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Solid-state electrochemiluminescence sensor based on RuSi nanoparticles combined with molecularly imprinted polymer for the determination of ochratoxin A
Accepted Manuscript Title: Solid-state Electrochemiluminescence sensor based on RuSi nanoparticles combined with Molecularly Imprinted Polymer for the determination of Ochratoxin A Author: Qingling Wang Miaomiao Chen Haiqing Zhang Wei Wen Xiuhua Zhang Shengfu Wang PII: DOI: Reference:
S0925-4005(15)30235-5 http://dx.doi.org/doi:10.1016/j.snb.2015.08.057 SNB 18912
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
5-6-2015 11-8-2015 13-8-2015
Please cite this article as: Q. Wang, M. Chen, H. Zhang, W. Wen, X. Zhang, S. Wang, Solid-state Electrochemiluminescence sensor based on RuSi nanoparticles combined with Molecularly Imprinted Polymer for the determination of Ochratoxin A, Sensors and Actuators B: Chemical (2015), http://dx.doi.org/10.1016/j.snb.2015.08.057 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.
Graphical Abstract (for review)
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Graphical abstract
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Schematic diagram of the molecularly imprinted polymer electrochemiluminescence
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sensor for the detecting of ochratoxin A
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Research Highlights
Highlights
1. A simple and convenient sensor for OTA was prepared. 2. The electrochemiluminescence sensor based on Ru(bpy)32+-doped silica nanoparticles has high
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sensitivity . 3. The electrochemiluminescence sensor combines with desirable selectivity of molecularly imprinted
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technology.
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4. The sensor showed good sensitivity and stability for OTA in corn sample.
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*Manuscript
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Solid-state Electrochemiluminescence sensor based on RuSi
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nanoparticles combined with Molecularly Imprinted Polymer
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for the determination of Ochratoxin A
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Qingling Wang b, Miaomiao Chen a, b, Haiqing Zhang a, Wei Wen a, b,
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Xiuhua Zhang a, b*, Shengfu Wanga, b
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Wuhan 430062, China
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Hubei Collaborative Innovation Center for Advanced Organic Chemical Materials, Hubei University,
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b
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Molecules, College of Chemistry and Chemical Engineering, Hubei University, Wuhan 430062, China
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Ministry-of-Education Key Laboratory for the Synthesis and Application of Organic Functional
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*Corresponding author. Fax: +86-27-88663043; Telephone: +86-27-50865309; E-mail address: [email protected] (X. Zhang)
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ABSTRACT An electrochemiluminescence (ECL) sensor based on Ru(bpy)32+-doped silica nanoparticles (RuSi
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NPs) combined with molecularly imprinted polymer (MIP) has been developed for the determination of
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ochratoxin A (OTA). The sensor was fabricated by remodification of RuSi NPs with a thin film of
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molecularly imprinted polymer, which provided the specific binding sites for OTA. The process of
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template elution and rebinding acted as a gate to control the flux of probes, which passed through the
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cavities and reacted with Ru(bpy)32+ immobilized on the electrode surface to emit ECL signal. The
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ECL intensity decreased with the OTA molecules rebound in MIP. The as-prepared sensor exhibited a
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very high sensitivity and excellent selectivity to the target molecule. The ∆IECL depended linearly on
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the logarithm of the OTA concentration in the range from 0.1 pg/mL to 14.76 ng/mL with lower
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detection limit of 0.027 pg/mL (S/N=3). The MIP-ECL sensor enabled the successful application of this
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method to determine OTA in corn.
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Keywords
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Electrochemiluminescence
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Ochratoxin A
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nanoparticles,
Molecularly imprinted
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sensor,
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1. Introduction Ochratoxin A (OTA) is a class of mycotoxin, which is a toxic and harmful substance produced by
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fungi [1]. The mechanism of poisoning of OTA has been studied, and it’s found that OTA exhibits
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unusual toxicokinetics [2]. OTA mainly invades liver and kidney, and the existence of OTA poses a
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threat to the health of animals and human [3]. In this sense, OTA in food and feed is urgently needed to
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be detected sensitively for the purpose of protecting animals and people. Up to now, various methods
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have already been established to assay OTA, such as thin-layer chromatography (TLC) [4], fluorimetry
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(FL) [5], electrochemistry [6], high performance liquid chromatography (HPLC) [7], capillary
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electrophoresis (CE) [8], chemiluminescence (CL) [9], and enzyme-linked immunosorbent assay
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(ELISA) [10]. Among them, TLC and CE methods exhibit very poor sensitivity. The electrochemical
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and FL methods can obtain higher sensitivity than that of TCL, but they lack selectivity. Though HPLC
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methods are sensitive and selective, they require expensive apparatus and complex procedures for
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sample pretreatment. A high sensitivity of ELISA can be achieved, but the poor chemical/physical
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stability of the antibodies or enzymes prevents their use in the harsh environments of acids or bases and
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organic solvents.
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Electrochemiluminescence (ECL) is a technique that can initiate light radiation during the
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high-energy electron-transfer reactions occurring at the surface of working electrode in electrochemical
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processes. ECL has received increasing attention in many fields, including environmental pollutant
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determination [11], pharmaceutical analysis [12], and immunoassays [13], because of its advantages of
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high sensitivity, wide linear range, simple operation and controllability. Ru(bpy)32+ is the most used
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ECL reagent due to its commercial availability, reversible electrochemical behavior, and high
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luminescence efficiency [14]. Unlike solution phase Ru(bpy)32+ ECL system, solid-state ECL offers
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many advantages, such as reduced the consumption of expensive ECL reagent Ru(bpy)32+, simplified
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experimental design, and enhanced the ECL signal [15]. Recently, many efforts have also been made to
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fabricate solid-state ECL sensors by immobilizing Ru(bpy)32+ or its derivatives on electrode surfaces,
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including Langmuir-Blodgett films (LB) [16], self-assembly (SA) [17], Nafion [18] and sol-gel method
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[19]. However, LB has poor membrane stability because of its physical adsorption. Self-assembled film
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of SA is easy to fall off from the electrode, and it is not conducive to the analysis application. Nafion
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has good chemical stability, but nafion films are dense, which contributes to the slow mass transfer rate.
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Sol-gel method has good chemical stability, light permeability and biocompatibility, and it has better
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stability than that of the first three methods. Silica-based materials were used due to their easy surface
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modification, biocompatibility and good electrochemical stability [20]. In addition, RuSi NPs were
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used as signal substances because of their high luminous efficiency, chemical stability and
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electrochemical reversibility.
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Molecularly imprinted polymers (MIPs) have been identified as artificially synthesized receptors
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and widely used in sensors [21], catalysis reactions [22] and separations [23], because MIPs can
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recognize and bind the desired target molecules with a high affinity and selectivity. The combination of
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the MIPs with the ECL detection would be able to produce a sensor with both high sensitivity and
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desirable selectivity, which could help to solve the problem related to the selectivity of ECL. There are
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a lot of literatures that had been reported about MIP-ECL sensors for various applications [24-26].
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In this work, we designed an ECL sensor based on Ru(bpy) 32+-doped silica nanoparticles (RuSi
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NPs) combined with molecularly imprinted polymer for the determination of OTA (figure 1). The RuSi
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NPs were synthesized by the method of water-in-oil (W/O) microemulsion, while chitosan (CS) was
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used to immobilize the RuSi NPs on the electrode. The sensor was fabricated by remodification of
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RuSi NPs with a thin film of molecularly imprinted polymer, which provided the specific binding sites
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for OTA, and the prepared sensor could used to detect OTA in corn sample.
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2. Experiment
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2.1. Chemicals and reagents
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Please, insert Fig. 1. here.
Ochratoxin A was obtained from cayman chemical company (United States). Tripropyl amine
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(TPrA), Triton X-100, cyclohexane, 1-hexanol, tetraethyl orthosilicate (TEOS), NH3·H2O, and acetone
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were obtained from Sigma-Aldrich (Beijing, China). Terpyridyl ruthenium was obtained from Green
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kaemmer (beijing, China). Corn samples (8.6 μg/kg ± 3.6 μg/kg, HPLC) were obtained from Biopure
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(New Zealand). All reagents were used of analytical grade and double distilled water was used to
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prepare sample solutions.
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2.2. Apparatus
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ECL signals were measured with MPI-E electrochemiluminescence analyzer (Xi’An Remax
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Electronic Science & Technology Co. Ltd., China). A three-electrode system was used in the
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measurements, with a glassy carbon electrode (GCE, 3 mm in diameter) as the working electrode, a
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saturated calomel electrode (SCE) as the reference electrode, and a platinum wire as the auxiliary
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electrode. Transmission electron microscopy (TEM) was performed using a JEOL JEM-100SX TEM
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facility. Fluorescence was measured with LS-55 fluorescent spectrophotometer (USA). Branson 2000
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ultrasonic cleaner (USA) was used to clean the electrodes. UV polymerization was implemented by
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ZF-I UV analyzer (Shanghai Guanghao Analysis Instrument Co. Ltd., shanghai, China).
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2.3. Synthesis of RuSi NPs
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The RuSi NPs were synthesized by a typical W/O microemulsion route [27]. 1.77 mL of Triton
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X-100, 7.5 mL of cyclohexane, 1.8 mL of 1-hexanol, and 340 μL 40 mM Ru(bpy)32+ were firstly mixed
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with constant magnetic stirring for 0.5 h to form the W/O microemulsion. Then 100 μL TEOS and 60
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μL NH3·H2O were added to the mixing solution, which reacted last for 24 hours. Acetone was then
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added to destroy the emulsion, followed by centrifuging and washing with ethanol and water three
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times respectively. At last, the orange-colored RuSi NPs were obtained.
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2.4. Preparation of MIP-ECL sensor
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Prior to modification, the GCE was polished with 0.3 and 0.05 mm alumina slurry and rinsed
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thoroughly with doubly distilled water between each polishing step. Then, it was washed successively
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with doubly distilled water and ethanol in an ultrasonic bath, and finally dried at room temperature.
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Firstly, RuSi NPs were dispersed in 0.1 M PBS (pH 7.0), then 1% chitosan was added to mix with
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constant magnetic stirring for 2 h to obtain RuSi/CS. The GC electrode was coated with 5 μL RuSi/CS,
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and the solvent was allowed to evaporate at room temperature. Secondly, the imprinted polymer was
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obtained by the original solution which was comprised of the template molecule of OTA, the functional
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monomer methacrylic acid (MAA), the crosslinking agent of ethylene glycol dimethacrylate (EDMA)
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and initiator azodiisobutyronitrile (AIBN). Dropped 2 μL the original solution on the modified
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electrode, and then induced by the wavelength of 245 nm ultraviolet light. The MIP-ECL sensor was
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obtained by eluting imprinted polymer film with ethanol. Except without OTA, the preparation process
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of the non imprinted polymer (NIP-ECL sensor) is same as the imprinted polymer (MIP-ECL sensor).
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3. Results and discussion
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3.1. Characterization of synthesized RuSi NPs
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As the morphology of NPs influences the spectral and electrochemical properties, NPs with a
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uniform size is one of the key steps to improve the ECL sensitivity and stability. Transmission electron
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microscopy (TEM) was used to research the morphology of RuSi NPs. As shown in figure 2A, it is
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clear that the as-prepared nanoparticles exhibited a uniform size and the diameter of the RuSi NPs was
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around 50 nm.
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3.2. Characterization of MIP-ECL sensor
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In virtue of the fluorescence of OTA, fluorescence spectrum (FLS) was selected to attest the
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process of elution and rebinding of the imprinted polymer. As shown in figure 2B, the imprinted film of
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initial preparation (curve a) had the highest fluorescence intensity, because it was prepared with a high
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concentration of OTA (20 μg/mL). After the imprinted film being eluted, the fluorescence intensity
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decreased (curve b), which verified that the OTA template were efficiently removed from the imprinted
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film in the elution process. When the imprinted polymer was immersed in the standard solution of 5
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ng/mL OTA for 5 min, the template molecule OTA could rebind with imprinted film, and the
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fluorescence intensity increased (curve c).
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Electrochemical impedance spectroscopy (EIS) is an effective method for monitoring the changes
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in the surface features of the modified electrodes in the assembly process. As shown in figure 2C,
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because of the presence of CS, the charge transfer resistance for RuSi/CS/GCE (curve b) showed an
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increase compared with bare GCE (curve a). The resistance increased obviously (curve c) when the
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surface of RuSi/CS/GCE was coated by imprinted polymer, because the imprinted polymer was
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nonconductive. However, after template molecule being eluted from the imprinted polymer, cavities
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could form and act as the channels for electron transfer, which made the resistance decrease (curve d).
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When the imprinted polymer was incubated with the template molecule, OTA rebound with imprinted
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film coated on the surface of sensor, the channels of electron transfer would be blocked and the
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resistance increased (curve e).
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Please, insert Fig. 2. Here. 3.3. ECL characterized for MIP-ECL sensor and NIP-ECL sensor. Experiments that aimed to find an appropriate potential used for the assay were carried out, and
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we found that the ECL signal was highest in 1.4 V range from 0 V to 1.6 V. The applied potential range
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of 0-1.4 V was selected for the assay of OTA. The concentration of TPrA was selected by referring to
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the published literatures [14, 27-29] and experiments. The results of experiments indicated that the
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ECL intensity and background signal all increased with the increasing of the concentration of TPrA.
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Considering that TPrA had low solubility in water, 2 mM TPrA was selected to be used for the assay.
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It was difficult for the reaction between RuSi NPs and TPrA when the MIP was coated on the
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modified electrode, because it was nonconducting and prevented RuSi NPs from contacting with TPrA.
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As shown in figure 2D, the peak value of initially prepared MIP-ECL sensor was very small (curve a),
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and the modified electrode coated with NIP (curve d) was same with MIP. After the removal of the
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template molecule from the sensor, there were some cavities appeared and were used as the channels
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for electron transportation, which resulted in that the intensity of ECL increased obviously (curve b).
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But the NIP-ECL sensor had no cavities, the intensity increased few (curve e). After rebinding, the
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cavities blocked, and the intensity decreased (curve c). The ECL results were in accordance with that of
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FLS and EIS.
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3.4. Optimization of Experimental Conditions
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Considering both luminescent efficiency and the adhesion of the film, the optimal proportion of
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RuSi NPs and chitosan is crucial to this MIP-ECL sensor. The optimization experiments were carried
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out in the absence of the imprinted film. As shown in figure 3A, it could be seen that the strong signal
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was achieved when the proportion of RuSi NPs and chitosan was 6:1.
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In order to optimize the elution time, the MIP-ECL sensor were washed in 2 mL ethanol at various
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extraction time, whereas the other elution parameters were kept constant, and then followed by ECL
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determination in 0.1 M PBS (pH 7.0) containing 2 mM TPrA. As shown in figure 3B, increasing of the
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elution time leads to the responses reached a plateau about 5 min and afterwards the increasing elution
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time has no considerable effect on the ECL response (curve a). 5 min was selected as optimum elution
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time accompanying with high luminous intensity and short analyzing time.
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The rebinding time was also one of the main parameters that must be investigated. The rebinding
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was performed in 2 mL of 5 ng/mL OTA solution. The detection of ECL intensity was performed every
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5 min from 0 to 25 min, and the results was shown in figure 3B (curve b). When the rebinding time
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increased, the ECL intensity decreased and remained constant at 5 min. As a result, 5 min was selected
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as the rebinding time in all of the following assays.
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Please, insert Fig. 3. Here.
Under the optimized conditions, the MIP-ECL sensor was used to detect OTA. The ECL intensity
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was measured before and after the MIP-ECL sensor rebound in OTA solutions, respectively. When the
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concentrations of OTA increased, more cavities were blocked by the template molecule of OTA, and
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the ECL intensity significantly decreased. The difference between ECL intensities before and after
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rebinding (∆IECL) was linear to the logarithm of the OTA concentration in the range from 0.1 pg/mL to
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14.76 ng/mL with lower detection limit of 0.027 pg/mL (S/N=3) (figure 4). The linear regression
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equation was ∆IECL = 116.6 log (c, ng/mL) + 750.4, with a coefficient of correlation r=0.999. The
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numerical values for the relative standard deviation at each concentration are 1.4%, 1.8%, 3.4%, 1.4%,
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3.0%, 2.7%, 1.3%, 1.5%, 1.7% and 0.4% from low concentration to high concentration, respectively.
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Please, insert Fig. 4. Here. 3.6. Selectivity and Stability To evaluate the selectivity of the MIP-ECL sensor, measurements of ECL intensity were
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performed in the rebinding procedure. Some mixed solutions were used to examine the selectivity of
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the designed MIP-ECL sensor; 0.76 ng/mL OTA was mixed with 7.6 ng/mL ochratoxin B (OTB), 7.6
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ng/mL deoxynivalenol (DON), and 10 mM glucose (GLU). OTA, OTB and DON belong to the class of
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mycotoxin, and OTB has similar structure to OTA. Meanwhile GLU is a kind of interfering substance
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exists in corn samples. Figure 5A showed that ECL intensities had almost no change after the MIP-ECL
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sensors were incubated with the mixed solutions for 5 min. The results indicated that there was little
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interference from coexistent substances, and the MIP-ECL sensor had high selectivity for the
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determination of OTA.
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To investigate the stability of the MIP-ECL sensor, ECL was recorded under continuously cyclic
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potential scanning range from 0 V to 1.4V for 10 cycles in 0.1 M PBS (pH 7.0) containing 2 mM TPrA.
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As shown in figure 5B, the ECL intensities did not appear any obvious changes with a relative standard
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deviation (RSD) of 1.9%. The results indicated that the MIP-ECL sensor had good stability, and these
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results were due to that the charge transfer in the imprinted film was fast and imprinted film was stable.
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The MIP-ECL sensors which were placed at room temperature for a week were detected, and the
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results showed that the ∆IECL reduced 2.1%. It could be seen that the MIP-ECL sensor exhibited
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excellent life expectancy. What’s more, the reproducibility of the MIP-ECL sensor was detected. It
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found that RSD of 6 electrodes which were obtained in the same way was 2.4%, which showed good
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sensor-to-sensor reproducibility.
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Please, insert Fig. 5. Here.
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3.7. Determination of OTA in corn sample The high sensitivity obtained with the MIP-ECL sensor makes it feasible to detect the presence of
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OTA in corns. The corns (0.5 g) which contained OTA were dissolved into the solution of 2 mL 75%
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(V/V) methanol/water, then ultrasound dispersion 2 h. After centrifugation at 12000 rpm for 10 min,
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the resulting supernatant was diluted ten times as the OTA sample, and then mixed with various
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concentrations of standard OTA. The prepared corn sample was detected by the MIP-ECL sensor, and it
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found that the concentration of OTA was 6.44 μg/kg, which was consistent with the result of HPLC. As
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shown in table 1, the recoveries of the MIP-ECL sensor ranged from 95.3% to 99.3%, and the RSD
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ranged from 5.2% to 6.1%. The results showed that the MIP-ECL sensor had good accuracy and
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practicability.
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4. Conclusions
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Please, insert Table 1. Here.
In summary, we have successfully demonstrated a novel ECL sensor based on Ru(bpy)32+-doped
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silica nanoparticles combined with molecularly imprinted polymer for the determination of OTA. The
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resulting sensor showed a high sensitivity and excellent selectivity to OTA in the range from 0.1 pg/mL
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to 14.76 ng/mL. In addition, combining MIP with ECL can improve the selectivity of the ECL sensor
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and overcome the interference to the template molecule. The MIP-ECL sensor is easy to make and has
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good stability, it can be applied for monitoring OTA in corn samples. Moreover, this strategy can be
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further expected to fabricate various sensitive and selective sensors for advanced applications.
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Acknowledgements
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This work was financially supported by the National Natural Science Foundation of China
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(No. 21075029, 21375033), the Natural Science Fund for Creative Research Groups of Hubei
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Province of China (No. 2011CDA111, 2014CFA015), the Key Project of the Natural Science
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Foundation of Hubei Province (No. 2015CFA124) and the Program for Excellent Youth Scholars
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of Innovative Research Team by Hubei Provincial Department of Education (No. T201101).
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multi-walled carbon nanotubes through a simple indirect method for the determination of 2,4-dichlorophenoxyacetic acid in environmental water, Appl. Surf. Sci. 284 (2013) 692– 699.
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[21] T. L. Panasyuk, V. M. Mirsky, S. A. Piletsky, O. S. Wolfbeis, Electropolymerized Molecularly
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Imprinted Polymers as Receptor Layers in Capacitive Chemical Sensors, Anal. Chem. 71 (1999)
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affinity and catalytic receptors prepared by molecular imprinting, Trends in Anal. Chem. 33 (2012)
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68–80. [23] M. Soleimani, S. Ghaderi, M. G. Afshar, S. Soleimani, Synthesis of molecularly imprinted polymer
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as a sorbent for solid phase extraction of bovine albumin from whey, milk, urine and serum,
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Microchemical 100 (2012) 1–7.
cr
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[24] J. Zhou, N. Gan, T.H. Li, F. T. Hu, X. Li, L. H. Wang, L. Zheng, A cost-effective sandwich
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electrochemiluminescence immunosensor for ultrasensitive detection of HIV-1 antibody using
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magnetic molecularly imprinted polymers as capture probes, Biosens. Bioelectron. 54 (2014)
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dual-template molecularly imprinted electrochemiluminescence immunosensor array using
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Ru(bpy)32+-Silica@Poly-L-lysine-Au composite nanoparticles as labels for near-simultaneous
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detection of tumor markers, Electrochim. Acta 139 (2014) 127–136.
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[26] S. H. Li, H. L. Tao, J. P. Li, Molecularly Imprinted Electrochemical Luminescence Sensor Based
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on Enzymatic Amplification for Ultratrace Isoproturon Determination, Electroanalysis 24 (2012)
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1664–1670.
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[27] M. S. Wu, L. J. He, J. J. Xu, H. Y. Chen, RuSi@Ru(bpy)32+/Au@Ag2S Nanoparticles
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Electrochemiluminescence Resonance Energy Transfer System for Sensitive DNA Detection, Anal.
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Chem. 86 (2014) 4559−4565.
332 333
[28] Y. Xu, X. B. Yin, X. W. He, Y. K. Zhang, Electrochemistry and electrochemiluminescence from a redox-active metal-organic framework, Biosens. Bioelectron. 68 (2015) 197-203.
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[29] D. F. Wang, Y. Y. Li, Z. Y. Lin, B. Qiu, L. H. Guo, Surface-Enhanced Electrochemiluminescence
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of Ru@SiO2 for Ultrasensitive Detection of Carcinoembryonic Antigen, Anal. Chem. 87 (2015)
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5966-5972.
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Table(s)
Tables Table 1. Detection of OTA in corn samples by the MIP-ECL sensor. OTA added to corn samples
OTA detected
Recovery
RSD(n=3)
（ng/mL)
(ng/mL)
(ng/mL)
(%)
(%)
0.3
0.459
99.3
5.2
0.6
0.733
95.3
6.1
0.9
1.053
99.1
5.3
0.161
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(RSD=1.976%)
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Corn samples
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Figure(s)
Figures: Fig. 1. Schematic diagram of the molecularly imprinted polymer electrochemiluminescence sensor for the detecting of ochratoxin A.
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Fig. 2. (A) TEM images of RuSi NPs. (B) FLS of the imprinted film (a) of initial preparation, (b) after elution and (c) after rebinding. (C) EIS of (a) Bare GCE, (b) RuSi/CS/GCE, (c) MIP/RuSi/CS/GCE, (d)
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MIP/RuSi/CS/GCE after template removal, (e) MIP/RuSi/CS/GCE after rebinding. (D) ECL curves of
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(a) MIP/RuSi/CS/GCE, (b) MIP/RuSi/CS/GCE after template removal, (c) MIP/RuSi/CS/GCE after rebinding, (d) NIP/RuSi/CS/GCE and (e) NIP/RuSi/CS/GCE after elution. Scan rate: 100 mV/s; scan
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range: from 0 to 1.4 V.
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Fig. 3. (A) Optimization of the proportion of RuSi NPs and chitosan, six volume proportions were investigated: 8:1, 6:1, 4:1, 2:1, 1:1, 1:2. (B) Optimization of (a) elution time for obtaining the MIP-ECL
scan range: from 0 to 1.4 V.
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sensor and (b) rebinding time of the MIP-ECL sensor immersed in 5 ng/mL OTA. Scan rate: 100 mV/s;
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Fig. 4. Calibration curves for OTA determination based on ∆IECL as the response signal. The inset showed the ∆IECL depended linearly on the logarithm of the OTA concentration in the range from 0.1
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pg/mL to 14.76 ng/mL under the optimized conditions. The error bars represent the standard deviation of three independent measurements. Scan rate: 100 mV/s; scan range: from 0 to 1.4 V.
Fig. 5. (A) Selectivity of the MIP-ECL sensor to OTA, OTB, DON, GLU. (B) Stability of ECL emission under continuous cyclic potential scan for 10 cycles from MIP-ECL sensor. Scan rate: 100 mV/s; scan range: from 0 to 1.4 V.
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Fig. 1.
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Fig. 2.
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Fig. 3.
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Fig. 4
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Fig. 5.
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Author Biographies
Biographies Qingling Wang is currently working toward the M.Sc. degree in College of Chemistry& Chemical Engineering, Hubei University. Her research interests include electrochemical luminescence for
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decteting of ochratoxin A.
Miaomiao Chen is an experimentalist in the Department of Chemistry and Chemical Engineering
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of Hubei University, China. She received M.Sc. degree in analytical chemistry from Hubei
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University in 2008. Her research interest is in the development of chemical sensors and biosensors.
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Haiqing Zhang is currently a post-graduate student in the Zhang group and studying for his Ph.D.
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degree. Her research interests include molecular imprinting for decteting of mycotoxin. Wei Wen earned his M.S.(2010) majoring in Analytical Chemistry from Hubei University, China. He
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is currently a post-graduate student in the Wang group and studying for his Ph.D. degree. His research interest is focused on the development of electrochemical biosensors.
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Xiuhua Zhang is an associate professor in the College of Chemistry and Chemical Engineering of Hubei University. He received M.Sc. degree in analytical chemistry and Dr. Eng. degree in materials
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science from Hubei University, in 2003 and 2008, respectively. His main current interest is in the development of sensors based on carbon nanotubes and on conducting polymers.
Shengfu Wang is a professor in the College of Chemistry and Chemical Engineering of Hubei University. He received M.Sc. and Ph.D. in analytical chemistry from Wuhan University (China) in 1992 and 2005, respectively. His main current interests are in bioelectrochemistry, nanoelectrochemistry, chemically modified electrodes, chemical sensors and biosensors.
Page 26 of 26
S0925-4005(15)30235-5 http://dx.doi.org/doi:10.1016/j.snb.2015.08.057 SNB 18912
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
5-6-2015 11-8-2015 13-8-2015
Please cite this article as: Q. Wang, M. Chen, H. Zhang, W. Wen, X. Zhang, S. Wang, Solid-state Electrochemiluminescence sensor based on RuSi nanoparticles combined with Molecularly Imprinted Polymer for the determination of Ochratoxin A, Sensors and Actuators B: Chemical (2015), http://dx.doi.org/10.1016/j.snb.2015.08.057 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.
Graphical Abstract (for review)
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Graphical abstract
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Schematic diagram of the molecularly imprinted polymer electrochemiluminescence
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sensor for the detecting of ochratoxin A
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Research Highlights
Highlights
1. A simple and convenient sensor for OTA was prepared. 2. The electrochemiluminescence sensor based on Ru(bpy)32+-doped silica nanoparticles has high
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sensitivity . 3. The electrochemiluminescence sensor combines with desirable selectivity of molecularly imprinted
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technology.
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4. The sensor showed good sensitivity and stability for OTA in corn sample.
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*Manuscript
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Solid-state Electrochemiluminescence sensor based on RuSi
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nanoparticles combined with Molecularly Imprinted Polymer
3
for the determination of Ochratoxin A
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Qingling Wang b, Miaomiao Chen a, b, Haiqing Zhang a, Wei Wen a, b,
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Xiuhua Zhang a, b*, Shengfu Wanga, b
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a
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Wuhan 430062, China
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Hubei Collaborative Innovation Center for Advanced Organic Chemical Materials, Hubei University,
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b
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Molecules, College of Chemistry and Chemical Engineering, Hubei University, Wuhan 430062, China
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Ministry-of-Education Key Laboratory for the Synthesis and Application of Organic Functional
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19 20 21 22 23 24
*Corresponding author. Fax: +86-27-88663043; Telephone: +86-27-50865309; E-mail address: [email protected] (X. Zhang)
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25
ABSTRACT An electrochemiluminescence (ECL) sensor based on Ru(bpy)32+-doped silica nanoparticles (RuSi
27
NPs) combined with molecularly imprinted polymer (MIP) has been developed for the determination of
28
ochratoxin A (OTA). The sensor was fabricated by remodification of RuSi NPs with a thin film of
29
molecularly imprinted polymer, which provided the specific binding sites for OTA. The process of
30
template elution and rebinding acted as a gate to control the flux of probes, which passed through the
31
cavities and reacted with Ru(bpy)32+ immobilized on the electrode surface to emit ECL signal. The
32
ECL intensity decreased with the OTA molecules rebound in MIP. The as-prepared sensor exhibited a
33
very high sensitivity and excellent selectivity to the target molecule. The ∆IECL depended linearly on
34
the logarithm of the OTA concentration in the range from 0.1 pg/mL to 14.76 ng/mL with lower
35
detection limit of 0.027 pg/mL (S/N=3). The MIP-ECL sensor enabled the successful application of this
36
method to determine OTA in corn.
37
Keywords
38
Electrochemiluminescence
39
Ochratoxin A
42 43
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ed Ru-silica
nanoparticles,
Molecularly imprinted
polymers,
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sensor,
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44 45 46 47
2
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1. Introduction Ochratoxin A (OTA) is a class of mycotoxin, which is a toxic and harmful substance produced by
50
fungi [1]. The mechanism of poisoning of OTA has been studied, and it’s found that OTA exhibits
51
unusual toxicokinetics [2]. OTA mainly invades liver and kidney, and the existence of OTA poses a
52
threat to the health of animals and human [3]. In this sense, OTA in food and feed is urgently needed to
53
be detected sensitively for the purpose of protecting animals and people. Up to now, various methods
54
have already been established to assay OTA, such as thin-layer chromatography (TLC) [4], fluorimetry
55
(FL) [5], electrochemistry [6], high performance liquid chromatography (HPLC) [7], capillary
56
electrophoresis (CE) [8], chemiluminescence (CL) [9], and enzyme-linked immunosorbent assay
57
(ELISA) [10]. Among them, TLC and CE methods exhibit very poor sensitivity. The electrochemical
58
and FL methods can obtain higher sensitivity than that of TCL, but they lack selectivity. Though HPLC
59
methods are sensitive and selective, they require expensive apparatus and complex procedures for
60
sample pretreatment. A high sensitivity of ELISA can be achieved, but the poor chemical/physical
61
stability of the antibodies or enzymes prevents their use in the harsh environments of acids or bases and
62
organic solvents.
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Electrochemiluminescence (ECL) is a technique that can initiate light radiation during the
64
high-energy electron-transfer reactions occurring at the surface of working electrode in electrochemical
65
processes. ECL has received increasing attention in many fields, including environmental pollutant
66
determination [11], pharmaceutical analysis [12], and immunoassays [13], because of its advantages of
67
high sensitivity, wide linear range, simple operation and controllability. Ru(bpy)32+ is the most used
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ECL reagent due to its commercial availability, reversible electrochemical behavior, and high
69
luminescence efficiency [14]. Unlike solution phase Ru(bpy)32+ ECL system, solid-state ECL offers
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many advantages, such as reduced the consumption of expensive ECL reagent Ru(bpy)32+, simplified
71
experimental design, and enhanced the ECL signal [15]. Recently, many efforts have also been made to
72
fabricate solid-state ECL sensors by immobilizing Ru(bpy)32+ or its derivatives on electrode surfaces,
73
including Langmuir-Blodgett films (LB) [16], self-assembly (SA) [17], Nafion [18] and sol-gel method
74
[19]. However, LB has poor membrane stability because of its physical adsorption. Self-assembled film
75
of SA is easy to fall off from the electrode, and it is not conducive to the analysis application. Nafion
76
has good chemical stability, but nafion films are dense, which contributes to the slow mass transfer rate.
77
Sol-gel method has good chemical stability, light permeability and biocompatibility, and it has better
78
stability than that of the first three methods. Silica-based materials were used due to their easy surface
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modification, biocompatibility and good electrochemical stability [20]. In addition, RuSi NPs were
80
used as signal substances because of their high luminous efficiency, chemical stability and
81
electrochemical reversibility.
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Molecularly imprinted polymers (MIPs) have been identified as artificially synthesized receptors
83
and widely used in sensors [21], catalysis reactions [22] and separations [23], because MIPs can
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recognize and bind the desired target molecules with a high affinity and selectivity. The combination of
85
the MIPs with the ECL detection would be able to produce a sensor with both high sensitivity and
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desirable selectivity, which could help to solve the problem related to the selectivity of ECL. There are
87
a lot of literatures that had been reported about MIP-ECL sensors for various applications [24-26].
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In this work, we designed an ECL sensor based on Ru(bpy) 32+-doped silica nanoparticles (RuSi
89
NPs) combined with molecularly imprinted polymer for the determination of OTA (figure 1). The RuSi
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NPs were synthesized by the method of water-in-oil (W/O) microemulsion, while chitosan (CS) was
91
used to immobilize the RuSi NPs on the electrode. The sensor was fabricated by remodification of
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RuSi NPs with a thin film of molecularly imprinted polymer, which provided the specific binding sites
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for OTA, and the prepared sensor could used to detect OTA in corn sample.
94 95
2. Experiment
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2.1. Chemicals and reagents
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Please, insert Fig. 1. here.
Ochratoxin A was obtained from cayman chemical company (United States). Tripropyl amine
98
(TPrA), Triton X-100, cyclohexane, 1-hexanol, tetraethyl orthosilicate (TEOS), NH3·H2O, and acetone
99
were obtained from Sigma-Aldrich (Beijing, China). Terpyridyl ruthenium was obtained from Green
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kaemmer (beijing, China). Corn samples (8.6 μg/kg ± 3.6 μg/kg, HPLC) were obtained from Biopure
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(New Zealand). All reagents were used of analytical grade and double distilled water was used to
102
prepare sample solutions.
103
2.2. Apparatus
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ECL signals were measured with MPI-E electrochemiluminescence analyzer (Xi’An Remax
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Electronic Science & Technology Co. Ltd., China). A three-electrode system was used in the
106
measurements, with a glassy carbon electrode (GCE, 3 mm in diameter) as the working electrode, a
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saturated calomel electrode (SCE) as the reference electrode, and a platinum wire as the auxiliary
108
electrode. Transmission electron microscopy (TEM) was performed using a JEOL JEM-100SX TEM
109
facility. Fluorescence was measured with LS-55 fluorescent spectrophotometer (USA). Branson 2000
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ultrasonic cleaner (USA) was used to clean the electrodes. UV polymerization was implemented by
111
ZF-I UV analyzer (Shanghai Guanghao Analysis Instrument Co. Ltd., shanghai, China).
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2.3. Synthesis of RuSi NPs
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The RuSi NPs were synthesized by a typical W/O microemulsion route [27]. 1.77 mL of Triton
5
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X-100, 7.5 mL of cyclohexane, 1.8 mL of 1-hexanol, and 340 μL 40 mM Ru(bpy)32+ were firstly mixed
115
with constant magnetic stirring for 0.5 h to form the W/O microemulsion. Then 100 μL TEOS and 60
116
μL NH3·H2O were added to the mixing solution, which reacted last for 24 hours. Acetone was then
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added to destroy the emulsion, followed by centrifuging and washing with ethanol and water three
118
times respectively. At last, the orange-colored RuSi NPs were obtained.
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2.4. Preparation of MIP-ECL sensor
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Prior to modification, the GCE was polished with 0.3 and 0.05 mm alumina slurry and rinsed
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thoroughly with doubly distilled water between each polishing step. Then, it was washed successively
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with doubly distilled water and ethanol in an ultrasonic bath, and finally dried at room temperature.
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Firstly, RuSi NPs were dispersed in 0.1 M PBS (pH 7.0), then 1% chitosan was added to mix with
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constant magnetic stirring for 2 h to obtain RuSi/CS. The GC electrode was coated with 5 μL RuSi/CS,
125
and the solvent was allowed to evaporate at room temperature. Secondly, the imprinted polymer was
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obtained by the original solution which was comprised of the template molecule of OTA, the functional
127
monomer methacrylic acid (MAA), the crosslinking agent of ethylene glycol dimethacrylate (EDMA)
128
and initiator azodiisobutyronitrile (AIBN). Dropped 2 μL the original solution on the modified
129
electrode, and then induced by the wavelength of 245 nm ultraviolet light. The MIP-ECL sensor was
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obtained by eluting imprinted polymer film with ethanol. Except without OTA, the preparation process
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of the non imprinted polymer (NIP-ECL sensor) is same as the imprinted polymer (MIP-ECL sensor).
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3. Results and discussion
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3.1. Characterization of synthesized RuSi NPs
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As the morphology of NPs influences the spectral and electrochemical properties, NPs with a
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uniform size is one of the key steps to improve the ECL sensitivity and stability. Transmission electron
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microscopy (TEM) was used to research the morphology of RuSi NPs. As shown in figure 2A, it is
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clear that the as-prepared nanoparticles exhibited a uniform size and the diameter of the RuSi NPs was
138
around 50 nm.
139
3.2. Characterization of MIP-ECL sensor
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In virtue of the fluorescence of OTA, fluorescence spectrum (FLS) was selected to attest the
141
process of elution and rebinding of the imprinted polymer. As shown in figure 2B, the imprinted film of
142
initial preparation (curve a) had the highest fluorescence intensity, because it was prepared with a high
143
concentration of OTA (20 μg/mL). After the imprinted film being eluted, the fluorescence intensity
144
decreased (curve b), which verified that the OTA template were efficiently removed from the imprinted
145
film in the elution process. When the imprinted polymer was immersed in the standard solution of 5
146
ng/mL OTA for 5 min, the template molecule OTA could rebind with imprinted film, and the
147
fluorescence intensity increased (curve c).
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Electrochemical impedance spectroscopy (EIS) is an effective method for monitoring the changes
149
in the surface features of the modified electrodes in the assembly process. As shown in figure 2C,
150
because of the presence of CS, the charge transfer resistance for RuSi/CS/GCE (curve b) showed an
151
increase compared with bare GCE (curve a). The resistance increased obviously (curve c) when the
152
surface of RuSi/CS/GCE was coated by imprinted polymer, because the imprinted polymer was
153
nonconductive. However, after template molecule being eluted from the imprinted polymer, cavities
154
could form and act as the channels for electron transfer, which made the resistance decrease (curve d).
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When the imprinted polymer was incubated with the template molecule, OTA rebound with imprinted
156
film coated on the surface of sensor, the channels of electron transfer would be blocked and the
157
resistance increased (curve e).
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158 159
Please, insert Fig. 2. Here. 3.3. ECL characterized for MIP-ECL sensor and NIP-ECL sensor. Experiments that aimed to find an appropriate potential used for the assay were carried out, and
161
we found that the ECL signal was highest in 1.4 V range from 0 V to 1.6 V. The applied potential range
162
of 0-1.4 V was selected for the assay of OTA. The concentration of TPrA was selected by referring to
163
the published literatures [14, 27-29] and experiments. The results of experiments indicated that the
164
ECL intensity and background signal all increased with the increasing of the concentration of TPrA.
165
Considering that TPrA had low solubility in water, 2 mM TPrA was selected to be used for the assay.
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It was difficult for the reaction between RuSi NPs and TPrA when the MIP was coated on the
167
modified electrode, because it was nonconducting and prevented RuSi NPs from contacting with TPrA.
168
As shown in figure 2D, the peak value of initially prepared MIP-ECL sensor was very small (curve a),
169
and the modified electrode coated with NIP (curve d) was same with MIP. After the removal of the
170
template molecule from the sensor, there were some cavities appeared and were used as the channels
171
for electron transportation, which resulted in that the intensity of ECL increased obviously (curve b).
172
But the NIP-ECL sensor had no cavities, the intensity increased few (curve e). After rebinding, the
173
cavities blocked, and the intensity decreased (curve c). The ECL results were in accordance with that of
174
FLS and EIS.
175
3.4. Optimization of Experimental Conditions
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Considering both luminescent efficiency and the adhesion of the film, the optimal proportion of
177
RuSi NPs and chitosan is crucial to this MIP-ECL sensor. The optimization experiments were carried
178
out in the absence of the imprinted film. As shown in figure 3A, it could be seen that the strong signal
179
was achieved when the proportion of RuSi NPs and chitosan was 6:1.
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In order to optimize the elution time, the MIP-ECL sensor were washed in 2 mL ethanol at various
181
extraction time, whereas the other elution parameters were kept constant, and then followed by ECL
182
determination in 0.1 M PBS (pH 7.0) containing 2 mM TPrA. As shown in figure 3B, increasing of the
183
elution time leads to the responses reached a plateau about 5 min and afterwards the increasing elution
184
time has no considerable effect on the ECL response (curve a). 5 min was selected as optimum elution
185
time accompanying with high luminous intensity and short analyzing time.
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The rebinding time was also one of the main parameters that must be investigated. The rebinding
187
was performed in 2 mL of 5 ng/mL OTA solution. The detection of ECL intensity was performed every
188
5 min from 0 to 25 min, and the results was shown in figure 3B (curve b). When the rebinding time
189
increased, the ECL intensity decreased and remained constant at 5 min. As a result, 5 min was selected
190
as the rebinding time in all of the following assays.
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191 3.5. ECL Response to OTA
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192
Please, insert Fig. 3. Here.
Under the optimized conditions, the MIP-ECL sensor was used to detect OTA. The ECL intensity
194
was measured before and after the MIP-ECL sensor rebound in OTA solutions, respectively. When the
195
concentrations of OTA increased, more cavities were blocked by the template molecule of OTA, and
196
the ECL intensity significantly decreased. The difference between ECL intensities before and after
197
rebinding (∆IECL) was linear to the logarithm of the OTA concentration in the range from 0.1 pg/mL to
198
14.76 ng/mL with lower detection limit of 0.027 pg/mL (S/N=3) (figure 4). The linear regression
199
equation was ∆IECL = 116.6 log (c, ng/mL) + 750.4, with a coefficient of correlation r=0.999. The
200
numerical values for the relative standard deviation at each concentration are 1.4%, 1.8%, 3.4%, 1.4%,
201
3.0%, 2.7%, 1.3%, 1.5%, 1.7% and 0.4% from low concentration to high concentration, respectively.
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202 203
Please, insert Fig. 4. Here. 3.6. Selectivity and Stability To evaluate the selectivity of the MIP-ECL sensor, measurements of ECL intensity were
205
performed in the rebinding procedure. Some mixed solutions were used to examine the selectivity of
206
the designed MIP-ECL sensor; 0.76 ng/mL OTA was mixed with 7.6 ng/mL ochratoxin B (OTB), 7.6
207
ng/mL deoxynivalenol (DON), and 10 mM glucose (GLU). OTA, OTB and DON belong to the class of
208
mycotoxin, and OTB has similar structure to OTA. Meanwhile GLU is a kind of interfering substance
209
exists in corn samples. Figure 5A showed that ECL intensities had almost no change after the MIP-ECL
210
sensors were incubated with the mixed solutions for 5 min. The results indicated that there was little
211
interference from coexistent substances, and the MIP-ECL sensor had high selectivity for the
212
determination of OTA.
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To investigate the stability of the MIP-ECL sensor, ECL was recorded under continuously cyclic
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potential scanning range from 0 V to 1.4V for 10 cycles in 0.1 M PBS (pH 7.0) containing 2 mM TPrA.
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As shown in figure 5B, the ECL intensities did not appear any obvious changes with a relative standard
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deviation (RSD) of 1.9%. The results indicated that the MIP-ECL sensor had good stability, and these
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results were due to that the charge transfer in the imprinted film was fast and imprinted film was stable.
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The MIP-ECL sensors which were placed at room temperature for a week were detected, and the
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results showed that the ∆IECL reduced 2.1%. It could be seen that the MIP-ECL sensor exhibited
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excellent life expectancy. What’s more, the reproducibility of the MIP-ECL sensor was detected. It
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found that RSD of 6 electrodes which were obtained in the same way was 2.4%, which showed good
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sensor-to-sensor reproducibility.
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Please, insert Fig. 5. Here.
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3.7. Determination of OTA in corn sample The high sensitivity obtained with the MIP-ECL sensor makes it feasible to detect the presence of
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OTA in corns. The corns (0.5 g) which contained OTA were dissolved into the solution of 2 mL 75%
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(V/V) methanol/water, then ultrasound dispersion 2 h. After centrifugation at 12000 rpm for 10 min,
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the resulting supernatant was diluted ten times as the OTA sample, and then mixed with various
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concentrations of standard OTA. The prepared corn sample was detected by the MIP-ECL sensor, and it
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found that the concentration of OTA was 6.44 μg/kg, which was consistent with the result of HPLC. As
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shown in table 1, the recoveries of the MIP-ECL sensor ranged from 95.3% to 99.3%, and the RSD
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ranged from 5.2% to 6.1%. The results showed that the MIP-ECL sensor had good accuracy and
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practicability.
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4. Conclusions
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Please, insert Table 1. Here.
In summary, we have successfully demonstrated a novel ECL sensor based on Ru(bpy)32+-doped
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silica nanoparticles combined with molecularly imprinted polymer for the determination of OTA. The
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resulting sensor showed a high sensitivity and excellent selectivity to OTA in the range from 0.1 pg/mL
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to 14.76 ng/mL. In addition, combining MIP with ECL can improve the selectivity of the ECL sensor
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and overcome the interference to the template molecule. The MIP-ECL sensor is easy to make and has
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good stability, it can be applied for monitoring OTA in corn samples. Moreover, this strategy can be
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further expected to fabricate various sensitive and selective sensors for advanced applications.
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Acknowledgements
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This work was financially supported by the National Natural Science Foundation of China
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(No. 21075029, 21375033), the Natural Science Fund for Creative Research Groups of Hubei
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Province of China (No. 2011CDA111, 2014CFA015), the Key Project of the Natural Science
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Foundation of Hubei Province (No. 2015CFA124) and the Program for Excellent Youth Scholars
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of Innovative Research Team by Hubei Provincial Department of Education (No. T201101).
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Table(s)
Tables Table 1. Detection of OTA in corn samples by the MIP-ECL sensor. OTA added to corn samples
OTA detected
Recovery
RSD(n=3)
（ng/mL)
(ng/mL)
(ng/mL)
(%)
(%)
0.3
0.459
99.3
5.2
0.6
0.733
95.3
6.1
0.9
1.053
99.1
5.3
0.161
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(RSD=1.976%)
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Corn samples
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Figure(s)
Figures: Fig. 1. Schematic diagram of the molecularly imprinted polymer electrochemiluminescence sensor for the detecting of ochratoxin A.
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Fig. 2. (A) TEM images of RuSi NPs. (B) FLS of the imprinted film (a) of initial preparation, (b) after elution and (c) after rebinding. (C) EIS of (a) Bare GCE, (b) RuSi/CS/GCE, (c) MIP/RuSi/CS/GCE, (d)
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MIP/RuSi/CS/GCE after template removal, (e) MIP/RuSi/CS/GCE after rebinding. (D) ECL curves of
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(a) MIP/RuSi/CS/GCE, (b) MIP/RuSi/CS/GCE after template removal, (c) MIP/RuSi/CS/GCE after rebinding, (d) NIP/RuSi/CS/GCE and (e) NIP/RuSi/CS/GCE after elution. Scan rate: 100 mV/s; scan
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range: from 0 to 1.4 V.
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Fig. 3. (A) Optimization of the proportion of RuSi NPs and chitosan, six volume proportions were investigated: 8:1, 6:1, 4:1, 2:1, 1:1, 1:2. (B) Optimization of (a) elution time for obtaining the MIP-ECL
scan range: from 0 to 1.4 V.
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sensor and (b) rebinding time of the MIP-ECL sensor immersed in 5 ng/mL OTA. Scan rate: 100 mV/s;
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Fig. 4. Calibration curves for OTA determination based on ∆IECL as the response signal. The inset showed the ∆IECL depended linearly on the logarithm of the OTA concentration in the range from 0.1
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pg/mL to 14.76 ng/mL under the optimized conditions. The error bars represent the standard deviation of three independent measurements. Scan rate: 100 mV/s; scan range: from 0 to 1.4 V.
Fig. 5. (A) Selectivity of the MIP-ECL sensor to OTA, OTB, DON, GLU. (B) Stability of ECL emission under continuous cyclic potential scan for 10 cycles from MIP-ECL sensor. Scan rate: 100 mV/s; scan range: from 0 to 1.4 V.
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Fig. 1.
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Fig. 2.
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Fig. 3.
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Fig. 4
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Fig. 5.
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Author Biographies
Biographies Qingling Wang is currently working toward the M.Sc. degree in College of Chemistry& Chemical Engineering, Hubei University. Her research interests include electrochemical luminescence for
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decteting of ochratoxin A.
Miaomiao Chen is an experimentalist in the Department of Chemistry and Chemical Engineering
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of Hubei University, China. She received M.Sc. degree in analytical chemistry from Hubei
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University in 2008. Her research interest is in the development of chemical sensors and biosensors.
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Haiqing Zhang is currently a post-graduate student in the Zhang group and studying for his Ph.D.
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degree. Her research interests include molecular imprinting for decteting of mycotoxin. Wei Wen earned his M.S.(2010) majoring in Analytical Chemistry from Hubei University, China. He
ed
is currently a post-graduate student in the Wang group and studying for his Ph.D. degree. His research interest is focused on the development of electrochemical biosensors.
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Xiuhua Zhang is an associate professor in the College of Chemistry and Chemical Engineering of Hubei University. He received M.Sc. degree in analytical chemistry and Dr. Eng. degree in materials
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science from Hubei University, in 2003 and 2008, respectively. His main current interest is in the development of sensors based on carbon nanotubes and on conducting polymers.
Shengfu Wang is a professor in the College of Chemistry and Chemical Engineering of Hubei University. He received M.Sc. and Ph.D. in analytical chemistry from Wuhan University (China) in 1992 and 2005, respectively. His main current interests are in bioelectrochemistry, nanoelectrochemistry, chemically modified electrodes, chemical sensors and biosensors.
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