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A solid-state electrochemical sensing platform based on a supramolecular hydrogel
Accepted Manuscript Title: A Solid-State Electrochemical Sensing Platform Based on a Supramolecular Hydrogel Authors: Li Fu, Aiwu Wang, Fucong Lyu, Guosong Lai, Jinhong Yu, Cheng-Te Lin, Zhong Liu, Aimin Yu, Weitao Su PII: DOI: Reference:
S0925-4005(18)30299-5 https://doi.org/10.1016/j.snb.2018.02.029 SNB 24124
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
16-9-2017 24-1-2018 2-2-2018
Please cite this article as: Li Fu, Aiwu Wang, Fucong Lyu, Guosong Lai, Jinhong Yu, Cheng-Te Lin, Zhong Liu, Aimin Yu, Weitao Su, A Solid-State Electrochemical Sensing Platform Based on a Supramolecular Hydrogel, Sensors and Actuators B: Chemical https://doi.org/10.1016/j.snb.2018.02.029 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A Solid-State Electrochemical Sensing Platform Based on a Supramolecular Hydrogel
Li Fu1*, Aiwu Wang2*, Fucong Lyu2, Guosong Lai3, Jinhong Yu5, Cheng-Te Lin5, Zhong Liu6, Aimin Yu3,4 and Weitao Su1 1
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Collage of Materials and Environmental Engineering, Hangzhou Dianzi University, Hangzhou, 310018, P.R. China 2
Center of Super-Diamond and Advanced Films (COSDAF), City University of Hong Kong, Hong Kong 3
Hubei Collaborative Innovation Center for Rare Metal Chemistry, Hubei Key Laboratory of Pollutant Analysis & Reuse Technology, Department of Chemistry, Hubei Normal University, Huangshi, 435002, P.R. China 4
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Department of Chemistry and Biotechnology, Faculty of Science, Engineering and Technology, Swinburne University of Technology, Hawthorn, VIC 3122, Australia
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Key Laboratory of Marine New Materials and Related Technology, Zhejiang Key Laboratory of Marine Materials and Protection Technology, Ningbo Institute of Material Technology and Engineering, Chinese Academy of Sciences, Ningbo 315201, P.R. China 6
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Key Laboratory of Comprehensive and Highly Efficient Utilization of Salt Resources, Qinghai Institute of Salt Lakes, Chinese Academy of Sciences, Xining 810008, China
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Corresponding authors: Li Fu, Aiwu Wang Email: [email protected]; [email protected]
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Graphical abstract
Highlights
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Supramolecular hydrogels of biopolymer chitosan and zinc ions were first time used as disposable electrochemical sensing platform. Chitosan hydrogel has been directly served as electrolyte. The current changes of zinc redox has been used for determination of hydroxyl radical or hydrogen peroxide. The linear detection range can be further manipulated via varying the depolymerization time. Proposed chitosan hydrogel could be used for disposable point of care and in-field testing applications
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Abstract: Supramolecular hydrogels formed from the biopolymer chitosan and zinc ions were used as disposable electrochemical sensing platforms for the determination of hydroxyl radicals and hydrogen peroxide for the first time. The enclosed chitosan hydrogel served as the electrolyte for the system. In the presence of hydroxyl radicals or hydrogen peroxide, the chitosan hydrogel underwent depolymerization, resulting in the release of zinc ions from complexes formed with the hydrogel. The changes in the current from the zinc ions were then monitored and used as a signal for determining the concentration of analyte. The proposed chitosan hydrogel sensing platform exhibited a comparable detection performance with wide linear ranges and low detection limits for the two analytes. The linear detection range could be further manipulated by varying the depolymerization time. Due to the low cost and simplicity of the preparation process, this enzyme- and DNAfree sensing platform possesses great potential for application in disposable point-of-care devices and in-field testing. Additionally, the application of the proposed chitosan hydrogel to antioxidant screening could be further explored.
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Keywords: Chitosan; Hydrogel; Electrochemical sensing platform; Depolymerization; Hydroxyl radical
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1. Introduction
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Chitosan is a unique cationic polysaccharide that is derived from chitin by deacetylation in an alkaline media [1]. Chitosan has attracted considerable attention owing to its commercial and possible biomedical uses. It can be applied as a seed primer and biopesticide in the agriculture field [2, 3]. It also has been used as an additive in a selfhealing polyurethane paint coating [4]. The excellent absorption ability of chitosan makes it a potential adsorbent for water treatment [5]. In the clinical field, the outstanding characteristics of chitosan, such as its biodegradability, biocompatibility and antigenicity, make it useful in tissue regeneration [6] and drug delivery applications [7]. In addition to its solution form, chitosan can also be prepared as a hydrogel via a crosslinking process by adding a suitable crosslinking agent [8, 9]. For example, a chitosan hydrogel can be 2
prepared via crosslinking with glycerol in an acidic medium [8]. Wu et al. [10] reported the preparation of a chitosan hydrogel by using genipin as the cross-linking agent. Recently, Sun et al. [9] reported an ultrafast hydrogelation process by adding transition metal ions into chitosan.
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Chitosan hydrogel is a 3D network structure that is capable on locking water in. The hydrogel possesses many new properties, such as an improved swelling behaviour [11] and responsiveness to stimuli [9]. The hydrogel form of chitosan could be manufactured in packed products, especially for disposable medical applications. So far, many studies have investigated the possibility of using chitosan-based hydrogels as wound dressings [12, 13]. For electrochemical applications, although chitosan has been widely applied as an electrode material due to its high permeability towards anions and excellent film-forming ability [14-18], few reports have focused on the electrochemical properties of chitosan hydrogel. Zhang and Ji [19] reported an enzyme-immobilized chitosan hydrogel for the electrochemical determination of phenol. Liu and co-workers demonstrated a trichloroacetic acid electrochemical sensor based on a silver nanoparticle-doped chitosan hydrogel film [20].
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The abovementioned works demonstrated that chitosan hydrogel could be used as a surface modifier to enhance the electrochemical properties of an electrode. We believe that chitosan hydrogel could also be used as a platform for sensing applications. A piece of chitosan hydrogel provides a closed aqueous environment that can function as an electrolyte. In this work, we prepared a chitosan hydrogel via a simple zinc ion crossinglinking method with a fast gelation rate. A complex was formed between the zinc ions and chitosan chains, which could then be continuously deconstructed by hydroxyl radicals. The as-prepared chitosan hydrogel was directly used as a sensing platform for determining hydrogen peroxide and hydroxyl radical for the first time. The concentrations of both analytes were proportional to the concentration of zinc ions freed from the complexes during the depolymerisation process. Since hydrogen peroxide and hydroxyl radical are two major reactive oxygen species (ROS) produced by humans and associated with health [21, 22], we believe that the proposed chitosan hydrogel provides an easy and disposable method with great potential for clinical analysis. Moreover, this work also shows that the proposed chitosan hydrogel could be potentially used for antioxidant screening.
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2. Experimental Chemicals and instruments Zinc acetate, acetic acid, ascorbic acid, uric acid, potassium ferrocyanide, sodium hydroxide and hydrogen peroxide were purchased from Aladdin Reagent Inc. Chitosan (deacetylation: 75%~80%, 50000-190000 Da) was purchased from Sigma-Aldrich. Hydrogen peroxide assay kit and hydroxyl free radical assay kit were purchased from Nanjing Jiancheng Bioengineering Institute. All other chemicals were analytical-grade reagents and were used without further purification. A Fenton solution was prepared in an 3
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aqueous solution containing potassium ferrocyanide and hydrogen peroxide at a molar ratio of 1:6. Milli-Q water (18.2 MΩ/cm) was used throughout the experiments. The morphologies the prepared samples were characterized using a field emission scanning electron microscope (FESEM, Philips XL-30 FeSEM, ZEISS SUPRA 40VP, Germany). The hydrogel sample used for SEM analysis was freeze dried. Powder X-ray diffraction (XRD) patterns were collected on an X-ray diffractometer (D2 Phaser) with Cu-Kα radiation (λ = 1.5405 Å). Weight-average molecular weight of hydrogel during the depolymerization has been tested by a gel permeation chromatography (LC-Tech GPC Vario, Dorfen) with an auto-sampler, a solvent delivery module and a fraction collector was used for the analysis. Salicylate assay was used for hydroxyl radical confirmation. Salicylic acid was added into hydrogel depolymerization process. After the depolymerization, the solution was filtered using an EPS filter paper followed by adding sodium tungstate (0.25 ml 10% (w/v) in H2O), and sodium nitrite (0.25 ml 0.5% (w/v) in H2O) were added. The solutions were left standing for five minutes, and then KOH (0.5 ml of 0.5 M) was added; exactly sixty seconds after the addition of KOH, the absorbance at 510 nm was determined. Fourier transform infrared (FTIR) spectroscopic studies were carried out with a Nicolet iS5 spectrometer. Chitosan hydrogel and depolymerized chitosan samples were freeze-dried for characterization. High performance liquid chromatography (HPLC) analysis was conducted using an Agilent 1100 HPLC spectrometer (detection channel of 214 nm) in 35 minutes. The mobile phase solution was an isocratic system containing 10% acetonitrile and 90% deionized water. All electrochemical measurements were performed using a CHI 832 electrochemical workstation with a conventional threeelectrode system comprised of platinum wire as the auxiliary electrode, a 3 M Ag/AgCl electrode as the reference and a glassy carbon electrode as the working electrode. All experiments were conducted at room temperature.
Preparation of chitosan-zinc ions hydrogel and its depolymerization
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The chitosan-zinc ion hydrogel was prepared based on our previous report with some modifications [9]. In a typical synthetic process, a certain amount of zinc acetate solution (30 mM) was added to 2 mL of a 1 wt % chitosan solution (in 1% acetic acid). After vigorous shaking for 30 s, 0.1 M NaOH was added dropwise until the gelation process was initiated. The pH of the final chitosan-zinc ion hydrogel (denoted chitosan hydrogel) was 6.5.
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Depolymerization was conducted by injecting a certain amount of hydrogen peroxide solution or Fenton solution into the chitosan hydrogel. Sonication in a bath was performed for 15 seconds to accelerate the diffusion process.
Electrochemical determination
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After depolymerization for a desired period, the working, reference and counter electrodes were inserted into the hydrogel for electrochemical measurements. Cyclic voltammetry (CV) was conducted from ‒1.6 V to 1.2 V with a scan rate of 100 mV/s. Differential pulse voltammetry (DPV) was conducted from ‒1.2 V to ‒0.7 V with an amplitude of 0.03 V, a pulse width of 0.05 s, a pulse period of 0.1 s and a quiet time of 5 s.
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3. Results and Discussion
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A schematic diagram of the chitosan hydrogel formation and depolymerization is shown in Figure 1A. A supramolecular complexation process was initiated between the Zn2+ and chitosan chains at a suitable pH. The formed hydrogel had a high water content with a critical gelation concentration of approximately 1.1 wt%. Note that chitosan hydrogels with different viscosities could be formed by manipulating the amount of zinc ions injected. A low viscosity was favourable for this work because it allowed for easy insertion of the electrodes and subsequent refilling of the gaps induced from the insertion process. Moreover, a low zinc ion concentration was also favourable for the electrochemical determination performance. Figure 1B and 1C compare the morphological changes from chitosan to the chitosan hydrogel. As expected, a smooth film structure was observed for chitosan. After rapid complexing with zinc ions, a novel, 3D, porous nanostructure was formed in the chitosan hydrogel. This structure is due to the successful formation of crosslinks between zinc ions and chitosan.
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Hydroxyl radicals could induce depolymerization of the chitosan hydrogel. Upon injection of hydrogen peroxide, hydroperoxide anions were produced, which are very unstable and decompose to form reactive hydroxyl radicals (•HO) [23]. The rupture of glycosidic bonds caused by a hydrogen abstraction reaction between the hydroxyl radical and chitosan polysaccharide chains was the main cause of the depolymerization process [24, 25]. A clear liquidation process was visually observed when a high concentration of hydrogen peroxide was injected into the chitosan hydrogel. The salicylate assay and EPR results were confirmed the generation of hydroxyl radical. Figure S1 shows DMPO spin-trapping EPR spectrum from chitosan hydrogel after the addition of 0.03 mM H2O2. As shown in the figure, the spectrum exhibits four splitting lines with a 1:2:2:1 intensity ratio, which is characteristic of the DMPO–OH adduct. Figure 1D shows the XRD patterns of the chitosan hydrogel before and after the addition of hydrogen peroxide. It can be seen that the XRD pattern of the chitosan hydrogel was is in good agreement with those of previous reports [26, 27]. After depolymerization for 30 min, the characteristic peaks of chitosan at 2θ =19.24◦, 2θ =30.05◦ and 2θ =40.22◦ clearly decreased and broadened, suggesting that the crystalline region of the chitosan was depolymerized to an amorphous phase. The depolymerization has been also confirmed by the SEM characterization. As shown in Figure S2, after 30 depolymerization, the porous nanostructure of the chitosan hydrogel was partially destroyed.
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The depolymerization process slowly decomposed the interwoven structure of the chitosan chains and zinc ions, resulting in low molecular weight chitosan-linked zinc ions. These units are more likely to diffuse to the electrode surface when an electric potential is applied. Thus, a higher current response from zinc ions could be expected after depolymerization of the hydrogel.
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Chitosan is constructed by copolymers of glucosamine (GlcN) and N-acetylglucosamine (GlcNAc) linked with b-1,4-glycosidic bonds. Under ideal condition, the depolymerization should release of both GlcN and GlcNAc. HPLC has been applied for analysing chitosan hydrogel and low molecular weight parts of chitosan. As shown in Figure S3, HPLC characterization showed main decomposed products are GlcNAc. Other products include GlcN and their dimer to teteamer. Moreover, the HPLC profile also indicated the depolymerized chitosan still remains some high molecular weight part. FTIR was further used for investigating the changes in the chemical structure of the chitosan hydrogel (Figure S4). Line width and frequency position of the band above 3000 cm-1 can be ascribed to the intermolecular crystal lattice, which depends on the hydrogen bonding network, and results in the incorporation of water [28]. This band slightly shifts at depolymerized chitosan compared with that of the original chitosan, suggesting the decrease in the ordered structure [29, 30]. This can be also confirmed by the decrease in the peak located at 1320.6 cm-1 for the spectrum of depolymerized chitosan compared with than of the original chitosan (corresponding to GlcNAc residues) [31]. Based on above results, the GlcNAc linked zinc ions mainly participated the redox reaction.
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Cyclic voltammetry was then employed to study the redox behaviour of the zinc ions in the chitosan hydrogel. Figure 2A depicts the cyclic voltammograms (CVs) of 2.5 mM zinc ions in an acetic acid-sodium hydroxide solution (curve a), the chitosan hydrogel (curve b) and the chitosan hydrogel after the addition of 0.01 and 0.03 mM hydrogen peroxide (curve c, d), recorded using a glassy carbon electrode (GCE). For the CV scan in the solution without chitosan hydrogel (curve a), zinc produced a reduction peak at approximately −1.13 V. Further lowering the scanning potential led to a reduction process associated with the electrodeposition of metallic zinc [32]. A pale grey film could be observed on the GCE surface. In the reverse scan, an anodic peak appeared at approximately −1.06 V that was associated with the dissolution of zinc. In comparison, the redox reaction of zinc ions was significantly suppressed in the chitosan hydrogel (curve b). The cathodic curve reveals that the initiation of zinc ion reduction shifted to −1.24 V, while the reverse scan shows that the oxidation of zinc appeared at −0.97 V. The remarkable decrease in both the cathodic and anodic currents can be ascribed to the supramolecular cross-linking between zinc ions and chitosan chains, which locks the zinc ions in place and consequently hinders their diffusion. The redox behaviour of zinc ions was partially restored after depolymerization of the chitosan hydrogel. Curve c in Figure 2A shows the CV of zinc ions in a chitosan hydrogel containing 0.01 mM H2O2. An increase in the reduction current was noticed during the scan, suggesting that more zinc ions reached the electrode. Meanwhile, a more distinct current rise was recorded in the reverse scan at −0.95 V, corresponding to the oxidation of 6
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zinc. The restoration of the redox activity was found to be affected by the H2O2 concentration. We also recorded a CV of zinc ions in a chitosan hydrogel containing 0.03 mM H2O2. Further increasing in both the cathodic and anodic currents were noticed (curve d), suggesting that a higher hydrogen peroxide concentration accelerated the depolymerization process and enhanced the amount of low molecular weight chitosanliked zinc ions that participated in the redox reaction. Moreover, the anodic peak exhibited a positive shift with respect to that in curve c due to the acceleration of the ion migration velocity in the electrolyte after chitosan depolymerization.
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In the above system, the current difference of the zinc ions before and after depolymerization of the hydrogel could be used as a signal for probing the concentrations of hydrogen peroxide and hydroxyl radicals in the system. Excess zinc ions could result in uncomplexed zinc ions in the hydrogel system and consequently increase the background current, while insufficient zinc ions would lead to failure in the hydrogel formation. Therefore, the initial zinc ion concentration in the chitosan hydrogel should play an important role in the detection sensitivity. Thus, the initial zinc ion concentration was first optimized by monitoring the oxidation current of zinc before and after the addition of 0.03 mM H2O2. The current difference (ΔI) was plotted against the initial zinc ion concentration in Figure 2B. It is worth noting that the zinc ion concentrations higher than 20 mM could result in overflow during the scan. Very small differences with high standard deviations were detected when high zinc ion concentrations were present in the hydrogel system. A perceptible and reliable difference was recorded for zinc ion concentrations down to 6 mM. Consecutive drops in the zinc ion concentration increased the current difference because fewer free zinc ions contributed to the background current. The optimized zinc ion concentration was found to be 2.5 mM. Smaller current differences were observed with further decreases in the zinc ion concentration even though small background currents were recorded. This result can be ascribed to the limited amount of the low molecular weight chitosan-liked zinc ions that were freed during depolymerization.
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Since hydrogen peroxide decomposition is a continual process, the depolymerization time also needed to be optimized. After injection of hydrogen peroxide, sonication in a bath for 15 s was performed to ensure a homogenous reaction. Figure 2C shows the current changes as a function of the depolymerization time. It is pertinent to note that the current response increased quickly at the beginning of the reaction and then continued increasing but at a slower rate. For practical purposes and to achieve better performance, the electrochemical investigations were conducted 15 min after the injection of hydrogen peroxide. Through the depolymerization of the chitosan hydrogel, the amount of hydrogen peroxide could be reflected by the current signal increase of zinc. Figure 3A shows the DPV responses, recorded using a GCE, to various concentrations of hydrogen peroxide injected into the chitosan hydrogel. A quiet time of 5 s was applied for pre-reduction of the zinc ions. Then, the oxidation of zinc was recorded and used to calculate the concentration of hydrogen peroxide. We found that the DPV oxidation peak current continuously increased with increasing hydrogen peroxide concentration. We further plotted the relationship 7
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between the DPV responses and the logarithmic values of the hydrogen peroxide concentrations (Figure 3B). The logarithmic relationship between the current responses against the hydrogen peroxide concentration could be ascribed to the depolymerization process kinetics. Therefore, the weight-average molecular weight (Mw) changes of the chitosan hydrogel during the H2O2 depolymerization process was recorded and plotted in Figure S5. It can be noticed that the Mw of chitosan hydrogel decreased steeply during the first 30 mins. Depolymerized chitosan collected at 2 h showed a near completion of reaction. This result indicated the certain parts of the chitosan (relative low molecular weight parts and lattice defect parts) will be easily depolymerized at the initial stage. Well-crystallized core parts are more resistance of hydroxyl radical attack. Therefore, the current response of zinc redox was increased significantly when H2O2 increased and then tended slowly when further increasing of H2O2 concentration. A good linear relationship was obtained in the range from 0.5 to 2000 μM with a correction coefficient of 0.997. The detection limit (LOD) of the proposed method was estimated as 0.14 μM based on a signal-to-noise ratio of 3, which is comparable to many previously reported enzymatic and nonenzymatic hydrogen peroxide electrochemical sensors [33-37]. Table 1 summaries the H2O2 detection details of the proposed solid-state sensing platform with other reported works.
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Hydroxyl radicals are one of the most common ROS and are primarily responsible for cellular disorders and cytotoxic effects [38]. So far, several methods have been developed for the detection of hydroxyl radicals, such as electron spin resonance spectroscopy [39], fluorescence spectroscopy [40], chromatography [41] and electrochemical methods [42, 43]. Among them, electrochemical methods have received great interest. The fabrication of a Fenton-mediated DNA damage system is the most common approach for the electrochemical determination of hydroxyl radicals. However, the high price of oligonucleotides and the long incubation process restrict the practical application of this method. Since the depolymerization process of chitosan hydrogel is caused by hydroxyl radicals [25, 44], we further tested the feasibility of the proposed chitosan hydrogel for the direct determination of hydroxyl radicals.
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It is well known that the concentration of hydroxyl radicals is equal to that of Fe2+ in the Fenton solution [45, 46]. Figure 4A depicts the DPV responses after various concentrations of the Fenton solution were injected into the chitosan hydrogel. As expected, the DPV response of zinc oxidation increased with increasing hydroxyl radical concentration after depolymerization for 15 min. The peak current was proportional to the logarithmic value of the hydroxyl radical concentration in the range of 4–100 nM, and the LOD was estimated to be as low as 1.98 nM. Moreover, we further found that the chitosan hydrogel could exhibit different detection ranges of hydroxyl radical by manipulating the depolymerization time. As shown in Figure 4B, a linear detection range of 50–500 nM was obtained was only 8 min of depolymerization was applied. However, larger deviations were observed when a short depolymerization was applied, which is due to the quick depolymerization process at the early stage. On the other hand, lengthening of the depolymerization time allowed this method to detect hydroxyl radicals in a low 8
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concentration range. This detection performance was comparable with that reported for DNA damage-based electrochemical sensors [42, 43]. The detailed analytical performance of the chitosan hydrogel in the detection of hydroxyl radicals was compared with that of other reports and summarized in Table 2. Although some of the reports showed a superior performance compared with that of the proposed chitosan hydrogel, the proposed sensing platform exhibited several new features, such as good manufacturability, absence of DNA, low cost and feasibility for point-of-care devices.
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Because hydroxyl radicals are highly reactive and occur naturally in cells and extracellular fluids but can be deactivated in the presence of antioxidants, we further studied the performance of the chitosan hydrogel upon the addition of antioxidants, and the results are shown in Figure 5. Ascorbic acid (AA) and uric acid (UA) are two common non-enzymatic antioxidants that have been used as ROS scavengers. As shown in the figure, the injection of AA and UA solutions significantly reduced the depolymerization rate. AA also exhibited a higher antioxidant activity than UA at the same concentration, which is in a good agreement with the activity described in other reports [47, 48]. This result suggests that the proposed chitosan hydrogel also has potential for screening and assessing the performance of antioxidants.
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In terms of an electrochemical sensing platform, selectivity is a very important feature for real applications. Some common interfering biological species (glucose (Glu), sucrose (Su), dopamine (DA) and acetaminophen (AP)) and ROS (1O2 and NaClO) were therefore investigated. Due to the large difference in the redox potential, the presence of Glu, Su and AP showed negligible changes in both H2O2 and hydroxyl radical detection. When 1O2 and NaClO were injected into the chitosan hydrogel in conjunction with hydroxyl radicals, the current exhibited a slight increase relative to that observed for hydroxyl radicals only. An RSD of 3.7% was obtained. The DA showed a RSD of 5.7 %. These results suggest that the proposed solid-state sensing platform has acceptable selectivity.
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The reproducibility was tested by examination of six individual freshly prepared chitosan hydrogels. The relative standard deviations (RSD) of the chitosan hydrogel in the determination of H2O2 and hydroxyl radicals were 2.6% and 2.8%, respectively. The operational stability of the chitosan hydrogel was studied through ten successive determinations of H2O2 and hydroxyl radicals, giving deviations of 3.6% and 4.1%, respectively, suggesting that the proposed solid-state sensor has good stability. The performance of the proposed sensing platform has been applied for comprising with the commercial kits and the results have been summarized in Table S1. It can be seen that the chitosan hydrogel could result in similar sensing performances commercial kits with slightly higher RSD. Similarly with chitosan, alginate is a polysaccharide could be degraded by H2O2 but with an opposite charge [49-54]. It can be expected that the interaction between metal ions with alginate will be stronger than that of the chitosan-metal ions complex. Based on the result
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shown in this work, a solid-state sensing platform based on alginate hydrogel is worth for further explore. 4. Conclusion
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In this study, we proposed the application of a supramolecular hydrogel as a new, disposable electrochemical sensing platform for the determination of both hydrogen peroxide and hydroxyl radicals. Hydroxyl radicals induced a depolymerization process that freed zinc ions from the complexes formed with the hydrogel. Then, the released zinc ion acted as an electrochemical probe for determining the concentrations of the analytes. Comparable linear detection ranges and LODs were obtained for the two analytes. The depolymerization time could be varied to accommodate a variety of analyte concentration requirements. Considering the proposed chitosan hydrogel is ready for use a sensing platform, it shows a high potential for point-of-care analysis and diagnosis. In addition, the proposed chitosan hydrogel shows potential for the evaluation of antioxidant activity.
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[58] S.-J. Li, Y. Xing, H.-Y. Yang, J.-Y. Huang, W.-T. Wang, R.-T. Liu, Electrochemical synthesis of a binary mn-co oxides decorated graphene nanocomposites for application in nonenzymatic H2O2 sensing, Int. J. Electrochem. Sci, 12 (2017) 6566-6576. [59] G.-w. He, J.-q. Jiang, D. Wu, Y.-l. You, X. Yang, F. Wu, Y.-j. Hu, A novel nonenzymatic hydrogen peroxide electrochemical sensor based on facile synthesis of copper oxide nanoparticles dopping into graphene sheets@ cerium oxide nanocomposites sensitized screen printed electrode, International Journal Of Electrochemical Science, 11 (2016) 8486-8498. [60] M. Liang, S. Jia, S. Zhu, L.H. Guo, Photoelectrochemical sensor for the rapid detection of in situ dna damage induced by enzyme-catalyzed fenton reaction, Environmental Science & Technology, 42 (2008) 635639. [61] L. Wu, Y. Yang, H. Zhang, G. Zhu, X. Zhang, J. Chen, Sensitive electrochemical detection of hydroxyl radical with biobarcode amplification, Analytica Chimica Acta, 756 (2012) 1-6. [62] Y. Yang, J. Zhou, Y. Zhang, Q. Zou, X. Zhang, J. Chen, Sensitive electrochemical detection of hydroxyl radical based on MBs–DNA–AgNPs nanocomposite, Sensors & Actuators B Chemical, 182 (2013) 504-509.
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Li Fu received his Ph.D from Swinburne University of Technology, Australia. Currently, he is an associate professor at College of Materials and Environmental Engineering, Hangzhou Dianzi University. His research interest involves electroanalysis, 2D materials based composite preparation and application.
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Aiwu Wang received his Bachelor degree in Materials Science and Engineering from University of Science and Technology Beijing in 2013 and his Master degree in Nanotechnology and Materials Engineering from City University of Hong Kong in 2014. He is now pursuing his Ph.D. in Prof. Yangyang Li group at the City University of Hong Kong.
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Fucong Lyu received his Bachelor degree in Applied Chemistry from Central South University in 2013. He is now pursuing his Ph.D. in Prof. Jian Lu’s group at the City University of Hong Kong. His research interest involves supramolecular hydrogel, energy storage materials and catalytic materials.
Guosong Lai received his BS degree in Chemistry from Hubei Normal University and MS degree in Analytical Chemistry from Central China Normal University in 2001 and 2008, respectively. In 2011, he received his Ph.D degree in Chemistry from Nanjing University, China. From August 2012 to December 2013, he worked as a Postdoctoral Fellow at Swinburne University of Technology, Australia. Currently he is a professor of chemistry at Hubei Normal University and named as Chutian Young Scholar in Hubei 13
Province of China. His research interests include the development and application of nano biosensors, immunoassay, bioanalytical chemistry and electroanalytical chemistry.
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Jinhong Yu received his Ph.D degree in material science at Shanghai Jiaotong University, China. Currently, he is an associate professor in Ningbo Institute of Materials Technology and Engineering, Chinese Academy of Sciences. His research focuses on design novel polymer-based composites with high thermal conductivity.
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Cheng-Te Lin received his Ph.D. degree in Materials Science and Engineering at National Tsing Hua University (Taiwan) in 2008. In 2012, he was a postdoc in Prof. Jing Kong's group at Massachusetts Institute of Technology (MIT, USA), and undertook the investigation of graphene aerogel supercapacitors. Subsequently, he took part in the project of highly thermally-conductive polymer composites with Prof. Gang Chen (MIT, USA). From 2014 June, he is working as a full professor at Ningbo Institute of Material Technology and Engineering. He is the recipient of "1000 Young Talents Plan" from China's government (2015), "Hundred Talents Program" from Chinese Academy of Sciences (2014), and so on. Dr. Lin has 70 publications on Nature Communications, Advanced Functional Materials, Journal of Materials Chemistry A, and Carbon etc. His research interests focus on the development of graphene-based applications, including CVD growth technique, functional composites, thermal interface materials, and biosensors.
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Zhong Liu is a researcher professor at Qinghai Institute of Salt Lakes, Chinese Academy of Sciences. He received his Ph.D. degree in Institute of Coal Chemistry, Chinese Academy of Sciences. His research works are focus on preparing the metal oxides-based nanostructures, especially in catalytic materials and sensing materials.
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Aimin Yu received her Ph.D in Chemistry at Nanjing University (China) in 1997. After being a Lecturer at the same university for three years, Dr Yu took a postdoctoral position at Max Planck Institute of Colloids and Interfaces (Germany). Dr Yu moved to Australia in 2003 and currently is an Associate Professor at Swinburne University of Technology, based in Melbourne Australia. Her research fields include: synthesis and functionalization of nanomaterials; preparation of nanostructured thin films; and development of biosensors and assay methods for biomedical applications.
Weitao Su is a professor of College of Materials and Environmental Engineering, Hangzhou Dianzi University. He received his Ph.D of Electronic Engineering from 14
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Shanghai Institute of Technical Physics (Chinese Academy of Sciences). His current research interests are 2D materials and tip enhanced Raman spectroscopy.
Table 1. Comparison of H2O2 analytical detection using electrochemical sensors. Detection limit (μM)
Reference
0.83
[55]
50-500
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1-2580 5-1200
0.5 0.8
5-18000 0.5 to 2000
2.1 0.14
[56]
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[59] This work
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MoS2/GRMWCNTs/GCE α-FeOOH NW/carbon fiber paper Co3O4/MCNFs/GCE Mn-Co oxides decorated graphene/GCE GS/CeO2-CuO/SPE Chitosan hydrogel
Linear detection range (μM) 5-145
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Modified electrodes
Detection limit (nM)
Reference
50000
[60]
3000
[61]
50-4000
10
[62]
0.125-625 4-100 or 50-500
― 1.98
[43] This work
5000-10000000
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SnO2/PDDA/GOX/PDDA/dsDNA film DNA-based biosensor MBs–DNA–AgNPs nanocomposite DNA modified sensor Chitosan hydrogel
Linear detection range (nM) ―
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Table 2. Comparison of hydroxyl radical analytical detection using electrochemical sensors.
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Figure Captions Figure 1. (A) Schematic illustration of functional mechanism of the chitosan hydrogel sensing platform. SEM images of (B) chitosan and (C) the chitosan hydrogel. (D) Normalized XRD patterns of the chitosan hydrogel before and after polymerization.
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Figure 2. (A) CV scans of 2.5 mM zinc ions in (a) an acetic acid-sodium hydroxide solution, (b) the chitosan hydrogel, (c) the chitosan hydrogel with 0.01 mM H2O2 and (d) the chitosan hydrogel with 0.03 mM H2O2. Effects of (B) the zinc ion concentration and (C) depolymerization time on the electrochemical responses of the chitosan hydrogel towards 0.3 mM H2O2. Figure 3. (A) DPV curves of the chitosan hydrogel after the injection of various concentrations of hydrogen peroxide. Curves a-k correspond to hydrogen peroxide at concentrations from 0.5 μM to 2000 μM. (B) Relationship of ΔI versus the logarithm of the hydrogen peroxide concentration.
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Figure 4. DPV curves of the chitosan hydrogel after injection of various concentrations of hydroxyl radicals with different depolymerization periods: (A) 0.004 to 1 μM hydroxyl radical with 15 min of depolymerization; (B) 0.05 to 5μM hydroxyl radical with 8 min of depolymerization.
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Figure 5. DPV response changes of the chitosan hydrogel to OH (40 nM) in the presence of AA (0.5 μM) and UA (0.5 μM).
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Figure 1. (A) Schematic illustration of functional mechanism of the chitosan hydrogel sensing platform. SEM images of (B) chitosan and (C) the chitosan hydrogel. (D) Normalized XRD patterns of the chitosan hydrogel before and after polymerization.
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Figure 2. (A) CV scans of 2.5 mM zinc ions in (a) an acetic acid-sodium hydroxide solution, (b) the chitosan hydrogel, (c) the chitosan hydrogel with 0.01 mM H2O2 and (d) the chitosan hydrogel with 0.03 mM H2O2. Effects of (B) the zinc ion concentration and (C) depolymerization time on the electrochemical responses of the chitosan hydrogel towards 0.03 mM H2O2.
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Figure 3. (A) DPV curves of the chitosan hydrogel after the injection of various concentrations of hydrogen peroxide. Curves a-k correspond to hydrogen peroxide at concentrations from 0.5 μM to 2000 μM. (B) Relationship of ΔI versus the logarithm of the hydrogen peroxide concentration.
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Figure 4. DPV curves of the chitosan hydrogel after injection of various concentrations of hydroxyl radicals with different depolymerization periods: (A) 0.004 to 1 μM hydroxyl radical with 15 min of depolymerization; (B) 0.05 to 5μM hydroxyl radical with 8 min of depolymerization.
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Figure 5. DPV response changes of the chitosan hydrogel to OH (40 nM) in the presence of AA (0.5 μM) and UA (0.5 μM).
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S0925-4005(18)30299-5 https://doi.org/10.1016/j.snb.2018.02.029 SNB 24124
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
16-9-2017 24-1-2018 2-2-2018
Please cite this article as: Li Fu, Aiwu Wang, Fucong Lyu, Guosong Lai, Jinhong Yu, Cheng-Te Lin, Zhong Liu, Aimin Yu, Weitao Su, A Solid-State Electrochemical Sensing Platform Based on a Supramolecular Hydrogel, Sensors and Actuators B: Chemical https://doi.org/10.1016/j.snb.2018.02.029 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A Solid-State Electrochemical Sensing Platform Based on a Supramolecular Hydrogel
Li Fu1*, Aiwu Wang2*, Fucong Lyu2, Guosong Lai3, Jinhong Yu5, Cheng-Te Lin5, Zhong Liu6, Aimin Yu3,4 and Weitao Su1 1
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Collage of Materials and Environmental Engineering, Hangzhou Dianzi University, Hangzhou, 310018, P.R. China 2
Center of Super-Diamond and Advanced Films (COSDAF), City University of Hong Kong, Hong Kong 3
Hubei Collaborative Innovation Center for Rare Metal Chemistry, Hubei Key Laboratory of Pollutant Analysis & Reuse Technology, Department of Chemistry, Hubei Normal University, Huangshi, 435002, P.R. China 4
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Department of Chemistry and Biotechnology, Faculty of Science, Engineering and Technology, Swinburne University of Technology, Hawthorn, VIC 3122, Australia
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Key Laboratory of Marine New Materials and Related Technology, Zhejiang Key Laboratory of Marine Materials and Protection Technology, Ningbo Institute of Material Technology and Engineering, Chinese Academy of Sciences, Ningbo 315201, P.R. China 6
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Key Laboratory of Comprehensive and Highly Efficient Utilization of Salt Resources, Qinghai Institute of Salt Lakes, Chinese Academy of Sciences, Xining 810008, China
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Corresponding authors: Li Fu, Aiwu Wang Email: [email protected]; [email protected]
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Graphical abstract
Highlights
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Supramolecular hydrogels of biopolymer chitosan and zinc ions were first time used as disposable electrochemical sensing platform. Chitosan hydrogel has been directly served as electrolyte. The current changes of zinc redox has been used for determination of hydroxyl radical or hydrogen peroxide. The linear detection range can be further manipulated via varying the depolymerization time. Proposed chitosan hydrogel could be used for disposable point of care and in-field testing applications
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Abstract: Supramolecular hydrogels formed from the biopolymer chitosan and zinc ions were used as disposable electrochemical sensing platforms for the determination of hydroxyl radicals and hydrogen peroxide for the first time. The enclosed chitosan hydrogel served as the electrolyte for the system. In the presence of hydroxyl radicals or hydrogen peroxide, the chitosan hydrogel underwent depolymerization, resulting in the release of zinc ions from complexes formed with the hydrogel. The changes in the current from the zinc ions were then monitored and used as a signal for determining the concentration of analyte. The proposed chitosan hydrogel sensing platform exhibited a comparable detection performance with wide linear ranges and low detection limits for the two analytes. The linear detection range could be further manipulated by varying the depolymerization time. Due to the low cost and simplicity of the preparation process, this enzyme- and DNAfree sensing platform possesses great potential for application in disposable point-of-care devices and in-field testing. Additionally, the application of the proposed chitosan hydrogel to antioxidant screening could be further explored.
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Keywords: Chitosan; Hydrogel; Electrochemical sensing platform; Depolymerization; Hydroxyl radical
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1. Introduction
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Chitosan is a unique cationic polysaccharide that is derived from chitin by deacetylation in an alkaline media [1]. Chitosan has attracted considerable attention owing to its commercial and possible biomedical uses. It can be applied as a seed primer and biopesticide in the agriculture field [2, 3]. It also has been used as an additive in a selfhealing polyurethane paint coating [4]. The excellent absorption ability of chitosan makes it a potential adsorbent for water treatment [5]. In the clinical field, the outstanding characteristics of chitosan, such as its biodegradability, biocompatibility and antigenicity, make it useful in tissue regeneration [6] and drug delivery applications [7]. In addition to its solution form, chitosan can also be prepared as a hydrogel via a crosslinking process by adding a suitable crosslinking agent [8, 9]. For example, a chitosan hydrogel can be 2
prepared via crosslinking with glycerol in an acidic medium [8]. Wu et al. [10] reported the preparation of a chitosan hydrogel by using genipin as the cross-linking agent. Recently, Sun et al. [9] reported an ultrafast hydrogelation process by adding transition metal ions into chitosan.
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Chitosan hydrogel is a 3D network structure that is capable on locking water in. The hydrogel possesses many new properties, such as an improved swelling behaviour [11] and responsiveness to stimuli [9]. The hydrogel form of chitosan could be manufactured in packed products, especially for disposable medical applications. So far, many studies have investigated the possibility of using chitosan-based hydrogels as wound dressings [12, 13]. For electrochemical applications, although chitosan has been widely applied as an electrode material due to its high permeability towards anions and excellent film-forming ability [14-18], few reports have focused on the electrochemical properties of chitosan hydrogel. Zhang and Ji [19] reported an enzyme-immobilized chitosan hydrogel for the electrochemical determination of phenol. Liu and co-workers demonstrated a trichloroacetic acid electrochemical sensor based on a silver nanoparticle-doped chitosan hydrogel film [20].
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The abovementioned works demonstrated that chitosan hydrogel could be used as a surface modifier to enhance the electrochemical properties of an electrode. We believe that chitosan hydrogel could also be used as a platform for sensing applications. A piece of chitosan hydrogel provides a closed aqueous environment that can function as an electrolyte. In this work, we prepared a chitosan hydrogel via a simple zinc ion crossinglinking method with a fast gelation rate. A complex was formed between the zinc ions and chitosan chains, which could then be continuously deconstructed by hydroxyl radicals. The as-prepared chitosan hydrogel was directly used as a sensing platform for determining hydrogen peroxide and hydroxyl radical for the first time. The concentrations of both analytes were proportional to the concentration of zinc ions freed from the complexes during the depolymerisation process. Since hydrogen peroxide and hydroxyl radical are two major reactive oxygen species (ROS) produced by humans and associated with health [21, 22], we believe that the proposed chitosan hydrogel provides an easy and disposable method with great potential for clinical analysis. Moreover, this work also shows that the proposed chitosan hydrogel could be potentially used for antioxidant screening.
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2. Experimental Chemicals and instruments Zinc acetate, acetic acid, ascorbic acid, uric acid, potassium ferrocyanide, sodium hydroxide and hydrogen peroxide were purchased from Aladdin Reagent Inc. Chitosan (deacetylation: 75%~80%, 50000-190000 Da) was purchased from Sigma-Aldrich. Hydrogen peroxide assay kit and hydroxyl free radical assay kit were purchased from Nanjing Jiancheng Bioengineering Institute. All other chemicals were analytical-grade reagents and were used without further purification. A Fenton solution was prepared in an 3
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aqueous solution containing potassium ferrocyanide and hydrogen peroxide at a molar ratio of 1:6. Milli-Q water (18.2 MΩ/cm) was used throughout the experiments. The morphologies the prepared samples were characterized using a field emission scanning electron microscope (FESEM, Philips XL-30 FeSEM, ZEISS SUPRA 40VP, Germany). The hydrogel sample used for SEM analysis was freeze dried. Powder X-ray diffraction (XRD) patterns were collected on an X-ray diffractometer (D2 Phaser) with Cu-Kα radiation (λ = 1.5405 Å). Weight-average molecular weight of hydrogel during the depolymerization has been tested by a gel permeation chromatography (LC-Tech GPC Vario, Dorfen) with an auto-sampler, a solvent delivery module and a fraction collector was used for the analysis. Salicylate assay was used for hydroxyl radical confirmation. Salicylic acid was added into hydrogel depolymerization process. After the depolymerization, the solution was filtered using an EPS filter paper followed by adding sodium tungstate (0.25 ml 10% (w/v) in H2O), and sodium nitrite (0.25 ml 0.5% (w/v) in H2O) were added. The solutions were left standing for five minutes, and then KOH (0.5 ml of 0.5 M) was added; exactly sixty seconds after the addition of KOH, the absorbance at 510 nm was determined. Fourier transform infrared (FTIR) spectroscopic studies were carried out with a Nicolet iS5 spectrometer. Chitosan hydrogel and depolymerized chitosan samples were freeze-dried for characterization. High performance liquid chromatography (HPLC) analysis was conducted using an Agilent 1100 HPLC spectrometer (detection channel of 214 nm) in 35 minutes. The mobile phase solution was an isocratic system containing 10% acetonitrile and 90% deionized water. All electrochemical measurements were performed using a CHI 832 electrochemical workstation with a conventional threeelectrode system comprised of platinum wire as the auxiliary electrode, a 3 M Ag/AgCl electrode as the reference and a glassy carbon electrode as the working electrode. All experiments were conducted at room temperature.
Preparation of chitosan-zinc ions hydrogel and its depolymerization
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The chitosan-zinc ion hydrogel was prepared based on our previous report with some modifications [9]. In a typical synthetic process, a certain amount of zinc acetate solution (30 mM) was added to 2 mL of a 1 wt % chitosan solution (in 1% acetic acid). After vigorous shaking for 30 s, 0.1 M NaOH was added dropwise until the gelation process was initiated. The pH of the final chitosan-zinc ion hydrogel (denoted chitosan hydrogel) was 6.5.
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Depolymerization was conducted by injecting a certain amount of hydrogen peroxide solution or Fenton solution into the chitosan hydrogel. Sonication in a bath was performed for 15 seconds to accelerate the diffusion process.
Electrochemical determination
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After depolymerization for a desired period, the working, reference and counter electrodes were inserted into the hydrogel for electrochemical measurements. Cyclic voltammetry (CV) was conducted from ‒1.6 V to 1.2 V with a scan rate of 100 mV/s. Differential pulse voltammetry (DPV) was conducted from ‒1.2 V to ‒0.7 V with an amplitude of 0.03 V, a pulse width of 0.05 s, a pulse period of 0.1 s and a quiet time of 5 s.
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3. Results and Discussion
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A schematic diagram of the chitosan hydrogel formation and depolymerization is shown in Figure 1A. A supramolecular complexation process was initiated between the Zn2+ and chitosan chains at a suitable pH. The formed hydrogel had a high water content with a critical gelation concentration of approximately 1.1 wt%. Note that chitosan hydrogels with different viscosities could be formed by manipulating the amount of zinc ions injected. A low viscosity was favourable for this work because it allowed for easy insertion of the electrodes and subsequent refilling of the gaps induced from the insertion process. Moreover, a low zinc ion concentration was also favourable for the electrochemical determination performance. Figure 1B and 1C compare the morphological changes from chitosan to the chitosan hydrogel. As expected, a smooth film structure was observed for chitosan. After rapid complexing with zinc ions, a novel, 3D, porous nanostructure was formed in the chitosan hydrogel. This structure is due to the successful formation of crosslinks between zinc ions and chitosan.
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Hydroxyl radicals could induce depolymerization of the chitosan hydrogel. Upon injection of hydrogen peroxide, hydroperoxide anions were produced, which are very unstable and decompose to form reactive hydroxyl radicals (•HO) [23]. The rupture of glycosidic bonds caused by a hydrogen abstraction reaction between the hydroxyl radical and chitosan polysaccharide chains was the main cause of the depolymerization process [24, 25]. A clear liquidation process was visually observed when a high concentration of hydrogen peroxide was injected into the chitosan hydrogel. The salicylate assay and EPR results were confirmed the generation of hydroxyl radical. Figure S1 shows DMPO spin-trapping EPR spectrum from chitosan hydrogel after the addition of 0.03 mM H2O2. As shown in the figure, the spectrum exhibits four splitting lines with a 1:2:2:1 intensity ratio, which is characteristic of the DMPO–OH adduct. Figure 1D shows the XRD patterns of the chitosan hydrogel before and after the addition of hydrogen peroxide. It can be seen that the XRD pattern of the chitosan hydrogel was is in good agreement with those of previous reports [26, 27]. After depolymerization for 30 min, the characteristic peaks of chitosan at 2θ =19.24◦, 2θ =30.05◦ and 2θ =40.22◦ clearly decreased and broadened, suggesting that the crystalline region of the chitosan was depolymerized to an amorphous phase. The depolymerization has been also confirmed by the SEM characterization. As shown in Figure S2, after 30 depolymerization, the porous nanostructure of the chitosan hydrogel was partially destroyed.
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The depolymerization process slowly decomposed the interwoven structure of the chitosan chains and zinc ions, resulting in low molecular weight chitosan-linked zinc ions. These units are more likely to diffuse to the electrode surface when an electric potential is applied. Thus, a higher current response from zinc ions could be expected after depolymerization of the hydrogel.
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Chitosan is constructed by copolymers of glucosamine (GlcN) and N-acetylglucosamine (GlcNAc) linked with b-1,4-glycosidic bonds. Under ideal condition, the depolymerization should release of both GlcN and GlcNAc. HPLC has been applied for analysing chitosan hydrogel and low molecular weight parts of chitosan. As shown in Figure S3, HPLC characterization showed main decomposed products are GlcNAc. Other products include GlcN and their dimer to teteamer. Moreover, the HPLC profile also indicated the depolymerized chitosan still remains some high molecular weight part. FTIR was further used for investigating the changes in the chemical structure of the chitosan hydrogel (Figure S4). Line width and frequency position of the band above 3000 cm-1 can be ascribed to the intermolecular crystal lattice, which depends on the hydrogen bonding network, and results in the incorporation of water [28]. This band slightly shifts at depolymerized chitosan compared with that of the original chitosan, suggesting the decrease in the ordered structure [29, 30]. This can be also confirmed by the decrease in the peak located at 1320.6 cm-1 for the spectrum of depolymerized chitosan compared with than of the original chitosan (corresponding to GlcNAc residues) [31]. Based on above results, the GlcNAc linked zinc ions mainly participated the redox reaction.
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Cyclic voltammetry was then employed to study the redox behaviour of the zinc ions in the chitosan hydrogel. Figure 2A depicts the cyclic voltammograms (CVs) of 2.5 mM zinc ions in an acetic acid-sodium hydroxide solution (curve a), the chitosan hydrogel (curve b) and the chitosan hydrogel after the addition of 0.01 and 0.03 mM hydrogen peroxide (curve c, d), recorded using a glassy carbon electrode (GCE). For the CV scan in the solution without chitosan hydrogel (curve a), zinc produced a reduction peak at approximately −1.13 V. Further lowering the scanning potential led to a reduction process associated with the electrodeposition of metallic zinc [32]. A pale grey film could be observed on the GCE surface. In the reverse scan, an anodic peak appeared at approximately −1.06 V that was associated with the dissolution of zinc. In comparison, the redox reaction of zinc ions was significantly suppressed in the chitosan hydrogel (curve b). The cathodic curve reveals that the initiation of zinc ion reduction shifted to −1.24 V, while the reverse scan shows that the oxidation of zinc appeared at −0.97 V. The remarkable decrease in both the cathodic and anodic currents can be ascribed to the supramolecular cross-linking between zinc ions and chitosan chains, which locks the zinc ions in place and consequently hinders their diffusion. The redox behaviour of zinc ions was partially restored after depolymerization of the chitosan hydrogel. Curve c in Figure 2A shows the CV of zinc ions in a chitosan hydrogel containing 0.01 mM H2O2. An increase in the reduction current was noticed during the scan, suggesting that more zinc ions reached the electrode. Meanwhile, a more distinct current rise was recorded in the reverse scan at −0.95 V, corresponding to the oxidation of 6
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zinc. The restoration of the redox activity was found to be affected by the H2O2 concentration. We also recorded a CV of zinc ions in a chitosan hydrogel containing 0.03 mM H2O2. Further increasing in both the cathodic and anodic currents were noticed (curve d), suggesting that a higher hydrogen peroxide concentration accelerated the depolymerization process and enhanced the amount of low molecular weight chitosanliked zinc ions that participated in the redox reaction. Moreover, the anodic peak exhibited a positive shift with respect to that in curve c due to the acceleration of the ion migration velocity in the electrolyte after chitosan depolymerization.
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In the above system, the current difference of the zinc ions before and after depolymerization of the hydrogel could be used as a signal for probing the concentrations of hydrogen peroxide and hydroxyl radicals in the system. Excess zinc ions could result in uncomplexed zinc ions in the hydrogel system and consequently increase the background current, while insufficient zinc ions would lead to failure in the hydrogel formation. Therefore, the initial zinc ion concentration in the chitosan hydrogel should play an important role in the detection sensitivity. Thus, the initial zinc ion concentration was first optimized by monitoring the oxidation current of zinc before and after the addition of 0.03 mM H2O2. The current difference (ΔI) was plotted against the initial zinc ion concentration in Figure 2B. It is worth noting that the zinc ion concentrations higher than 20 mM could result in overflow during the scan. Very small differences with high standard deviations were detected when high zinc ion concentrations were present in the hydrogel system. A perceptible and reliable difference was recorded for zinc ion concentrations down to 6 mM. Consecutive drops in the zinc ion concentration increased the current difference because fewer free zinc ions contributed to the background current. The optimized zinc ion concentration was found to be 2.5 mM. Smaller current differences were observed with further decreases in the zinc ion concentration even though small background currents were recorded. This result can be ascribed to the limited amount of the low molecular weight chitosan-liked zinc ions that were freed during depolymerization.
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Since hydrogen peroxide decomposition is a continual process, the depolymerization time also needed to be optimized. After injection of hydrogen peroxide, sonication in a bath for 15 s was performed to ensure a homogenous reaction. Figure 2C shows the current changes as a function of the depolymerization time. It is pertinent to note that the current response increased quickly at the beginning of the reaction and then continued increasing but at a slower rate. For practical purposes and to achieve better performance, the electrochemical investigations were conducted 15 min after the injection of hydrogen peroxide. Through the depolymerization of the chitosan hydrogel, the amount of hydrogen peroxide could be reflected by the current signal increase of zinc. Figure 3A shows the DPV responses, recorded using a GCE, to various concentrations of hydrogen peroxide injected into the chitosan hydrogel. A quiet time of 5 s was applied for pre-reduction of the zinc ions. Then, the oxidation of zinc was recorded and used to calculate the concentration of hydrogen peroxide. We found that the DPV oxidation peak current continuously increased with increasing hydrogen peroxide concentration. We further plotted the relationship 7
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between the DPV responses and the logarithmic values of the hydrogen peroxide concentrations (Figure 3B). The logarithmic relationship between the current responses against the hydrogen peroxide concentration could be ascribed to the depolymerization process kinetics. Therefore, the weight-average molecular weight (Mw) changes of the chitosan hydrogel during the H2O2 depolymerization process was recorded and plotted in Figure S5. It can be noticed that the Mw of chitosan hydrogel decreased steeply during the first 30 mins. Depolymerized chitosan collected at 2 h showed a near completion of reaction. This result indicated the certain parts of the chitosan (relative low molecular weight parts and lattice defect parts) will be easily depolymerized at the initial stage. Well-crystallized core parts are more resistance of hydroxyl radical attack. Therefore, the current response of zinc redox was increased significantly when H2O2 increased and then tended slowly when further increasing of H2O2 concentration. A good linear relationship was obtained in the range from 0.5 to 2000 μM with a correction coefficient of 0.997. The detection limit (LOD) of the proposed method was estimated as 0.14 μM based on a signal-to-noise ratio of 3, which is comparable to many previously reported enzymatic and nonenzymatic hydrogen peroxide electrochemical sensors [33-37]. Table 1 summaries the H2O2 detection details of the proposed solid-state sensing platform with other reported works.
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Hydroxyl radicals are one of the most common ROS and are primarily responsible for cellular disorders and cytotoxic effects [38]. So far, several methods have been developed for the detection of hydroxyl radicals, such as electron spin resonance spectroscopy [39], fluorescence spectroscopy [40], chromatography [41] and electrochemical methods [42, 43]. Among them, electrochemical methods have received great interest. The fabrication of a Fenton-mediated DNA damage system is the most common approach for the electrochemical determination of hydroxyl radicals. However, the high price of oligonucleotides and the long incubation process restrict the practical application of this method. Since the depolymerization process of chitosan hydrogel is caused by hydroxyl radicals [25, 44], we further tested the feasibility of the proposed chitosan hydrogel for the direct determination of hydroxyl radicals.
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It is well known that the concentration of hydroxyl radicals is equal to that of Fe2+ in the Fenton solution [45, 46]. Figure 4A depicts the DPV responses after various concentrations of the Fenton solution were injected into the chitosan hydrogel. As expected, the DPV response of zinc oxidation increased with increasing hydroxyl radical concentration after depolymerization for 15 min. The peak current was proportional to the logarithmic value of the hydroxyl radical concentration in the range of 4–100 nM, and the LOD was estimated to be as low as 1.98 nM. Moreover, we further found that the chitosan hydrogel could exhibit different detection ranges of hydroxyl radical by manipulating the depolymerization time. As shown in Figure 4B, a linear detection range of 50–500 nM was obtained was only 8 min of depolymerization was applied. However, larger deviations were observed when a short depolymerization was applied, which is due to the quick depolymerization process at the early stage. On the other hand, lengthening of the depolymerization time allowed this method to detect hydroxyl radicals in a low 8
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concentration range. This detection performance was comparable with that reported for DNA damage-based electrochemical sensors [42, 43]. The detailed analytical performance of the chitosan hydrogel in the detection of hydroxyl radicals was compared with that of other reports and summarized in Table 2. Although some of the reports showed a superior performance compared with that of the proposed chitosan hydrogel, the proposed sensing platform exhibited several new features, such as good manufacturability, absence of DNA, low cost and feasibility for point-of-care devices.
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Because hydroxyl radicals are highly reactive and occur naturally in cells and extracellular fluids but can be deactivated in the presence of antioxidants, we further studied the performance of the chitosan hydrogel upon the addition of antioxidants, and the results are shown in Figure 5. Ascorbic acid (AA) and uric acid (UA) are two common non-enzymatic antioxidants that have been used as ROS scavengers. As shown in the figure, the injection of AA and UA solutions significantly reduced the depolymerization rate. AA also exhibited a higher antioxidant activity than UA at the same concentration, which is in a good agreement with the activity described in other reports [47, 48]. This result suggests that the proposed chitosan hydrogel also has potential for screening and assessing the performance of antioxidants.
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In terms of an electrochemical sensing platform, selectivity is a very important feature for real applications. Some common interfering biological species (glucose (Glu), sucrose (Su), dopamine (DA) and acetaminophen (AP)) and ROS (1O2 and NaClO) were therefore investigated. Due to the large difference in the redox potential, the presence of Glu, Su and AP showed negligible changes in both H2O2 and hydroxyl radical detection. When 1O2 and NaClO were injected into the chitosan hydrogel in conjunction with hydroxyl radicals, the current exhibited a slight increase relative to that observed for hydroxyl radicals only. An RSD of 3.7% was obtained. The DA showed a RSD of 5.7 %. These results suggest that the proposed solid-state sensing platform has acceptable selectivity.
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The reproducibility was tested by examination of six individual freshly prepared chitosan hydrogels. The relative standard deviations (RSD) of the chitosan hydrogel in the determination of H2O2 and hydroxyl radicals were 2.6% and 2.8%, respectively. The operational stability of the chitosan hydrogel was studied through ten successive determinations of H2O2 and hydroxyl radicals, giving deviations of 3.6% and 4.1%, respectively, suggesting that the proposed solid-state sensor has good stability. The performance of the proposed sensing platform has been applied for comprising with the commercial kits and the results have been summarized in Table S1. It can be seen that the chitosan hydrogel could result in similar sensing performances commercial kits with slightly higher RSD. Similarly with chitosan, alginate is a polysaccharide could be degraded by H2O2 but with an opposite charge [49-54]. It can be expected that the interaction between metal ions with alginate will be stronger than that of the chitosan-metal ions complex. Based on the result
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shown in this work, a solid-state sensing platform based on alginate hydrogel is worth for further explore. 4. Conclusion
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In this study, we proposed the application of a supramolecular hydrogel as a new, disposable electrochemical sensing platform for the determination of both hydrogen peroxide and hydroxyl radicals. Hydroxyl radicals induced a depolymerization process that freed zinc ions from the complexes formed with the hydrogel. Then, the released zinc ion acted as an electrochemical probe for determining the concentrations of the analytes. Comparable linear detection ranges and LODs were obtained for the two analytes. The depolymerization time could be varied to accommodate a variety of analyte concentration requirements. Considering the proposed chitosan hydrogel is ready for use a sensing platform, it shows a high potential for point-of-care analysis and diagnosis. In addition, the proposed chitosan hydrogel shows potential for the evaluation of antioxidant activity.
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Author Biographies
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[58] S.-J. Li, Y. Xing, H.-Y. Yang, J.-Y. Huang, W.-T. Wang, R.-T. Liu, Electrochemical synthesis of a binary mn-co oxides decorated graphene nanocomposites for application in nonenzymatic H2O2 sensing, Int. J. Electrochem. Sci, 12 (2017) 6566-6576. [59] G.-w. He, J.-q. Jiang, D. Wu, Y.-l. You, X. Yang, F. Wu, Y.-j. Hu, A novel nonenzymatic hydrogen peroxide electrochemical sensor based on facile synthesis of copper oxide nanoparticles dopping into graphene sheets@ cerium oxide nanocomposites sensitized screen printed electrode, International Journal Of Electrochemical Science, 11 (2016) 8486-8498. [60] M. Liang, S. Jia, S. Zhu, L.H. Guo, Photoelectrochemical sensor for the rapid detection of in situ dna damage induced by enzyme-catalyzed fenton reaction, Environmental Science & Technology, 42 (2008) 635639. [61] L. Wu, Y. Yang, H. Zhang, G. Zhu, X. Zhang, J. Chen, Sensitive electrochemical detection of hydroxyl radical with biobarcode amplification, Analytica Chimica Acta, 756 (2012) 1-6. [62] Y. Yang, J. Zhou, Y. Zhang, Q. Zou, X. Zhang, J. Chen, Sensitive electrochemical detection of hydroxyl radical based on MBs–DNA–AgNPs nanocomposite, Sensors & Actuators B Chemical, 182 (2013) 504-509.
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Li Fu received his Ph.D from Swinburne University of Technology, Australia. Currently, he is an associate professor at College of Materials and Environmental Engineering, Hangzhou Dianzi University. His research interest involves electroanalysis, 2D materials based composite preparation and application.
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Aiwu Wang received his Bachelor degree in Materials Science and Engineering from University of Science and Technology Beijing in 2013 and his Master degree in Nanotechnology and Materials Engineering from City University of Hong Kong in 2014. He is now pursuing his Ph.D. in Prof. Yangyang Li group at the City University of Hong Kong.
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Fucong Lyu received his Bachelor degree in Applied Chemistry from Central South University in 2013. He is now pursuing his Ph.D. in Prof. Jian Lu’s group at the City University of Hong Kong. His research interest involves supramolecular hydrogel, energy storage materials and catalytic materials.
Guosong Lai received his BS degree in Chemistry from Hubei Normal University and MS degree in Analytical Chemistry from Central China Normal University in 2001 and 2008, respectively. In 2011, he received his Ph.D degree in Chemistry from Nanjing University, China. From August 2012 to December 2013, he worked as a Postdoctoral Fellow at Swinburne University of Technology, Australia. Currently he is a professor of chemistry at Hubei Normal University and named as Chutian Young Scholar in Hubei 13
Province of China. His research interests include the development and application of nano biosensors, immunoassay, bioanalytical chemistry and electroanalytical chemistry.
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Jinhong Yu received his Ph.D degree in material science at Shanghai Jiaotong University, China. Currently, he is an associate professor in Ningbo Institute of Materials Technology and Engineering, Chinese Academy of Sciences. His research focuses on design novel polymer-based composites with high thermal conductivity.
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Cheng-Te Lin received his Ph.D. degree in Materials Science and Engineering at National Tsing Hua University (Taiwan) in 2008. In 2012, he was a postdoc in Prof. Jing Kong's group at Massachusetts Institute of Technology (MIT, USA), and undertook the investigation of graphene aerogel supercapacitors. Subsequently, he took part in the project of highly thermally-conductive polymer composites with Prof. Gang Chen (MIT, USA). From 2014 June, he is working as a full professor at Ningbo Institute of Material Technology and Engineering. He is the recipient of "1000 Young Talents Plan" from China's government (2015), "Hundred Talents Program" from Chinese Academy of Sciences (2014), and so on. Dr. Lin has 70 publications on Nature Communications, Advanced Functional Materials, Journal of Materials Chemistry A, and Carbon etc. His research interests focus on the development of graphene-based applications, including CVD growth technique, functional composites, thermal interface materials, and biosensors.
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Zhong Liu is a researcher professor at Qinghai Institute of Salt Lakes, Chinese Academy of Sciences. He received his Ph.D. degree in Institute of Coal Chemistry, Chinese Academy of Sciences. His research works are focus on preparing the metal oxides-based nanostructures, especially in catalytic materials and sensing materials.
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Aimin Yu received her Ph.D in Chemistry at Nanjing University (China) in 1997. After being a Lecturer at the same university for three years, Dr Yu took a postdoctoral position at Max Planck Institute of Colloids and Interfaces (Germany). Dr Yu moved to Australia in 2003 and currently is an Associate Professor at Swinburne University of Technology, based in Melbourne Australia. Her research fields include: synthesis and functionalization of nanomaterials; preparation of nanostructured thin films; and development of biosensors and assay methods for biomedical applications.
Weitao Su is a professor of College of Materials and Environmental Engineering, Hangzhou Dianzi University. He received his Ph.D of Electronic Engineering from 14
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Shanghai Institute of Technical Physics (Chinese Academy of Sciences). His current research interests are 2D materials and tip enhanced Raman spectroscopy.
Table 1. Comparison of H2O2 analytical detection using electrochemical sensors. Detection limit (μM)
Reference
0.83
[55]
50-500
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1-2580 5-1200
0.5 0.8
5-18000 0.5 to 2000
2.1 0.14
[56]
U
[57] [58]
[59] This work
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MoS2/GRMWCNTs/GCE α-FeOOH NW/carbon fiber paper Co3O4/MCNFs/GCE Mn-Co oxides decorated graphene/GCE GS/CeO2-CuO/SPE Chitosan hydrogel
Linear detection range (μM) 5-145
N
Modified electrodes
Detection limit (nM)
Reference
50000
[60]
3000
[61]
50-4000
10
[62]
0.125-625 4-100 or 50-500
― 1.98
[43] This work
5000-10000000
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SnO2/PDDA/GOX/PDDA/dsDNA film DNA-based biosensor MBs–DNA–AgNPs nanocomposite DNA modified sensor Chitosan hydrogel
Linear detection range (nM) ―
D
Modified electrodes
M
Table 2. Comparison of hydroxyl radical analytical detection using electrochemical sensors.
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Figure Captions Figure 1. (A) Schematic illustration of functional mechanism of the chitosan hydrogel sensing platform. SEM images of (B) chitosan and (C) the chitosan hydrogel. (D) Normalized XRD patterns of the chitosan hydrogel before and after polymerization.
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Figure 2. (A) CV scans of 2.5 mM zinc ions in (a) an acetic acid-sodium hydroxide solution, (b) the chitosan hydrogel, (c) the chitosan hydrogel with 0.01 mM H2O2 and (d) the chitosan hydrogel with 0.03 mM H2O2. Effects of (B) the zinc ion concentration and (C) depolymerization time on the electrochemical responses of the chitosan hydrogel towards 0.3 mM H2O2. Figure 3. (A) DPV curves of the chitosan hydrogel after the injection of various concentrations of hydrogen peroxide. Curves a-k correspond to hydrogen peroxide at concentrations from 0.5 μM to 2000 μM. (B) Relationship of ΔI versus the logarithm of the hydrogen peroxide concentration.
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Figure 4. DPV curves of the chitosan hydrogel after injection of various concentrations of hydroxyl radicals with different depolymerization periods: (A) 0.004 to 1 μM hydroxyl radical with 15 min of depolymerization; (B) 0.05 to 5μM hydroxyl radical with 8 min of depolymerization.
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Figure 5. DPV response changes of the chitosan hydrogel to OH (40 nM) in the presence of AA (0.5 μM) and UA (0.5 μM).
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Figure 1. (A) Schematic illustration of functional mechanism of the chitosan hydrogel sensing platform. SEM images of (B) chitosan and (C) the chitosan hydrogel. (D) Normalized XRD patterns of the chitosan hydrogel before and after polymerization.
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Figure 2. (A) CV scans of 2.5 mM zinc ions in (a) an acetic acid-sodium hydroxide solution, (b) the chitosan hydrogel, (c) the chitosan hydrogel with 0.01 mM H2O2 and (d) the chitosan hydrogel with 0.03 mM H2O2. Effects of (B) the zinc ion concentration and (C) depolymerization time on the electrochemical responses of the chitosan hydrogel towards 0.03 mM H2O2.
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Figure 3. (A) DPV curves of the chitosan hydrogel after the injection of various concentrations of hydrogen peroxide. Curves a-k correspond to hydrogen peroxide at concentrations from 0.5 μM to 2000 μM. (B) Relationship of ΔI versus the logarithm of the hydrogen peroxide concentration.
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Figure 4. DPV curves of the chitosan hydrogel after injection of various concentrations of hydroxyl radicals with different depolymerization periods: (A) 0.004 to 1 μM hydroxyl radical with 15 min of depolymerization; (B) 0.05 to 5μM hydroxyl radical with 8 min of depolymerization.
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Figure 5. DPV response changes of the chitosan hydrogel to OH (40 nM) in the presence of AA (0.5 μM) and UA (0.5 μM).
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