Controlled electrophoretic deposition of multifunctional nanomodules for bioelectrochemical applications

Biosensors and Bioelectronics 24 (2008) 55–59

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Controlled electrophoretic deposition of multifunctional nanomodules for bioelectrochemical applications Srujan Kumar Dondapati 1 , Pablo Lozano-Sanchez ∗ , Ioanis Katakis Bioengineering and Bioelectrochemistry Group, Dept. d’Enginyeria Qu´ımica, Escola T`ecnica Superior d’Enginyeria Qu´ımica, Universitat Rovira i Virgili, Av. Pa¨ısos Catalans 26, 43007 Tarragona, Spain

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Article history: Received 22 February 2008 Received in revised form 13 March 2008 Accepted 14 March 2008 Available online 29 March 2008 Keywords: Electrophoretic deposition Layer-by-layer deposition Glucose sensing Gold nanoparticles Redox polymer Glucose oxidase Microelectrodes

a b s t r a c t The design of an electrochemical glucose sensing device formed by the electrodeposition of multifunctional Au nanoparticles is reported here as a novel concept for an enhanced generic sensing platform. Initially gold nanoparticles (Au) were alternatively coated with a layer of positively charged redox polymer (ORP) and a negatively charged glucose oxidase (GOX) layer alternatively using layer-by-layer methodology to form multifunctional Au/ORP/GOX/ORP particles. The modification and stability of the Au nanoparticles was monitored by using UV–vis spectroscopy and zeta-potential measurements. The modified Au nanoparticles were electrophoretically deposited onto an electrode to produce an electrochemical glucose sensing device. A considerable influence of electrophoretic deposition time and potential was found on the sensing platform response. Preliminary responses to glucose addition showed an enhanced performance by applying an electrophoretic deposition potential of +1.2 V vs. Ag/AgCl for 30 min. The observed response in the case of microelectrode geometry was in the range of mA cm2 . This work also shows that the presence of a second outer ORP layer on the functionalised Au nanoparticles improved the response. © 2008 Elsevier B.V. All rights reserved.

1. Introduction The incorporation of an enzyme as a highly specific catalytic recognition element, coupled with electrochemical (amperometric or potentiometric) transduction of the recognition event, provides the basis for constructing enzyme-based biosensors, biomedical devices and enzymatic bioreactors (Maestre et al., 2005; Dondapati et al., 2006; Mir et al., 2007). Multi-analyte biosensors have an increasingly important application in the field of medical diagnostics, especially in low-cost self-care devices. For this application to be feasible, there is a need to develop simplified generic fabrication methods. When the same principle is used for creation and detection, it is possible to integrate features in the devices that have significant cost lowering effects. A general method was sought that would selectively immobilize proteins with absolute control over orientation and density and that does not require synthetic modification or purification before immobilization. Electrophoretic deposition has been widely applied to fine particle technologies

∗ Corresponding author. E-mail address: [email protected] (P. Lozano-Sanchez). 1 Present address: Photonics and Optoelectronics Group, Department of Physics, ¨ Munchen, ¨ ¨ Ludwig-Maximilians-Universtitat Amalienstr. 54, D-80799 Munchen, Germany. 0956-5663/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2008.03.022

like advanced ceramic materials. Slowly this technique has been gaining increasing interest in the field of biomedical applications (Wang and Hu, 2003). Electrochemically controlled co-deposition of enzymes with other proteins such as collagen and bovine serum albumin (BSA) has been reported (Johnson et al., 1994). Using this electrophoretic deposition approach, controlled immobilization of GOX on a platinum electrode has been achieved in the presence of a non-ionic detergent, Triton X-100 producing a multilayer films (Matsumoto et al., 2002). Layer-by-layer electrodeposition of redox polymer/enzyme composition films on screen-printed carbon electrodes for fabrication of reagentless enzyme biosensors has been used for producing a very stable and rigid films (Gao and Yang, 2004). A glucose biosensor based on the one-step coelectrodeposition of an ORP and GOX on a Au electrode surface has been developed (Fei et al., 2003). Electrodeposition of hydrated redox polymers and co-electrodeposition of enzymes through coordinate crosslinking has been proved to produce stable enzymes films with excellent catalytic activity (Gao et al., 2002). The purpose of these strategies is to immobilize enzyme in a highly active state. Nanoscale particles offer a variety of interesting properties, and there is growing interest in their assembly into higher ordered structures that can be used in a variety of biosensor applications. Metal and semiconductor nanocrystals have tunable properties (e.g., optical, electronic, and magnetic) that depend on particle size, interparticle spacing, and higher order structure (Jena et al.,

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Scheme 1. Schematic of the Au nanoparticle modification and electrode construction.

2006; Raschke et al., 2003, 2004; Peng et al., 2005). Nanoparticles can display four unique advantages over macroelectrodes when used for electroanalysis: enhancement of mass transport, catalysis, high effective surface area and control over electrode microenvironment. Despic and Pavlovic (1984) introduced for first time the electrophoretic deposition technique for the deposition of Au nanoparticles on the electrode surface. Since then, this technique was used for the patterning of nanoparticles on surfaces for different applications (Crumbliss et al., 1992; Trau et al., 1996; Bharathi et al., 2001; Wei et al., 2005). Based on this technique Crumbliss et al. (1992) have developed biosensors for peroxide or glucose have been developed. We have previously reported the use of the electrophoretic deposition of oligonucleotide modified nanomodules for obtaining DNA arrays (Campas et al., 2006). However little has been done to exploit this technique through depositing enzyme-modified nanoparticles to specific locations by controlled deposition potential. In this work we report controlled electrophoretic deposition followed by investigation of the sensitivity and specificity of enzyme-modified nanomodules. Layer-by-layer (LBL) assembly is a simple technique that allows the construction of thin films by sequential exposure of polycationic and polyanionic solutions in the nanoscale. This approach has been used for the construction highly stable enzyme biosensors (Calvo et al., 2001; Ferreyra et al., 2003). Recently LBL approach has been used to produce highly stable nanocapsules by modifying Au nanoparticles with positive and negative polyelectrolytes just controlling the electrostatic interactions (Schneider and Decher, 2004). In this work stable multifunctional nanomodules are reported by modifying Au nanoparticles with ORP (as electrochemical functionality) and GOX (as biofunctionality) using similar layer-by-layer approach as depicted in Scheme 1. The methodology shown in this work also reveals good potential control in the electrophoretic deposition of the modified nanoparticles. Additionally the response of these modified multifunctional nanomodules to glucose was also studied on microelectrode configurations to reveal a great enhancement of the signal recorded, which shows the potential of this modified nanomodules to be integrated in microelectrode arrays for the production of electrochemical sensing devices or multifunctional modified nanoreactors. 2. Experimental 2.1. Reagents and materials Glucose Oxidase was purchased from Biozymes, UK. The rest of chemicals used were purchased from Sigma–Aldrich, and used as received. Gold working electrodes of 0.5 mm and 50 ␮m diameter were fabricated by resin-sealing (Mecaprex M42, PRESI, France) gold wire (Advent, Oxford, UK) within a glass capillary. Next the electrodes were polished using different grades alumina slurries in a polishing cloth (Buehler). All solutions were made with purified distilled water obtained from a Milli-Q water system.

Absorption spectra were measured with an HP 8453 spectrophotometer. Zeta-potential measurements were done using zeta sizer (MALVERN, UK). Electrochemical experiments were performed using an AUTOLAB PGSTAT 10 potentiostat (Eco Chemie, Netherlands) in a conventional three-electrode cell. An Ag/AgCl electrode (BAS, UK) was used as reference electrode and a platinum gauze as counter electrode. 2.2. Preparation of the Au/ORP/GOX/ORP nanoparticles Osmium redox polymer was prepared as described previously (Cregg and Heller, 1991). Layer-by-layer formation of the ORP/GOX/ORP structure on the gold nanoparticles was done in a step by step manner starting with the modification of gold nanoparticles with cationic ORP followed by the modification with GOX and finally again with ORP. Gold nanoparticles (∼30 nm) were synthesized using citrate reduction method (Frens, 1973). After washing the nanoparticles three times with distilled water to eliminate impurities, the concentrated pellet collected was diluted 1:20 by adding 10 mg mL−1 cationic ORP solution drop by drop of while stirring and then left 12 h to react at room temperature in darkness with continuous stirring. After washing three times with distilled water. The Au/ORP concentrated pellet was diluted 1:20 by adding 10 mg mL−1 of GOX in distilled water solution while stirring and then left 12 h to react at room temperature in darkness with continuous stirring. Given that the isoelectric point of GOX is 4.2, the enzyme in the aqueous media has negative charge, which is essential for the attachment to the ORP modified nanoparticles. The modification with the outer ORP layer was achieved by again diluting 1:20 in ORP solution the Au/ORP/GOX modified nanoparticles, leaving 12 h reaction and washing three times with distilled water. Scheme 1 outlines the different steps involved and the final arrangement in an electrode format. 2.3. Electrode modification The modified gold nanoparticles were electrophoretically deposited onto gold electrodes using a two electrode system (5 mm diameter platinum as counter electrode opposite to a 0.5 mm diameter gold as working electrode) separated by a distance of 1 mm. All depositions were carried out from identical aliquots of a parent solution containing the nanoparticles suspended in milliQ water. All resulting electrodes were thoroughly rinsed with distilled water and dried with nitrogen. The electrophoretic deposition of the modified nanoparticles was studied applying anodic (positive) and cathodic (negative) potentials. In order to investigate the effect of the deposition time and potential upon the response of the nanomodules-modified electrodes to glucose, electrodeposition of modified gold nanoparticles was carried out at different potentials and time periods. Electrochemical characterisation of the modified electrodes was done by cyclic voltammetry scanning from +0.1 V to +0.5 V at a scan rate of 0.1 V s−1 in 0.1 M PBS pH 7.0 to check the

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Bare gold nanoparticle showed a zeta-potential of −0.45 V due to the presence of citrate ions on the surface, while the same nanoparticles modified with the first layer of positive ORP showed a zeta-potential of +0.28 V. After modification with anionic GOX, a charge reversal was observed as expected to −0.23 V. After the second anionic ORP modification, the zeta-potential returned to +0.26 V. UV–vis spectra and zeta-potential measurements suggests that Au nanoparticles were modified with ORP and GOX in a sequential manner successfully. 3.2. Effect of potential on electrode response

Fig. 1. Cyclic voltammograms in 0.1 M PBS pH 7.0 of 0.5 mm diameter nanomodulemodified gold electrodes. Typical example of nanomodules deposited under the optimised conditions of 30 min at +1.2 V. The electrodes were modified with bare nanoparticles, with ORP-GOX and with ORP-GOX-ORP layers. Room temperature 22 ± 1 ◦ C.

quasi-reversible behaviour of ORP, see Fig. 1, where it is compared when there is only an internal ORP layer (inner), and when there are two ORP layers, one internal (inner) and one external (outer). Performance of the sensing platform was monitored at +0.4 V by injecting different concentrations of glucose. 3. Results and discussion 3.1. Charaterisation of Au colloid/ORP/GOX/ORP particles Fig. 2 shows the UV–vis spectral data obtained from the Au colloid coated with ORP and GOX in a sequential manner. Qualitative analysis of the peak signals showed that initially Au nanoparticles gave a characteristic surface plasmon resonance absorption peak at  = 523 nm. Later when the Au nanoparticles were modified with ORP there is a characteristic peak due to ORP close to  = 300 nm, which might be due to the ␲–␲* transition of the bipyridine groups. Later when the Au colloid/ORP was modified with GOX the peaks corresponding to ORP were absent due to the attachment of GOX on the surface of particles. When the Au colloids/ORP/GOX were further modified with a second layer of ORP, the peak corresponding to the ORP was again observed. Moreover Au nanoparticles did not show any aggregation as observed from the absence of change in the characteristic surface plasmon peak characteristic of Au nanoparticles.

Fig. 2. UV–vis spectroscopy of the Au nanoparticles modified layer-by-layer with ORP/GOX/ORP.

The electrophoretic deposition of the modified nanoparticles was studied applying anodic and cathodic potentials. The results showed a response of the nanoparticles-modified electrodes to glucose addition almost 70 times higher from electrodes modified by applying +1.2 V than those modified by applying −1.2 V. Looking at these results, the mechanism controlling the electrophoretic deposition seemed controlled by the gold nanoparticle and by the ORP outer layer, which it has been reported to be easily deposited at positive potentials (Gao et al., 2002). Fig. 3(A) shows the effect of the applied electrodeposition potential on the response to the glucose. The maximum current is obtained after electrophoretic deposition at +1.2 V over those currents obtained after +1.6 V applied potential, suggesting that +1.2 V is potential at which maximum amount of enzyme is stable and active. Cyclic voltammetry in 0.1 M PBS was performed and showed again a loss of redox behaviour after +1.6 V deposition potential. At +1.6 V the electrophoretic deposition rate is too fast not allowing the formation of pseudo-ordered or pseudo-homogeneous films, leading to the formation of large nanoparticle agglomerates that limit the formation of more regular monolayers hindering a more effective response from the modified electrode (Matsumoto et al., 2002). In addition, the large aggregation of the GOX-modified particles may also lead to the denaturation of the enzyme activity. Modified gold nanoparticles deposited at 0 and 0.8 V showed no response to glucose, which points towards the absence of non-specific adsorption of the modified gold nanoparticles at low potentials. 3.3. Effect of electrodeposition time on electrode response The effect of electrodeposition time on the glucose response of the nanomodule-modified electrodes was investigated. Fig. 3(B)

Fig. 3. Steady-state amperometric responses to glucose measured at 0.4 V in 0.1 M PBS pH 7.0 under stirring from nanomodule-modified gold electrodes. (A) Response after deposition for 30 min and 0, +0.4, +0.8, +1.2 and +1.6 V deposition potential. (B) Response after 15, 30 and 60 min deposition time and +1.2 V deposition potential. 0.5 mm diameter gold electrodes. Room temperature, 22 ± 1 ◦ C.

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tion to explore its performance and estimate the integrability of the multifunctional nanomodules in microelectrode array platforms (Fig. 5). The amperometric response to glucose was measured showing a clear enhancement expected due the enhanced radial diffusion typical of microelectrodes (Foster, 1994). The enhancement of the current also indicates that the controlled electrophoretic deposition carried out to modify the microelectrode had not affected the enhanced mass transport mechanism typical of microelectrodes, fundamental for its implementation in multiarrays platforms. Work is in progress to improve the response by increasing the enzyme layers and also by optimising the physical parameters. 4. Conclusions Fig. 4. Steady-state amperometric responses to successive injections of glucose measured at 0.4 V in 0.1 M PBS pH 7.0 under stirring from nanomodule (with and without outer ORP layer) modified gold electrodes. Nanomodules deposited for 30 min at +1.2 V. 0.5 mm diameter gold electrodes. Room temperature, 22 ± 1 ◦ C.

shows response current as a function of deposition time, the longer deposition time showed a drastic decrease in the glucose response that was also attributed to an excessive aggregation effect and an enzyme activity loss (Matsumoto et al., 2002). Nonetheless the level of amperometric response of the multifunctionalised nanomodules to glucose was considered more than acceptable. 3.4. Effect of the presence of outer redox layer on the electrode response The effect of the presence of outer ORP layer on the glucose response was studied on electrodes deposited for 30 min at +1.2 V. As seen in Fig. 4, the fast response from electrodes deposited with particles having the outer ORP layer produced higher currents upon the sequential addition of glucose. The presence of an extra ORP layer may certainly enhance the electron transfer showing a better response at the electrode, this can also be deducted from Fig. 1. Alternatively it was also thought that an outer ORP layer stabilises the GOX as well as protecting it from a loss of the activity due to the direct contact with the electrode surface. 3.5. Response from the microelectrode The response from the multifunctional gold nanomodules was measured in a 50 ␮m diameter gold microelectrode configura-

This work describes for the first time the production of independent multifunctional bioelectrochemical modules at a nanoscale level. Layer-by-layer modification technique allowed a fine tuning of the components that are part of this nanomodules, The controlled electrophoretic deposition allows the integration of different nanomodules in the same platform, which opens a large window of opportunities to integrate these multifunctional nanomodules into biosensors arrays or even considering them independent nanoreactors. Important aspects optimised are the layer-by-layer approach for preventing the loss of biomolecule activity yet producing a stable glucose response at a nanomodule scale. It is equally important for optimisation of the electrophoretic deposition parameters, which shows how fine the tuning of the conditions that integrate all the techniques used must be to ensure the performance enhancement. On the other hand the wide optimisation range that those technique present also shows the potential to extrapolate the preliminary work shown here to other bioelectrochemical enzymatically catalysed systems, where the utilisation of the multifunctional nanomodules can imply a profitable performance improvement. Acknowledgements This work has been carried out with financial support from the Commission of the European communities specific RTD IST-20021-001837 HEALTHY AIMS and QLRT-2001-01678 INNOCUOUS. It does not necessarily reflect its views and in no way anticipates the Commission’s future policy in this area. The authors want to acknowledge Universitat Rovira i Virgili for Mr. S.K. Dondapati Ph.D. studentship. References

Fig. 5. Steady-state amperometric responses to successive injections of glucose measured at 0.4 V in 0.1 M PBS pH 7.0 under stirring from nanomodule modified gold electrodes and microelectrodes (0.5 mm and 50 ␮m diameter). Nanomodules deposited for 30 min at +1.2 V. Room temperature, 22 ± 1 ◦ C.

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