High performance metal oxide based sensing device using an electrode with a solid/liquid/air triphase interface
superhydrophobicity, triphase interface, metal oxide, electrocatalyst, sensing device
High performance metal oxide based sensing device using an electrode with a solid/liquid/air triphase interface Jun Zhang1,2,§, Xia Sheng1,§, Jian Jin2, Xinjian Feng1 (), and Lei Jiang3 1 College of Chemistry, Chemical Engineering and Materials Science, Soochow University, Suzhou 215123, China 2 Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, Suzhou 215123, China 3 School of Chemistry and Environment, Beihang University, Beijing 100191, China § Jun Zhang and Xia Sheng contributed equally to this work. Received: 12 December 2016 Revised: 13 January 2017 Accepted: 3 February 2017 © Tsinghua University Press and Springer-Verlag Berlin Heidelberg 2017 superhydrophobicity, triphase interface, metal oxide, electrocatalyst, sensing device ABSTRACT The wetting properties of an electrode surface are of significant importance to the performance of electrochemical devices because electron transfer occurs at the electrode/electrolyte interface. Described in this paper is a low-cost metal oxide electrocatalyst (CuO)-based high-performance sensing device using an enzyme electrode with a solid/liquid/air triphase interface in which the oxygen level is constant and sufficiently high. We apply the sensing device to detect glucose, a model test analyte, and demonstrate a linear dynamic range up to 50 mM, which is about 25 times higher than that obtained using a traditional enzyme electrode with a solid/liquid diphase interface. Moreover, we show that sensing devices based on a triphase assaying interface are insensitive to the significant oxygen level fluctuation in the analyte solution. 1 Introduction Inspired by the surface morphologies of natural non-wetting surfaces [1, 2], artificial substrates with superhydrophobicity have been fabricated and have affected many industrial processes [3–6], such as selfcleaning, drag reduction, and heat transfer [7–12]. One of the unique properties of a superhydrophobic surface is that it can trap gases inside micro-/nanostructured porous substrates that are in contact with an aqueous solution and form a solid/liquid/gas triphase interface [2, 3, 12]. The superhydrophobic surface is different from conventional hydrophobic or hydrophilic surfaces and offers the opportunity to solve the gasshortage problem that exists in many gas-involving reaction systems [13–16]. The advancement of high-performance sensing devices is currently of concern to our society because, for example, over 420 million adults worldwide are diabetic and require stringent glucose level monitoring. In the presence of O2, almost all oxidases catalyze oxidation of their substrates and produce H2O2. Nano Research DOI 10.1007/s12274-017-1510-x Address correspondence to firstname.lastname@example.org | www.editorialmanager.com/nare/default.asp 2 Nano Res. Anodic measurements of H2O2 formation at the surface of electrocatalyst-modified electrodes have been widely used for determining analyte levels and developing sensing devices [17–20]. Compared to precious metals, metal oxides, such TiO2, ZnO, Fe2O3, and CuO, are a class of low-cost, promising H2O2 electrocatalysts and have been employed in the fabrication of oxidasebased sensing devices for the detection of chemical and biological agents (e.g., glucose, lactate, and H2O2) [21–26]. Numerous studies of metal oxides have led to sensing devices with high sensitivities. However, the linear dynamic upper limit of these devices, which is a criterion for the fabrication of reliable sensing devices, is still restricted to approximately 2–10 mM (for glucose), which is well below the clinically required level of ~30 mM. Previous efforts have focused on the effect of electrocatalysts (morphology and composition) on the device performance. In contrast, we show (using CuO as an example) that it is not the electrocatalyst, but instead the low concentration and diffusion rate of O2 in the electrolyte that limit the oxidase kinetics (H2O2 formation), and thus the linear dynamic range of metal oxide-based sensing devices. Further, we solved this limitation by integrating CuO into a superhydrophobic substrate with a solid/liquid/air triphase interface, where oxygen is sufficient and constant. A linear detection upper limit of 50 mM (about 25 times higher than that of a diphase electrode system) was achieved by applying such a triphase electrode-based sensing device to detect glucose, a model test agent. Importantly, we show that such a triphase electrode is also insensitive to the significant oxygen level variation in an analyte solution. The design principle is generally applicable to the development of metal oxide-based high-performance biosensors. 2 Experimental 2.1 Chemicals Glucose oxidase (GOx, EC 220.127.116.11, 126,000 U/g) was obtained from Toyobo Co., Ltd. (Japan). Copper(II) sulfate pentahydrate and DL-lactic acid were purchased from Sigma (USA). Sodium hydroxide, sodium sulfate, glucose, chitosan, acetic acid, glutaraldehyde (25%), and ethanol were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). 2.2 Preparation of the triphase CuO/GOx electrode A carbon fiber mesh filled with carbon powder (~20 nm) was cleaned by sonication in water and ethanol (15 min each) and then immersed in a poly(tetrafluoroethylene) (PTFE) suspension (2 wt.%) for 10 min. It was removed from the suspension, dried in air, and then annealed at 350 °C for 30 min. CuO was fabricated on the substrate using our previously reported approach  followed by annealing in air at 400 °C. Solutions of GOx (1 wt.% in a phosphate buffer solution, PBS), chitosan (1 wt.% in acetic acid), and glutaraldehyde (25 wt.% in deionized water) at a volume ratio of 10:10:3 were mixed thoroughly. The mixture was then drop-casted onto the CuO catalyst-coated substrate with an area of 0.5 cm2. After drying naturally, it was used as the working electrode. Normal electrodes, i.e., hydrophilic fluorine-doped tin oxide (FTO) electrodes, were prepared in the same manner, except no PTFE treatment was applied. 2.3 Characterization methods Surface morphologies of the CuO catalyst-modified electrode substrates were characterized using fieldemission scanning electron microscopy (FE-SEM, HITACHI-S4800, Japan). Surface morphologies of the oxidase/chitosan composite film-coated electrode substrate were characterized using environmental scanning electron microscopy (E-SEM, FEI Quanta 400 FEG, USA). X-ray diffraction (XRD) analysis was performed using an X-ray powder diffractometer (X’Pert PRO, PANalytical, Almelo, Netherlands). Electrochemical experiments were performed using a CHI 660E workstation (CH Instruments, Inc., USA). The working electrode, Ag/AgCl reference electrode, and Pt wire counter electrode were inserted into a cell containing 5 mL of a 0.2 M PBS (pH 7.2). A stirring rate of 400 rpm was employed during the measurements. A schematic diagram of the setup is shown in Fig. S1 in the Electronic Supplementary Material (ESM). For the solutions with different oxygen concentrations, the buffered solutions were each purged with argon/ oxygen for 40 min prior to the bioassay. Cyclic www.theNanoResearch.com∣www.Springer.com/journal/12274 | Nano Research Nano Res. 3 voltammetry experiments were performed at a sweep speed of 50 mV/s, allowing a constant background current to be obtained while adding the desired concentration of glucose. The current difference was recorded, and it was used to plot the current– concentration curves. 3 Results and discussion The triphase enzyme electrode was fabricated by immobilizing glucose oxidase (a model enzyme) film onto a CuO-modified porous superhydrophobic carbon fiber substrate, as illustrated in Fig. 1(a). The analyte solution can wet the top enzyme and electrocatalyst layers but cannot penetrate into the porous substrate because of its superhydrophobicity. As a result, the air phase is trapped underneath the liquid phase, forming a solid/liquid/air triphase interface where sufficient oxygen can diffuse directly from the air phase to the oxidase reaction zone. Thus, the oxidase kinetics is no longer limited by the oxygen level, and large amounts of H2O2 production are ensured for glucose level determination. Figure 1(b) shows an FE-SEM image of the porous substrate after being filled with the nanostructured carbon powder and treated with PTFE (a low surface energy material). The micro-/nano-composite surface structure and the low surface energy materials endow superhydrophobicity to the substrate. The insert of Fig. 1(b) shows a nearly spherical water droplet placed on the substrate with a water contact angle (CA) of 151° ± 2°, indicating superhydrophobicity. The CuO electrocatalyst was fabricated on the topmost layer of the substrate by first depositing CuO onto the substrate using our recently reported robust electrochemical deposition approach , followed by annealing in air at 400 °C. The XRD pattern in Fig. 1(d) shows that the as-fabricated electrocatalyst can be indexed to a monoclinic-structured CuO (JCPDS 05-0661). CuO has a rough surface with a diameter Figure 1 (a) Schematic representation of the triphase electrode. The oxidase/chitosan composite film is immobilized on the superhydrophobic substrate that is modified with CuO. With the superhydrophobic substrate, sufficient oxygen can diffuse directly from the air phase to the oxidase reaction zone, which means that the oxidase kinetics is no longer limited by the oxygen level and ensures a large amount of H2O2 production for glucose level measurements. (b) FE-SEM images (top views) of the porous carbon fiber substrate. The insert in (b) is a spherical water droplet placed on the substrate with a water CA of 151° ± 2°, indicating a superhydrophobicity. (c) Low magnification FE-SEM image of a CuO electrocatalyst that was immobilized at the surface of the substrate. The insert in (c) is a water droplet placed on the CuO-modified substrate with a CA of 41° ± 2°, indicating a hydrophilic feature. (d) Powder XRD patterns of the CuO samples. (e) High-magnification FE-SEM image of the CuO electrocatalyst. (f) E-SEM image (top view) of the thin GOx/chitosan-covered electrode. | www.editorialmanager.com/nare/default.asp 4 Nano Res. range from 50 to 100 nm (Fig. 1(e)). The insert in Fig. 1(c) shows a water droplet placed on the substrate after CuO deposition with a water CA of 41° ± 2°, indicating that the surface of the electrode becomes hydrophilic. A mixture of GOx and chitosan was then drop-casted onto the as-deposited hydrophilic electrocatalyst surface and dried naturally. Figure 1(e) shows an E-SEM image of the electrode surface covered with a thin GOx/chitosan film. Unlike a normal diphase electrode that is fabricated by immobilizing CuO and GOx on a hydrophilic FTO glass substrate, when the triphase enzyme electrode is in contact with an analyte aqueous solution, the liquid can wet the top enzyme layer but cannot penetrate into the underlying microporous superhydrophobic substrate. This leads to the formation of an assaying three-phase interface where liquid, solid, and air coexist. The as-fabricated CuO-based triphase enzyme electrode was then used to detect glucose. Cyclic voltammograms (CVs) (Fig. 2(a)) of the CuO/GOx-based triphase electrode show that the electrooxidation current increases steadily with the increase of the glucose concentration up to 100 mM. The calibration plot of the oxidation current vs. glucose concentration (Fig. 2(b)) shows that the triphase electrode exhibits a linear range up to 50 mM (R2 = 0.99), which is more than 25 times higher than that which can be achieved using a traditional diphase electrode (up to about 2 mM) (inserts in Fig. 2(b) and Fig. S2 in the ESM). Figure S3(a) in the ESM shows CVs of the triphase enzyme electrode recorded in a 10 mM glucose solution at different scan rates (10–300 mV/s). The anodic peak current increases steadily with the potential scan rate, and it is linearly correlated to the square root of the scan rates, V1/2 (Fig. 2(c)), indicating that the electrochemical process of the triphase electrode is controlled by glucose diffusion. In sharp contrast, as shown in Fig. 2(d) and Fig. S3(b) in the ESM, when a traditional diphase electrode is operated in a 10 mM glucose solution, the anodic peak current is linearly correlated to the scan rate, V (Fig. 2(d)), implying that the electrochemical process is surface-reaction controlled at relatively high concentrations of the analyte solution. This explains why the dynamic range of a triphase electrode is much higher than that of a diphase electrode. Figure 2 (a) CVs corresponding to the electrooxidation of glucose on the triphase electrode with the increase of glucose concentration up to 100 mM. (b) Calibration plot derived from the CVs at 0.60 V vs. Ag/AgCl. Insert of (b) is the detection range achieved on a traditional diphase electrode. (c) The dependence of the oxidation peak current of glucose (10 mM) on the scan rate, V1/2, using a triphase electrode at 0.65 V vs. Ag/AgCl. (d) Plot of peak current vs. scan rate, V, using a diphase electrode at 0.65 V vs. Ag/AgCl. www.theNanoResearch.com∣www.Springer.com/journal/12274 | Nano Research Nano Res. 5 Generally, there are two reactions that occur at the electrode surface while detecting an analyte: (1) an oxidase-catalyzed bioreaction that produces H2O2 with the consumption of O2, and (2) an electrooxidation reaction of H2O2 at the surface of the CuO electrocatalyst that gives an electrode current output. The biosensor needs to be operated under an analyte diffusion-controlled process to possess a wide linear detection range; thus, a high electrode activity (high oxidase and electrocatalyst kinetics) is required. To understand whether the diphase electrode process is limited by CuO electrocatalyst kinetics or oxidase kinetics, experiments were performed to examine the catalytic ability of CuO toward H2O2 oxidation. CVs of the CuO-based diphase electrode (FTO) (Fig. 3(a) and Fig. S4(a) in the ESM) show that the electrooxidation current increases steadily with the increase of the H2O2 concentration up to 50 mM. Further, when the traditional diphase electrode is operated in a 10 mM H2O2 solution (as shown in Fig. 3(b) and Fig. S4(b) in the ESM), the anodic peak current is linearly correlated to the square root of the scan rate, V1/2 (Fig. 3(b)), implying a H2O2 diffusion-controlled electrode process. The results confirm that the kinetics of CuO toward H2O2 electrooxidation is very high and will not be the limiting factor of the diphase electrode process if sufficient H2O2 can be produced during the oxidasecatalyzed bioreaction. In a traditional diphase enzyme electrode system, the oxidase kinetics is limited because the concentration and mass transfer rate of O2 in the analyte solution are very low. This consequently restricts the enzymatic reaction kinetics (the formation of H2O2) and thus the linear detection range. In marked contrast, sufficient oxygen at the triphase electrode surface can be supplied directly from the air phase with negligible resistance; thus, the oxidase kinetics are no longer oxygen level-limited, and large amounts of H2O2 can be produced in proportion to the glucose concentration so that a wide detection range and good sensitivity are achieved. We further studied the influence of oxygen level fluctuations in solution on the detection accuracy of the electrode because oxygen directly participates in the oxidase catalytic reaction, and its level is significantly variable in the analyte solution. Prior to the assay, oxygen was removed from the solution or saturated by bubbling with argon or oxygen for 40 min, respectively. As shown in Fig. 4(a), the electrooxidation current of the diphase electrode increases with respect to the oxygen level at the same glucose concentration, indicating that the oxygen level variation has a strong impact on the detection accuracy. Higher oxygen levels would increase the H2O2 formation at the diphase electrode surface and the electrode current response. In sharp contrast, the CVs for the triphase electrode are almost the same for the solutions with and without soluble oxygen (Fig. 4(b)). Oxygen at the triphase electrode is supplied directly from the air phase with a diffusion constant that is four orders of magnitude higher than that for the liquid phase ; thus, the oxygen level at the electrode/liquid interface will be air phase-dependent and insensitive to the oxygen level in the solution. Therefore, the detection accuracy of the triphase CuO-based enzyme electrode is insensitive to the oxygen level variation in the analyte solution. Figure 3 Performance of the CuO electrocatalyst toward H2O2 electrooxidation on a diphase electrode. (a) Calibration plot derived from the CVs at 0.6 V. (b) Plot of the peak current vs. scan rate, V1/2, using a diphase electrode at 0.65 V vs. Ag/AgCl. | www.editorialmanager.com/nare/default.asp 6 Nano Res. In addition to the wide linear detection range, sensitivity, and accuracy, the other important aspects of the triphase electrode were further characterized. Figures S5(a) and S5(b) in the ESM show, respectively, measurements of 10 mM glucose using five separately fabricated triphase electrodes and 100 time continuous measurements using 10 mM glucose and one electrode. The relative standard deviations are 1.3% and 2.3%, respectively, and indicate the acceptable reproducibility, repeatability, and short-term stability of the electrode. Figure S6 in the ESM shows that the electrode responds rapidly within 10 s upon the addition of glucose and has a detection limit of 0.75 μM (based on the signalto-noise ratio, S/N = 3). The results demonstrate that the triphase CuO-based amperometric biosensor provides an outstanding performance. 4 Conclusions In summary, we fabricated a CuO-based highperformance solid/liquid/air triphase electrode for amperometric biosensor applications with a wide linear detection range, high sensitivity, and good detection accuracy. Sufficient oxygen at the triphase assaying interface means that both the electrocatalyst kinetics and the oxidase kinetics are no longer restricted, which consequently lead to an analyte diffusioncontrolled electrochemical process, wide linear detection range, and good sensitivity. Further, the constant oxygen level at the electrode surface makes the electrode response insensitive to the oxygen level fluctuations in the liquid phase. This study reveals that, in contrast to previous tremendous efforts and progress in electrocatalyst exploration, rational electrode-architecture construction is actually of great importance because both rapid electron transport and mass (e.g., oxygen) transfer are commonly required for high-performance device applications. Acknowledgements This work is financially supported by the National Natural Science Foundation of China (No. 21371178), the Jiangsu Province Science Foundation for Distinguished Young Scholars (No. BK20150032), and the Chinese Thousand Youth Talents Program (No. YZBQF11001). Electronic Supplementary Material: Supplementary material (electrochemical measurement curves) is available in the online version of this article at https:// doi.org/10.1007/s12274-017-1510-x. References  Barthlott, W.; Neinhuis, C. Purity of the sacred lotus, or escape from contamination in biological surfaces. Planta 1997, 202, 1–8.  Cassie, A. B. D.; Baxter, S. Wettability of porous surfaces. 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Tsinghua University Press
Jun Zhang,Xia Sheng,Jian Jin,Xinjian Feng,Lei Jiang, High performance metal oxide based sensing device using an electrode with a solid/liquid/air triphase interface. NanoRes.2017, 10(9): 2998–3004