IMMUNOSENSORS FOR ATRAZINE DETECTION

IN RED WINE SAMPLES

Enrique Valera, Ángel Rodríguez

Micro and Nano Technologies Group (MNTg), Departament d’Enginyeria Electrònica

Universitat Politècnica de Catalunya,C/. Jordi Girona 1-3 Campus Nord, Mòdul C4, Barcelona 08034, Spain

Javier Ramón-Azcón, Francisco J. Sanchez, M.-Pilar Marco

Applied Molecular Receptors Group (AMRg), IIQAB-CSIC

CIBER of Bioengineering, Biomaterials and Nanomedicine, Jordi Girona 18-26, 08034 Barcelona, Spain

Keywords: Immunosensor; Interdigitated μ-electrodes, Atrazine; Impedance spectroscopy, Conductive measurements,

Wine matrix effect, Food safety.

Abstract: Two novel immunosensors, one impedimetric and other one conductimetric, for atrazine detection in red

wine samples have been developed. Impedimetric immunosensor is based on an array of interdigitated μ-

electrodes (IDµEs) and bioreagents specifically developed to detect this pesticide. Conductimetric

immunosensor incorporates additionally gold nanoparticles. Bioreagents were covalently immobilized on

the surface of the electrodes (interdigital space). In both cases the biochemical determination of atrazine is

possible without any redox mediator. For the case of the impedimetric immunosensor, the detection method

is based on impedimetric measurements (in a wide range of frequencies), whereas in the case of the

conductimetric immunosensor the detection method is based on conductimetric measurements (DC

measurements).The potential of the impedimetric immunosensor to analyze atrazine in complex sample

matrices, such as red wine, have been evaluated. This immunosensor can detect atrazine with limits of

detection in the order sub-ppb, far below the maximum residue level (MRL) (50 µg L

−1

) established by

European Union (EU) for residues of this herbicide in the wine grapes.

1 INTRODUCTION

In the recent years, modern chemical analysis has

been revolutionized by the electrochemical

biosensors because of their accuracy, technical

simplicity, high efficiency, possibility of portability

and miniaturization, and because they offer fast

(instantaneous) response times, allow a rapid and

permanent control and a direct transduction of the

biomolecular recognition event into electronic

signals (Murphy, 2006; Pumera, 2007; Wang, 2006).

An important disadvantage of the

electrochemical sensors is that the impedance

changes due to biomolecular recognition are

generally very small and it can not reach the

necessary detection limits required by the

legislation. However, this disadvantage can be

solved by applying techniques such as

electrochemical impedance spectroscopy (EIS) or

including labels that amplify the signal.

By means of EIS is possible to record

information on biorecognition events, occurring at

the electrode surfaces, inducing impedance changes

(Guan et al., 2004; Katz and Willner, 2003), that can

be directly measure, allowing the development of

label-free biosensing devices.

On the other hand, many types of labels are used

to amplify biorecognition events. Between the

labels, the gold nanoparticles are some of the most

recently used (Pumera, 2007; Zhang, 2007), because

its unique properties at nanoscale dimension.

In this work we report the description of two

immunosensors for atrazine detection. One

immunosensor is label-free, whereas the other one is

labelled with gold particles (40 nm). In the case of

the label-free immunosensor, impedimetric

measurements (impedance spectroscopy) are used as

detection method. In the second case, due the

presence of the gold particles, conductimetric

measurements (DC measurements) could be used.

226

Valera E., Rodríguez Á., Ramón-Azcón J., J. Sanchez F. and Marco M. (2009).

IMMUNOSENSORS FOR ATRAZINE DETECTION IN RED WINE SAMPLES .

In Proceedings of the International Conference on Biomedical Electronics and Devices, pages 226-230

DOI: 10.5220/0001542002260230

 SciTePress

In order to demonstrate the applicability of the

devices developed, wine production has been

selected as scenario for the proof-of-concept study.

Reasons for this selection are because wine is a high

value product (with a great economic relevance in

EU) and because their strong matrix effect. In fact, if

the sensors are demonstrated to red wine samples,

their use can be extrapolated to many other matrices.

Likewise, we will prove that the impedimetric

immunosensor can respect the Maximum Residue

Level (MRL) established by EU (European Union)

for residues of this herbicide in wine grapes (50 µg

-1

2 EXPERIMENTAL

2.1 Instrumentation

Impedimetric and conductive measurements were

carried out at room temperature in a probe station

(Faraday cage) KARL SUSS. Impedance analyses

were performed using an Agilent 4294A Precision

Impedance Analyzer and conductive measurements

were performed using an Agilent 4156C

Semiconductor Parameter Analyzer. The

competitive curves were analyzed with a four-

parameter logistic equation using the software

SoftmaxPro v2.6 (Molecular Devices) and GraphPad

Prism version 4.00 for Windows (GraphPad

Sofware, San Diego California USA). Data shown

correspond to the average of at least three replicates

per concentration of atrazine.

2.2 Arrays of Interdigitated

μ-electrodes

The immunosensors developed are based on a two

coplanar non-passivated interdigitated metallic μ-

electrodes. Thin Au/Cr (200 nm thickness)

interdigitated μ–electrodes with 10 µm pitch were

patterned on a Pyrex 7740 glass substrate (purchased

from Präzisions Glas&Optik GmbH, 0.7 mm (±0.05)

thickness). For the immunosensor measurements,

electrode arrays were constructed consisting on six

IDµEs organized on a 0.99 cm

area. The Pyrex

substrate was first cleaned using absolute ethanol.

Following metal deposition was performed by

sputtering and the interdigitated μ-electrodes were

patterned by a photolithographic metal etching

process. The chromium layer, much thinner than the

gold layer, was deposited prior the gold to improve

adhesion to the Pyrex substrate.

2.3 Immunosensors Functionalization

Biofunctionalization with 2d-BSA was done

selectively on the surfaces of the gold electrodes.

The chemical recognition layer was covalent

immobilized on the interdigital space. For this

purpose the array of IDµEs were treated as follows.

2.3.1 Surface Cleaning

Before functionalization, the IDµEs samples were

first cleaned with a combination of ethanol:Mili-Q

water 70:30, absolute ethanol absolute, piranha

solution and absolute ethanol.

2.3.2 Surface Activation

After the pre-treatment explained above, surface

activation took place in two steps to modify

selectively the gold electrodes and the Pyrex

substrate.

Activation of gold surfaces was readily and

specifically performed using thiol-chemistry. N-

acetylcysteamine was used to cover the gold

electrodes and to protect the sensor from undesired

non-specific absorptions. Thus, the surface texture

of the IDµE defines the template for deposition of

layers, since the gold fingers have been deposited on

a solid support such as glass with the necessary

controlled geometry. In a second step, the Pyrex was

derivatized with 3-glycidoxypropyl trimethoxysilane

(GPTS). The epoxy group provided the necessary

reactivity for further attachment of the bioreagents

through a nuchelophylic attack of functional groups

of the biomolecule such as the amino groups of the

lysine residues (Luzinov, 2000). The immunosensor

surface functionalization is schematically shown in

Figure 1.

2.3.3 Antigen Immobilization

Covalent immobilization of the pesticide antigen 2d-

BSA was performed on the interdigitated μ-

electrodes surface via the amino groups of the lysine

residues by reaction with the epoxy groups of the

surface.

2.4 Competitive Assay

The assay of detection, common for both

immunosensors, relies on the immunochemical

competitive reaction between the atrazine residues

and the immobilized antigen on IDµEs for a small

amount of the specific antibody (primary antibody).

The detection of a small number of molecules of

IMMUNOSENSORS FOR ATRAZINE DETECTION IN RED WINE SAMPLES

227

atrazine is performed under competitive conditions

involving the competition between the free pesticide

(analyte) and a fixed amount of coated antigen for a

limited amount (low concentration) of primary

antibody (Ab

). At the end of the reaction the

amount of Ab

captured on the IDµE surface and

hence the free antigen (analyte) is determined.

Finally, and only in the case of the

conductimetric immunosensor, a secondary labelled

with gold antibody (Ab

) is captured in order to

amplify the conductive signal. The immunosensor

assay is schematically shown in Figure 2.

2.5 Impedance Measurements

Impedance measurements were carried out at room

temperature. No redox mediator was used in the

devices presented in this work. The two electrodes

were covered by a diluted PBS solution with a

conductivity of 1.6 µS cm

−1

and connected to the

input of an Agilent 4294A Precision Impedance

Analyzer by means of standard probe tips.

Measurements were taken in the 40 Hz to 1MHz

frequency range using 0V of polarization potential

and a modulation voltage of 25mV amplitude. All

impedance measurements were performed in a

Faraday cage.

2.6 Conductance Measurements

As for impedance measurements, the conductance

measurements were also carried out at room

temperature without any redox mediator and in a

Faraday cage. The two electrodes were covered by a

diluted PBS solution with a conductivity of 1.6 µS

−1

and connected to the input of an Agilent 4156C

Semiconductor Parameter Analyzer by means of

standard probe tips. Conductivity was measured to

+25 mV, from +22.5 to +27.5 mV sweep bias. These

conductive measurements were performed after the

incubation step of the secondary antibody labelled

with gold.

3 ATRAZINE DETECTION

In the case of the impedimetric immunosensor, the

quantitative tool that seems adequate to provide

sensitivity graphs is the impedance measurement in

a wide frequency range and the fitting of the Nyquist

plots of impedance spectra to an equivalent circuit.

Then, the atrazine concentration should finally

be related to the values of at least some of the

parameters of the equivalent circuit. The equivalent

circuit used was previously reported (Valera, 2007)

and the resistance of the solution (Rs) was chosen as

parameter of analysis.

Interdigitated μ-electrode (IDµE)

IDµEs protection with N-Acetylcysteamine

inter-digits space functionalization with GPTS

Figure 1: Schematic diagram of: i) protection of

interdigitated μ-electrodes with N-acetylcysteamine; and

ii) immunosensor surface functionalization with GPTS.

NH2

SO2NH

NH2

SO2NH

NH2

SO2NH

NH2

SO2NH

NH2

SO2NH

NH2

SO2NH

NH2

SO2NH

NH2

SO2NH

coated antigen

primary

specific

antibody

NH2

SO2NH

NH2

SO2NH

NH2

SO2NH

NH2

SO2NH

NH2

SO2NH

NH2

SO2NH

NH2

SO2NH

NH2

SO2NH

NH2

SO2NH

NH2

SO2NH

NH2

SO2NH

NH2

SO2NH

NH2

SO2NH

NH2

secondary

antibody

labelled wit

gold

nano

articles

Figure 2: Schematic diagram of the complete assay system

performed on the IDµEs for the immunosensors

developed.

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228

For the conductimetric immunosensor, the

measurement of the conductance after the capture of

the secondary labelled with gold antibody could be

used as detection method, as it was previously

demonstrated using PBS in the competitive assay

(Valera, 2008). Then, the atrazine concentration

would be related to the amount of gold nanoparticles

present on the immunosensor.

The impedimetric response of the immunosensor

in red wine is shown in Figure 3.

To validate the sensing approach, our

immunosensors were also characterized by means of

chemical affinity methods. In Figure 4 the results

obtained from the immunosensor are compared with

the results of an ELISA assay on the same IDµEs

devices but using a chemically raised colorimetric

signal. In order to comparatively show the

immunosensor performance, in Figure 4 the

normalized values of the change in the Rs as a

function of the atrazine concentration as well as the

normalized results of the ELISA assay are plotted.

It is important to remark that the ELISA assay

performed on the IDµE device show a similar

analytical profile that the obtained using microtitrer

plates. Therefore, we could be confident that our

immunosensor really reflects the selective binding

event. The more important analytical features of the

atrazine assays (immunosensors and ELISA) are

shown in Table 1.

As it can be seen in Table 1, using the

impedimetric immunosensor is possible to detect

atrazine in sub-ppb concentrations (0.19 µg L

−1

These good results are directly related to the

advantages of the impedimetric device presented

such as the use of IDµEs and the competitive assay

based on the antibodies variation.

Comparing these results with the recent

literature, the impedimetric immunosensor presented

in this work have been demonstrated to be more

sensitive that other biosensors based on

impedimetric methods for the atrazine detection

(Helali, 2006; Hleli, 2006; Fredj, 2008), specially

taking into account that our immunosensor have

been tested using red wine samples.

-4

-3

-2

-1

400

800

1200

1600

2000

Impedimetric immunosensor

Red wine

[Atrazine, μg L

-1

]

Rs (

)

Figure 3: Impedimetric response when the immunosensor

are used to detect atrazine. Reprinted from Biosensors and

Bioelectronics, 23, J. Ramón-Azcón et al., An

impedimetric immunosensor based on interdigitated

microelectrodes (IDµE) for the determination of atrazine

residues in food samples, 1367–1373, (2008), with

permission from Elsevier.

-4

-3

-2

-1

100

Impedimetric

Immunosensor

ELISA

[Atrazine, μg L

-1

]

Normalized signals

Figure 4: Normalized calibration curves of the optimized

atrazine immunoassay and the impedimetric

immunosensor presented. Measures were taken in diluted

PBS solution.

Table 1: Features of the atrazine assays in red wine

samples.

Impedimetric

Immunosensor

ELISA

, µg L

-1

1.876±0.23 1.93±0.02

LOD, µg L

-1

0.19 0.09

0.86 0.99

Reprinted from Biosensors and Bioelectronics, 23, J.

Ramón-Azcón et al., An impedimetric immunosensor

based on interdigitated microelectrodes (IDµE) for the

determination of atrazine residues in food samples, 1367–

1373, (2008), with permission from Elsevier.

IMMUNOSENSORS FOR ATRAZINE DETECTION IN RED WINE SAMPLES

229

4 CONCLUSIONS

Two immunosensors, one impedimetric and other

one conductimetric, for the atrazine detection in red

wine samples have been developed. Both devices are

based on an array of IDµEs and in bioreagents

specifically developed. Impedimetric immunosensor

is able to detect atrazine in red wine at sub-ppb

concentrations, far below the Maximum Residue

Level (MRL, 50 µg L

−1

) required by EC. However,

this result could be improved using the

conductimetric device, which includes secondary

antibodies labelled with gold particles that increase

the conductive signal.

ACKNOWLEDGEMENTS

This work has been partially supported by the

Ministry of Science and Technology (Contract

number TEC2007-67081) and FEDER funds. The

MNT group is a consolidated Grup de Recerca de la

Generalitat de Catalunya since the year 2001

(expedient 00329). The AMR group is a

consolidated Grup de Recerca de la Generalitat de

Catalunya and has support from the Departament

d’Universitats, Recerca i Societat de la Informació la

Generalitat de Catalunya (expedient 2005SGR

00207).

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