types of biosensors manufac-

tures and probed in this work.

Kinetic characteristics, namely the apparent Mi-

chaelis constant (K

app

) and maximum current (I

app

), calculated for each type of the biosensors are

summarized in Table 1.

BIODEVICES 2009 - International Conference on Biomedical Electronics and Devices

450

Table 1: Kinetic characteristics of SWCNT-based biosen-

sors with different enzymes.

Biosen-

sor type

app

max

app

μA

n r

s-ADH 305.4 42 7 0.9913

s-GDH 5 27 12 0.9945

m-GDH 0.11 10 8 0.9986

GOx 5.8 260 7 0.9870

Results in Table 1 shows that enzymes possess

different kinetics of action. From the functional

standpoint, there are also different, i.e. (s-) soluble

types operate in cytoplasma and (m-) membrane-

bound is tightly bound to the outer surface of the

cytoplasmic membrane (Matsushita et al., 2003). It

has been shown that they are different enzymes with

different pH-optima, molecular weights and sub-

strate specificity.

Figure 4: The calibration curves of the biosensors based

on direct immobilization of s-PQQ-GDH on SWCNT (1)

and with changeable s-PQQ-GDH film attached to

SWCNT electrode (2). C

is glucose concentration in the

solution.

In our previous paper (Laurinavicius et al., 2004)

has been demonstrated that due to the immobiliza-

tion the active center of enzyme can be distorted that

leads to different affinity to substrates as in the case

of the native enzyme. Aiming to evaluate the affinity

of enzymes operating in heterogeneous biosensing

systems, the selectivity to clinically important meta-

bolites such as glucose, lactose, galactose and mal-

tose were investigated for all types of the proposed

biosensors. The responses to individual metabolites

were represented by the signal ratio with respect to

the detection of 100 % glucose. The results are

summarized in Table 2.

In order to understand an influence of the en-

zyme immobilization method of the of s-PQQ-GDH

on the the selectivity and main kinetic parameters of

the biosensor we investigated the electrodes based

on SWCNT and carbon paste electrodes (CE) and

manufactured by the method previously described in

(Razumiene et al., 2006). The K

app

and I

max

app

pa-

rameters for three types of the electrodes are sum-

marized in Table 3.

The I

max

app

for all substrates can be explained by

the s-PQQ-GDH catalyzed oxidation of main sub-

strates such as glucose, lactose, and galactose with

almost the same rate ratio for all probed types of

biosensors (Table 3). However, the kinetic parame-

ters are individual for the biosensors with differently

immobilized enzymes (Table 3). The increase in

app

results in extension of the interval of the linear

calibration curve. We associate it with diffusion

limited access of the substrate.

The stability of the s-PQQ-GDH and GOx based

biosensors was investigated during couple of weeks.

The responses to the standard glucose solution (5

mM) were periodically recorded at room tempera-

ture equal to about 25 °C during these experiments.

The residual response of the probed biosensors was

not less than about 80 % of initial magnitude over

the period of the tests.

4 CONCLUSIONS

Original technology was developed and probed for

manufacturing of prototype biosensors with change-

able selectivity. The template of the electrodes has

been prepared on the basis of SWCNT conductive

layer deposited on the polycarbonate membrane.

The attachable flexible matrix with immobilized

enzymes was proved functionally acceptable for

catalysis of biochemical reactions and detection of

these reactions. Vertical arrangement of the SWCNT

in the electrodes was related to the areas of high

electric conductivity of the electrodes that was as-

sumed essential for functioning of the biosensors.

Using glucose oxidase, two types of pyrroloqui-

noline quinone dependent glucose dehydrogenases

(namely s-PQQ-GDH, m-PQQ-GDH) and water-

soluble aldose sugar dehydrogenase s-PQQ-ADH

four versions of the prototype biosensors were

manufactured and investigated in this work. In the

tests, the responses to clinically important metabo-

lites such as glucose, lactose, galactose, arabinose,

manose and glucose-6-phosphate were measured. It

was

NOVEL COMBINED TEMPLATE FOR AMPEROMETRIC BIOSENSORS WITH CHANGEABLE SELECTIVITY

451

Table 2: Responses to different substrates of SWCNT-based biosensors.

Biosensor type Glucose Lactose Galactose Manose Arabinose Glucose-6-

phosphate

s-ADH 100 61 120 7 102 60

s-GDH 100 95 99 87 68 15

m-GDH 100 0 76 57 72 91

GOx 100 0 3 1 0 0

Table 3: Kinetic parameters of SWCNT-based and CE-based biosensors with differently immobilised s-PQQ-GDH.

Substrate

CE, enzymes on tery-

lene

SWNT, enzymes on

terylene

enzymes adsorbed on

max

app

μA

app

max

app

μA

app

max

app

μA

app

Glucose 5.2 4.8 23.4 9 0.37 5.7

Lactose 4.7 8.1 21.7 7.2 0.3 8

Galactose 3.6 10.1 10.4 4.2 0.24 4

proved that the prototype biosensors are sufficiently

stable so that can be acceptable for practical use.

ACKNOWLEDGEMENTS

The study was partly supported by the Lithuanian

State Science and Studies Foundation contracts no.

N-08007 and N-09/2008. It was also partly sup-

ported by COST programme contract no. 31V-119.

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