Functionalized single-walled carbon nanotubes/polypyrrole composites for amperometric glucose biosensors
© Raicopol et al.; licensee Springer. 2013
Received: 19 May 2013
Accepted: 2 July 2013
Published: 9 July 2013
This article reports an amperometric glucose biosensor based on a new type of nanocomposite of polypyrrole (PPY) with p-phenyl sulfonate-functionalized single-walled carbon nanotubes (SWCNTs-PhSO3−). An environmentally friendly functionalization procedure of the SWCNTs in the presence of substituted aniline and an oxidative species was adopted. The nanocomposite-modified electrode exhibited excellent electrocatalytic activities towards the reduction or oxidation of H2O2. This feature allowed us to use it as bioplatform on which glucose oxidase (GOx) was immobilized by entrapment in an electropolymerized PPY/SWCNTs-PhSO3− film for the construction of the glucose biosensor. The amperometric detection of glucose was assayed by applying a constant electrode potential value necessary to oxidize or reduce the enzymatically produced H2O2 with minimal interference from the possible coexisting electroactive compounds. With the introduction of a thin film of Prussian blue (PB) at the substrate electrode surface, the PPY/GOx/SWCNTs-PhSO3−/PB system shows synergy between the PB and functionalized SWCNTs which amplifies greatly the electrode sensitivity when operated at low potentials. The biosensor showed good analytical performances in terms of low detection (0.01 mM), high sensitivity (approximately 6 μA mM−1 cm−2), and wide linear range (0.02 to 6 mM). In addition, the effects of applied potential, the electroactive interference, and the stability of the biosensor were discussed. The facile procedure of immobilizing GOx used in the present work can promote the development of other oxidase-based biosensors which could have a practical application in clinical, food, and environmental analysis.
KeywordsFunctionalization Carbon nanotubes Conductive polymers Nanocomposite Amperometric biosensor Sensitivity Selectivity
Research and development in electrochemical biosensors have gained increasing importance as analytical tools in the last years, since electrochemical biosensors have advantageous properties such as the simplicity of use, potential miniaturization, and low cost, in comparison with well-established, lab-based methods. However, a number of problems are still present, preventing the total success in the sensor market, so nanocomposite materials may play an important role for improving their properties .
Conducting polymers (CPs) are especially amenable to the development of electrochemical biosensors by providing biomolecule immobilization and rapid electron transfer. The combination of known CP substrates with carbon nanotubes (CNTs) may generate composites with new and interesting properties, providing higher sensitivity and stability of the immobilized molecules, thus constituting the basis for new and improved analytical devices for biomedical and other applications . The enhanced response can be attributed to several factors such as the improved electron transfer within the polymeric matrix from the presence of CNTs, the direct electron transfer from the active site of the enzymes to the electrode through the CNTs bridging them, and the enhanced accessibility of the enzyme catalytic sites for the substrate due to highly open reticular morphology of the nanocomposite film. Surface functionalization of CNTs can greatly enhance their utility in the formation of composites by aiding in dispersability and ensuring efficient interactions between the SWCNTs and the host materials . In this regard, the development of simple and cost-effective chemical procedures for covalent functionalization of CNTs is a matter of increasing importance . In our research an environmentally friendly functionalization procedure of the SWCNTs was adopted. The reaction was performed ‘on water’ in the presence of a substituted aniline and an oxidative species similar to that described by Price and Tour  with obtainment of p-phenyl sulfonate-functionalized SWCNTs (SWCNTs-PhSO3−). Running reactions on water can reduce harmful waste and reaction times while increasing yields and reaction rates .
Among the various conducting polymers, films of PPY and derivatives have good conductivity, selectivity, stability, and efficient polymerization at neutral pH . Enzymes and, in particular, oxidases, have been preferentially chosen for the entrapment in PPY matrices, but other biomolecules are also potential targets. In general, glucose oxidase (GOx) is selected as a model enzyme due to its low cost, stability, and practical utility. The oxidases act by oxidizing the substrate and then returning to their original active state by transferring electrons to molecular oxygen, so the final products of these enzymes are the oxidized form of the substrate and, as a side product, hydrogen peroxide (H2O2). Both the measurement of oxygen consumption and H2O2 production can provide information about the concentration of the enzyme substrate (glucose). Methods based on the measurement of H2O2 have been greatly preferred in the recent years to those based on the reduction of oxygen. However, a great drawback in this approach is represented by the high overpotential needed for H2O2 oxidation (greater than +0.6 V vs. Ag/AgCl reference electrode). At this relatively high potential, there may be interferences from other oxidable species such as ascorbic acid, uric acid, and acetaminophen. One of the most common ways to overcome this problem has been the use of another enzyme, namely, horseradish peroxidase (HRP) which catalyzes the reduction of H2O2 and allows the direct electron transfer between its active site and the electrode surface . This approach, although exhibiting good sensitivity and accuracy, suffers from some important shortcomings such as high cost, low stability, and the limited binding of HRP to solid surfaces. For this reason, the electrochemical inorganic mediators , able to catalyze the oxidation or reduction of hydrogen peroxide, have been preferred to HRP and have been used for the assembling of oxidase-based biosensors. This results in a decrease of the applied potential and the consequent avoidance of many electrochemical interferences. In this perspective, Prussian blue (PB), which has high electrocatalytic activity, stability, and selectivity for H2O2 electroreduction, has been extensively studied and used for H2O2 detection .
Incorporating a thin PB film into the PPY/GOx/SWCNTs-PhSO3− nanocomposite, the obtained hybrid shows synergistic augmentation of the response current for glucose detection. The effects of applied potential on the current response of the composite-modified electrode toward glucose, the electroactive interference, and the stability were optimized to obtain the maximal sensitivity. The resulting biosensor exhibits high sensitivity, long-term stability, and freedom of interference from other co-existing electroactive species.
Chemicals and instrumentation
Single-walled carbon nanotubes (>90% C, >77% C as SWCNTs) were obtained from Aldrich (Sigma-Aldrich Corporation, St. Louis, MO, USA). Glucose oxidase (type X-S from Aspergillus niger, 250,000 μg−1) was purchased from Sigma. Pyrrole (98%, Aldrich), D-(+)-glucose (≥99.5%), ascorbic acid, uric acid, and acetaminophen were used as received (Sigma). All other chemicals were of analytical grade. Electrochemical experiments were performed using a 128N Autolab potentiostat and a conventional three-electrode system with a platinum-modified electrode (disk-shaped with diameter of 2 mm; Metrohm Autolab B.V., Utrecht, the Netherlands) as the working electrode, a platinum wire as the counter electrode, and Hg/Hg2Cl2 (3 M KCl) as reference electrode (purchased from Metrohm). Unless otherwise stated, all experiments were carried out at room temperature in pH 7.4 phosphate buffer solution (0.1 M phosphate). Amperometric determination of glucose was carried out at different applied potentials under magnetic stirring.
Single-walled carbon nanotubes functionalization
Preparation of the modified electrode
Firstly, a PB film was electropolymerized at the Pt electrode surface in an unstirred fresh 2 mM K3Fe(CN)6 + 2 mM FeCl3. 6H2O in 0.1 M KCl + 1 mM HCl aqueous solution by cyclic voltammetry in the potential range of −0.2 to 1.0 V at a scan rate of 0.1 V s−1.
Different amounts of the functionalized nanotubes (usually 1 mg/mL) were dispersed in bidistilled water by sonication for 1 h. The selected amount of GOx (1 mg/mL) was then added to the CNTs solution. Afterwards, pyrrole was added (at a concentration of 0.5 M) to the GOx and SWCNTs-PhSO3− mixture, and the electropolymerization was performed at current densities of 0.1, 0.2, or 0.5 mA cm−2 for different times. The electropolymerization was carried out at pH 7.4. After the electropolymerization, the composite film (PPY/GOx/SWCNTs-PhSO3−/PB) was subjected to overoxidation by cycling the potential from −0.2 to 1 V for 50 cycles at 0.1 V s−1 in a phosphate buffer solution at pH 7.4. For comparison, PPY/GOx/SWCNTs-PhSO3−, PPY/GOx/PB, and PPY/GOx films have been also obtained.
Results and discussion
Raman spectroscopy characterization
The Raman spectra of electrochemically deposited PPY/GOx/SWCNTs-PhSO3− composite are strongly dependent on different parameters such as electrodeposition time or density current. In some samples of PPY/GOx/SWCNTs-PhSO3− composite (higher current densities used for electrodeposition), the Raman spectra are quite modified from the CNT spectra: the lines corresponding to the breathing mode disappear. This maybe because the PPY was too thick in the used samples. Further work is in progress in order to characterize the samples and correlate their properties with the electrochemical parameters used during synthesis.
Influence of electroactive interferents on glucose response at PPY/GOx/SWCNTs-PhSO 3 − /PB/Pt electrode
Concentration (physiological normal, mM)
iGlu + interf/iGluaat E= 0 V
The storage stability of the biosensor was also studied. The steady-state response current of 2 mM glucose was determined every 2 days. When not in use, the biosensor was stored in 0.1 M phosphate buffer pH 7.4 at 4°C. The results show that the steady-state response current only decreases by 12% after 30 days measurements, which indicates that the enzyme electrode was considerably stable.
A new type of PPY/GOx/SWCNTs composite has been obtained in a one-step preparation procedure by electropolymerization of pyrrole in the presence of enzyme and functionalized SWCNTs. For SWCNTs-PhSO3− synthesis, an environmentally friendly functionalization procedure was adopted. The reaction was performed on water in the presence of sulfanilic acid and tert-butyl nitrite. The functionalized SWCNTs were characterized using spectroscopic and microscopic methods.
The studies undertaken in this article demonstrate that the new electrochemically synthesized PPY/GOx/functionalized SWCNTs nanocomposite can be used for the fabrication of electrochemical glucose biosensors with attractive performance. The nanocomposite biosensor exhibits high sensitivity and low detection limits even at an applied potential of 0 V vs. Hg/Hg2Cl2 (3 M KCl). The performance in glucose determination is better than that of much more biosensor assemblies based on similar components. The glucose biosensor shows good analytical characteristics such as low detection limit (0.01 mM), high sensitivity (approximately 6 μA mM−1 cm−2), wide linear range (0.02 to 6 mM), and good stability under the optimized experimental conditions. The selectivity of the biosensor is greatly improved due to the lower operation potential afforded by the catalytic ability of the presence of both PB film and SWCNTs. The PPY/GOx/SWCNTs-PhSO3−/PB hybrid material has a potential to provide operational access to a large group of oxidase enzymes for designing a variety of biosensing devices.
This work was supported by CNCS-UEFISCDI, project PN II-RU number 15/05.08.2010, code TE_153.
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