Scholarly article on topic 'Gold nanoparticle-modified ultramicroelectrode arrays: A suitable transducer platform for the development of biosensors'

Gold nanoparticle-modified ultramicroelectrode arrays: A suitable transducer platform for the development of biosensors Academic research paper on "Chemical sciences"

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{"ultramicroelectrode array / gold-nanoparticle / Horseradish peroxidase / amperometric biosensor / catechol"}

Abstract of research paper on Chemical sciences, author of scientific article — Jahir Orozco, César Fernández-Sánchez, Cecilia Jiménez-Jorquera

Abstract This work reports on the performance of gold ultramicroelectrode arrays (UMEAs) modified with gold nanoparticles (GNP) for the development of biosensors. As a proof of concept an amperometric peroxidase-based biosensor for the detection of phenolic compounds was developed. GNPs were electrodeposited on to the UMEA surface, thereby increasing its active area up to 100 times but without affecting its inherent electrodic properties. Horseradish peroxidase enzyme (HRP) was covalently immobilized over the electrodes by means of a thiol self-assembled monolayer (SAM). The resulting biosensors were applied to the amperometric detection of catechol. The use of GNP-modified UMEAs increased the sensitivity of the biosensor compared with the values achieved with bare UMEAs and microelectrode based biosensors.

Academic research paper on topic "Gold nanoparticle-modified ultramicroelectrode arrays: A suitable transducer platform for the development of biosensors"

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Procedía Chemistry

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Procedía Chemistry 1 (2009) 666-669

www.elsevier.com/locate/procedia

Proceedings of the Eurosensors XXIII conference

Gold nanoparticle-modified ultramicroelectrode arrays: A suitable transducer platform for the development of biosensors

Jahir Orozco*, César Fernández-Sánchez and Cecilia Jiménez-Jorquera

This work reports on the performance of gold ultramicroelectrode arrays (UMEAs) modified with gold nanoparticles (GNP) for the development of biosensors. As a proof of concept an amperometric peroxidase-based biosensor for the detection of phenolic compounds was developed. GNPs were electrodeposited on to the UMEA surface, thereby increasing its active area up to 100 times but without affecting its inherent electrodic properties. Horseradish peroxidase enzyme (HRP) was covalently immobilized over the electrodes by means of a thiol self-assembled monolayer (SAM). The resulting biosensors were applied to the amperometric detection of catechol. The use of GNP-modified UMEAs increased the sensitivity of the biosensor compared with the values achieved with bare UMEAs and microelectrode based biosensors.

Keywords: ultramicroelectrode array, gold-nanoparticle, Horseradish peroxidase, amperometric biosensor, catechol

1. Introduction

Ultramicroelectrode arrays have gained importance in electrochemical analysis and sensor technology over the past two decades mainly due to the greatly enhanced electrochemical response that they exhibit compared with conventional microelectrodes [1, 2]. They have been extensively used for the anodic stripping voltammetric detection of heavy metals [1, 3]; however their use for the development of biosensors has been scarce. Regarding gold nanoparticles, they enable the electronic or optical transduction of a wide variety of biological phenomena and provide a microenvironment similar to that of the redox proteins in native systems, thus allowing immobilization of proteins without affecting their bioactivity [4]. In this context, GNPs were electrodeposited onto a UMEA surface, thus increasing its surface area while keeping its inherent electrode features [2].

This work aims to demonstrate the enhanced voltammetric performance of biosensors fabricated using GNP-modified UMEAs compared to gold bare microelectrodes and UMEAs. HRP was covalently immobilised on the transducers surface by means of SAMs. Considering the blocking effect of such SAMs on the electron transfer process of the catechol target analyte, initial experiments were carried out to study the formation and partial

Corresponding author: Jahir Orozco, Instituto de Microelectrónica de Barcelona (IMB-CNM), CSIC, Campus UAB, Bellaterra, 08193, Spain. Tel: +34-935947700, Fax: +34-935801496. E-mail address: jahir.orozco@imb-cnm.csic.es

Instituto de Microelctrónica de Barcelona (IMB-CNM), CSIC, Campus UAB, Bellaterra, 08193, Spain

Abstract

1876-6196/09/$- See front matter © 2009 Published by Elsevier B.V. doi:10.1016/j.proche.2009.07.166

desorption of them. Selective desorption was carried out in order to get defective SAMs and an enhanced catechol electron transfer reaction. This step was unnecessary when working with GNP-modified UMEAs.

Responses of the different biosensor approaches were recorded. A catalytic current was clearly observed in all cases, which could be ascribed to the electrochemical reduction of the o-quinone species generated in the HRP reaction with catechol. A significant increase in sensitivity was attained when working with the GNP-modified UMEAs, which demonstrates that this transducer platform and the resulting biosensor approach could be seen as a rapid and simple alternative for environmental analysis compared to available standardised analytical methods [5].

2. Experimental Section

Peroxidase Type II from Horseradish 181 Umg-1, catechol, 20-nm gold nanoparticle stock solution containing 0.01% HAuCl4 and 0.02% sodium azide, hydrogen peroxide, and dithiobis-N-succinimidyl propionate (DTSP) were purchased from Sigma-Aldrich Química S.A. (Spain). All other chemicals were of analytical grade. Solutions were prepared using 18 MQ.cm deionised water.

Voltammetric experiments were performed at room temperature using a type III ^-Autolab potentiostat [1], controlled with GPES 4.7 (General Purpose Electrochemical System) software package. Measurements were carried out with a three electrode cell consisting of a UMEA working electrode or, alternatively, a microelectrode, an on-chip counter electrode and a Ag/AgCl/10% (w/v) KNO3 reference electrode (Metrohm 0726 100). A miniaturised Ag/AgCl pseudo-reference electrode RC6 (50-mm length, 1.5-mm external diameter from World Precision Instruments) was used for electrodeposition experiments.

Au microelectrodes and ultramicroelectrode arrays (UMEAs) used in this work were fabricated at the IMB-CNM facilities with standard photolithographic techniques using Si/SiO2/metal structures, as described elsewhere [2]. Both microelectrode and UMEAs included a counter gold electrode on chip, separated 0.5 mm from the working electrode.

Microelectrodes and UMEAs were initially cleaned and activated following the protocols previously established in [2]. Cyclic voltammograms in a 0.1 M KNO3 background solution containing 1 mM catechol were then recorded in a potential window between +0.35 V and +0.15 V, at 0.1 V.s-1, in order to evaluate the electrochemical surface active area. Electrodeposition of GNPs on the electrode surface was also carried out following a procedure described elsewhere [1]. Cyclic voltammetric scans in a potential window between -0.2 V and +1.4 V in 0.1 M H2SO4 /0.1 M KNO3 at a scan rate of 0.1 V.s-1 were carried out to estimate the microscopic surface area of the UMEAs [2].

SAMs were prepared by immersing the Au substrates in a 4 mM DTSP solution, dissolved in DMSO, for 1 h at room temperature. Then, the modified electrodes were thoroughly rinsed with DMSO and water to remove any weakly-adsorbed thiol molecules. The surface coverage was estimated from the peak areas of the cyclic voltammetric peaks ascribed to the reductive desorption of the adsorbed thiols [6]. These measurements were carried out in a N2-saturated 0.5 M KOH solution. The potential was scanned between -0.3 V and -1.3 V, at 50 mVs-1. The percentage of bare electrochemical surface was also estimated by cyclic voltammetric measurements carried out in a 0.1 M KNO3 solution containing 1 mM catechol, in the same fashion as above.

HRP was immobilized on to the SAM-modified electrodes by immersing them in 0.05 M phosphate buffer saline solution pH 7.4 (PBS), containing 10 mg.ml-1 HRP, overnight at 4°C. Following a washing step in PBS, the electrodes were immersed in a PBS solution containing 0.05 % Tween 20 for 1 h under stirring, in order to remove any adsorbed HRP molecules from the electrode surface.

2. Results and discussion

Gold microelectrodes were initially modified with SAMs fallowing the conditions explained in Experimental Section. Figure 1 shows the voltammetric responses corresponding to the thiol desorption from the different transducer platforms. Voltammograms recorded in a deoxygenated 0.5 M KOH solution with SAM-coated microelectrodes show three cathodic peaks at -0.6 V, -0.9 V and -1.1 V (Fig. 1a), related to the reductive desorption of the corresponding gold thiolated species from a polycrystalline Au surface. The presence of more than one desorption peak might be related to the thiol binding strength differences at either the exposed Au crystalline faces

[7] or at Au domain and domain edges or boundaries [8]. In the case of SAM-coated UMEAs three waves can be distinguished (Fig. 1b), one well-defined peak at -0.9 V and a couple of small shoulders at -1.0 V and -0.8 V.

Electro deposition of GNP highly increased the UMEAs surface area without affecting the electrode voltammetric response. Cathodic desorption of SAMs at GNP-modified UMEAs gave rise to well-defined peaks at potentials of -0.73 V, -0.93 V and -1.15 V. The peaks indicate that the new surface contains also terrace and step edges and also slightly differences in affinity towards the thiol molecules. Potential peaks were slightly more negative than those recorded with the bare UMEAs and microelectrodes, which might be related to a stronger interaction of the thiolated molecules with the electrodeposited GNP, compared with the thiol reaction with bulk gold, as previously reported [9].

Selective desorption of thiols was carried out in order to get a defective SAM, with a higher number of bare domains, and thus an enhanced catechol electron transfer reaction at the electrode solution interface. This step was unnecessary when working with GNP-modified UMEAs.

E / V ra Ag/AgCl E / V ra Ag/AgCl E / V ra Ag/AgCl

Fig. 1. Linear sweep voltammograms corresponding to the reductive desorption of a DTSP SAM at (a) microelectrode, (b) UMEA and (c) GNP-modified UMEA. Signals were recorded in N2-saturated 0.5 M KOH solution.Scan rate: 0.05 Vs-1

To test the performance of the platform to develop a biosensor, HRP was covalently attached to the different transducer approaches. HRP/DTSP/microelectrode, HRP/DTSP/UMEA and HRP/DTSP/GNP-modified UMEA were interrogated by cycling voltammetry. Catechol was chosen as a redox mediator and as a target analyte. No direct electron transfer between the heme group of the HRP structure and the electrode took place. However, anodic and cathodic faradaic currents -due to the catechol electrodic process- when the three different devices were immersed in PBS containing 0.5 mM catechol, were recorded. When H2O2 was added to the catechol solution, a large cathodic current was recorded in all cases, thus evidencing the catalytic process that takes place. Figure 2 shows the higher catalytic current density obtained with the HRP/DTSP/GNP-modified UMEA (Figure 2c) compared with those from the other two HRP-biosensor platforms (Figures 2a and 2b, respectively). Amperometric experiments were carried out at a set potential of -0.1 V vs. Ag/AgCl, selected on the basis of the catalytic currents recorded previously. The dynamic response of the electrodes was linear in a concentration range from 0.05 mM to 0.4 mM. Again, the improved amperometric response of the HRP/DTSP/GNP-modified UMEA with respect to the others was clearly shown. This was the expected behaviour considering the advantageous electrodic properties of the GNP-UMEA devices. The corresponding calibration parameters for this biosensor approach, in the linear concentration range studied, shows a sensitivity of 228.6 ^Acm-2mM-1. This value is around 3-fold and 80-fold higher than the values obtained with the HRP/DTSP/UMEA and the HRP/DTSP/microelectrode, respectively. The RSD values, obtained with three different biosensors of each configuration, were always below 10 %, which indicate the good reproducibility attained. The repeatability was below 6 % (n=5) in all cases.

Overall, it was showed the feasibility of the obtained platforms for the immobilization of biomolecules and the superior performance achieved when the UMEAs devices were modified with gold nanoparticles.

Fig. 2. Biosensor cyclic voltammetric responses of (a) HRP/DTSP/microelectrode, (b) HRP/DTSP/UMEA, and (c) HRP/DTSP/GNP-modified UMEA. Inset, amplified signal a. Signals were recorded in pH 7.4, 0.05 M PBS, 0.5 mM catechol and 0.1 mM H2O2. Scan rate: 0.005 Vs-1

-0.3 -0.2 -0.1 0.0 0.1 0.2 0.3 0.4

E / V vs Ag/AgCl

3. Conclusions

GNP-modified UMEAs are shown to be an excellent platform for the development of biosensors. The comparative study carried out with microelectrodes reveals the higher sensitivities that can be attained when working with UMEA-based devices. The simple electrodeposition process of GNP further improves the biosensor performance very highly. The benefits of this approach were demonstrated by developing a HRP biosensor for the detection of catechol, chosen as a model analyte, and bearing in mind the potential use of such platforms in the development of environmental applications.

Acknowledgements

J. Orozco was supported by a research studentship from FPI Program, MEC, Spain. C. Fernández-Sánchez thanks the MICINN and CSIC for the award of a Ramon y Cajal Contract. Funding from the TEC2007-68012-C03-02/MIC and the CSD2006-12.4615 Consolider Ingenio 2010 projects from MEC (Spain) is also acknowledged.

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3. Beni V, Arrigan D, Microelectrode Arrays and Microfabricated Devices in Electrochemical Stripping Analysis. Cuerrent Analytical Chemistry 2008;4:(3), 229-241.

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5. Ong CP, Lee HK, Li SFY, Optimization of mobile-phase-composition for high-performance liquid-chromatographic analysis of 11 priority substituted phenols. Journal of Chromatography 1989;464:405-410.

6. Yoon L, Lee S, Lennox RB, Electrochemical desorption of n_alkylthiol SAMs on policrystalline gold: studies using a ferrocenylalkylthiol probe. Langmuir 2007;23:292-296.

7. Strutwolf J, O'Sullivan CK, Microstructures by selective desorption of self-assembled monolayer from polycrystalline gold electrodes. Electroanalysis 2007;19:1467-1475.

8. Lee LYS, Lennox RB, Electrochemical desorption of n-alkylthiol SAMs on polycrystalline gold: Studies using a ferrocenylalkylthiol probe. Langmuir 2007;23:292-296.

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