Elsevier

Biosensors and Bioelectronics

Volume 23, Issue 3, 31 October 2007, Pages 332-340
Biosensors and Bioelectronics

Carbon nanotube/polysulfone screen-printed electrochemical immunosensor

https://doi.org/10.1016/j.bios.2007.04.021Get rights and content

Abstract

The simple and efficient method for preparing sensitive carbon nanotube/polysulfone/RIgG immunocomposite is described. The membrane of the modified disposable screen-printed electrochemical immunosensor is based on phase inversion method. Carbon nanotube/polysulfone membrane acts both as reservoir of immunological material and transducer while offering high surface area, high toughness and mechanical flexibility. The comparison with graphite/polysulfone/RIgG immunosensors shows a much higher sensitivity for those prepared with carbon nanotubes coupled with polysulfone (PSf). The membrane was characterized by scanning electron microscopy/energy dispersive X-ray analysis (SEM/EDX), laser profilometer and by atomic force microscopy (AFM). The purity of the materials was evaluated by thermogravimetric analysis (TGA). The roughness value is doubled when MWCNTs are used instead of graphite into the PSf membranes and the incorporation of antibodies enhances the dispersion of the carbon with the polymeric membrane reducing the roughness in all cases.

This biosensor was based on the competitive assay between free and labelled anti-RIgG for the available binding sites of immobilized rabbit IgG (RIgG). The RIgG was incorporated into the polysulfone membrane by a phase inversion method. Horse radish peroxidase (HRP) enzyme was used as label and hydroquinone as mediator. The detection limit for competitive assay was determined to be 1.66 μg/ml. the linear range of anti-RIgG from 2 to 5 μg/ml and the C50 was found at 3.56 μg/ml. The sensitivity is five times higher for MWCNT than for graphite electrodes, showing lower unspecific adsorption.

Introduction

In the era of proteomics, the methods for identification and quantification of proteins are highly needed. The ability to measure the protein concentration by cost-effective and timely means is highly demanded by diagnostic and biosecurity applications (Wang, 2006, Kingsmore, 2006). Electrochemical immunoassays are highly suitable for high-throughput screening since they require minute sample size and minimal power consumption and thus they are greatly suited for point-of-care or field decentralized testing.

Since their discovery in 1991 (Iijima, 1991), carbon nanotubes (CNT) have attracted considerable interest because of their peculiar shape. Carbon nanotubes offer unique mechanical and electronic properties combined with their chemical stability. Recently, there has been a great interest in the development of carbon nanotube-based sensors and biosensors (Gooding et al., 2003, Guiseppi-Elie et al., 2002, Joshi et al., 2005, Musameh et al., 2002, Merkoci et al., 2005, Pumera, 2007, Pumera et al., 2007, Sánchez et al., 2007, Wang et al., 2003, Wang, 2005, Zhao et al., 2003). The metallic conductivity of multiwall carbon nanotubes (MWCNT) and their electrocatalytic properties are greatly suitable for the development of electrochemical biosensors. The crucial step in the design of biosensors is the immobilization of biological reagents onto the electrode surface. Carbon nanotube biocomposites with Teflon (Wang and Musameh, 2003), epoxy (Perez et al., 2005), chitosan (Liu et al., 2006) or polypyrole (Wang and Musameh, 2005), were prepared in order to improve the robustness of CNT electrodes and to facilitate the immobilization of enzyme biosensors.

There has been little research done on CNT-based electrochemical immunosensors (Yu et al., 2005, O’Connor et al., 2004, Viswanathan et al., 2006). Amperometric immunosensor based on the adsorption of antibodies onto perpendicularly oriented assemblies of single wall carbon nanotubes was developed (Yu et al., 2005, O’Connor et al., 2004) and electrochemical immunosensors for cholera toxin were based on poly (3,4-ethylenedioxythiophene)-coated carbon nanotubes (Viswanathan et al., 2006).

Polysulfone (PSf) has been widely used in last years for different applications in chemistry (Baker, 2004), in membrane separation (Macanás and Muñoz, 2005) and in sensors field (Sánchez and Fàbregas, 2007, Sánchez et al., 2007, Prieto-Simón and Fàbregas, 2006, González-Bellavista et al., 2006) Polysulfone is a polymer that shows high resistance in extreme pH conditions, good adhesion, the susceptibility to incorporate biological molecules and a good thermal stability. These properties, in addition to the good conductivity of MWCNT, make this a good composite for electrochemical detection. Therefore, we decided to explore the possibility of using the MWCNT/PSf composite as a platform for the development of electrochemical sensors and biosensors. It is also possible to modify the chemical nature of the polysulfone matrix in order to optimize the membrane composition and to incorporate antibodies following an easy inversion phase technique (Mulder, 2000).

This paper reports on electrochemical biosensors based on polysulfone membrane encapsulating multiwall carbon nanotubes and immunoreagents layered on disposable screen-printed electrodes. This membrane is printed by serigraphy onto an electrode built on a polycarbonate sheet. Rabbit IgG is used as model antibody being easily labelled with enzymes. HRP is a very used enzyme for immunological analysis as label, being an easy and cheap reagent to be found commercially. Direct and competitive immunoassays are carried out and the electrochemical response of HRP is followed by the addition of hydrogen peroxide to the solution. Different carbon/PSf membranes compositions were studied and characterized. Such screen-printed immunosensors are highly suitable for easy preparation, rapid response and low-cost mass-production. After a thoroughly literature research we found no report on disposable CNT-based immunosensor.

Section snippets

Apparatus

Amperometric experiments were performed with a Bioanalytical system (BAS) LC-4C amperometric controller in connection to a BAS X-Y recorder. Cyclic voltammograms were recorded with the AUTOLAB PGSTAT10 Electrochemical Analyzer (Eco Chemie BV, The Netherlands). The working electrodes were made by screen printing using a Dek248 semi-automatic system (Asflex S.A. Int., Spain). The squeegees used were soft polymer-type and the pressure applied during the printing process was set to 7 kg/cm2. A

Atomic force microscopy

Fig. 1 compares surfaces of four different carbon/PSf electrodes. The 3D images demonstrate that both MWCNT9/PSf and MWCNT50/PSf have extremely high roughness compared with the other materials (Fig. 1A and B). This fact can lead the electrode to a low reproducibility of the response. Either MWCNT9 or MWCNT50 was difficult to dissolve in PSf/DMF suspension when preparing the composite membrane, so their dispersion in PSf is not easy. In case of MWCNT200/PSf, higher values than graphite are

Conclusions

The experiments described above indicate that MWCNT can be used, better than graphite, to prepare attractive soft immunocomposites for amperometric immunosensing. MWCNT200 was selected as the best material in connection with polysulfone polymer for this bio-composite membrane due to its higher sensibility, easy to dissolve homogenously into the PSf/DMF solution, suitable surface roughness and mechanical and physical properties. MWCNT9 and MWCNT50 presented very high roughness, were difficult to

Acknowledgements

S.S. and E.F. would like to thank Spanish Ministry of Education and Science (MAT2003-01253 and CTQ2006-15681-C0) for its financial support. M.P. was supported by the Japanese Ministry for Education, Culture, Sports, Science and Technology (MEXT) through ICYS program. Authors are indebted to Dr. Maria José Esplandiu (GSB, UAB, Spain) for AFM measurements.

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