Materials Science and Engineering C 40 (2014) 228–234

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Fluorescent boronic acid terminated polymer grafted silica particles synthesized via click chemistry for affinity separation of saccharides Zhifeng Xu ⁎, Peihong Deng, Siping Tang, Junhua Li Department of Chemistry and Materials Science, Hengyang Normal University, Hengyang 421008, PR China Key Laboratory of Functional Organometallic Materials of Hunan Province University, Hengyang 421008, PR China

a r t i c l e

i n f o

Article history: Received 2 December 2013 Received in revised form 22 February 2014 Accepted 17 March 2014 Available online 2 April 2014 Keywords: Boronic acid Saccharide Fluorescence Click chemistry Affinity binding

a b s t r a c t Boronic acids are important for effective separation of biological active cis-diols. For the purpose of constructing a new type of saccharide-sensitive material which can not only provide convenient separation but also improve the access of boronic acid to guest molecules, the fluorogenic boronic acid terminated, thermo-sensitive polymers (BA-polyNIPAm) were grafted to an alkyne modified silica gel through the exploitation of click chemistry. The BA-polyNIPAm grafted silica gel (BA-polyNIPAm-SG) was characterized by FT-IR, fluorescence spectra, fluorescence microscopy, elemental analysis (EA), thermal gravimetric analysis (TGA), scanning electron microscope (SEM) and so on. BA-polyNIPAm-SG displayed affinity binding ability for saccharides under physiological pH value and allowed saccharides to be conveniently separated from solution. The maximum binding capacities for fructose and glucose are 83.2 μmol/g and 70.4 μmol/g polymer, respectively. The intensity of fluorescence emission of BA-polyNIPAm-SG increased with the increasing of fructose concentration. The present study provides a new kind of composite material which contains moveable and flexible grippers for recognizing and binding guest molecules. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Carbohydrate widely exists in nature, which is treated as one of the three substantial units as protein and nucleic acid due to their roles as structural materials, sources of energy, biological functions and environmental analytes. For biomedical applications, it is highly desirable that saccharide-sensitive systems respond at physiological pH in aqueous media [1–6]. Boronic acid derivatives can bind to cis-diols tightly through ester formation. This binding property allows boronic acid to be a competitive chelator for diol appended molecules such as saccharides in water. Therefore, boronic acid derivatives have been widely exploited for the design of functional materials for detection and affinity separation of saccharides over the past decade [7–22]. In practical application, challenges such as the recovery and recycling of boronic acid, improving access to the target molecules, etc. still remain to be addressed. Currently, there are two main approaches used to address these concerns, namely immobilization of boronic acid ligands onto a suitable supporter [23–25] and synthesis of boronic acid containing polymers [26–32].

⁎ Corresponding author at: Department of Chemistry and Materials Science, Hengyang Normal University, Hengyang, 421008, PR China. Tel.: +86 734 8486630; fax: +86 734 8486629. E-mail address: [email protected] (Z. Xu).

http://dx.doi.org/10.1016/j.msec.2014.03.066 0928-4931/© 2014 Elsevier B.V. All rights reserved.

Various boronic acid ligands have been developed in the past. The common feature of these ligands is that they contain one boronic acid moiety able to bind cis-diols, and one functional group (e.g. amino, thiol, or polymerizable vinyl group) that can be used for immobilization on solid support [23]. In a previous work, Uddin et al. [24] developed a fluorescent clickable boronic acids derivative, i.e., 3(2-azido-acetylamino) phenylboronic acid (APBA). More interestingly, when this boronic acid was conjugated to an alkyne-functionalized material via click reaction (which changed the terminal azide into a triazole ring), the immobilized boronic acid remained the favorable fluorescence response. In another work of Xu et al. [25], APBA was conjugated to a thermo-responsive polymer, poly(N-isopropylacrylamide) (polyNIPAm) by click reaction. This boronic acid terminated, thermosensitive poymer (BA-polyNIPAm) has been used for separation of saccharides from water. In order to separate BA-polyNIPAm from aqueous solution by filtration, the temperature must be kept above its lower critical solution temperature (LCST). This is sometimes an inconvenience in practical application. For constructing a new type of saccharide-sensitive material, we intend to anchor the thermo-responsive polymer (BA-polyNIPAm) on a suitable supporter. By doing this, the boronic acid “gripper” is linked to the supporter with a flexible arm. Therefore, the “grippers” (i.e. binding sites) of the material are moveable and flexible, which should lead to improved accessibility and binding capacity. On the other hand, immobilization on a solid support can provide convenient separation

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(e.g. a simple centrifugation step or a filtration step) and thus, allowing boronic acid to be easily recovered for repeated use. Due to the feature of high surface area, convenient to be modified, proper physical properties and cost-effective, silica materials have been used as a variety of supporters matrices in separation, solid phase extraction, catalysis, binding assays, etc. The copper-catalyzed azide-alkyne cycloaddition (CuAAC) is a prototypical example of click reaction that has been recognized as a facile and versatile chemistry for bioconjugation and functionalization of polymeric architectures [33–38]. Therefore, we intend to anchor BA-polyNIPAm on the surface of silica gel through the exploitation of click chemistry. In this work, the bromine of BA-polyNIPAm has been transformed into azide group by means of nucleophilic displacement reaction to give the clickable polymer (BA-polyNIPAm-N3). Then BA-polyNIPAmN3 was grafted to an alkyne modified silica via click reaction. The saccharides affinity binding and the corresponding fluorescence response of the prepared material were achieved in aqueous media under physiological pH conditions.

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rate, 1 MHz at 16 bit; preamplifier gain, 5 ×; output amplifier, conventional. An Alltech HPLC system (USA) equipped with an ELSD 2000 detector and an Alltech prevail carbohydrate analytic column (250 mm × 4.6 mm) were used for determining the concentrations of fructose and glucose. The mobile phase was prepared with methanol and water (80:20, v/v). The flow rate of mobile phase was 1.0 ml/min. The column temperature was set at 85 °C and sample injection volume was 20 μl. BA-polyNIPAm was synthesized according to the reported procedures [25]. The synthetic route, the FT-IR, 1H NMR spectra and UV/Vis of BA-polyNIPAm and the reaction intermediates can be seen in the Supporting Information. The molecular weight of the synthesized polyNIPAm was determined by MALDI-TOF mass spectrometry (Supporting Information). Based on the result of MALDI-TOF measurement, the average degree of polymerization of polyNIPAm is calculated to be about 15. Direct determination of the molecular weight of BA-polyNIPAm was not successful, as this polymer gave a rather poor ionization results under the experimental condition.

2. Experimental section 2.2. Synthesis of BA-polyNIPAm-N3 2.1. Materials and characterization Aminopropyl silica gel (particle size 15–35 μm, Fluka Chemika, 09297), 3-Aminophenylboronic acid hemisulfate, bromoacetyl bromide, CuSO4, CuBr (98%), sodium ascorbate, sodium azide, Tris(2dimethylaminoethyl)amine (Me6TREN), propargylamine, propargyl chlorofomate, 2-bromoisobutyryl bromide, D-fructose, D-glucose dimethylsulfoxide-d6 (99.9 at.% D) and chloroform-d (≥ 99.8 at.% D) were purchased from Sigma-Aldrich. CuBr was stirred overnight in acetic acid, filtered, washed with acetone and dried in vacuo before use. N-isopropylacrylamide (NIPAm) was purchased from Acros and recrystallized from toluene/hexane (2:1, v/v). 2-propanol, tetrahydrofuran (THF) and N,N-dimethylformamide (DMF) were purchased from Sigma-Aldrich and used without further purification. Ultrapure water (18.2 MΩ cm) obtained from an ELGA LabWater System (Vivendi Water Systems Ltd.) was used throughout the experiments. All other solvents purchased from commercial resources were of analytical grade. Attenuated total reflection (ATR) infrared spectra were recorded using a Perkin-Elmer FTIR instrument (Perkin-Elmer Instruments). UV–vis absorption spectra were recorded with a Beckman Coulter DU 800 UV/Vis Spectrophotometer. Fluorescence emission was measured using a QuantaMaster C-60/2000 spectrofluorometer (Photon Technology International, Lawrenceville, NJ, USA). 1H NMR spectra were recorded on a 400 MHz Superconducting Magnet NMR Spectrometer (Bruker B-ACS60). The elemental analysis was measured on a Vario EL CHNS Elemental Analyzer (Elementar, Germany). The surface morphologies of the particles were observed with a scanning electron microscope (SEM; JEOL JSM-6610LV). The thermal gravimetric analysis (TGA) was performed using a TG 209F3 (Netzsch, Germany) at heating rate 10 °C/min under N2 atmosphere. The samples were heated from 40 °C to 800 °C. MALDI-TOF Mass Spectra were acquired using a 4700 Proteomics Analyzer (Applied Biosystems/MDS SCIEX, USA) in the positive reflector mode. The samples were dissolved in THF and the concentration was 0.2 mg/ml. The matrix solution consisted of 50% (v/v) acetonitrile in water, 5 mg/ml α-cyano-4-hydroxy cinnamic acid and 0.1% (v/v) phosphoric acid. The matrix solution was mixed with sample on a stainless target plate. Typically, 0.5 μl of sample was mixed with 0.5 μl matrix solution spiked with two internal standard peptides (m/z = 904.468 and m/z = 2465.199). The two internal standards allowed accurate mass calibration with a mass deviation less than 20 ppm. For the fluorescence microscopy measurement, the synthesized materials were deposited on a glass slide and observed under a Nikon Eclipse E400 epifluorescence microscope equipped with a CCD camera. The conditions of measurement were: exposure time, 0.2 s; readout

BA-polyNIPAm (0.18 g), NaN3 (32.5 mg, 0.5 mmol) and DMF (10 ml) were added to a 100 ml round-bottom flask. The reaction mixture was allowed to stir at 50 °C for 48 h. After the reaction, most of the DMF was removed. The residue was diluted with THF, and then passed through a neutral alumina column to remove residual sodium salts. The solid product was then precipitated from diethyl ether and dried by vacuum. 2.3. Synthesis of alkyne immobilized silica To a solution of triethylamine (0.50 ml) in THF (12 ml) at ice-water temperature, aminopropyl silica gel (0.50 g) and propargyl chlorofomate (0.21 ml, 2.0 mmol) were sequentially added. The reaction mixture was warmed to room temperature and stirred overnight. The silica particles were isolated by centrifugation, thoroughly washed with water and methanol. The particles were then dried by vacuum. 2.4. Linking BA-polyNIPAm-N3 to the alkyne-silica BA-polyNIPAm-N3 (0.15 g), alkyne-silica(0.20 g), CuBr (0.0144 g, 0.10 mmol) and DMF (15.0 ml) were added to a 100 ml dried flask. The mixture was deoxygenated by bubbling with nitrogen for 40 min. Then Me6TREN (0.0276 g, 0.12 mmol) was added. The mixture was heated to 85 °C and magnetically stirred under nitrogen atmosphere for 48 h. After the reaction, the particles were collected by centrifugation and washed thoroughly with water and methanol until no fluorescence could be observed from the supernatant. Then, the particles were dried by vacuum. 2.5. Etching silica gel from BA-polyNIPAm-SG by hydrofluoric acid To remove the silica gel from BA-polyNIPAm-SG, 0.200 g of BApolyNIPAm-SG was transferred to a plastic tube and stirred in 12.0 ml of diluted hydrofluoric acid at room temperature for 12 h. After the reaction, water was removed by bubbling with nitrogen. The residue was treated by THF and the insoluble residue was removed by filtration. The solid polymers were precipitated from diethyl ether, collected by centrifugation, washed with pure methanol and then dried in a vacuum chamber. 2.6. Measurement of fluorescence response to addition of saccharides To a set of 15 ml calibrated test tubes, 4.0 mg of BA-polyNIPAm-SG, 0.5 ml of 0.20 M phosphate buffer solution (PBS) (pH 7.4), and a given

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Scheme 1. Schematic representation of synthesis of BA-polyNIPAm-SG.

concentration of fructose solution were sequentially added. The mixture was then diluted to 2.0 ml with ultrapure water, and mixed thoroughly. The fluorescence tests were carried out after the mixture has been shaken for 2 h. To maintain a stable particle suspension, the samples were stirred with a built-in magnetic stirrer during the fluorescent measurement.

were 1.0, 2.0, 3.0, 4.0, 5.0 and 6.0 mM, respectively. After being shaken for 2 h, the particles were isolated by centrifugation. The concentration of saccharide in the supernatant was determined by HPLC. The amount of saccharide bound to particles was calculated by subtracting the concentration of free saccharide from the initial saccharide concentration. The average data of triplicate independent results were used for the following discussion.

2.7. Separation of fructose and glucose by affinity binding 3. Results and discussion To a set of 15 ml calibrated test tubes, 5.0 mg of particles (aminopropyl silica or BA-polyNIPAm-SG), 0.75 ml of 0.20 M PBS (pH 7.4) and a given concentration of fructose or glucose solution were sequentially added. The mixture was then diluted to 3.0 ml with ultrapure water, and mixed thoroughly. The initial concentrations of saccharide

Fig. 1. FT-IR spectra of aminopropyl silica, alkyne silica and BA-polyNIPAm-SG.

3.1. Preparation and characterization The approach for preparation of BA-polyNIPAm-SG is shown in Scheme 1. BA-polyNIPAm was successfully synthesized according to the reported procedures. The bromine at the end of BA-polyNIPAm chain has been easily transformed into azide group via the high efficient nucleophilic displacement reaction. The characteristic IR absorption band of N3 group in BA-polyNIPAm-N3 can be found at 2112 cm−1 (Supporting Information). For preparation of “clickable” silica, alkyne groups were introduced on surface of aminopropyl silica by reacting propargyl chlorofomate with the amino groups of silica. BA-polyNIPAm was then grafted to the alkyne modified silica through copper-catalyzed azide-alkyne cycloaddition. FT-IR spectra (Fig. 1) were also used to characterize the organically modified silica gel. Compared with aminopropyl silica, the FT-IR spectrum of alkyne-immobilized silica demonstrates the characteristic amide band at 1706 cm− 1 and amide II band at 1529 cm−1. After the click reaction, the BA-polyNIPAm-SG shows characteristic IR bands for C_O stretching vibration (1712 cm− 1 corresponding to ester bonds) and amide (1660 cm−1 and 1531 cm−1). In the previous works [24,25], Uddin et al. found that the fluorescence characteristic of the clickable boronic acid APBA remains even

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Wavelength(nm) Fig. 2. Fluorescence emission spectra of BA-polyNIPAm-SG (1) and aminopropyl silica gel (2) (2.0 mg/ml) in PBS at pH 7.4 (λex = 380 nm).

after it has been conjugated to the alkye-modified support materials (e.g., agarose gels [24] and thermo-responsive polymer [25]) through click reaction. In the present work, BA-polyNIPAm-SG was dispersed in PBS at pH 7.4, and its fluorescence intensity was measured. As a control, the aminopropyl silica gel was treated under the same conditions followed by fluorescence measurement. The samples were stirred with a built-in magnetic stirrer during the fluorescent measurement. As can be seen in Fig. 2, BA-polyNIPAm-SG displayed a maximum fluorescence emission at 450 nm when excited at 380 nm, while the

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solution of aminopropyl silica gel displayed a very weak fluorescence emission. The fluorescence test results can further verify the successful grafting of BA-polyNIPAm onto silica gel. The fluorescence property of BA-polyNIPAm-SG was also confirmed using fluorescence microscopy. As can be seen in Fig. 3, BA-polyNIPAm-SG exhibited strong fluorescence. Obtained from the fluorescence microscopy measurement, the fluorescence intensity of the grafted silica gel is about 8 times higher than that of the aminopropyl silica. The scanning electron microscope (SEM) was used to characterize the particles morphologically (Fig. 3). Compare to the aminopropyl

Fig. 3. Fluorescence microscopy images (1,-2) and SEMs (3, 4) of BA-polyNIPAm-SG (1, 3) and aminopropyl silica (2, 4). The maximum fluorescence intensities are approximately 6.5 × 105 in (1) and 8.1 × 104 in (2).

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Fig. 5. (a) Fluorescence spectra of BA-polyNIPAm-SG (2.0 mg/ml) in 50 mM PBS buffer, pH 7.4, measured in the presence of increasing amount of fructose (λex = 380 nm). (b) Influence of pH value on the fluorescence emission intensity of BA-polyNIPAm-SG (2.0 mg/ml) in 50 mM PBS buffer. The pH was adjusted by adding concentrated hydrochloric acid or NaOH solution (8.0 M). The fluorescence intensity was measured at 450 nm (λex = 380 nm).

silica particles, surface morphologies of BA-polyNIPAm-SG particles indicate an improving surface roughness. We can see that there are many aggregated polymers (indicated by arrows) attached on the surface of BA-polyNIPAm-SG particles. To determine the content of the grafted polymers in BA-polyNIPAmSG, hydrofluoric acid was used to remove silica gel from BA-polyNIPAmSG particles. After BA-polyNIPAm-SG (0.200 g) has been treated with hydrofluoric acid, an amount of 0.0612 g residue polymers was left, implying that the polymers content in the grafted silica gel is about 30.6%. The results of elemental analysis of the materials are listed below: aminopropyl silica: C 5.27, H 1.19, N 1.22%; alkyne silica: C 8.21, H 1.42, N 1.15%; BA-polyNIPAm-SG: C 19.14, H 2.91, N 4.47%. Based on the nitrogen content in aminopropyl silica, the density of aminopropyl in the aminopropyl silica used in this work was calculated to be 0.871 mmol/g.

The stability and the composition of the three different particles were investigated by TGA analysis (Fig. 4). When the temperature reached 800 °C, the weight loss of aminopropyl silica and alkyne silica were 10.69% and 15.59%, respectively. The weight loss (approximately 4%) below 250 °C can be attributed to the evaporation of residual organic solvent and water. Based on the TGA results, the density of alkyne immobilized on the silica is calculated to be about 0.7113 mmol/g. It means that about 81.67% of the aminopropyl groups were converted into alkyne groups. The TGA curve of BA-polyNIPAm-SG (Fig. 4, curve 3) shows that when the temperature reached 800 °C, the weight loss of BApolyNIPAm-SG was 33.10%. Subtracting the weight loss caused by the evaporation of residual organic solvent and water (approximately 4%), the organic component content in the grafted silica gel is approximately 29.10%. This result is very close to the value obtained from hydrofluoric acid treated experiment. According to the results of MALDI-TOF

Fig. 6. Hypothetical mechanism of fluorescence change in response to cis-diol compounds or pH value.

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experiments, the average molecular weight of the polymer chain (BA-polyNIPAm-N 3) is about 2000. Based on this, the density of the BA-polyNIPAm chain grafted on silica gel is calculated to be 0.1166 mmol/g. It means that about 16.39% of alkyne groups on silica gel have been converted into polymers chains after the click reactions. 3.2. Change of fluorescence emission of BA-polyNIPAm-SG in response to fructose or pH value Earlier studies [25] demonstrate that the intensity of fluorescence emission of BA-polyNIPAm increased after it has bound cis-diol compounds. To investigate if the fluorescence response to cis-diol compounds remained after the polymer has been grafted to silica gel via click reaction, we measured the fluorescence spectra of BA-polyNIPAm-SG in PBS buffer (pH 7.4) in the presence of different amount of fructose. Fig. 5a shows the change of fluorescence emission when increasing amount of fructose was added into the particles dispersed solution. Clearly, the intensity of fluorescence emission of BA-polyNIPAm-SG increased with the increasing of fructose concentration. Calibration curve for fructose was obtained using fluorescence intensity measured at 450 nm. In the 0–10.0 mM range of initial concentration of fructose, the calibration curve is linear. The linear regression equation is Y = 117863.2 + 5416.9x (R = 0.9936), where Y is the intensity of fluorescence emission (a.u.) and x is the concentration of fructose (mM). We repeated this experiment 3 times to test the reproducibility of fluorescence calibration plots. All the results of the experiments are well in accordance with the linear regression equation. We also investigated the influence of pH value on the fluorescence emission intensity of BA-polyNIPAm-SG. As can be seen in Fig. 5b, the fluorescence intensity of BA-polyNIPAm-SG in water decreased when the pH was increased in the range of 3–10. According to the literature [9], phenylboronic acid in water can exist as several interchangeable coordination structures, and the proportion of the different structures can be influenced by the solution pH and the concentration of cis-diols added in the solution. Based on our fluorescence measurement results (Fig. 5a and b) and the literature information, we propose a possible mechanism to explain the fluorescence change of APBA in response to cis-diol compounds or pH value (Fig. 6), where among the different solution structures of the terminal boronic acid, only the structures containing the dative B\N bond can have strong fluorescence emission. According to this mechanism, binding fructose or decreasing pH value have the same effect of increasing the proportion of the fluorescent boronic acid. It is worthy noting that in this work, the corresponding fluorescence response was observed under physiological pH conditions, which makes the fluorescence-responsive materials suitable for analysis of biological sample.

In this work, the binding performances of BA-polyNIPAm-SG for fructose and glucose were investigated. The amount of fructose or glucose bound by BA-polyNIPAm-SG was determined by equilibrium binding experiments. The binding isotherms of saccharides to the silica were determined in the 0–6.0 mM range of initial concentration of saccharides. After BA-polyNIPAm-SG has bound saccharides through covalent interactions between the boronic acid groups and cis-diol groups, the silica particles were isolated by centrifugation. The concentration of saccharide in the supernatant was determined by HPLC. The amount of saccharide bound to BA-polyNIPAm-SG was then calculated by subtracting the concentration of free saccharide from the initial saccharide concentration. For comparison, aminopropyl silica was used as a control to evaluate the non-specific binding. As shown in Fig. 7, the amounts of saccharides bound to BA-polyNIPAm-SG increased with the increasing concentrations of saccharides. The figure shows that the binding reached saturation when the initial concentration of saccharide was 4.0 mM and that the maximum binding capacities are 83.2 μmol/g polymer for fructose and 70.4 μmol/g polymer for glucose. Obviously, the maximum binding for fructose is higher than that of glucose. This phenomenon can be explained by the fact that fructose binds to phenylboronic acid with an affinity higher than glucose [11]. Because aminopropyl silica alone did not show obvious binding to saccharides, the saccharides binding achieved by BA-polyNIPAm-SG can only be attributed to the specific interaction offered by the boronic acid. 4. Conclusions In this work, the fluorogenic boronic acid terminated, thermosensitive polymers (BA-polyNIPAm) were grafted to an alkyne modified silica through the exploitation of click chemistry. The BA-polyNIPAm grafted silica displayed affinity binding ability for saccharides under physiological pH value. After binding saccharides, it can be conveniently separated from solution by centrifugation. Due to the interesting fluorescence property of the boronic acid, the intensity of fluorescence emission of the synthesized boronic acid terminated polymer grafted silica particles was found to increase when increasing amount of fructose was added. The current effort presents a new kind of composite material which contains moveable and flexible grippers for recognizing and binding guest molecules. This kind of materials should find potential applications in affinity separation, adsorption, catalysis, drug delivery, etc. In addition, this work has presented an effective method for anchoring functional polymers on the surface of solid matrix. Acknowledgments The authors thank the Foundation of Collaborative Innovation of Hengyang Normal University (No. 12XT02), the Open Funds of Key Laboratory of Functional Organometallic Materials of ordinary university in Hunan province (No. 12K125 and No. 09K02) and the National Natural Science Foundation of China (No. 21102040 and No. 21105024) for the financial support to the research. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.msec.2014.03.066. References [1] K.T. Kim, J.J.L.M. Cornelissen, R.J.M. Nolte, J.C.M. van Hest, Polymeric monosaccharide receptors responsive at neutral pH, J. Am. Chem. Soc. 131 (2009) 13908–13909. [2] X. Kang, J. Wang, H. Wu, I.A. Aksay, J. Liu, Y. Lin, Glucose oxidase–graphene–chitosan modified electrode for direct electrochemistry and glucose sensing, Biosens. Bioelectron. 25 (2009) 901–905.

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Zhifeng Xu received his M.S. degree in Chemistry from Central China Normal University, Wuhan, China in 1999. In 2006, he received his Ph.D. degree in Chemistry at Sun YatSen University, Guangzhou, China. The same year he joined Hengyang Normal University as a teacher in the Department of Chemistry and Material Science. He has worked as a visiting Scientist in Sweden from Oct. 2011 to Oct. 2012 at Lund University. Currently, he is a professor at Hengyang Normal University. His main research interest is design and preparation of novel functional materials for affinity separation.

Peihong Deng received her B.S. degree in Chemistry from Guangxi University, Nanning, China in 1997. She received her M.S. degree in Chemistry from Xiangtan University, Xiangtan, China in 2000. She joined Hengyang Normal University as a teacher in the Department of Chemistry and Material Science in 2001. Currently, she is an associate professor and engaged in teaching of chemistry at Hengyang Normal University. Her main research interest is construction and application of electrochemical sensors.

Siping Tang received her M.S. degree in Chemistry from Hunan Normal University, Changsha, China in 2001. In 2010, she received her Ph.D. degree in Chemistry at Sun Yat-Sen University, Guangzhou, China. The same year she joined Hengyang Normal University as a teacher in the Department of Chemistry and Material Science. Currently, she is an associate professor at Hengyang Normal University. Her research interest is focused on organic analysis and separation.

Junhua Li received his B.S. degree in Chemistry from Yangtze University, Jingzhou, China in 2003. In 2006, he received his M.S. degree in Chemistry at Central China Normal University, Wuhan, China. He joined Hengyang Normal University as a teacher in the Department of Chemistry and Material Science in 2006. His research interests cover chemical modified electrodes and chemical sensors.