Research Article - (2015) Volume 6, Issue 2

Impedance Spectroscopy of Supported Multiwalled Carbon Nanotubes for Immunosensor Applications

Imen Hafaiedh1*, Sami Ameur2 and Adnane Abdelghani1
1Nanotechnology Laboratory, National Institute of Applied Science and Technology Centre, Carthage University, Urbain Nord, Bp676, 1080 Charguia Cedex, Tunisia
2Higher Institute of Agronomic Sciences of Chatt-Meriem, University of Sousse, BP 47 ; 4042 Chatt-Meriem, Tunisia
*Corresponding Author: Imen Hafaiedh, Nanotechnology Laboratory, National Institute of Applied Science and Technology centre, Urbain Nord, Bp676, 1080 Charguia Cedex, Tunisia, Tel: +216 703 829, Fax: +216 70 704 329 Email:

Abstract

In this work, we used carboxyl multiwall carbons nanotubes for immunosensor application. The carbon nanotubes functionalisation can be achieved with Pressure Vapor Deposition (PVD) technique. The obtained carbon nanotubes can be immobilized on gold electrode with the airbrushing technique. The morphology of the deposited carboxyl carbon nanotubes was observed with Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM). The electrochemical characterization of the carboxyl multiwall carbon nanotubes was achieved with cyclic voltammetry and impedance spectroscopy. It shows a higher resistance and a good stability in water interface. For anti-rabbit IgG immobilization, the carboxyl carbon nanotubes were activated with EDC/NHS and non-specific sites was blocked with Bovine Serum Albumin (BSA). The development of immunosensor for IgG detection was observed with the impedance spectroscopy. The results show that the immunosensor response depends on the conductivity and the large surface-tovolume ratio attained with multiwall carbon nanotubes. The response of the developed immunosensor was reproducible, with higher stability and with limit detection of 10 pg/ml antigen.

Keywords: Carboxyl multiwalled carbon nanotubes; Immunosenors; Impedance spectroscopy; Cyclic voltammetry

Introduction

Owing to their nano-dimensions, rich electronic states, large surface area, high mechanical strength, and excellent chemical and thermal stability, carbon nanotubes have attracted a great deal of interest [1]. Among the many potential applications [1-3], carbon nanotubes have recently become promising functional materials for the development of advanced biosensors [2] and gas sensors [4-6] with novel features.

There are multiple possibilities for surface modification [7] of carbon nanotubes, but functionalization with antibodies has gained significant importance in recent years. Antibodies not only provide the required biocompatibility [8] but also alter the electrical and optical properties of nanotubes and make their surface sensitive to surroundings [8,9].

In previous work [10-12], we used carboxyl modified thiol monolayers for immunosensor applications. We used impedance spectroscopy and quartz microbalance as transduction techniques. The sample preparation was time consuming and we obtained a higher limit detection [10-12].

In this work, we used carboxyl multiwall carbon nanotubes for immunosensor application. The obtained carbon nanotubes can be immobilized on gold electrode with the airbrushing technique. The morphology of the deposited carboxyl carbon nanotubes was observed with Electron Scanning Microscopy (ESCM) and Transmission Electron Microscopy (TEM). The electrochemical characterization of the carboxyl multiwall carbon nanotubes were achieved with cyclic voltammetry and impedance spectroscopy. The results show that the immunosenosr response depends on the conductivity and the large surface-to-volume ratio attained with multiwall carbon nanotubes. The response of the developed immunosensor was reproducible, with higher stability and with limit detection of 10 pg/ml antigen.

Materials and Methods

Reagents

1-ethyl-3-(3-(dimethylamino)-propyl)carbodiimide (EDC), N-1-ethyl-3-(3-(dimethylamino)-propyl)carbodiimide (EDC), N-hydroxy succinimide (NHS) (Aldrich) and Albumin from Bovine Serum (BSA) were obtained from sigma Aldrich. The Goat anti-rabbit IgG and Rabbit IgG were purchased from Pierce Biotechnology. The MWCNTs (Nanocyl, Belgium) were grown by PVD (Pressure Vapor Deposition) with purity higher than 95%. Nanotubes were upto 50 micrometer in length and their outer and inner diameters ranged from 3 nm to 15 nm and 3 nm to 7 nm, respectively.

The buffer solution used for all experiments was phosphate buffered saline (PBS) containing 140 mM NaCl, 2.7 mM KCl, 0.1 mM Na2HPO4, 1.8 mM KH2PO4, pH=7 and the redox couple Fe(CN)−36 / Fe(CN)−46 at a 5 mM concentration. All reagents were of analytical grade and ultrapure water (resistance 18.2 MΩ.cm−1) produced by a MilliporeMilli-Q system was used.

Carboxyl MWCNT synthesis

The MWCNT used in the experiment were obtained from Nanocyl, S.A. They were synthesized by chemical vapor deposition and their purity was higher than 95%. The nanotubes were upto 50 microns in length and their outer and inner diameters ranged from 3 to 15 nm and 3 to 7 nm, respectively.

A uniform functionalization with carboxylic group was applied to the as provided carbon nanotubes in order to improve their dispersion and surface reactivity. More details can be found in reference [2]. In the second processing step, the functionalized carbon nanotubes were dispersed in an organic vehicle (dimethylformamide), ultrasonically stirred during 20 min at room temperature and subsequently airbrushed onto alumina sensor substrates while controlling the resistance of the resulting film during the deposition. These substrates consist of gold electrode. The active films coated the membrane areas through a shadow mask. During deposition, substrates were kept at 100°C, which ensured a fast evaporation of the solvent.

Gold Cleaning and Functionalization

The gold electrodes (1cm × 1cm) were fabricated at the National Center of Microelectronics of Barcelona (Spain). Evaporated gold (~300 nm thickness) was deposited on silicon, using a titanium underlayer (~30 nm thickness) as substrate. Before modification, the gold electrodes were cleaned in acetone solution for 10 min with ultrasonic bath. After that, they were dried under a nitrogen flow and then dipped for 10 min into “piranha solution” 7:3 (v/v) 96% H2SO4/30% H2O2. Finally, the gold substrates were rinsed 2 to 3 times with ultrapure water and immediately immersed in an ethanol solution. After cleaning, the functionalized multiwall carbon nanotubes were deposited on gold electrode with the Airbrushing technique at 90°C [5]. Immediately the substrate was treated for two hours at 120°C. More details concerning the synthesis and deposition of the gold labeled multiwall carbon nanotubes could be found [6].

After this, the modified gold electrode was immersed in a solution of EDC (0.4 mM) and NHS (0.1 mM) for 1 h. A 500 μl of Goat antirabbit IgG solution with a concentration of 5 μg/ml was deposited on the treated electrode for 1 hour. Then, a 500 μl of 1% BSA solution was added on the substrate for 30 min to block the free spaces between the antibody and multiwall carbon nanotubes [10-13]. Antigen at different concentrations was incubated on immobilized antibody for 30 minutes at room temperature [11-13]. Figure 1 shows the schematic representation of the fabrication process of the multilayer structure of the immunosensor.

nanomedicine-nanotechnology-fabrication-multilayer-immunosensor

Figure 1: Schematic representation of the fabrication process of the multilayer system of the immunosensor on the gold electrode.

Electrochemical Set-Up

Cyclic Voltammetry: Cyclic voltammetry was performed at room temperature in a conventional voltammetric cell with a three electrode configuration using Autolab impedance anlayzer (Ecochemie, Netherland). The gold electrode (0.16 cm2) was used as working electrode, platinum (1 cm2) and Ag/Ag- Cl electrodes were used as counter and reference electrodes respectively. All the electrochemical measurements were carried out in PBS at pH 7.0 with 5 mM Fe(CN)−36 / Fe(CN)−46 and in Faraday cage [13-20].

Impedance Spectroscopy: In a number of reports [12-20], it has been shown that impedance spectroscopy is a useful tool to characterise the compactness amphiphilic films on solid state surfaces. A capacitor is formed between the conducting electrode and the electrolyte. The frequency dependent capacitance C of the complex stratified surface is related to the absolute value of the complex electric impedance Z (measured in Ohms) by the equation:

Equation

Where f is the frequency (in Hz) at which |Z| is measured.

The surface of the device with a supported film in contact with the aqueous phase exhibits a complex capacitance composed of atleast three sub-layers. If the complex impedance Zi of the sub-layers i can be determined by analysis of the measured impedance in terms of an electrical equivalent circuit, the thickness of the sub-layers (i) can be determined according to equation:

Equation

ε0 is the vaccum dielectric constant (8.85×10-14 F/cm2), εI is the dielectric constant of the layer i and A is the area of the surface. To fit the measured spectra with the impedance spectra out of ideal elements, we replaced the ideal elements with the constant phase elements (CPE) :

Equation

The frequency exponent is α=1 and K=1/C for an ideal capacitance, and α=0 and K=R for an ideal resistance, respectively. The exponent α could be obtained, when the membrane capacitance (or layer capacitance) was replaced by a constant phase element ZCPE. The deviation of the exponent α from the ideal values is attributed to the inhomogeneities of the analyzed layer, like defects or roughness. The measured spectra of the impedance and phase were analyzed in terms of electrical equivalent circuits using an Zview software. The electric parameters of the system were calculated with the computer program and the fit error was kept under a maximum of 10%.

The impedance measurement was performed in the frequency range of 0.05 Hz to 100 kHz, using a modulation voltage of 10 mV. More details on electrochemical impedance spectroscopy can be found in reference [15-20].

Results and Discussion

Characterization of multiwall carbon nanotubes layer

Morphological characterization of CNT films: The morphology of the functionalized MWCNT films was investigated with scanning electron microscopy and transmission electron microscopy (Figure 2). The MWCNT nanopores of two-dimensional arrays with different size and with homogenous distribution were observed.

nanomedicine-nanotechnology-image

Figure 2: (a) ESCM image (b) TEM image.

Cyclic voltammetry: Cyclic voltammetry is an electrochemical technique which can be used to study the kinetic of redox reactions of materials, their insulating and conducting properties. Cyclic voltammograms of the gold electrode figure 3a, shows a reversible wave which is the typical behavior of gold surface in the presence of the redox couple. As is shown in (Figure 3a), the cyclic voltammogram defines the characteristics of a diffusion controlled redox process observed at the cleaned bare gold electrode. After modification of the gold surface with the functionalized multiwall carbon nanotube layer, the current decrease due to the insulating properties of the functionalized MWCNTs layer (Figure 3b). The success immobilization of multiwall carbon nanotube layer was confirmed with the current decreases.

nanomedicine-nanotechnology-voltammograms-electrode-behavior

Figure 3: (a): Cyclic voltammograms of the gold electrode shows a reversible wave which is the typical behavior of gold surface in the presence of the redox couple; (b): Cyclic voltammograms of the gold electrode after MWCNTs deposition shows an decrease of the current.

Impedance Measurement: Electrochemical Impedance Spectroscopy (EIS) is an effective tool for probing the feature surface-modified electrode while controlling its electrical properties [15-20]. This technique was used for example for the characterization of impedance behavior of SAM modified gold electrode [15-18]. Figure 4 shows Nyquist plots for gold electrode before and after MWCNTs deposition at 200 mV with redox couple, where Re (z) is the real part and Im (z) is the imaginary part of the complex impedance Z. The diameter of semicircle corresponds to the charge transfer resistance of the electrode/ electrolyte interface. The impedance spectra should be fitted using the electric model presented in figure 5 (Randle’s equivalent circuit). The CPE is the Constant Phase Element impedance of the gold/electrolyte interface, Rm is the charge transfer resistance in low frequency range and W is the impedance due to the mass transfer of the redox species to the electrode described by Warburg (Table 1). The resistance in high frequency ranges Rs is the resistance of the electrolyte, the contacts and connections.

nanomedicine-nanotechnology-nyquist-impedance-electrode

Figure 4: (a): Nyquist impedance plots for gold electrode with redox couple at 200 V; (b): Nyquist impedance plots for gold electrode after MWCNTs deposition with redox couple at 200m V.

nanomedicine-nanotechnology-electric-model

Figure 5: Electric model.

  Rs (Ω.Cm2) Rm (Ω.Cm2) CPE (F.cm2) W (Ω.Cm2)
Gold 259,456 134,980 2,197.10-5 1645,035
Gold+ MWCNTs 259,124 7185,340 5,132.10-5 293,132
Gold+ MWCNTs 261,034 221,654 1,034.10-5 1734,181
Gold+ MWCNTs 261,112 490,045 1,980.10-5 1801, 445
Gold+MWCNTs+anti- IgG+BSA+ Ag (10pg/ml) 260,305 555,009 0,238.10-5 1635,005

Table 1: The electrical parameters of Randle’s circuit obtained with the fitting program. The fit error was kept under a maximum of 10%.

The increase of the charge transfer resistance after MWCNTs immobilization (Figure 4b) is due to the higher conductivity decreases at the gold-electrolyte interface. This confirms the results obtained with cyclic voltammetry. The charge transfer resistance before and after MCWNTs deposition were extracted from the fitted data (Table 1).

Anti-IgG immobilization on multiwall carbon Nanotubes

Cyclic voltammetry: Figure 6 shows the cyclic voltammograms of the gold electrode with MWCNTs (Figure 5a), with immobilized anti- IgG (Figure 6b) and with BSA (Figure 6c). The immobilization of anti- IgG induce an increase in the current due to the iso-electric point of the used antibody. The deposition of BSA blocking layer induce a decrease in the current.

nanomedicine-nanotechnology-voltammograms-electrode-gold

Figure 6: (a): Cyclic voltammograms of the gold electrode with MWCNTs; (b): Cyclic voltammograms of the gold elec- trode with MWCNTs after Antibody deposition; (c): Cyclic voltammograms of the gold electrode with MWCNTs Ab and BSA.

Impedance spectroscopy: Figure 7 shows the Nyquist plot of the gold electrode with MWCNTs (Figure 7a), with immobilized anti-IgG (Figure 7b) and with BSA (Figure 7c). The immobilization of anti- IgG induce a decrease in the charge transfer resistance with confirm the cyclic voltammetry results. The deposition of BSA blocking layer induces an increase in the charge transfer resistance which confirm the cyclic voltammetry results.

nanomedicine-nanotechnology-impedance-gold-electrode

Figure 7: (a): Nyquist impedance plots for gold electrode with MWCNTs at 200 mV; (b): Nyquist impedance plots for gold electrode with MWCNTs after Antibody immobilization at 200 mV; (c): Nyquist impedance plots for gold electrode with MWCNTs BSA immobilization at 200 mV.

The Constant phase element impedance, the charge transfer resistance in low frequency range and the warburg impedance for each deposition steps was shown in table 1.

Immunosensor application: Figure 8 shows the impedance spectra of the gold electrode functionalized with anti-IgG (with BSA as blocking layer) before and after injection of IgG concentrations. The semicircle diameter in the Nyquist plot seems to increase with the antigen concentration, implying that more amount of antigen was linked to the interface. When the concentration of antigen was increased over to 100 pg/ml, the change of impedance spectra become gradually slow, showing that the immobilization of the antibody on a gold electrode trends to be saturated.

nanomedicine-nanotechnology-impedance-electrode-antigen

Figure 8: Nyquist impedance plots for gold electrode with MWCNTs after antigen injections.

In order to obtain the calibration data set, the values of electrode resistance variation ΔRm versus the antigen concentration were plotted in figure 9 (curve a). The change of membrane resistance is calculated following the equation:

nanomedicine-nanotechnology-calibration-curve-detection

Figure 9: Calibration curve for specific and non-specific detection.

ΔRm = Rm(Ab) - Rm(Ab-Ag)

Where Rm(Ab) is the value of electrode resistance as antibody immobilized on the electrode, Rm(Ab-Ag) is the value of the electrode resistance after antigen binding to antibody. As we can see in the figure 9a, the plot is linear and presents saturation for higher IgG concentration. A detection limit of 10pg/ml antigen was obtained. These results show that the immunosensor response depends on the conductivity and the large surface-to-volume ratio attained with multiwall carbon nanotubes [21,22].

To confirm that the above observed impedance changes generated from the result of specific antibody-antigen interaction, the sensor was exposed to the solution of sheep IgG that are expected to bind nonspecifically. Figure 9 (curve b) shows the variation of the electrode resistance versus the non-specific antigen concentration. As we can see, the sensor was not subjected to the non-specific binding and applicable to the selective determination of IgG.

Conclusion

In this work, we used gold labeled functionalized multiwall carbon nanotubes for immunosensors applications. The labeled carboxyl carbon nanotubes can be immobilized on gold electrode with the Airbrushing technique. The stability and the molecular structure of the labeled multiwall carbon nanotubes were characterized with cyclic voltammetry, impedance spec-troscopy, Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM). For Anti-IGg immobilization, the labeled carboxyl carbon nanotubes were activated with EDC/NHS. The developed biosensor can detect IGg concentration in the range 10 pg ml-1–100 pg ml-1. The results show that the Immunosensor response depends on the conductivity and the large surface-to-volume ratio attained with multiwall carbon nanotubes. The response of the developed Immunosensor was reproducible and with higher stability.

References

Citation: Hafaiedh I, Abdelghani A (2012) Impedance Spectroscopy of Supported Multiwalled Carbon Nanotubes for Immunosensor Applications. J Nanomedic Nanotechnol 6:275.

Copyright: © 2012 Hafaiedh I, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.