Carbon Nanotube Modified Screen Printed Electrodes: Pyranose Oxidase Immobilization Platform for Amperometric Enzyme Sensors

Here, a novel enzymatic biosensor was developed using multiwalled carbon nanotube including screen printed electrodes (MWCNT-SPE). Pyranose oxidase (PyOx) was immobilized on the electrode surface by way of gelatin membrane and then cross-linked using glutaraldehyde. Glucose was detected at -0.7 V (vs. Ag/AgCl) by watching consumed oxygen in enzymatic reaction after addition substrate. After optimization of pH and enzyme loading, the linearity was found in the range of 0.1–1.0 mM of glucose. After that, the effect of MCNT on the current was tested. Also the enzymatic biosensor including glucose oxidase instead of pyranose oxidase was prepared and the biosensor response followed for glucose. Furthermore, this system was tested for glucose analysis in soft drinks.

Carbon nanotubes (CNTs) are members of the carbon-based nanomaterials offering unique mechanical, electronic and chemical stability properties [13]. CNTs are constructed from sp 2 carbon units and can be several nanometers in diameter and many microns in length [3,14]. When CNTs have been used as an electrode, they have a characteristic to mediate electron-transfer reactions with electroactive species [15]. In our previous studies, CNTs-modified carbon paste electrodes were prepared and pyranose oxidase was immobilized to design glucose biosensors [16]. In another study, both enzymes α-glucosidase (AG) and pyranose oxidase (PyOx) were immobilized on graphite electrode surface covered with chitosan-CNTs and amperometric maltose detection was carried out [17].
Here, multi walled carbon nanotube-modified SPE (MWCNT-SPE) was developed and the immobilization of PyOx as a model enzyme was carried out. Gelatin was used as an immobilization matrix. PyOx was added to gelatin network and finally amine groups of gelatin and enzyme were cross-linked with glutaraldehyde. The optimization of working conditions were performed by using Dglucose as a substrate of enzymatic reaction. And then analytical characterization was performed. After optimization, the proposed MWCNT-SPE/PyOx biosensor was used to determine glucose in soft drinks. Multi walled carbon nanotubes (MWCNTs) were from Aldrich (www.sigmaaldrich.com) with the following features: external diameter: 10-20 nm, internal diameter (ID): 5-10 nm, lenght: 0.5-200 µm. In this work the raw material was subjected to a purification treatment based on nitric acid: 200 mg of MWCNTs are dispersed in 200 ml of acqueous 3M HNO3 obtained by diluting HNO3 65% w/w (d=1.400±0.010 g/ml RPE Carlo Erba, Milano). The pot is immersed in an ultrasonic bath for 15 minutes to achieve a homogeneous dispersion and then let warming on a heating plate for 3 hours below its boiling point. The solution is then washed with distilled water up to reaching pH about 7; MWCNTs are separated by settling and the precipitate is desiccated in an oven kept at 37±1°C until all the hydration water is evaporated.

Chemicals and reagents
Glucose content of real samples was analyzed by commercial kits based on Trinder reaction [29] and the proposed MWCNT-SPE/PyOx biosensors.

Apparatus
Electrochemical experiments were carried out with a PalmSens electrochemical measurement system (Palm Instruments, Houten, The Netherlands). PyOx immobilized MWCNT-SPE was used as the working electrode. The electrodes were inserted into a conventional electrochemical cell (10 mL).

Preparation of SPEs
SPEs were prepared by printing on 0.3-0.5 mm thick PVC substrate using screen printing machine (Fleischle; Brackenheim, Germany). Carbon pastes for SPEs (from Gwent Electronic Materials TM ) were modified, by mixing the paste with activated powder of MWCNTs, in approximately 5% weight ratio which is the highest amount can be added to the graphite paste without losing the rheological properties (mainly viscosity) for printing. Such MWCNTs are not soluble in water therefore a minimum amount of terpineol (few mimcroliters) was used to help mixing. Screen printing of the working electrode (WE) was carried out along with the graphite tracks (Fig. 1A).
The following process to deposit Ag/AgCl paste as a reference electrode (Fig. 1B) and dielectric (Fig. 1C) are also schematically reported in the sequence of Fig. 1 [7].

Measurements
All the measurements were monitored chronoamperometrically at -0.7 V by SPE (versus Ag/AgCl) at room temperature (~ 25 o C) (working buffer was phosphate buffer (PB) solution (50 mM, pH 7.5)] Signal time was 100 s. The electrochemşcal response was registered as current (µA).

Results
Because of some advantages such as low cost, versatility, and miniaturization in screen-printing technology, the disposable amperometric biosensors based on SPEs has increasing potential. The alteration of SPEs with CNTs has enabled the production of sensitive and stabile sensors. For this aim, Lin et al. developed a new disposable biosensor by co-immobilization of acetycholinesterase/choline oxidase on CNTs-SPE and tested the detection of toxic compounds [30]. In another study, SPEs were fabricated on polypropylene sheets and modified by CNTs.
Pyrrole quinoline quinine glucose dehydrogenase (PQQ-GDH) was used for biosensor preparation to measure glucose [31]. In this paper, the design and application of a disposable biosensor based on MWCNT-SPEs modified with PyOx was described. CNT modification was carried out by mixing the paste with activated powder of MWCNTs. After immobilization of PyOx on the MWCNT-SPEs surface, optimization and analytical characterization studies were performed.

Effect of enzyme activity
The performance properties of the biosensors depend on the enzyme activity; so that the enzyme loading within the gelatin matrix was investigated. Different amounts of PyOx (0.2, 0.3 and 0.4 Unit) were studied and the dynamic ranges for glucose with different MWCNT-SPE/PyOx in working buffer were shown in Fig. 2. Optimum amount was found to be 0.3 U of enzyme activity. Use of higher enzyme amount resulted in a decrease in the MWCNT-SPE/PyOx response. The presence of a larger amount of enzyme causes diffusion problems for the oxygen as well as substrate transfer to the bioactive layer and therefore a lower current response were obtained. For further experiments, 0.3 U was chosen.

Influence of pH
pH effect on the amperometric response in PB solution at different pH values (50 mM, 6.5-8.2) in the presence of 0.6 mM glucose was shown in Fig. 3. The highest sensitivity was achieved at pH 7.5. This behavior was very similar to the activity profile vs pH of the free enzyme [32].  The influence of the MWCNT on the current has been compared with blank graphite based SPEs without MWCNTs. Non-modified SPE/PyOx biosensors were fabricated, and calibrated for glucose. Higher signals were observed with MWCNT-SPE/PyOx biosensor as shown in Fig. 4, where calibration curve for glucose in both cases are reported. The repeatability of the biosensor has been tested by 5 replicate determinations of glucose standard solution (0.6 mM). The average value (x), standard deviation (S.D) and variation coefficient (c.v) was calculated as ± 0.02 mM and 3%, respectively.
For a period of 6 h, 13 measurements of 0.6 mM glucose were carried out using the biosensor to determine operational stability. The enzymatic activity decreased 40 % at the end of 6h. It is mentioned in introduction part, PyOx catalyses the oxidation of glucose without selecting anomeric forms [33]. Additionally GOx, instead of PyOx, was used to prepare MWCNT-SPE/GOx biosensors and the influence of GOx on the current characteristics towards to glucose was investigated. Calibration curves for both types of MWCNT-SPE based on PyOx and GOx were examined. When comparing the data obtained for GOx with the PyOx biosensor, a higher responses were found in the latter case (Fig. 5). In this part, substrate specificity of the MWCNT-SPE/PyOx was examined by using various potential interfering sugar compounds such as xylose, galactose, mannose and maltose at the same concentration (0.6 mM). The biosensors response obtained for Glc was fixed as 100% and compared to the currents acquired for the other compounds (Fig.  6).

Measurements on real samples
MWCNT-SPE/PyOx was applied to analyze fruit juice samples. In addition, Trinder method was utilized as the reference method to evaluate Glc concentration data obtained from MWCNT-SPE/PyOx system. To determine the amount of glucose in fruit juice by means of MWCNT-SPE/PyOx biosensor, not treated samples were added to the buffer solution in the same way as previously added standard solutions of Glc. Then, signals were recorded and data were directly calculated from calibration curve. Acquired values were checked against to spectrophotometric method. Table 1 summarizes the results belong to real samples of soft drink. As shown in the tables, yield values were found to be closer which means that the system has not been influenced by the nature of the sample.

Discussion and Conclusion
In this study, PyOx was immobilized on the surface of the carbon nanotube-modified SPEs by means of gelatin. After optimizing the pH and enzyme loading, MWCNT-SPE/PyOx biosensor was calibrated for glucose detection. The addition of MWCNT to the carbon paste used for printing electrodes resulted in increased sensitivity with respect to electrodes without CNT. Higher sensitivity was also verified for PyOx with respect to GOx immobilized on the same nanostructured electrode surfaces. The use of PyOx instead of GOx for obtaining a novel glucose biosensor resulted in a larger range of substrates allowed to be detected too. For these reasons the MWCNT-SPE/PyOx can be fruitfully used in enzymatic biofuel cells as an alternative to GOx because many other sugars than glucose can be oxidised by PyOx (e.g., lignocellulose hydrolysate). In this paper preliminary determination of sugars in soft drinks was tested obtaining good performance and recovery with respect to a reference spectrophotometric method. mode. Biosens. Bioelectron., 21 (2006), 1591-1598.