Tin Oxide Nanorod Array-Based Electrochemical Hydrogen Peroxide Biosensor
SnO2 nanorod array grown directly on alloy substrate has been employed as the working electrode of H2O2 biosensor. Single-crystalline SnO2 nanorods provide not only low isoelectric point and enough void spaces for facile horseradish peroxidase (HRP) immobilization but also numerous conductive channels for electron transport to and from current collector; thus, leading to direct electrochemistry of HRP. The nanorod array-based biosensor demonstrates high H2O2 sensing performance in terms of excellent sensitivity (379 μA mM−1 cm−2), low detection limit (0.2 μM) and high selectivity with the apparent Michaelis–Menten constant estimated to be as small as 33.9 μM. Our work further demonstrates the advantages of ordered array architecture in electrochemical device application and sheds light on the construction of other high-performance enzymatic biosensors.
KeywordsNanostructure SnO2 Nanorod array Biosensor
The determination of hydrogen peroxide (H2O2) has attracted great attention because H2O2 plays an important role in food industry, clinical diagnosis and environmental monitoring . The H2O2 detection is also of special interest in terms of tracking many biological targets such as glucose and lactose. Among various analytical techniques, electrochemical enzyme biosensor is most tempting due to its simplicity, high selectivity of the biological recognition elements and high sensitivity of electrochemical transduction process [2, 3, 4]. To achieve high biosensor performance, it is very necessary to fabricate novel electrode materials for both effective immobilization of enzyme (without losing the enzymatic bioactivity) and fast electron transport from enzyme to metallic electrode [5, 6, 7]. Over the past decades, nanostructured materials have emerged as promising electrode material candidates because of their regular structure, high active surface area for protein binding and good chemical and thermal stability . However, most electrodes using nanostructures for the direct determination of biomolecules such as H2O2 still have several problems unsolved. For example, the fabrication of electrodes conventionally involves casting nanostructure powders with insulating polymer binders onto current collector [1, 2, 3, 4, 8]. In this case, the response time and sensitivity of biosensor may be significantly limited by the electron transport through numerous interparticle contact areas. In addition, the sensing based on biomolecule oxidation is always at very high working potential, at which the interference from electroactive substrates co-existing in the sample media cannot be avoided.
Very recently, a perfect electrode architecture consisting of single-crystalline (ZnO, TiO2, Si, etc.) nanorod/nanowire/nanotube arrays grown directly on metal substrate was proposed to address the above impasse [5, 6, 7, 9]. These aligned one-dimensional (1D) nanostructures not only provide large-area scaffolds for enzyme immobilization but also direct channels for electron transport from redox enzymes to current collector. In several cases, this architecture leads to the realization of enzymatic direct electrochemistry that typically operates at negative potentials . Tin oxide (SnO2), a wide bandgap semiconductor, is well known for its excellent gas sensitivity [10, 11] and its use in fabricating transparent conductive glasses . SnO2 nanowires and their arrays have also been investigated for photoluminescence , lasing , field emission , transistors, solar cells  and lithium ion batteries [10, 17]. In particular, SnO2 is biocompatible [18, 19, 20], cheaper than Si, more stable than ZnO in physiological environment and more conductive than TiO2. Despite these, there is no report yet on applying SnO2 nanorod/nanowire arrays in electrochemical biosensors.
Herein, we present the use of direct-grown SnO2 nanorod arrays as a new platform for biosensing at low potential (-0.45 V), taking H2O2 detection as a case study. The low isoelectric point of SnO2 (4.0 ~ 5.0) facilitates the homogeneous immobilization of horseradish peroxidase (HRP; isoelectric point: 7.2) through electrostatic interaction. The facile HRP loading combined with ordered array architecture endows the constructed biosensor with very low detection limit and high sensitivity.
SnO2 nanorod arrays were synthesized by a facile hydrothermal method . Typically, a transparent solution of Sn(OH)62− was first prepared by mixing 1.17 g SnCl4·5H2O and 2.0 g NaOH; it was then transferred into a Teflon-lined autoclave (80 ml) with a Fe-based alloy substrate present, which was subsequently heated to 200°C for 24 h. After the reaction, the obtained SnO2 arrays on alloy substrate further underwent an annealing treatment at 400°C for 2 h in Ar gas. The product was characterized using powder X-ray diffraction (XRD) (Bruker D-8 Avance), transmission electron microscopy (TEM) (JEM-2010FEF, 200 kV), scanning electron microscopy (SEM) (JSM-6700F, 5.0 kV), and Raman spectroscope (Witech CRM200, 532 nm).
For a typical process of protein immobilization, 10 μl HRP solution (15 mg ml−1, 0.01 M PBS, pH 7.0) was dropped onto 0.75 × 0.75 cm2 SnO2 array alloy electrode surface. After the evaporation of water, the electrode was stored at 4°C. Prior to the measurement, the electrode was rinsed in 0.01 M PBS to remove the unimmobilized HRP, a 5 μl 0.5 w% Nafion solution was further introduced to form a tight membrane on the surface. The electrochemical properties were examined with a electrochemical workstation (CHI) utilizing a conventional three-electrode system, which consists of a SnO2 array on alloy as the working electrode, a Pt wire as the counter electrode, and a saturated calomel electrode (SCE) as the reference. The electrolyte was 0.1 M pH 7.0 N2-saturated PBS solution. A continuous stream of N2 was introduced into the cell above the liquid surface to maintain an inert atmosphere over the testing solution.
Results and Discussion
Comparison of analytical performances of H2O2 biosensors using different substrate materials for enzyme immobilization
Sensitivity (μA mM−1 cm−2)
Detection limit (μM)
C- TiO2 nanotube
ZnO-C nanowire array
SnO2 nanorod array
The detection reproducibility of the developed sensor was also examined by repeated detections of H2O2. When 4 μM H2O2 was measured continuously for eight assaying runs, a relative standard deviation (RSD) of 7.5% was obtained. In addition, the storage stability of the HRP-modified array electrode was tested by measuring the current responses to 4 μM H2O2 at intervals of 5 days during one month when the biosensor was stored in the refrigerator at 4°C. It was discovered that the so prepared electrode could retain about 92% electrocatalytic activity to H2O2 after one month storage. Other advantages of the SnO2 array-based biosensor are the low detection potential (−0.47 V) and high HRP enzyme selectivity, which could eliminate the interference of other substrates such as citric acid, ascorbic acid, sucrose and glucose . The addition of these common interfering species into the PBS showed negligible influence on H2O2 determination.
In sum, a novel biosensor based on immobilization of HRP on SnO2 nanorod array electrode has been developed. The three-dimensional (3D) array architecture provides a favorable microenvironment around HRP to retain the enzymatic bioactivity, while the vertically aligned single-crystalline SnO2 nanorods ensure numerous direct electron transport channels to electrode. Accordingly, the HRP/array electrode is demonstrated for determination of H2O2 at an applied potential of −0.47 V with excellent sensitivity, low detection limit and high selectivity. 3D microelectrode fabricated by direct growth offers a relatively straightforward means to produce biosensors on a mass scale and represents a useful platform for electroanalysis applications.
This work was financially supported by National Natural Science Foundation of China (No. 50872039; 50802032).
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