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Molecularly Imprinted Sensor Based on an Enzyme Amplifier for Ultratrace Oxytetracycline Determination Jianping Li,* Fuyang Jiang, and Xiaoping Wei College of Chemistry and Bioengineering, Guilin University of Technology, Guangxi, 541004, China A novel strategy for preparing highly sensitive, molecularly imprinted sensors based on enzyme amplifiers was proposed for oxytetracycline (OTC) determination. A molecularly imprinted polymer (MIP) film was used as an artificial antibody to interact with OTC and horseradish peroxidase-labeled OTC (HRP-OTC). Oxytetracycline was determined according to the competition reaction. The molecularly imprinted sensor was characterized by alternating current (ac) impedance spectroscopy, differential pulse voltammetry (DPV), and cyclic voltammetry (CV). The DPV technique was performed to verify the voltammetric behavior of the molecularly imprinted sensor. At the concentration of 0-1 × 10-7 mol/L, OTC could be determined with a detection limit of 6.49 × 10-10 mol/ L. The MIP artificial immunosensor showed high sensitivity, selectivity, and reproducibility. Determination of OTC in samples showed good recovery. With its high selective recognition and affinity, the molecular imprinting technique (MIT) has attracted increased attention, and molecularly imprinted polymers (MIPs) have been widely explored for numerous applications.1-6 The binding sites of MIPs were characterized by affinities and selectivities similar to antigen-antibody systems.7 For this reason, molecularly imprinted materials have been dubbed “artificial antibodies”.8 An MIP has unique advantages over natural biological receptors in terms of physical and chemical stability, ease of preparation, low cost, and applicability in harsh environmental conditions.9 Thus, MIPs have been widely explored in the field of analytical chemistry, especially in studies on solid-phase extraction,10 stationary phases for high* To whom correspondence should be addressed. Phone: +86 773 5895622. E-mail:
[email protected]. (1) Mosbach, K.; Ramstro ¨m, O. Nat. Biotechnol. 1996, 14, 163–170. (2) Haupt, K. Analyst 2001, 126, 747–756. (3) Ye, L.; Mosbach, K. Chem. Mater. 2008, 20, 859–868. (4) Muldoon, M. T.; Stanker, L. H. Anal. Chem. 1997, 69, 803–809. (5) Haupt, K.; Mosbach, K. Chem. Rev. 2000, 100, 2495–2504. (6) Roche, P. J. R.; Ng, S. M.; Narayanaswamya, R.; Goddard, N.; Page, K. M. Sens. Actuators, B 2009, 139, 22–29. (7) Fernandez-Gonzalez, A.; Guardia, L.; Badia-Laino, R.; Diaz-Garcia, M. E. Trends Anal. Chem. 2006, 25, 949–957. (8) Kitade, T.; Kitamura, K.; Konishi, T.; Takegami, S.; Okuno, T.; Ishikawa, M.; Wakabayashi, M.; Nishikawa, K.; Muramatsu, Y. Anal. Chem. 2004, 76, 6802–6807. (9) Yang, Z.; Zhang, C. Sens. Actuators, B 2009, 142, 210–215. (10) Tang, K.; Chen, S.; Gu, X.; Wang, H.; Dai, J.; Tang, J. Anal. Chim. Acta 2008, 614, 112–118.
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performance liquid chromatography (HPLC),11 capillary electrochromatography,12 and enzyme-linked sorbent for colorimetric and chemiluminescence detection.13,14 MIPs were also used to prepare biotin-specific receptors for biotin reorganization based on biotin-horseradish peroxidase (HRP) conjugate binding.15 MIPmodified electrodes, usually produced by direct determination of electroactive molecules and indirect determination of nonelectroactive molecules,16 have been widely explored in biological analysis,17-20 medical analysis,21,22 environmental analysis,23,24 and so on. However, the sensitivities of MIP sensors were unsatisfactory. Owing to this, methods for improving sensitivity were attempted, which led to the introduction of an enzyme amplifier into the MIP sensor in this study. We constructed a novel sensor for determination in which oxytetracycline (OTC) was chosen as sample based on MIPs and enzyme amplifier. Molecularly imprinted film was modified on the electrode surface, and OTC was linked to the cavities constructed by binding sites of molecularly imprinted film. Owing to the stereoscopic hindrance effect of HRP, some of those cavities are designated only for OTC, whereas others could interact with both OTC and horseradish peroxidase-labeled OTC (HRP-OTC). In Scheme 1, a step named “isolation” was introduced to occupy vacant binding cavities and avoid the OTC in samples interacting with cavities that HRP-OTC cannot occupy due to the stereo hindrance effect. After “isolation”, all cavities were occupied. The (11) Baggiani, C.; Baravalle, P.; Giraudi, G.; Tozzi, C. J. Chromatogr., A 2007, 1141, 158–164. (12) Priego-Capote, F.; Ye, L.; Shakil, S.; Shamsi, S. A.; Nilsson, S. Anal. Chem. 2008, 80, 2881–2887. (13) Surugiu, I.; Ye, L.; Yilmaz, E.; Dzgoev, A.; Danielsson, B.; Mosbach, K.; Haupt, K. Analyst 2000, 125, 13–16. (14) Surugiu, I.; Danielsson, B.; Ye, L.; Mosbach, K.; Haupt, K. Anal. Chem. 2001, 73, 487–491. (15) Piletska, E.; Piletsky, S.; Karim, K.; Terpetschnig, E.; Turner, A. Anal. Chim. Acta 2004, 504, 179–183. (16) Zhao, J.; Li, J. P.; Jiang, F. Y. Chin. J. Anal. Chem. 2009, 37, 1219–1222. (17) Diltemiz, S. E.; Hu ¨ r, D.; Ersoz, A.; Denizli, A.; Say, R. Biosens. Bioelectron. 2009, 25, 599–603. (18) Wan, Y.; Zhou, Y.; Sokolov, J.; Rigas, B.; Levon, K.; Rafailovich, M. Biosens. Bioelectron. 2008, 24, 162–166. (19) Kirat, K. E.; Bartkowski, M.; Haupt, K. Biosens. Bioelectron. 2009, 24, 2618– 2624. (20) Li, J. P.; Zhao, J.; Wei, X. P. Sens. Actuators, B 2009, 140, 663–669. (21) Xu, X.; Zhou, G.; Li, H.; Liu, Q.; Zhang, S.; Kong, J. Talanta 2009, 78, 26–32. (22) Huang, C. Y.; Tsai, T. C.; Thomasc, J. L.; Leed, M. H.; Liue, B. D.; Lin, H. Y. Biosens. Bioelectron. 2009, 24, 2611–2617. (23) Li, J. P.; Jiang, F. Y. Chem. Lett. 2010, 39, 478–479. (24) Zhu, Q. Z.; Haupt, K.; Knopp, D.; Niessnera, R. Anal. Chim. Acta 2002, 468, 217–227. 10.1021/ac100667m 2010 American Chemical Society Published on Web 06/22/2010
Scheme 1. Scheme of the Molecular Imprinting Technique
sensor was then incubated in an HRP-OTC solution to utilize HRP-OTC as a replacement for OTC and to occupy those cavities for both OTC and HRP-OTC. When the artificial immunosensor was dipped into samples, the OTC in samples replaced HRP-OTC and the amount of HRP immobilized on HRP-OTC was reduced. This can be detected by the current changes in the hydroquinonehydrogen peroxide-phosphate buffer solution (PBS, pH ) 7.2). Hence, OTC concentrations in samples were determined by comparing the peak current from calibration curves of the sensor. OTC was determined, and high sensitivities of the MIP electrodes were obtained. EXPERIMENTAL PROCEDURES Materials. All reagents were of analytical grade and used without further purification. Oxytetracycline was obtained from Acros, U.S.A.; HRP-OTC was purchased from Zhengzhou Biocell Biotechnology Co., Ltd., China; hydroquinone, o-phenylenediamine (OPD), hydrogen peroxide, and potassium ferricyanide were purchased from Sinopharm Group Chemical Reagent Co., Ltd., China. Double-distilled water was used for preparation of all solutions and for washing. Apparatus and Electrodes. Electroanalytical measurements such as differential pulse voltammetry (DPV) and cyclic voltammetry (CV) were performed at 25 °C on a CHI660C electrochemical workstation with a standard three-electrode cell (Shanghai Chenhua Instruments, Shanghai, China) connected to a personal computer. The classical three-electrode system consisted of a KCl saturated Ag/AgCl electrode as reference electrode, a platinum wire electrode as auxiliary electrode, and an MIP-modified gold electrode (d ) 2 mm) as working electrode. Alternating current impedance spectroscopy was performed at 25 °C on an AutoLab PGSTAT302 (Eco Chemie, Utrecht, The Netherlands). Preparation of MIP and Non-molecular imprinted Polymer (nMIP) Modified Electrodes. MIPs can be constructed by electropolymerization, chemical grafting, photopolymerization, and the molecular self-assembled approach.25-27 In the current study, (25) Piletsky, S. A.; Turner, A. P. F. Electroanalysis 2002, 14, 317–323. (26) Piletsky, S. A.; Piletska, E. V.; Chen, B.; Karim, K.; Weston, D.; Barrett, G.; Lowe, P.; Turner, A. P. F. Anal. Chem. 2000, 72, 4381–4385. (27) Sergeyeva, T. A.; Matuschewski, H.; Piletsky, S. A.; Bendig, J.; Schedler, U.; Ulbricht, M. J. Chromatogr., A 2001, 907, 89–99.
electropolymerization was used as an easy-to-implement method for polymer deposition and integration because, in comparison to other techniques, it has the potential to regulate the thickness and the density of the polymer layer through polymerization conditions (e.g., applied voltage, cyclic scans).28 Both MIP and nMIP were constructed by electropolymerization of OPD on the surface of the gold electrode, using CV with the potential range between 0.0 and +0.8 V. Prior to electropolymerization, the surface of the gold electrode was polished with a microcloth (chamois leather) and 1.0, 0.3, and 0.05 µm aqueous slurry of alumina. This was followed by washing alternately with water, alcohol, and HNO3 (50% in volume). Thirty cycles of CV at 50 mV/s in an acetate buffer solution (pH ) 5.2, 25 °C) containing 0.03 mol/L OTC and 0.09 mol/L OPD were performed. An nMIP electrode was also prepared in every case under the same experimental conditions with no additional OTC. After electropolymerization, the MIP and nMIP electrodes were washed by HNO3 (50% in volume) for 12 h to remove imprinting molecules and adsorbates on the surface of the imprinted membrane. Meanwhile, the MIP electrode was washed by HNO3 (20% in volume) for 15 min to remove imprinting molecules. As a result, an electrode with an imprinted membrane that has stereo cavities for OTC was obtained. Isolation, Incubation, and Competition. The MIP electrode was immersed in 10 mL of 5 × 10-4 mol/L OTC solution for 15 min to isolate all vacant binding cavities in the MIP. It was incubated in 2 mL of 5 µg/mL HRP-OTC solution for 12 min to facilitate the recognition of HRP-OTC and the replacement of OTC by HRP-OTC in the cavities. Finally, the MIP electrode was placed into 10 mL of sample solution containing 2-100 nmol/L OTC for 12 min at room temperature (25 °C) to allow competition reaction to occur. Electroanalytical Measurements. Electrochemical measurements to characterize the MIP film were carried out in the supporting electrolyte of 0.01 mol/L K3[Fe(CN)6] solution containing 0.5 mol/L KCl at room temperature (25 °C). Cyclic voltammetry was performed from -0.2 to 0.6 V at a scan rate of 50 mV/s. Alternating current impedance spectroscopy was (28) Gabai, R.; Sallacan, N.; Chegel, V.; Bourenko, T.; Katz, E.; Willner, I. J. Phys. Chem. 2001, 105, 8196–8202.
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Figure 1. Electropolymerization of o-phenylenediamine in 0.1 mol/L acetate buffer solution.
performed at a potential of 0.190 V over the frequency range of 100 mHz to 100 kHz using an alternating voltage of 5 mV. Differential pulse voltammetry was performed in the supporting electrolyte of 0.01 mol/L PBS (pH ) 7.2) containing 1.5 mmol/L hydroquinone and 3.0 mmol/L H2O2 at room temperature (25 °C) over a potential range of -0.4 to 0.6 V, at a scan rate of 50 mV/s and a pulse amplitude of 50 mV. RESULTS AND DISCUSSION Cyclic Voltammetry Study of MIPs-OPD Electropolymerization. Electropolymerization of OPD was carried out by CV scanning in a 0.1 mol/L acetate buffer solution (pH ) 5.2, 25 °C). Results are shown in Figure 1. The currents decreased with increasing numbers of cycles. The highest current was obtained in the first scan. Oxidation of OPD was recorded as a distinct and irreversible peak at a peak potential of 0.35 V. When the number of cycles was increased to 30, the current density of the oxidation peak became smaller, which indicated film formation on the electrode surface. No reduction peak was observed during the polymerization. These results demonstrated the growth of an insulating OTC-MIP-OPD film on the Au electrode. Molecular Recognition by MIP-Modified Film. Cyclic voltammetry results of the MIP film were recorded in 0.01 mol/L K3[Fe(CN)6] solution containing 0.5 mol/L KCl. This was done to confirm whether or not OTC has been embedded in the MIP film. During this procedure, K3[Fe(CN)6] was used as the mediator between imprinted electrodes and substrate solutions. Figure 2A shows the relationship between peak current and surface modification conditions of the gold electrode. For the MIP-Au electrode, the decrease of oxidation-reduction peak current from curve a to curve b can be attributed to the film produced which covered the surface of the Au electrode. After the first template removal (curve c), a visible oxidation-reduction peak was obtained. The decrease of peak current from curve c to curve d can be attributed to the obstruction of the access of K3[Fe(CN)6] through the MIP film after OTC rebinding. This can be explained by the interaction between the OTC and the MIP film, which determines the electron transfer of the [Fe(CN)6]3-/[Fe(CN)6]4- ion pair on the electrode surface. 6076
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Figure 2. Cyclic voltammetry results of the electrode in 0.01 mol/L K3[Fe(CN)6] solution containing 0.5 mol/L KCl: (a) bare gold electrode, (b) MIP-Au electrode, (c) MIP-Au electrode after template removal, (d) MIP-Au electrode after rebinding, (e) bare gold electrode, (f) nMIP-Au electrode, and (g) nMIP-Au electrode after the removal template step.
In contrast, for the nMIP-Au electrode (Figure 2B), the oxidation-reduction current decreased dramatically from curve e to curve f because the OPD film covered the surface of the Au electrode polymerized in the absence of OTC where no cavities with binding sites were obtained. The change of oxidation-reduction current was negligible after the removal template step (curve g). This indicated that the nMIP-Au electrode was unselective and failed to recognize OTC. Alternating Current Impedance Measurement. Alternating current impedances were measured to confirm that the MIP film was properly produced. Figure 3 shows the changes in the MIP-Au electrode. The increased resistance from curve a to curve b could be attributed to the produced film that covered the surface of the Au electrode. The decrease of resistance from curve b to curve c could be attributed to the removal of template (OTC) from the MIP film. The increased resistance from curve c to curve d signified that OTC was rebounded to the film. This verified that the MIP film has good capability to distinguish the target molecule. Optimizing for Isolation, Incubation, and Competition. In this investigation, isolation was carried out in 10 mL of 5 × 10-4 mol/L OTC solution. During the experiment, DPV was per-
Figure 3. Alternating current impedances of MIPs film: (a) bare gold electrode, (b) MIP electrode, (c) MIP electrode after template removal, and (d) MIP electrode after rebinding.
Figure 4. Relationship between incubation time and concentration of HRP-OTC: (a) 3, (b) 5, and (c) 7 µg/mL.
formed for 3 min. Differential pulse voltammetric currents decreased gradually up to the 8 min mark and then remained constant. Thus, 8 min was selected as the isolation time. Incubation time was defined as the time HRP-OTC reached the imprinted membrane and replaced the OTC combined with cavities in the MIP. Incubation was operated separately in solutions containing 3, 5, and 7 µg/mL HRP-OTC, respectively. Differential pulse votammetric curve was recorded for incubation after 3 min. In Figure 4, the incubation time decreased with the increase in HRP-OTC concentration. This demonstrates that reaction can be accomplished with an incubation time of 12 min in 5 µg/mL HRP-OTC solution. As a result, 5 µg/mL HRP-OTC solution and incubation time of 12 min were selected for all subsequent assays. Competition is the process in which OTC in samples reaches the imprinted membrane and replaces the HRP-OTC combined with cavities in the MIP. Competition was operated in 2 × 10-9 to ∼1.0 × 10-7 mol/L OTC solution, and DPV was performed after competition for 3 min. In Figure 5, the DPV current decreased with the incubation time up to the 12 min mark and remained constant subsequently. Interaction reached equilibrium after 12 min. As a result, 12 min was selected as the competition time to ensure OTC will replace HRP-OTC. Calibration Curve. To adequately rebind OTC, DPV results were recorded after the MIP film electrode was dipped in the
Figure 5. Effects of incubation reaction time (a) and competition reaction time (b) on response signals: (a) 5 µg/mL HRP-OTC; (b) 5 × 10-8 mol/L OTC.
Figure 6. DPV results of the MIP sensor after incubation in different concentrations of OTC: (a f l) 0, 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 10 × 10-8 mol/L OTC, respectively.
solution containing OTC in different concentrations for 12 min. Figure 6 shows the dependence of the DPV oxidation current on OTC concentration. Due to the increasing number of binding sites in the film occupied by OTC molecules, the peak current decreased with the increase in OTC concentration. A calibration curve between the oxidation peak current and the OTC concentration is exhibited in Figure 6. The linear calibration graphs of oxidation peak current (I) versus OTC concentration (c) can be described by the following equation: I (10-6 A) ) 22.3372 - 2.1588c (10-8 mol/L) (r ) 0.9989) at the OTC concentration linear range of 0-1.0 × 10-7 mol/L. The curve bent toward the parallel of concentration axis in a highconcentration range up to 10-6 mol/L. The detection limit of 6.49 × 10-10 mol/L was calculated according to the equation DL ) 3δb/K, where DL is the detection limit at the 95% confidence level, δb is the standard deviation of the blank measurements (n ) 20), and K is the slope of the calibration curve. This indicates that the MIP sensor based on an enzyme amplifier is one of the most sensitive means of monitoring OTC compared with other methods.29,30 Selectivity of the Sensor. Some OTC analogues and polycyclic compounds were used to examine the selectivity of the (29) Hu, X.; Pan, J.; Hu, Y.; Huo, Y.; Li, G. J. Chromatogr., A 2008, 1188, 97– 107. (30) Chen, L.; Liu, J.; Zeng, Q.; Wang, H.; Yu, A.; Zhang, H.; Ding, L. J. Chromatogr., A 2009, 1216, 3710–3719.
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designed MIP electrode. The responses of tetracycline (TC), chlortetracycline (CTC), propranolol hydrochloride (PH), and anthraquinone (AP) were compared with OTC. The order of recognition selectivity and the ability of replacing HRP-OTC were OTC > TC > CTC > AP > PH. Moreover, 1.25 × 10-6 mol/L TC, 2 × 10-6 mol/L CTC, 6 × 10-6 mol/L AP, and 7.5 × 10-6 mol/L PH generated negligible changes (less than 5% in relative error) in the DPV currents compared with 5 × 10-8 mol/L OTC. Reproducibility and Stability of the Sensor. The reproducibility of the sensors was tested by detecting the peak current of 5 × 10-8 mol/L OTC in 0.01 mol/L PBS (pH ) 7.2) containing 1.5 mmol/L hydroquinone and 3.0 mmol/L hydrogen peroxide at room temperature (25 °C) five times with five sensors prepared under the same conditions. Prior to detection, incubation of the sensors was carried out through which the following results for the five sensors were obtained: 13.24, 13.37, 13.17, 13.33, and 13.88 µA. Relative standard deviation (RSD) of 2.1% was obtained indicating good sensor-to-sensor reproducibility. The RSD obtained from separate determination of 5 × 10-8 mol/L OTC for each of the five sensors ranged from 2.6% to 3.8%. Good reproducibility of each sensor was indicated after elution and rebinding of the template. The long-term stability of the sensors is an important factor to consider. To ensure stability, the sensors were dipped in doubledistilled water at 4 °C after each use. No apparent decrease in response to 6.0 × 10-8 mol/L OTC was found after 2 weeks. Over the next week, the current response decreased by about 10%; after a month, it decreased by about 17%. Determination of OTC in Milk Samples. Milk samples were purchased from the market. Prior to determination, the pretreatment of precipitate proteins was performed by the method introduced by Jeon et al., which used the McIlvaine buffer solution precipitate method.31 Subsequently, the obtained solution was treated by freeze-centrifugation (8000 rpm/min, 4 °C, 15 min) to remove fattiness. Then, 2 mL of the supernatant fluid was added into 8 mL of water to obtain the test solution. After incubation, the MIP electrode was competed in the test solution for 12 min. Given that no OTC was detected in the samples by both the proposed MIP sensor and HPLC,30 OTC solutions of known concentrations were added into the samples before pretreatment (31) Jeon, M.; Kim, J.; Paeng, K. J.; Park, S. W.; Paeng, I. R. Microchem. J. 2008, 88, 26–31.
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Table 1. Sample Analysis Results
samples
found mol/L
added mol/L
total found mol/L (n ) 10)
RSD %
recovery %
1 2 3
not detected not detected not detected
4.00 × 10-8 6.00 × 10-8 8.00 × 10-8
4.06 × 10-8 5.91 × 10-8 7.86 × 10-8
1.54 2.52 2.49
101.5 98.5 98.2
of precipitate proteins. As shown in Table 1, recoveries of this sensor range from 98.2% to 101.5%, and the RSD was less than 3.0%. These are good indicators of recovery and practicability. Total assay time with the proposed procedure is not more than 40 min excluding the purification step. The major advantage of this assay is that it is fast, economical, and simple compared with other methods reported.29,30 CONCLUSIONS An MIP film electrochemical artificial immunosensor used to indirectly detect OTC has been constructed, and a novel determination method using this sensor has been developed. High sensitivity to OTC was obtained owing to the presence of the enzyme amplifier of HRP. Good selectivity was obtained due to the reorganization of molecularly imprinted cavities. The sensor can be easily fabricated at low cost and has shown relevant analytical performance. ACKNOWLEDGMENT The authors gratefully acknowledge the financial support from the National Science Foundation of China (No. 20665003), the Key Project of Science and Technology of the Ministry of Education of China (No. 207087), and the Natural Science Foundation of Guangxi Province (No. 0728214). NOTE ADDED AFTER ASAP PUBLICATION This paper was published on June 22, 2010 with an error in the label of the axes of Figure 6 and in the text describing this figure. The corrected version was published on July 14, 2010.
Received for review March 13, 2010. Accepted June 7, 2010. AC100667M
