Article pubs.acs.org/ac
Highly Sensitive Molecularly Imprinted Electrochemical Sensor Based on the Double Amplification by an Inorganic Prussian Blue Catalytic Polymer and the Enzymatic Effect of Glucose Oxidase Jianping Li,* Yuping Li, Yun Zhang, and Ge Wei College of Chemistry and Bioengineering, Guilin University of Technology, Guangxi 541004, China
ABSTRACT: A novel strategy to improve the sensitivity of molecularly imprinted polymer (MIP) sensors was proposed. An electrocatalytic Prussian blue (PB) film was electrochemically polymerized on an electrode surface to fabricate an MIP electrochemical sensor using oxytetracycline (OTC) as a template. The OTC determination relied on a competition reaction between OTC and glucose-oxidase-labeled OTC and the catalytic reduction of hydrogen peroxide by the modified PB film. Experimental results show that double amplification, which is based on the catalysis of inorganic PB films and the enzymatic effect of glucose oxidase, can remarkably increase the assay sensitivity. The main experimental conditions (including electrocatalysis of the PB film, pH effects, incubation and competition times, and anti-interference) were optimized. This novel MIP sensor can offer an femtomole detection limit for OTC. In addition, the feasibility of its practical applications has been demonstrated in the analysis of a series of real milk samples.
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he development of new techniques to fabricate molecularly imprinted sensors that have selective recognition properties toward template molecules has been receiving increasing interest.1 Electrochemical sensors have certain advantages such as low cost, simple preparation, easy miniaturization and automation, high stability, and so on.2 Molecularly imprinted electrochemical sensors (MIECS) combine the strong points of both molecularly imprinted polymers (MIPs) and electrochemical sensors.3 They hold an enormous potential for environmental monitoring and assessment,4,5 biological analysis,6−8 pharmaceutical analysis,9,10 residue detection of pesticides and veterinary drugs,11,12 and so on. In general, the working principle of MIECS is based on the detection of changes in various electrochemical signals such as conductance,13 potential,14 electric capacity (impedance),15 and current,16,17 which are caused by template molecules embedded in the imprinted membrane. Among them, the current-type MIECSs are frequently used because they are more sensitive than others. However, the previously reported sensitivity of such type of MIECS is dissatisfactory, mainly because of the limited imprinting cavities on the electrode surfaces that result in weak electrochemical response signals. Recently, some efforts © 2012 American Chemical Society
have been dedicated to improving the sensitivity of MIECS using conductive polymers;18−21 however, little noticeable improvements have been obtained. In our previous studies,11,22,23 several MIECSs with enhanced detection sensitivities have been developed on the basis of the combination of a molecular imprinting technique and enzyme amplifiers. Nevertheless, the increased intensity of the signals is still highly restricted by the limited number of imprinting cavities on the electrode surfaces that also act as channels for electron transport. In addition, the process generally requires electronic media. Prussian blue (PB) has been considered an “artificial enzyme peroxidase”24 because it shows high selectivity and catalytic activity for the reduction of hydrogen peroxide (H2O2). For instance, Karyakin et al.25 developed a PB-based enzyme biosensor. The lower applied potential can effectively avoid the effects of potential interfering substances and, in turn, improve the assay selectivity.26,27 More importantly, as an inorganic conductive film, a PB film can replace electronic media to Received: October 9, 2011 Accepted: January 13, 2012 Published: January 13, 2012 1888
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Scheme 1. Schematic Diagram of the Preparation and Competition Assay Principle of the Prussian-Blue (PB)-Based Oxytetracycline (OTC) Molecularly Imprinted Electrochemical Sensors (MIECS)
prepared from chemicals of analytical grade. Double-distilled water was used throughout the experiments. Preparation of MIP and Non-MIP (nMIP) Modified Sensors. Both MIP and nMIP were constructed via the electropolymerization of PB on the surfaces of Pt electrodes. Prior to the preparation of the modified electrodes, the surface of the Pt electrode was polished with successively finer-grade aqueous alumina slurries (1.0, 0.3, and 0.05 μm grain sizes) on a microcloth (chamois leather), followed by alternately washing with 2 mol/L KOH, concentrated H2SO4, ethyl alcohol, and deionized water. The deposition procedure for the PB-MIP film was performed via electrooxidation at a working potential of +0.36 V for 40 s in a solution (pH = 2) containing 0.2 mol/L polypyrrole, 3 mmol/L FeCl3, 3 mmol/L K3[Fe(CN)6], 1 mmol/L OTC, and 0.1 mol/L KCl. CVs (20 cycles) in the −0.1 V to +0.5 V potential range were then performed in the above solution at a scanning rate of 0.05 V/s. After carefully washing with deionized water, the modified electrode was transferred into a PBS solution (pH = 7) for electrochemical activation at a working potential of −0.05 V for 600 s. CVs (10 cycles) were then performed in the −0.05 V to +0.36 V potential range. The modified electrode was washed with deionized water and dried at room temperature (25 °C). An nMIP sensor was prepared under the same experimental conditions but without the addition of OTC. After electropolymerization, the MIP and nMIP sensors were washed with ethanol (50% in volume) for 15 min to remove the template molecules and adsorbates on the surface of the imprinted membranes. Blocking, Incubation, and Competition. After removing the template molecules, the PB-MIP sensor was placed in 5 mL of a 0.3 μmol/L OTC solution for 10 min to block all OTC cavities in the MIP membrane. The sensor was then further incubated in 1 mL of an 8 μg/mL GOD-OTC solution for 15 min to allow for the competitive adsorption of the specific recognition sites for OTC by GOD-OTC. The sensor was subsequently immersed in samples with different OTC concentrations for 15 min, to allow for the competitive binding of the OTC analytes to the specific sites that had been occupied by GOD-OTC. To investigate the selectivity of the PB-MIP sensor, the OTC samples containing different foreign
directly produce electrochemical signals to improve sensitivity. A series of PB-based chemical and biological sensors with high detection sensitivities and selectivities have been developed.28−32 However, no PB-based MIECS has been reported to date. In the current study, a PB-based MIECS that uses oxytetracycline (OTC) as a template molecule is originally proposed (Scheme 1). The molecularly imprinted PB polymers (PB-MIP) not only selectively recognize the template molecule OTC, but also catalyze the electroreduction of H2O2, which is generated by the enzymatic reaction of glucose oxidase (GOD). The detection sensitivity can be significantly improved via double amplification, which is based on the inorganic catalytic polymer characteristic of PB and the enzymatic effect of GOD.
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EXPERIMENTAL SECTION
Apparatus. The pH measurements were conducted using a PHS-3D digital pH meter (Shanghai Precision & Scientific Instrument Co. Ltd., Shanghai, China). X-ray diffraction (XRD) studies were performed using a PANalytical X'Pert PRO powder diffractometer with Cu Kα radiation (Netherlands). Electrochemical measurements, including cyclic voltammetry (CV) and differential pulse voltammetry (DPV), were performed using an electrochemical workstation (CHI 660C, Shanghai Chenhua Instrument Co. Ltd., Shanghai, China) connected to a personal computer. The classical three-electrode system consisted of an MIP-modified Pt electrode (2 mm diameter) as the working electrode, a potassium chloride (KCl)-saturated Ag/AgCl electrode as the reference electrode, and a Pt wire electrode as the auxiliary electrode. Reagents and Chemicals. Oxytetracycline (OTC), tetracycline (TC), chlortetracycline (CTC), doxycycline (DC), and minocycline (MINO) were purchased from Acros. Glucose-oxidase-labeled OTC (GOD-OTC) was obtained from Zhengzhou Biocell Biotechnology Co., Ltd. (China). H2O2, Dglucose, ferric chloride, KCl, potassium ferricyanide or K3[Fe(CN)6], and polypyrrole were purchased from Sinopharm Group Chemical Reagent Co., Ltd. (China). Phosphatebuffered saline (PBS, pH = 7) was prepared using 0.1 mol/L K2HPO4 and 0.1 mol/L KH2PO4. All reagents used were 1889
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substances (i.e., lactic acid, ascorbic acid, vitamin A, vitamin E, and calcium, magnesium, zinc, and iron ions) and the relevant analogues of OTC (i.e., TC, DC, MINO, and CTC) were assayed using the same method. Comparative experiments were then conducted according to the same process, except that a bare Pt electrode and an nMIP sensor were used instead of the MIP sensor. Electrochemical Measurements. Electrochemical measurements, including CV and DPV, were performed for characterization and OTC determination. The scanning potential ranges were set from −0.3 V to +0.5 V for CV and from +0.5 V to −0.3 V for DPV in PBS containing 0.1 mol/L KCl and 50 μmol/L D-glucose at room temperature (25 °C), respectively.
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Figure 2. Cyclic voltammograms (CVs) of the (a) bare and PB-MIPmodified Pt electrode in the (a) absence or (b and c) presence of H2O2 in 0.1 mol/L PBS (pH = 6, containing 0.1 mol/L KCl).
RESULTS AND DISCUSSION Electropolymerization of the PB-MIP Film. The electropolymerization of PB-MIP on the Pt electrode was conducted in a 0.1 mol/L KCl solution by CV at room temperature (25 °C). Each of CV curves exhibits a couple of well-defined, reversible redox with peaks at +215 and +177 mV. Moreover, the peak currents increase with an increasing number of scanning cycles (up to 13 cycles), at which steady current values are observed. XRD Studies. The formation of the PB-MIP film on the electrode surface was further confirmed via XRD. Figure 1
various modified electrodes were recorded in 0.1 mol/L PBS containing 0.1 mol/L KCl and 50 μmol/L D-glucose after they had been incubated in an OTC-GOD solution (Figure 3).
Figure 3. Differential pulse voltammograms (DPVs) of (a, a′) the bare Pt electrode, (b, b′) the non-MIP (nMIP) sensor, and (c, c′) the MIP sensor after incubation of glucose-oxidase-labeled OTC (GOD-OTC) (a, b, and c) and competition with 0.3 μmol/L OTC (a′, b′, and c′), respectively.
Significantly low reduction current responses of H2O2 can be observed for either the bare or the nMIP-modified electrode (curve a or b, respectively), suggesting that only a few GODOTC conjugates have been physically adsorbed on the bare and nMIP electrodes because of the lack of molecular imprinting cavities in OTC. Moreover, the current signal obtained on the nMIP-modified electrode (Figure 3b) is slightly stronger than that obtained on the bare electrode (Figure 3a), presumably because of the higher number of nonspecific adsorptions of GOD-OTC conjugates that occurred on the nMIP film. Meanwhile, a high reduction current response of H2O2 is clearly observed at the PB-MIP modified electrode (Figure 3c). This result is due to the selective recognition and capture of the OTC template by a number of molecular imprinting cavities in the PB-MIP film. These captured OTC molecules can be accordingly replaced by GOD-OTC during GOD-OTC incubation. Subsequently, the H2O2 generated from the catalysis of glucose by GOD is further reductively catalyzed by the PB film, producing an obvious DPV current signal. To further investigate the performance of the three types of electrodes in recognizing the OTC analytes in the sample, the current responses were recorded after the sensors were
Figure 1. X-ray diffractograms (XRDs) of the PB-MIP (a) before and (b) after template removal.
shows the XRD patterns of the PB-MIP before (curve a) and after (curve b) the removal of the template molecules. Before the template removal, the well-defined peaks at 17.6°, 24.8°, 28°, 30°, 35.2°, and 39.6° (Figure 1, curve a) indicate the formation of PB crystalline structures. Meanwhile, the peaks in the vicinity of 28° and 30° disappear after template removal, possibly because of the elution of OTC.33 Electrochemical Behavior of the PB Film. The CVs of 5 mmol/L H2O2 on both the bare Pt electrode and the PB-MIPmodified electrode are shown in Figure 2. No redox peaks can be observed on the bare Pt electrode with or without H2O2 (Figure 2a,b), whereas a couple of well-defined reversible redox peaks appear on the electropolymerized PB-MIP-modified electrode in the presence of H2O2 (Figure 2c). The results not only further confirm the formation of PB-MIP on the surface of the Pt electrode, but also demonstrate the excellent catalytic activity of the PB film in the reduction of H2O2. Molecular Recognition of OTC by the PB-MIP Film. In the comparative study, the DPVs of the bare electrode and the 1890
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successively incubated in the GOD-OTC and 0.3 μmol/L OTC solutions (Figure 3). Interestingly, an obvious decrease in the reduction current response of H2O2 on the MIP sensor is observed (Figure 3c′), possibly because the rebinding of the OTC molecules to the imprinting cavities in the PB-MIP film can significantly reduce the amount of GOD-OTC on the electrode surface. The H2O2 yield in the enzymatic reaction of GOD is accordingly reduced, thereby resulting in the evidently decreased DPV current response. On the other hand, negligible changes in the current responses are observed on the bare electrode and the nMIP sensor before and after their incubation in the OTC sample (Figure 3a′,b′). Effects of KCl and pH. During the sweep process, the electrochemical reduction of the PB film needs the cations to balance the charge and maintain neutrality. Therefore, the presence of K+ is highly necessary to obtain a reversible voltammetric response of the PB film. Throughout the experiments, 0.1 mol/L KCl was added to PBS as the supporting electrolyte. Both the enzyme and the catalytic activities of the PB film are highly affected by the pH values. The effects of pH on the current response of H2O2 catalyzed by PB-MIP were evaluated in the 5.5−8.0 pH range. The highest current response was obtained with PBS at pH 7.0. Normally, PB films are stable in acidic and neutral solutions, but unstable in an alkaline solution. Thus, the neutral PBS at pH 7.0 was used in the succeeding experiments. Optimization of the Incubation and Competition Times. In the current study, the incubation time is defined as the equilibrium time needed for the substitution of the OTC template in PB-MIP by GOD-OTC. The modified electrodes were incubated in an 8 μg/mL GOD-OTC solution at different incubation times. The results show that the response currents increase as the incubation time increases from 0 to 15 min (Figure 4a), after which the responses reach a plateau. Thus,
recommended as the optimized competition time in the subsequent experiments. Calibration Curve. A series of samples with varying OTC concentrations were investigated under the optimal experimental conditions to determine the analytical capabilities of the developed sensors. The resultant DPVs were then recorded and are shown in Figure 5. The reduction currents decrease
Figure 5. DPVs of the PB-MIP sensor after incubation in samples with varying OTC concentrations: (a−n) 0, 0.005, 0.01, 0.05, 0.08, 0.1, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and 1.0 μmol/L OTC, respectively. The inset shows the resulting calibration curve.
with increasing OTC concentration (Figure 5). The calibration curve consists of two linear sections (Figure 5, inset). The calibration curve between the reduction peak current (ΔI) and the OTC concentration (C) can be described by the equation ΔI (μA) = −111.12C − 0.4209 (r = 0.9972), at the 0 μmol/L to 0.1 μmol/L OTC concentration linear range, and by the equation ΔI (μA) = −48.412C − 6.977 (r = 0.9985), at the 0.1 μmol/L to 1.0 μmol/L range. The occurrence of the two linear ranges may be attributed to the synthetic effect of the enzymatic production of H2O2 and its subsequent diffusion to the rough PB polymer film for reduction. However, the exact mechanism remains unknown,34−36 and further investigation is needed to form a definite conclusion. A detection limit of 230 fmol/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 = 12), and K is the slope of the calibration curve. Reproducibility and Stability. The reproducibility of the sensor was investigated by assaying 0.5 μmol/L OTC in 0.1 mol/L PBS (pH = 7.0) containing 0.1 mol/L KCl and 50 μmol/L D-glucose five times, using five sensors prepared under the same conditions. A standard deviation (STD) of 2.17 was obtained, indicating a good sensor-to-sensor reproducibility. Moreover, the reproducibility of the PB-MIP sensor was examined using the 0.5 μmol/L OTC for five successive runs; the STD obtained was 1.14. The long-term stability of the sensor, which depends on both the stability of the PB film and the activity of the OTC-labeled GOD, is also an important factor for practical applications. To ensure stability, the sensor was stored dry in a refrigerator (4 °C) when not in use. The results show that, after 45 days, the response of the sensor to 0.5 μmol/L OTC decreased by only 3.98% compared with the initial response. Moreover, the PBMIP sensor can be regenerated for the subsequent OTC detection after incubating the used MIP-modified electrode in a GOD-OTC solution. In the current experiment, the developed OTC PB-MIP sensor can be reused approximately 100 times.
Figure 4. Effects of (a) the incubation time and (b) the competition time on the sensor responses.
the incubation time chosen for the subsequent experiments is 15 min. The competition time is the equilibrium time needed for the substitution of the GOD-OTC that entered the cavities of MIP by the OTC analyte in the samples. The current responses show no increase after a 15 min substitution reaction (Figure 4b). That is, the competition time of 15 min may be enough for the competition reactions between the OTC analyte and the GOD-OTC in MIP to complete. Thus, 15 min is 1891
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Selectivity of the MIP Sensor. To evaluate the selectivity of the PB-MIP sensor, its responses in the detection of OTC in the presence of foreign substances, namely, lactic acid, ascorbic acid, vitamin A, vitamin E, and calcium, magnesium, zinc, and iron ions, were investigated. The DPV current results show that 0.1 mol/L lactic acid, 50 μmol/L vitamin A or E, and 0.5 mmol/L calcium, magnesium, zinc, and iron ions produced negligible interference in the determination of 0.5 μmol/L OTC. Some analogues (TC, DC, MINO, and CTC) were also used as interferences to examine the selectivity. The changes in the current response obtained from 14 μmol/L TC, 15 μmol/L DC, 15 μmol/L MINO, and 22.5 μmol/L CTC decreased by 0.46, 0.34, 0.29, and 0.37 μA, respectively. These changes are negligible compared with that obtained from 0.5 μmol/L OTC, which exhibited a difference of 35.48 μA. Moreover, these foreign substances and analogues were analyzed using the bare electrode and the nMIP-modified one. The current responses obtained are similar to those obtained from the electrodes used for OTC detection (data not shown), suggesting that the PBMIP sensor developed in the current study possesses satisfactory analytical selectivity. Determination of OTC in Milk Samples. To further demonstrate its potential in practical applications, this novel PB-MIP sensor was applied to the detection of OTC in four real milk samples. The method used for the pretreatment of these samples was reported elsewhere.22,37 In brief, the McIlvaine buffer solution was used to precipitate milk protein. The supernate was then subjected to freeze-centrifugation (8000 rpm/min, 4 °C, 15 min) to remove the fats. Finally, 2 mL of the supernatant fluid was diluted to 10 mL to obtain the test solution. After incubation in a GOD-OTC solution, the PB-MIP sensor was immersed in each test solution containing a prefilled OTC concentration for 15 min. Recovery tests were then conducted via the standard addition method. The obtained recoveries range from 96.7% to 104.9%, and the RSD is less than 2.0%, thereby validating the good recovery and practicability of the developed sensor. Another set of milk samples was assayed using the developed MIP sensor. A regression equation, ΔI (μA) = −108.98C − 0.2916 (r = 0.9980), was obtained in the linear concentration range of 0 μmol/L to 0.1 μmol/L OTC. The detection limit for OTC was estimated as 300 fmol/L, which is slightly higher than that obtained from the pure OTC samples, because of the interferences present in the milk background to some degree. However, an improvement by at least 1 order of magnitude in the detection limit was obtained compared with those of highperformance liquid chromatography,38 ultraperformance liquid chromatography,39 and electrochemical technique with enzymatic amplification.22 Moreover, the linear dynamic range was widened by >10-fold, demonstrating the excellent detection capability of the designed sensing scheme.
detection of OTC. The developed PB-assisted MIECS showed excellent analytical performance (e.g., high sensitivity, good selectivity, and good reproducibility) and has great potential for further development as a general and promising alternative sensor for a wide range of applications such as environmental monitoring, biological analysis, and pharmaceutical analysis. Further research is now being undertaken by our group.
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AUTHOR INFORMATION
Corresponding Author
*Phone: +86 773 5895622. E-mail:
[email protected].
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ACKNOWLEDGMENTS The authors gratefully acknowledge the financial support received from the National Nature Science Foundation of China (21165007, 21105017) and the Innovation Project of Guangxi Graduate Education (2011105960703M19).
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REFERENCES
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CONCLUSIONS In the current study, an MIECS based on a PB film electropolymerized on an electrode surface has been successfully developed for the first time. In the proof-of-concept experiments, OTC was chosen as the template molecule. The detection of OTC relied on a competition reaction between OTC and GOD-labeled OTC coupled with the subsequent catalytic reduction of H2O2 (generated from the enzymatic reaction of GOD) by the PB film. The double amplification based on the inorganic catalytic PB film and the enzymatic effect of GOD has been verified to favor the highly sensitive 1892
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