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300 Int J Nanotechnol., Vol 8, Nos 3/4/5, 2011 Electrochemical biosensor for glucose detection using zinc oxide nanotetrapods Nguyen Thu Loan, Luu Manh Quynh, Ngo Xuan Dai and Nguyen Ngoc Long* Faculty of Physics, Hanoi University of Science, Vietnam National University, 334 Nguyen Trai Road, Thanh Xuan District, Hanoi, Vietnam E-mail: ngthuloan@gmail.com E-mail: luumanhquynh@hus.edu.vn E-mail: dainx@vnu.edu.vn E-mail: longnn@vnu.edu.vn *Corresponding author Abstract: Wurtzite structural zinc oxide (ZnO) nanotetrapods were synthesised by a thermal evaporation method These ZnO nanotetrapods were used for the construction of an electrochemical biosensor for detecting glucose In the biosensor, the ZnO nanotetrapods acted as supporting materials for glucose oxidase (GOx) enzyme loading because of the large surface area to volume ratio The results indicated that the biosensor with the structure of [polystyrene mixed with ZnO powder/GOx/ZnO nanotetrapod powder/Au electrode] exhibited the lowest glucose detection limit (~0.5 mM) These results demonstrate that zinc oxide nanostructures have potential applications in biosensors Keywords: biosensor; nanotetrapods; zinc oxide; glucose Reference to this paper should be made as follows: Loan, N.T., Quynh, L.M., Dai, N.X and Long, N.N (2011) ‘Electrochemical biosensor for glucose detection using zinc oxide nanotetrapods’, Int J Nanotechnol., Vol 8, Nos 3/4/5, pp.300–311 Biographical notes: Nguyen Thu Loan received her BSc in Physics from Hanoi University of Science in 2009 Now, she is an MSc student in Osaka University, Japan Luu Manh Quynh received his MSc in Biophysics from Eotvos Lorand University of Science, Budapest, Hungary, in 2005 In September 2005, he joined the Protein Aggregation Study under Physical effects Group of Semmelweis University of Medicine as a PhD student until June 2008 At present, he works in the Centre for Materials Science, Hanoi University of Science, Vietnam National University His research area is the application of nanostructures in biology Ngo Xuan Dai obtained BSc in Materials Science from Hanoi University of Science in 2002 In 2008, he received the MSc in Theoretical Physics from Hanoi University of Education, Vietnam Since 2002, he has been working as a Researcher and Teaching Assistant in the Faculty of Physics, Hanoi University of Science, Vietnam National University His research focuses on ZnO and ZnS nanostructures Copyright © 2011 Inderscience Enterprises Ltd Electrochemical biosensor for glucose detection 301 Nguyen Ngoc Long received his PhD in Solid State Physics from Saint-Petersburg University, Russia, in 1972 He was promoted to the rank of Associate Professor in 1991 at Hanoi University He is a former Director of the Centre for Materials Science, Hanoi University of Science, Vietnam National University His research interests include the optical properties of semiconductors, synthesis, characterisation and application of nanostructures based on semiconductor and metal materials Introduction Detection of biomolecules by using different techniques has attracted much attention because of its importance in science, life and healthcare According to International Union of Pure and Applied Chemistry (IUPAC) recommendations, a biosensor is a self-contained integrated receptor-transducer device, which is capable of providing selective quantitative or semi-quantitative analytical information using a biological recognition element [1] There are two essential elements in the biosensor: First, the bioreceptors or a biorecognition element (such as antibodies, DNA, enzymes and cellular components of living systems) with powerful molecular recognition capability Second, the transducer element (such as optical, magnetic, piezoelectric and electrochemical transducers), which possesses the capability to translate the interactions of the biorecognition element into a measurable signal Among the electrochemical biosensors, the amperometric biosensor is a good and practical approach to detect biomolecules This is one of the most intensely investigated biosensors It utilises the enzyme-loaded electrode to detect biomolecules because enzymes are highly selective and quickly responsive to specific biomolecules [2,3] There have been numerous reports on amperometric biosensors in which the immobilisation of enzymes on electrodes is generally considered to be a key factor in fabrication [4–7] Nanomaterials can play an important role in adsorption of biomolecules due to their high specific area and size comparable with the biomolecules Besides, the enzyme’s bioactivity can be retained thanks to the desirable microenvironment, and electron transfer between the active sites of enzyme and the electrode can also be enhanced [8] In particular, one-dimensional (1D) nanostructures provide natural channels that are beneficial to electron transfer [9] Therefore, the study of biosensors using 1D nanoscale semiconductors as the matrix has emerged as a promising research topic ZnO is a wide and direct bandgap II-VI semiconductor with a rather large bandgap of 3.37 eV at room temperature ZnO has been traditionally used as a transparent conductor in optoelectronic devices such as displays and solar cells [10] One-dimensional ZnO nanorods have been used to fabricate many nanodevices such as room temperature lasers, transistors, field emitters and gas sensors [11–13] On the other hand, ZnO is a biocompatible material with a high isoelectric point of 8.7–10.3 [14], which makes it suitable for adsorption of proteins with low isoelectric points, as the protein immobilisation is primarily driven by electrostatic interaction [9,15] Moreover, ZnO nanostructures have unique advantages, including the highly effective surface area, non-toxicity, chemical stability, electrochemical activity and high electron conductivity Hence, they are promising for biosensor applications 302 N.T Loan et al Due to the pivotal role of glucose in physiological processes, much effort has been devoted to the development of methods for detecting glucose in food and biological matrices Recently, Wang et al [16] and Sun et al [17] reported on the preparation of glucose biosensors using ZnO nanocombs, ZnO nanorods and ZnO nanodisks In this study, we fabricated an electrochemical biosensor for detecting glucose using ZnO nanotetrapods as supporting materials for immobilising glucose oxidase (GOx) enzyme The operation of the electrochemical biosensor is based on the fact that the enzyme glucose oxidase catalyses the oxidation of glucose forming gluconic acid and hydrogen peroxide, whose coupling with oxygen generates an electron flow proportional to the number of glucose molecules The polystyrene (PS) polymer solution mixed with ZnO nanotetrapods was used to make conductive polymer membranes These ZnO nanotetrapod modified biosensors were investigated for the detection of glucose using a cyclic voltammetry This fabricating method is suggested to be a suitable and promising route for the development of glucose biosensors Experiment 2.1 Synthesis of ZnO nanotetrapods using the vapour phase transport method ZnO nanotetrapods were synthesised by a vapour transport process (Figure 1) Some pieces of high-purity zinc metal (Zn) and the cleaned SiO2/Si substrates were placed inside a small quartz tube The Zn pieces were located at the closed end, while the substrates were arranged towards the opened end of the tube The small tube was inserted in a longer quartz tube and then all these tubes were inserted into a horizontal electric-resistance furnace so that the Zn source material at middle of the furnace was heated at 900°C for 10 After that, the furnace was naturally cooled down to room temperature and the white products were collected on the SiO2/Si substrates and on the inside wall of the longer quartz tube Figure Experiment set-up of synthesis of ZnO nanotetrapods by using vapour phase transport method 2.2 Characterisation of the morphologies and structures of ZnO nanotetrapods The morphology of the as-prepared ZnO nanotetrapods was characterised by using a scanning electron microscope (JSM 5410 LV, JEOL, Japan) The composition of the samples was determined by an energy-dispersive X-ray (EDX) spectrometer (EDS, OXFORD ISIS 300) attached to the JEOL-JSM 5410 LV scanning electron microscope The crystalline structure of the ZnO nanotetrapods was analysed by using an X-ray Electrochemical biosensor for glucose detection 303 diffractometer (Siemens D5005, Bruker, Germany) with Cu-Kα1 (λ = 0.154056 nm) irradiation 2.3 Preparation of the biosensor The GOx solution was prepared by dissolving 20 mg GOx in mL of phosphate buffer solution (PBS, pH = 7.2) and kept at –20°C in a refrigerator It was naturally unfrozen when in use The polystyrene (PS) polymer was completely dissolved in CH2Cl2 solution to make the polymer membrane solution In order to make a conductive polymer membrane, a wt% of the synthesised ZnO nanotetrapods was put into this PS polymer solution and ultrasonicated for 30 The glucose biosensor was fabricated by the following steps (Figure 2) The ZnO nanotetrapods were first transferred to the surface of a gold electrode with a diameter of mm and then a µL GOx (200 U/g) solution was dropped onto the ZnO nanotetrapod layer After that, the structure of [GOx /ZnO/Au electrode] was dried to evaporate water Finally, a µL PS solution mixed with ZnO nanotetrapods was used to cover the surface of the structure of [GOx/ZnO/Au electrode] It quickly dried to form a film, which made the GOx/ZnO nanotetrapod layer attach tightly to the gold electrode’s surface Thus, we obtained a biosensor with the structure of [PS mixed with ZnO powder/GOx/ZnO nanotetrapod powder/Au electrode] Figure A schematic diagram for the construction of ZnO nanotetrapod glucose biosensor 2.4 Measurement of the biosensor The performance of the [PS mixed with ZnO/GOx /ZnO/Au electrode] biosensor was measured at room temperature Cyclic voltammetric measurements were recorded using an autolab (PGSTAT 302N, Eco Chemie, Netherlands) with a three-electrode cell The mentioned gold electrode with ZnO nanotetrapods was used as a working electrode A platinum electrode and an Ag/AgCl saturated electrode were used as counter electrode and reference electrode, respectively 304 N.T Loan et al Results and discussion 3.1 Morphologies and structures of ZnO nanotetrapods The SEM images shown in Figure illustrate the tetrapod-like form of the ZnO products Each tetrapod is composed of four ZnO micro/nanorods (legs of tetrapods) with two types of shapes and sizes Figure SEM images of ZnO nanotetrapods, obtained (a, b) on SiO2/Si substrates and (c, d) on the wall of quartz tube The ZnO tetrapods collected on the SiO2/Si substrates have long cylindrical legs with a diameter of about 300–350 nm and a length of several tens of micrometres (Figure 3(a) and (b)), whereas those collected on the wall of the quartz tube have legs, which are shorter and sharper with a length of 10 micrometres (Figure 3(c) and (d)) The XRD pattern of the synthesised ZnO products is shown in Figure 4(a), where all the diffraction peaks can be well indexed to the typical wurtzite-type ZnO crystals with the lattice constants of a = 3.249 Ǻ and c = 5.206 Ǻ, which match well the standard XRD data for the wurtzite ZnO crystals (JCPDS card, No 79-2205) No other crystalline forms, such as Zn metal or other impurities, were found As shown in Figure 4(b), all the EDX peaks are attributed to the binding energy of the Zn and O elements There is no sign of other elements Electrochemical biosensor for glucose detection Figure 305 (a) XRD pattern and (b) EDX spectrum of the ZnO nanotetrapod products (see online version for colours) (a) (b) 3.2 Performance of the glucose biosensor Cyclic voltammograms (CVs) were performed to investigate the characteristics of the biosensor and the influence of each component on its performance The electrochemical solution is the phosphate buffer solution (PBS, pH = 7.2), into which various amounts of glucose were added Potential was scanned from –0.2 V to 0.8 V (vs the Ag/AgCl electrode) at the rate of 50 mV/s and all the experiments were carried out at room temperature First, we studied the role of GOx enzyme in the glucose biosensor The CVs of the biosensor with the structure of [PS/ZnO/Au electrode] without using GOx and with the structure of [PS/GOx /ZnO/Au electrode] using GOx at the scanning rate of 50 mV/s as a function of glucose concentration are shown in Figure As can be seen from Figure 5(a), two very weak peaks at 0.14 V and 0.30 V were observed in the CV curves of the [PS/ZnO/Au electrode] biosensor These two peaks are related to the reduction and 306 N.T Loan et al oxidation of ZnO itself [16,17] Besides, the CVs weakly depended on the glucose concentration Figure The CVs of the biosensor (a) with the structure of [PS/ZnO/Au electrode] without using GOx and (b) with the structure of [PS/GOx /ZnO/Au electrode] using GOx at the scanning rate of 50 mV/s as a function of glucose concentration (see online version for colours) (a) (b) In the case of the [PS/GOx /ZnO/Au electrode] biosensor, the CVs strongly depended on the glucose concentration Figure shows the current values at the potential of 0.8 V as a function of glucose concentration for the biosensor without GOx and with GOx enzyme For the biosensor using GOx, the current rapidly increased from 0.9 µA to 2.4 µA while the glucose concentration increased from mM to mM Electrochemical biosensor for glucose detection Figure 307 The current values at the potential of 0.8 V as a function of glucose concentration for the biosensor without GOx and with GOx enzyme (see online version for colours) The role of GOx enzyme can be explained by the fact that the enzyme catalyses the oxidation of glucose to gluconic acid Here, the enzyme acts as a biorecognition element As soon as the enzyme recognises the glucose molecules, it acts as a catalyst to produce gluconic acid and hydrogen peroxide from glucose and oxygen from the air: GO x Glucose + O + H O → Gluconic acid + H O At the electrode: O2 + 2e− + 2H+ = H2O2 The electrode easily recognises the number of electron transfers due to hydrogen peroxide/oxygen coupling Hence, the electron flow is proportional to the number of glucose molecules present in the electrochemical solution The role of ZnO micro/nanotetrapods is clearly represented in Figure Figure 7(a)–(c) shows the CVs of the biosensors with the structure of [pure PS/GOx/Au electrode], the structure of [conductive PS/GOx/Au electrode] and the structure of [conductive PS/GOx/ZnO/Au electrode], respectively As seen from Figure 7(a), when the potential was scanned from –0.2 V to 0.8 V at the rate of 50 mV/s, in the case of the biosensor without both the ZnO layer in the biosensor construction and the ZnO powder mixed in the PS solution (the [pure PS/GOx/Au electrode] biosensor), the measured current varied from −2 µA to µA and weakly depended on glucose concentration In the case of the biosensor without the ZnO layer in the biosensor construction but with the ZnO powder mixed in the PS solution (the [conductive PS/GOx/Au electrode] biosensor), the measured current varied from −15 µA to µA (Figure 7(b)) Lastly, as shown in Figure 7(c), for the biosensor with both ZnO layer and ZnO in the PS solution (the [conductive PS/GOx/ZnO/Au electrode] biosensor), the measured current varied in wide range from −10 µA to 25 µA It is noted from Figure 7(b) that two peaks at 0.15 V and 0.28 V were observed in the CV curve of the [conductive PS/GOx/Au electrode] biosensor These two peaks are related to reduction and oxidation of ZnO itself In Figure 7(c) for the [conductive PS/GOx/ZnO/Au electrode] biosensor was observed only the peak at 0.21–0.30 V relating to the reduction of ZnO, because the strong increase of the current in the range 0.4–0.8 V 308 N.T Loan et al covers the peak relating to the oxidation of ZnO It is important that the current values of the previously mentioned two peaks (Figures 7(b) and (c)) and the current values at the potential of 0.8 V (Figure 7(c)) strongly depend on glucose concentration Glucose concentration dependence of the currents at the peak relating to the reduction of ZnO for the [conductive PS/GOx/Au electrode] biosensor and for the [conductive PS/GOx/ZnO/Au electrode] biosensor and glucose concentration dependence of the currents at 0.8 V potential for the [conductive PS/GOx/ZnO/Au electrode] biosensor are shown in Figure 8(a) and (b), respectively Figure Cyclic voltammograms for: (a) the structure of [pure PS/GOx/Au electrode]; (b) the structure of [conductive PS/GOx/Au electrode] and (c) the structure of [conductive PS/GOx/ZnO/Au electrode] Concentrations of glucose are noted in the figures (see online version for colours) (a) (b) (c) Electrochemical biosensor for glucose detection Figure 309 (a) Glucose concentration dependence of the currents at the peak relating to reduction of ZnO (1) for the [conductive PS/GOx/Au electrode] biosensor and (2) for the [conductive PS/GOx/ZnO/Au electrode] biosensor; (b) Glucose concentration dependence of the currents at 0.8 V potential for the [conductive PS/GOx/ZnO/Au electrode] biosensor (see online version for colours) (a) (b) From the above-mentioned results, it is revealed that the biosensors present in the ZnO layer in the construction are more sensitive to glucose concentration This proves that the ZnO nanotetrapods have acted as supporting materials that are good for glucose oxidase (GOx) enzyme immobilisation due to their high isoelectric point and the large surface to volume ratio On the other hand, the ZnO nanotetrapods exhibited an electrochemical activity and high electron conductivity The influences of different polymer membranes on the sensitivity of the electrochemical biosensor were studied Chitosan is a good conductive polymer with –OH and NH2 tags in its chemical construction and at a pH value of around the chitosan membrane is stable, and is usually used as an ion-transfer membrane We used chitosan as conductive polymer membranes, replacing the conductive PS membranes The results showed that when the glucose concentration varied in the range of 0–3 mM, the current at the potential of 0.8 V increased from 2.6 µA to 3.8 µA for 310 N.T Loan et al the chitosan membranes Meanwhile, the current at the potential of 0.8 V varied in the range from 10 µA to 23.8 µA for the conductive PS membranes Conclusion In this paper, wurtzite structural zinc oxide (ZnO) nanotetrapods were synthesised by a thermal evaporation method These ZnO nanotetrapods were used for the construction of an electrochemical biosensor for detecting glucose The ZnO layer in the biosensor construction played an especially important role In this biosensor, the ZnO nanotetrapods acted as supporting materials for glucose oxidase (GOx) enzyme loading because of the large surface 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(2006) ? ?Zinc oxide nanocomb biosensor for glucose detection? ??, Appl Phys Lett., Vol 88, p.233106 17 Sun, X.W., Wang, J.X and Wei, A (2008) ? ?Zinc oxide nanostructured biosensor for glucose detection? ??,... version for colours) (a) (b) (c) Electrochemical biosensor for glucose detection Figure 309 (a) Glucose concentration dependence of the currents at the peak relating to reduction of ZnO (1) for the... glucose concentration increased from mM to mM Electrochemical biosensor for glucose detection Figure 307 The current values at the potential of 0.8 V as a function of glucose concentration for