Scholarly article on topic 'Amperometric Sensor for Tetracycline Determination Based on Molecularly Imprinted Technique'

Amperometric Sensor for Tetracycline Determination Based on Molecularly Imprinted Technique Academic research paper on "Chemical sciences"

Share paper
Academic journal
Procedia Environmental Sciences
OECD Field of science
{Tetracycline / "Molecularly imprinted polymer" / "Electrochemical sensor" / "Amperometric determination"}

Abstract of research paper on Chemical sciences, author of scientific article — Huimin Zhao, Hongtao Wang, Xie Quan, Feng Tan

Abstract Tetracycline (TC), a widely used broad spectrum antibiotic, is excreted to environment seriously. Because of its harmful effects, it is essential to establish an effective method for TC determintation. In this work, we fabricated an electrochemical sensor for TC detection based on molecularly imprinted technique. The molecularly imprinted polymer was thermalpolymerized on Ti substrate electrodeposited with micro-nano Pt cluster (MIP-Pt/Ti). The linear range was in a TC concentration range from 0.1 to 10 mg L-1, and the detection limit was 0.026 mg L-1 (S/N = 3). The current change of TC on MIP-Pt/Ti electrode was 10 and 14 times than that of CTC and CAP, respectively. The results indicated that this electrochemical sensor exhibited good sensitivity and selectivity for TC.

Academic research paper on topic "Amperometric Sensor for Tetracycline Determination Based on Molecularly Imprinted Technique"

Available online at

Environmental Sciences



Procedía Environmental Sciences 18 (2013) 249 - 257

2013 International Symposium on Environmental Science and Technology (2013 ISEST)

Amperometric Sensor for Tetracycline Determination Based on Molecularly Imprinted Technique

Huimin Zhaoa, Hongtao Wanga,b, Xie Quana*, Feng Tana

aKey Laboratory of Industrial Ecology and Environmental Engineering (MOE), School of Environmental Science and Technology,

Dalian University of Technology, Dalian 116024, China bFushun Research Institute of Petroleum and Petrochemicals, Dandong Road 31, Fushun 113001, China

Tetracycline (TC), a widely used broad spectrum antibiotic, is excreted to environment seriously. Because of its harmful effects, it is essential to establish an effective method for TC determintation. In this work, we fabricated an electrochemical sensor for TC detection based on molecularly imprinted technique. The molecularly imprinted polymer was thermalpolymerized on Ti substrate electrodeposited with micro-nano Pt cluster (MIP-Pt/Ti). The linear range was in a TC concentration range from 0.1 to 10 mg L-1, and the detection limit was 0.026 mg L-1 (S/N = 3). The current change of TC on MIP-Pt/Ti electrode was 10 and 14 times than that of CTC and CAP, respectively. The results indicated that this electrochemical sensor exhibited good sensitivity and selectivity for TC.

© 2013 The Authors. Published by Elsevier B.V.

Selection and /peer-review under responsibility of Beij ing Institute of Technology.

Keywords: tetracycline; molecularly imprinted polymer; electrochemical sensor; amperometric determination

1. Introduction

Tetracycline antibiotics as one kind of pharmaceuticals and personal care products (PPCPs) are widely distributed environmental pollutants. They are used extensively in veterinary and aquaculture medicine, excreted to environment with a combination of intact and metabolized pharmaceuticals [1, 2]. The residues of these antibiotics have been identified in water, soil and other environment [3]. Tetracycline (TC) is a typical kind of tetracycline antibiotics, which is used for against a wide variety of microorganisms or as additives to enhance growth of food-producing animals [4], due to its broad spectrum activity and low price. However, the abuse of TC and other antibacterial in aquaculture and farming industry has certain drawbacks, such as antibiotic residues in water and food, provoking allergic

* Corresponding author.

E-mail address:


1878-0296 © 2013 The Authors. Published by Elsevier B.V.

Selection and peer-review under responsibility of Beijing Institute of Technology.

doi: 10.1016/j.proenv.2013.04.032

symptoms in human. Therefore, the identification and detection of those pollutants in environment is important.

Traditional methods for determination of TC include microbiological inhibition tests, immunoassays, and chemical-physical methods, e.g. gas/liquid chromatographic (GC/LC) analysis and capillary electrophoresis (CE) [5-7]. Among them, microbiological tests are relatively slow and nonspecific, and immunoassays are usually expensive. While the chemical-physical methods depend on large equipment and complicated procedure for sample pretreatment, they are of improper for on-site detection. Currently, electrochemical methods had attracted many attentions on organic compounds quantification due to their speediness and convenience [8, 9]. Masawat et al. [10] described the electrochemical analysis of TCs including tetracycline, chlortetracycline and oxytetracycline in pharmaceutical products and foods using gold screen-printed electrode combining with flow injection system. However, the process is time consuming and expensive. In view of these disadvantages, there is need for a simple, sensitive and selective method for the determination of tetracycline.

Molecularly imprinted technique is one of artificially generated recognition methods. It has been proved to be an efficient way to provide specific functionalized sites [11-13]. Because of its unique properties of predetermination, selectivity and specific affinity for the template [14, 15], this technique has been applied in chiral discrimination [16], sensors [17], solid phase extraction (SPE) [18] and antibody and receptor mimic [19]. A number of studies have been focused on the selective binding of organic compounds by molecularly imprinted polymer (MIP) for application in sensing [20, 21]. Whitcombe et al. [22] prepared a kind of hybrid electrochemical sensor for dopamine and catechol determination. The layer of poly(N-phenylethylene diamine methacrylamide) on the Au substrate could facilitate the transfer of electrons from recognition sites in MIP to the electrode surface. Therefore, the response of the sensor could be improved by facilitating the transfer of electrons and increasing the cavities in the molecularly imprinted polymer electrode.

In the present work, a simple approach was proposed to achieve the direct transfer of the electrons from recognition sites to electrode surface and increase the binding sites in MIP electrode. The Pt cluster electrodeposited on the Ti sheet was used as substrate, on which the MIP layer was direct polymerized. In this way, the conduction of electrons from the recognition sites in MIP to electrode surface could be facilitated. The electrochemical characterization, sensitivity and selectivity of the sensor to TC were investigated.

2. Experimental section

2.1. Chemical and reagents

The analytical standard of tetracycline and ethylene glycol dimethacrylate (EGDMA) were purchased from Sigma-Aldrich and A Johnson Matthey, respectively. Chlortetracycline hydrochloride (CTC) and chloramphenicol (CAP) were supplied by national institute for the control of pharmaceutical and biological products of China. Methacylic acid (MAA) and 2,2'-azobisisobutyronitrile (AIBN) were obtained from Bodi chemical holding Co., Ltd. and Tianjin Damao chemical reagent factory of China, respectively. All the other chemicals are of analytical grade and used as received. All the solutions were prepared using ultrapure water obtained by a Milli-Q purification system.

2.2. MIP-Pt/Ti membrane fabrication

The micro-nano Pt cluster deposited on titanium sheet (Pt/Ti) was used as substrate. It was prepared by direct electrodeposition process with titanium sheet as substrate. The electrolyte was 2 g L-1 H2PtCl4

solution containing 1.2 mM hydrochloric acid. The electrodeposition was carried out under -0.35V for 30 min in a three-electrode system with a Pt foil as the counter electrode and a saturated calomel electrode (SCE) as the reference electrode.

The fabrication procedures of MIP electrode (MIP-Pt/Ti) through thermalpolymerization were illustrated in Fig. 1. MAA and EGDMA were used as monomer and crosslinker, respectively. The polymerization was initialled by the free radical provided by AIBN at 60 °C, and a thin layer of molecularly imprinted polymer would be formed on the surface of Pt/Ti substrate. Under optimized condition, a solution of template (0.1g) and initiator (0.07g) was prepared in methanol (5 mL) in a 25 mL centrifuge tube, followed by addition of EGDMA (12.8 mmol) and MAA (2.7 mmol). After the mixture was sonicated for 5 min, the Pt/Ti substrate was immersed in the mixture for 15 s and then it was sealed in a centrifuge tube. After removal of oxygen by purged with nitrogen for 5 min, the tube was inserted into a thermostating bath at 60X2 to initial the polymerization and maintained for 24 h to obtain the MIP electrode. The removal or rebinding processes between the templates and the polymers were also indicated in Fig. 1. The TC molecules in the polymer could be removed by continuous eluting with ethanol and water. The binding of the other molecules was blocked by the shape and size of imprinted cavities and the hydrogen bonds formed in the polymer, which ensure the selective binding of this material to TC molecule.

* KJr-Jr . I

'k . Thermalpolymerization

■ oh ai, » _p.

? 9i99r M"1"°,'№!4h II

/ / °x>\ \ Removal

/ Rebinding

Ti substrate K Pt ciustur

Fig. 1. Schematic illustration for fabrication and formation procedures of MIP-Pt/Ti through thermalpolymerization.

To evaluate the molecular recognition properties of imprinted materials, the nonimprinted electrode (NIP-Pt/Ti) as a control sample was also synthesized under the same conditions, but in the absence of the template. Before used for the determination, the removing of the template in MIP-Pt/Ti or NIP-Pt/Ti electrode was obtained by washing the electrode with ethanol and water for four hours sequentially (according to the results of elution experiment, data not shown here).

2.3. Characterizations and measurements

The electrochemical measurements were carried out in 10 mL phosphate-buffered saline (PBS: NaH2PO4 and Na2HPO4, pH=7.4) and at potential bias of 0.8V. After the blank current reaching the

steady state, TC solutions with different concentrations were injected into the cell. 3. Results and discussion

3.1. Computer simulation of molecular imprinting

In order to study the interaction in the polymer, computer design has been suggested as a rational method to simulate the polymer structure [23, 24]. Accordingly, the computational design results with MAA as functional monomer were shown in Fig. 2. The results showed that the hydrogen bond interactions can be formed between -COOH in two MAA molecules and -CONH2 or -OH in one TC molecule, which were presented by dotted lines in Fig. 2. The interaction energies between the template and the functional monomer were calculated by the following equation in vacuum

AE = E (template —monomer) — E(template ) — ^ E(monomer) (1)

The highest binding energy was achieved at the conformation of Fig. 2 and estimated to be -152 kJ mol-1.

When the obtained MIP was used to extract TC molecules from water, the specific cavities and hydrogen bonds in the imprinted polymer will provide selective recognition for TC.

Fig. 2. Optimized conformation of the most stable complex of TC with MAA molecules. Dotted lines represent hydrogen bonds between TC and functional groups of monomers.

3.2. Characterization of MIP-Pt/Ti electrode

FTIR spectra of nonimprinted and imprinted polymer were showed in Fig. 3. The strong absorption peak at 1720 cm1 and 1150 cm1 were attributed to -C=O and C-O stretching vibration absorbance, respectively. At 1635 cm1, the weak absorbance peak of C=C suggests that most MAA had been cross-linked. No obvious bonds are present in the region 3100-3000 cm1 indicating the absence of C-H groups in imprinted polymeric surface layer, which also suggests that both MAA and EGDMA are completely polymerized [25]. The characteristic signals of NIP were almost the same with the MIP which means that both imprinted and non-imprinted membrane has the same chemical composition. The intensity of stretching vibration peak of -C=O (1720cm1) in NIP decreased compared with that of MIP indicating that the template molecules were assembled with monomer via the hydrogen-bonded interaction in preparing of imprinted polymer.

4000 3500 3000 2500 2000 1500 1000 500 Wavenumber/ cm 1

Fig. 3. FTIR spectra of imprinted polymer (a) and nonimprinted polymer (b).

The morphologies of Pt cluster on Ti substrate (Pt/Ti) by electrodeposition and MIP-Pt/Ti electrode are shown in Fig. 4. It could be seen that the diameter of Pt cluster was less than 1 |xm, which could provide high specific surface area for MIP and facilitate the conduction of electrons from the recognition sites in the MIP to the electrode surface. After thermalpolymerization, the Pt/Ti substrate was covered by a layer of imprinted polymer.

Fig. 4. SEM images of Pt cluster on Ti substrate by electrodeposition (Pt/Ti) (a) and MIP coated on Pt/Ti by thermalpolymerization (MIP-Pt/Ti) (b).

3.3. Cyclic voltammetric behavior

Cyclic voltammograms of the imprinted electrodes in 2.5 mmol L"1 [Fe(CN)6]3"/4" were shown in Fig. 5. Both Pt/Ti and MIP-Pt/Ti shown well-defined redox peaks. The cathodic and anodic peak current (Ipc and Ipa) of Pt/Ti were 1.89mA and -2.06 mA, respectively, which was attributed to the large specific surface area and good conductivity of the micro-nano Pt cluster. It could facilitate the transfer of electrons and accumulate much more [Fe(CN)6]3-4- on the electrode surface. After polymerized the molecularly imprinted polymer on Pt/Ti, both the conductivity of the electrode and the redox sites for [Fe(CN)6]3-/4-decreased due to the block of the polymer. Ipc and Ipa of MIP-Pt/Ti also decreased to 1.04 mA and -1.77 mA as shown in curve b of Fig. 5.

Fig. 5. Cyclic voltammograms of Pt/Ti (a), and MIP-Pt/Ti (b). Data was recorded in 2.5 mmolL-1 [Fe(CN)6]3"'4" with 0.1 mol L-1 KCl at 20mVs-1 with two circles. Insert shows CV curves of Ti in electrolyte.

3.4. Electrochemical behavior of MIP-Pt/Ti sensor

To prove whether the imprinted sites in the polymer could bind the target molecule, the amperometric method was explored. The responses of MIP-Pt/Ti and NIP-Pt/Ti electrode in the presence of TC were shown in Fig. 6. Before and after the injection of target compounds, the current was recorded as I0 and I;, respectively. The feedback signal is given by

M = I1- Io (2)

After electrochemical detection, the electrode was washed with ethanol and water about 10 min for the next detection.

For MIP-Pt/Ti electrode (Fig. 6(B)), the injection of 1 mg L-1 TC resulted in the increase of current, which was ascribed to the binding of the target molecules by the cavities in the imprinted polymer [26,27]. Meanwhile, the current change for the NIP-Pt/Ti electrode as indicted in Fig. 6(B) was much smaller than that of MIP-Pt/Ti electrode. The negligible change could be ascribed to the physical adsorption of the organic molecules on NIP-Pt/Ti electrode. The results demonstrated that the good response to TC on MIP electrode.

Fig. 6. Electrochemical behaviors of MIP-Pt/Ti (A) and NIP-Pt/Ti (B) in the presence of 0 and 1 mg L-1 TC (PBS pH 7.4, 0.8 V versus SCE). Insert shows change of the current from 180 to 250s particularly.

3.5. Sensitivity of MIP-Pt/Ti sensor

A calibration curve for TC detection on MIP-Pt/Ti sensor was determined. Current changes versus TC concentrations (as shown in Fig. 7) exhibit a linear response over the range of concentration from 0.1 to 10 mg L-1. The limit of detection (LOD) was estimated to be 0.026 mg L-1(S/N = 3). Compared with MIP-Pt/Ti electrode, the signal observed on NIP-Pt/Ti control electrode can be neglected when TC concentration is below 10 mg L-1, clearly demonstrating the formation of the specific binding sites on MIP-Pt/Ti electrode and the adsorption ability for TC molecules.

• MIP-Pt/Ti ■ NIP-Pt/Ti

AI = 0.075 + 0.462 [TC] R - 0.996

o l-f* * : ; .,.'.,.!

0 5 10 15 20 25 30

[TC] / mg L

Fig. 7. Calibration plot for MIP electrode of AI versus TC concentration and corresponding data for NIP control electrode with TC concentration range from 0.1 to 30 mg L-1.

3.6. Selectivity of MIP-Pt/Ti sensor

To examine the selectivity of the MIP-Pt/Ti sensor, CTC and CAP were chosen as interfering molecules and their structure were shown in Fig. 8, which were also used regularly as spectrum antibiotic in veterinary or aquaculture. CTC or CAP with 2.5 mg L-1 caused small current changes on MIP-Pt/Ti electrode as shown in Fig. 9. The current change of TC on MIP-Pt/Ti electrode is 10 and 14 times than that of CTC and CAP, respectively, suggesting high selectivity of MIP-Pt/Ti sensors. AI caused by the addition of CTC was larger than that of CAP, which could be attributed to the close structural resemblance of the CTC to TC. The current changes of TC, CTC and CAP on NIP-Pt/Ti electrode are

/Cl HN--С-CH

I II ^

CbN-(v /)-С-CH о


also showed in Fig. 9 and the changes are negligible.

Fig. 8. Structures of compounds used in this study: CTC (a), CAP (b) and TC (c).


Fig. 9. Responses of 2.5 mg L-1 TC, CTC and CAP on MIP and NIP electrode. 4. Conclusions

In this work, a sensitive and selective electrochemical sensor for the determination of tetracycline by molecularly imprinted technique was demonstrated. The shape and size of imprinted cavities in the polymer provided selective binding of template molecules through hydrogen bonds. In the amperometric determination of TC, the linear range was in a TC concentration range from 0.1 to 10 mg L-1, and the detection limit was 0.026 mg L-1 (S/N = 3). The potentially interfering compounds, including CTC and CAP, had minimal response on the MIP electrode. This electrochemical sensor exhibited good sensitivity and selectivity for tetracycline determination.


This work was supported by Hi-Tech Research and Development Program of China (No. 2007AA06Z406), National Natural Science Foundation of China (No. 20877012), Program for New Century Excellent Talents in University (NCET-08-0079) and Program for Changjiang Scholars and Innovative Research Team in University (IRT0813).


[1] Baguera A J, Jensen J, Krogh P H. Effects of the antibiotics oxytetracycline and tylosin on soil fauna. Chemosphere, 2000, 40 (7): 751--757.

[2] Matamoros V, Arias C, Brix H, Bayona J. Removal of pharmaceuticals and personal care products (PPCPs) from urban wastewater in a pilot vertical flow constructed wetland and a sand filter. Environ. Sci. Technol., 2007, 41 (23): 8171--8177.

[3] Richardson B J, Lam Paul K S, Martin M. Emerging chemicals of concern: pharmaceuticals and personal care products (PPCPs) in Asia, with particular reference to southern China. Mar. Pollut. Bull., 2005, 50 (9): 913--920.

[4] Hassani M, Lázaro R Pérez C, Condón S, Pagán R. Thermostability of oxytetracycline, tetracycline, and doxycycline at ultrahigh temperatures. J. Agric. Food Chem., 2008, 56 (8): 2676--2680.

[5] Pellinen T, Bylund G, Virta M, Niemi A, Karp M. Detection of traces of tetracyclines from fish with a bioluminescent sensor strain incorporating bacterial luciferase reporter genes. J. Agric. Food Chem., 2002, 50 (17): 4812--4815.

[6] Oka H, Ito Y, Matsumoto H. Chromatographic analysis of tetracycline antibiotics in foods. J. Chromatogr. A, 2000, 882 (1): 109--133.

[7] Schenck F J, Callery P S. Chromatographic methods of analysis of antibiotics in milk, J. Chromatogr. A, 1998, 812 (1): 99--109.

[8] Pejcic B, Eadington P, Ross A. Environmental monitoring of hydrocarbons: A chemical sensor perspective. Environ. Sci. Technol., 2007, 41 (18): 6333--6342.

[9] Zhang M G, Gorski W. Electrochemical sensing platform based on the carbon nanotubes/redox mediators-biopolymer system, J. Am. Chem. Soc., 2005, 127 (7), 2058--2059.

[10] Masawat P, Slater J M. The determination of tetracycline residues in food using a disposable screen-printed gold electrode (SPGE). Sens. Actuators B: Chem., 2007, 124 (1): 127--132.

[11] Vlatakis G, Andersson L I, Müller R, Mosbach K. Drug assay using antibody mimics made by molecular imprinting. Nature, 1993, 361: 645--647.

[12] Wei S T, Mizaikoff B. Recent advances on noncovalent molecular imprints for affinity separations. J. Sep. Sci., 2007, 30 (11): 1794--1805.

[13] Zhang H Q, Ye L, Mosbach K. Non-covalent molecular imprinting with emphasis on its application in separation and drug development. J. Mol. Recognit., 2006, 19 (4): 248--259.

[14] Haupt K, Mosbach K. Molecularly imprinted polymers and their use in biomimetic sensors. Chem. Rev., 2000, 100 (7): 2495-2504.

[15] Sellergren B. Imprinted polymers with memory for small molecules, proteins, or crystals. Angew. Chem., Int. Ed., 2000, 39 (6): 1031--1037.

[16] Sekine S, Watanabe Y, Yoshimi Y, Hattori K, Sakai K. Influence of solvents on chiral discriminative gate effect of molecularly imprinted poly(ethylene glycol dimethacrylate-co-methacrylic acid). Sens. Actuators B: Chem., 2007, 127 (2): 512--517.

[17] Percival C J, Stanley S, Galle M, Braithwaite A, Newton M I. Molecular-imprinted, polymer-coated quartz crystal microbalances for the detection of terpenes. Anal. Chem., 2001, 73 (17): 4225--4228.

[18] Djozan D, Ebrahimi B. Preparation of new solid phase micro extraction fiber on the basis of atrazine-molecular imprinted polymer: Application for GC and GC/MS screening of triazine herbicides in water, rice and onion. Anal. Chim. Acta, 2008, 616 (2): 152--159.

[19] Jenkins A L, Bae S Y. Molecularly imprinted polymers for chemical agent detection in multiple water matrices. Anal. Chim. Acta, 2005, 542 (1): 32--37.

[20] Li C Y, Wang C F, Wang C H, Hu S S, Development of a parathion sensor based on molecularly imprinted nano-TiO2 self-assembled film electrode. Sens. Actuators B: Chem., 2006, 117 (1): 166--171.

[21] Blanco-López M C, Gutiérrez-Fernandez S, Lobo-Castañón M J, Miranda-Ordieres A J, Tunon-Blanco P. Electrochemical sensing with electrodes modified with molecularly imprinted polymer films. Anal. Bioanal. Chem., 2004, 378 (8) 1922--1928.

[22] Lakshmi D, Bossi A, Whitcombe M J, Chianella I, Fowler S A, Subrahmanyam S, Piletska E V, Piletsky S A. Electrochemical sensor for catechol and dopamine based on a catalytic molecularly imprinted polymer-conducting polymer hybrid recognition element. Anal. Chem., 2009, 81(9): 3576--3584.

[23] Chianella I, Karim K, Piletska E V, Preston C, Piletsky S A. Computational design and synthesis of molecularly imprinted polymers with high binding capacity for pharmaceutical applications-model case: Adsorbent for abacavir. Anal. Chim. Acta, 2006, 559 (1): 73--78.

[24] Lv Y, Lin Z, Tan T, Feng W, Qin P Y, Li C. Application of molecular dynamics modeling for the prediction of selective adsorption properties of dimethoate imprinting polymer. Sens. Actuators B: Chem., 2008, 133 (1): 15--23.

[25] Wang X J, Xu Z L, Feng J L, Bing N C, Yang Z G. Molecularly imprinted membranes for the recognition of lovastatin acid in aqueous medium by a template analogue imprinting strategy. J. Membr. Sci., 2008, 313 (1): 97--105.

[26] Cai W S, Gupta R B. Molecularly-imprinted polymers selective for tetracycline binding. Sep. Purif. Technol., 2004, 35 (3): 215--221.

[27] Trotta F, Baggiani C, Luda M P, Drioli E, Massari T. A molecular imprinted membrane for molecular discrimination of

tetracycline hydrochloride. J. Membr. Sci., 2005, 254 (1): 13--19.