Atrazine Sensor Based on Molecularly Imprinted Polymer-Modified

Anal. Chem. 2003, 75, 4882-4886

Atrazine Sensor Based on Molecularly Imprinted Polymer-Modified Gold Electrode Reo Shoji,† Toshifumi Takeuchi,‡ and Izumi Kubo*,†

Department of Bioengineering, Faculty of Engineering, Soka University, 1-236 Tangi-cho, Hachioji, Tokyo 192-8677, Japan, and Graduate School of Science and Technology, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe 657-8501, Japan

Molecularly imprinted polymers (MIP) have been elucidated to work as artificial receptors. In our present study, a MIP was applied as a molecular recognition element to a chemical sensor. We have constructed an atrazine sensor based on a MIP layer selective for atrazine and its electrochemical reduction on gold electrode. The atrazine sensor was fabricated by directly polymerizing the atrazine-imprinted polymer composed from methacrylic acid and ethylene glycol dimethacrylate onto the surface of a gold electrode. By introducing LiCl into the MIP, atrazine was reduced below -800 mV vs Ag/AgCl reference electrode, at pH 3. The cathodic current of atrazine depended on the concentration of atrazine at the range of 1-10 µM. The sensor exhibited a selective response to atrazine. A nonimprinted polymer-modified electrode did not show selective response to atrazine, thus implying that the imprinted polymer acts as recognition element of atrazine sensor. Biological recognition molecules such as enzymes, antibodies, and receptors have been studied and utilized for selective sensing. Artificial receptors such as molecularly imprinted polymers (MIPs) are currently under investigation in numerous applications.1-3 The MIPs are comparable to biological receptors in respect to specificity, circumventing, however, the problem of expensive and complicated biosynthesis and the instabilities often displayed by biological receptors or antibodies; making MIPs a promising tool for analytical chemistry.4 MIPs are being applied to various analytical methods. They have been mainly utilized as a substitute for antibodies because of their specific affinity. They have been applied to competitive antigen binding assays using radioactively labeled ligand.5 Analytes such as theophylline,6,7 diazepam,8 morphine,9 Leu-enkephalin,9 * To whom correspondence should be addressed: (e-mail) [email protected]; (fax) +81-426-91-9312. † Soka University. ‡ Kobe University. (1) Mosbach, K. Trends Biochem. Sci. 1994, 19, 9-14. (2) Shea, K. J. Trends Polym. Sci. 1994, 2, 166-173. (3) Piletsky, S. A.; Piletska, E. V.; Bossi, A.; Karim, K.; Lowe, P.; Turner, A. P. F. Biosens. Bioelectron. 2001, 16, 701-707. (4) Matsui, J.; Kato, T.; Takeuchi, T.; Suzuki, M.; Yokoyama, K.; Tamiya, E.; Karube, I. Anal. Chem. 1993, 65, 2223-2224. (5) Andersson, L. I. Anal. Chem. 1996, 68, 111-117. (6) Vlatakis, G.; Andersson, L. I.; Muller, R.; Mosbach, K. Nature 1993, 361, 645-647. (7) Yoshimi, Y.; Ohdaira, R.; Iiyama, C.; Sakai, K. Sens. Actuators, B 2001, 73, 49-53.

4882 Analytical Chemistry, Vol. 75, No. 18, September 15, 2003

and atrazine10 can be determined by such competitive binding assays. This method can also be applied to the determination of bioactive chemicals such as drugs, herbicides, and pesticides. Another application of MIPs is solid-phase extraction for affinity chromatography.10-12 In such applications, fine particles of MIPs were utilized as a solid phase to concentrate and separate chemicals from structurally analogous molecules. Herbicides such as atrazine or prometryn have been determined by this method. Recently MIPs have been utilized as a molecular recognition membrane or layer on chemical sensing systems. Proposed applications are the combination of MIPs and transducers such as quartz crystal microbalances,13 Surface plasmon resonance devices,14 field-effect devices,15 conductometry,16 or impedometric determination.17 These sensing systems are based on the characteristic alteration of MIP membrane or layer caused by binding of the imprinted molecule. When the bound analyte itself reacts on the surface of the solid support on which MIPs are prepared, the analyte can be detected by its reaction providing for a simple and rapid reaction system. In this study, we aimed to establish a simple and rapid analytical system based on MIPs. For that purpose, we propose an electrochemical sensor utilizing the amperometric determination method of bound analyte to the MIPs by electrochemical reaction. To establish such a sensing system, atrazine was chosen as model analyte, as atrazine-imprinted polymers have already been studied intensively in nonamperometric systems by Sergeyeva et al.,16 Luo et al.,18 and Matsui et al.12 These studies utilized MIPs prepared from methacrylic acid (MAA) and ethylene glycol dimethacrylate (EDMA) with good sensitivity compared to other triazines. (8) Senhoidt, M.; Mosbach, K.; Andersson, I. Anal. Lett. 1997, 30, 1809-1821. (9) Andersson, L. I.; Muller, R.; Vlatakis, G.; Mosbach, K. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 4788-4792. (10) Muldoon, M. T.; Stanker, L. H. J. Agric. Food Chem. 1995, 43, 1424-1427. (11) Matsui, J.; Doblhoff-Dier, O.; Takeuchi, T. Chem. Lett. 1995, 489. (12) Matsui, J.; Miyoshi, Y.; Dobihoff-Dier, O.; Takeuchi, T. Anal. Chem. 1995, 67, 4404-4408. (13) Kugimiya, A.; Yoneyama, H.; Takeuchi, T. Electroanalysis 2000,12, 13221326. (14) Kugimiya, A.; Takeuchi, T. Biosens. Bioelectron. 2001, 16, 1059-1062. (15) Hedborg, E.; Winquist, F.; Lundstrom, I.; Andersson, L. I.; Mosbach, K. Sens. Actuators, A 1993, 37-38, 796-799. (16) Sergeyeva, T. A.; Piletsky, S. A.; Brovko, A. A.; Slinchenko, E. A.; Sergeeva, L. M.; El’skaya, A. V. Anal. Chim. Acta 1999, 392, 105-111. (17) Delaney, T. P.; Mirsky, V. M.; Ulbricht, M.; Wolfbeis, O. S. Anal. Chim. Acta 2001, 435, 157-162. (18) Luo, C.; Liu, M.; Mo, Y.; Qu, J.; Feng, Y. Anal. Chim. Acta 2001, 428, 143-148. 10.1021/ac020795n CCC: $25.00

© 2003 American Chemical Society Published on Web 08/13/2003

Electrochemical reduction of atrazine has been reported by Pospisil et al.19 According to Pospisil, s-triazine herbicides such as atrazine can be reduced with a mercury electrode in aqueous solution despite poor aqueous solubility. This led us to the consideration that an electrode modified with MIPs for atrazine would indicate selective response to atrazine electrochemically. In this study, we describe an atrazine sensing system based on electrochemical reduction and a molecularly imprinted polymer layer on gold electrode as a recognition element. On the surface of the electrode modified with atrazine-imprinted polymer, atrazine binds to the imprinted site while other chemicals do not bind or bind to a lesser degree. The bound atrazine can then be reduced electrochemically at the electrode surface. In this paper, we describe the fabrication procedure of an atrazine sensor prepared from a gold electrode and atrazine MIPs and the electrochemical characteristics of the MIP-modified electrode. The efficiency of the MIPs as molecular recognition element is also discussed. EXPERIMENTAL SECTION Materials. Atrazine (2-chloro-4-(ethylamino)-6-(isopropylamino)1,3,5-triazine) was obtained from Kanto Chemicals (Tokyo Japan). simazine (2-chloro-4, 6-bis(ethylamino)-1,3,5-triazine), and MCC (mechyl-3, 4-dichlorocarbanilate) were purchased from Wako Pure Chemical Industry as standard chemicals for herbicide testing. EDMA, MAA, and dimethylformamide (DMF) were distilled prior to use in order to remove stabilizers. Other chemicals were laboratory grade and used without further purification. Preparation of MIP-Modified Electrode. The electrode used in this study was QA-A9-Au and purchased from Seiko EG&G (Chiba, Japan), originally used in commercial quartz crystal microbalance (QCM) sensors, and fabricated by deposition of gold on both sides of the quartz crystal. The shape of the electrode is flat and therefore suitable for direct polymerization of MIPs on its surface. The surface of the electrode was cleaned for 30-60 s by sputtering of ionized gas with use of an ion coater (IB-2, Eiko Engineering). Immediately after the cleaning procedure, the electrode was immersed in an ethanol/water (4:1, v/v) solution containing allyl mercaptan (53 µM) and 1-butanethiol (13 µM) for 2 h or more, to introduce vinyl groups onto the surface of the gold electrode (Figure 1a). It was dried with nitrogen gas. The template atrazine (12 mg) was dissolved in 500 µL of DMF and 15 µL of functional monomer MAA, A total of 250 µL of the cross-linking monomer EDMA and 5 mg of the initiator 2,2′azobisisobutyronitrile was added and mixed. The surface of the mixture was purged with nitrogen gas for 10 s. Immediately after the electrode was dried with nitrogen gas, it was placed on a glass plate. The reaction mixture was dropped onto the surface of the gold electrode and then a strip of cover glass was put over the droplet. The polymerization was initiated by UV light irradiation at 4 °C in a cold room (Figure 1b). These conditions were maintained for 2 h. The blank polymer-modified electrodes were prepared using the same reaction mixture without addition of the template. The blank polymer is not imprinted, and this electrode can be used as the nonimprinted polymer (NIP)modified control electrode. (19) Pospisil, L.; Trskova, R.; Fuoco, R.; Colombini, M. P. J. Electroanal. Chem. 1995, 395, 189-193.

Figure 1. Preparation procedure of MIP-modified electrode: (a) modification of a gold electrode surface with allyl mercaptan and 1-butanethiol, (b) polymerization by UV light irradiation, (c) removal of atrazine from the MIP, and (d) introduction of electrolyte.

The polymer-coated electrode was washed in methanol/acetic acid (7:3 v/v) for 2 h or more to remove the template (Figure 1c). To introduce electrolyte into the polymer layer, the polymercoated electrodes were immersed in 0.1 M supporting electrolyte such as LiCl dissolved in DMF prior to the electrochemical measurement (Figure 1d). The polymer-coated electrode was compared with or without supporting electrolyte. Apparatus. Electrochemical measurements were performed with a computer-controlled voltammetry analyzer (BAS: CV-50W, Tokyo, Japan) and a three-electrode electrochemical cell. The polymer-modified electrodes were used as working electrodes. Ag/AgCl (3 M NaCl) reference electrodes and platinum wire auxiliary electrodes were purchased from Bioanalytical Systems (BAS). Electrochemical measurements were performed with these three electrodes. All the electrochemical measurements were carried out in a glass vial containing 10 mL of deoxygenated solution (0.1 M KCl) at room temperature. Evaluation of Sensor Response. All the solutions used here were fully deaerated prior to the electrochemical reaction. The polymer-modified electrode was placed in the vial filled with 10 mL of 0.1 M KCl. After the atrazine solution was injected into the vial, the electrodes were incubated 10 min or more, which is enough for atrazine to diffuse into the polymer and bind. Prior to the electrochemical experiments study, we additionally evaluated the time necessary for uptake of atrazine from the solution with use of the MIP films prepared on a slide glass by UV-visible spectroscopy. These uptake studies showed that and 10-min incubation time were sufficient enough for uptake. Cyclic voltammetry was carried out to reduce atrazine. After the measurement, the polymer-modified electrode was washed with methanol/acetic acid to remove atrazine in the MIP. RESULTS AND DISCUSSION Electrochemical Reduction of Atrazine. In this study, we aimed to prepare a MIP-modified electrode. Gold electrodes were the appropriate candidates for the purpose, because modification of the gold electrode surface has been intensively studied utilizing Analytical Chemistry, Vol. 75, No. 18, September 15, 2003

4883

Table 1. Response to 10 µM Atrazine with Different Electrolytes electrolyte

current decrease (µA)

TEAClO4 LiCLO4 LiCl

4.24 -2.72 -18.42

Figure 2. Cyclic voltammograms in 0.1 M KCl at a scan rate of 100 mV/s for a bare gold electrode in the absence and in the presence of atrazine.

Au-S bonds.20-23 At first electrochemical reduction of atrazine was examined with the use of an gold electrode by cyclic voltammetry. Figure 2 shows the cathodic current of the electrode with or without atrazine. In the presence of atrazine, an increase of cathodic current was observed and this increase depended on the concentration of atrazine. Other studies have shown that atrazine in aqueous media is reduced electrochemically on mercury electrodes at potentials lower than 0.9 V in polarographic measurement mode.19 Our results indicate that atrazine is also electrochemically reduced on gold electrodes at almost same potential as on a mercury electrode (Figure 2). The small shoulder observed around -0.85 V in the experiment without atrazine is considered to be caused by proton reduction at pH 3.0, as this shoulder could be observed neither at pH 6.0 nor at pH 9.0 at this potential. Electrochemical Reduction of Atrazine with the MIPModified Electrode. At first the MIP-modified electrode, which was washed thoroughly in methanol/acetic acid after polymerization (Figure 1c), was immersed in 0.1 M KCl, and cathodic current was measured by cyclic voltammetry as in bare gold electrode. However, with this procedure, the MIP-modified electrode did not show any significant cathodic current to atrazine. Even after exposure of the MIP-modified electrode to atrazine for as long as 60 min, cathodic current increased only slightly. Very possiblly the MIP membrane is so hydrophobic that the electrolyte in the aqueous solution does diffuse into the membrane. To enable penetration of the mebrane, the electrolyte should be soluble in DMF, used for the preparation of MIPs. LiCl, lithium perchlorate, and tetraethylammonium perchlorate (TEAClO4) were examined as candidate electrolytes. To introduce these electrolytes into the MIP, the MIP-modified electrode was immersed in DMF containing each electrolyte (0.1 M) for 1 h. After the treatment, the electrode was immersed in 0.1 M KCl aqueous solution and cyclic voltammetric studies were performed. As shown in Table 1, the MIP containing TEAClO4 did not indicate electrochemical reduction of atrazine and LiClO4 showed slight reduction of atrazine. (20) Bardea, A.; Katz, E.; Buckmann, A. F.; Willner, I., J. Am. Chem. Soc. 1997, 119, 9114-9119 (21) Nakaminami, T.; Ito, S.; Kuwabata, S.; Yoneyama, H. Anal. Chem. 1999, 71, 4278-4283. (22) Kubo, I.; Nagai, IEICE Trans. Electron. 2000, E83-C, 1035-1039. (23) Watanebe, S.; Kubo, I. Electrochemistry 2002, 70, 258-263.

4884 Analytical Chemistry, Vol. 75, No. 18, September 15, 2003

Figure 3. Cyclic voltammograms in 0.1 M KCl (pH 3.0) at a scan rate of 100 mV/s for the MIP-modified electrode in the absence and in the presence of atrazine.

Among these electrolytes, the MIP-modified electrode containing LiCl showed electrochemical reduction as expected with the cathodic current of the MIP-modified electrode depending on the concentration of atrazine. This could be explained by the small pore size created during the polymerization with atrazine and porogen (DMF) not allowing TEAClO4 or LiClO4 to penetrate the structure. The molecular size of LiCl is the smallest among examined supporting electrolytes and disperses easily into the MIP. In further experiments, the MIP-modified electrode was immersed in 0.1 M LiCl/DMF as in Figure 1d. The electrochemical reduction of atrazine was performed in 0.1 M KCl. As shown in Figure 3, no cathodic peak could be detected in the absence of atrazine. A cathodic current was observed below -0.8 V in the presence of atrazine. The MIPmodified electrode prepared here was considered to be free from template, because the MIP-modified electrode with remaining template showed the reduction current of atrazine. The cathodic peak potential is lower for the MIP-modified electrode than for the bare gold electrode. Generally speaking, the reduction peak is shifted when there is a layer limiting free diffusion; this may be attributed to the limiting diffusion to the MIP layer. The current was measured as the difference in current from the current measured in the absence of atrazine. The current difference was dependent on the concentration of atrazine. As all the solution was fully deaerated in the electrochemical experiment, the reductive current normally caused by dissolved oxygen was negligible. Other hydrophilic substances are excluded as they do not easily diffuse into the hydrophobic MIP layer. Some hydrophobic and electroactive substances could cause interferences and are considered below (see Selectivity). Effect of pH. The effect of pH on the current decrease was examined in the range from pH 3.0 to 9.0. The pH of the sample solution was adjusted to the given value by HCl or by NaOH

Figure 4. Effect of pH on the reductive current of atrazine with the MIP-modified electrode. Current differences at -800 mV in 0.1 M KCl were plotted.

Figure 6. Comparison of electrochemical response of herbicides. Current difference at -800 mV in the presence and absence of each herbicide was plotted. Table 2. Comparison of the Selectivity of Atrazine-Imprinted Polymera

atrazine simazine prometryn ametryn triazineg

Figure 5. Typical calibration curve of atrazine. Current differences at -800 mV in 0.1 M KCl (pH 3.0) were plotted.

solution. The response to atrazine at pH 3.0 was 3 or 4 times as large as the response at pH 6 or 9 (Figure 4). Therefore, further experiments were carried out at pH 3.0. Pospisil et al. have reported the influence of pH on electrochemical reduction of atrazine, linking this effect to a protonation equilibrium preceding the electron-transfer reaction.1 The number of protons participating in the rate-determining step was determined to be one in the range pH 3 to pH 2.19

Our result is consistent with Pospisil’s. Calibration Curve. From these results, atrazine was determined at the optimum condition. The relation between current difference and the concentration of atrazine was examined. The current difference at -800 mV was plotted against the concentration of atrazine. Figure 5 shows a typical calibration curve. The current difference increased to an atrazine concentration up to 10 µM. The linear relation ship was observed between the concentration of atrazine and the current difference at the range examined. Each concentration was measured four times or more, and the five electrodes were prepared and compared. The slopes

capacity factorb

conductometryc

cross reactivity of TSMd acoustic sensor

100 78 30 32 1

100 16 16

100 55 17

18

14

amperometrye 100 28 58f 2.5

a Data were nomalized by the data of atrazine as 100. b Relative capacity factor was calculated from the retention time of MIP column; see ref 12. c Conductometric change with use of MIP, Selectivity was calculated from the data in ref 16. d Thickness-shear mode (TSM) acoustic sensor. Cross reactivity at 10 µM was listed. See ref 18. e Present method. Current decreases at -800 mV in 0.1 M KCl (pH 3.0) of herbicides (10 µM) were compared. f Since peometryn was instable at acidic pH, it was compared at pH 9.0. g Triazine is listed for its structural interest.

of the calibration curve of each electrode were in the range between -2.3 and -4.0 µA/µM. The difference of the slopes is most probably due to the diffusion limits caused by the difference of the MIP layer thickness. For the production of the prototype MIP electrodes, the thickness was not controlled and their thickness was in the range between 50 and 80 µm. Thicker MIP resulted in more limiting diffusion of atrazine. The difference in the slopes of calibration curves should be minimized by control of the thickness of the MIPs. Selectivity. The selectivity of the MIP-modified electrode to other herbicides was checked. Among various herbicides, simazine and MCC were tested, due to their electrochemical activity and structural similarity. The current decrease of each reagent at 10 µM was compared. The current decreases of the MIP-modified electrode to MCC and simazine was much smaller than that to atrazine (Figure 6). Selective recognition of atrazine-imprinted polymers composed from MAA and EDMA has been investigated by several groups (Table 2) and compared to this work. Matsui and co-workers evaluated the selectivity of MIP particles by retention time in chromatography.12 Sergeyeva et al. investigated a conductometric sensor using a MIP membrane.16 Luo et al. constructed a thickness-shear mode acoustic sensor based on a film containing MIP particles.18 All the MIPs investigated until now showed selectivity to atrazine compared to triazines such as simazine, prometryn, triazine, and ametryn. Among these trizines, Analytical Chemistry, Vol. 75, No. 18, September 15, 2003

4885

Figure 7. Comparison of the atrazine-imprinted polymer-modified electrode (MIP), the nonimprinted-modified electrode (NIP), and a bare gold electrode. Current differences at -800 mV in 0.1 M KCl (pH 3.0) containing 10 µM herbicide were compared.

prometryn and triazine showed 10-30% binding to MIPs compared to atrazine. On the other hand, simazine showed relatively higher binding as compared to other triazines. However, in this study, the response of simazine was 28% to atrazine although simazine is reduced electrochemically in acidic condition at higher level than trizazine.24 Non-MIP-Modified Electrode. To confirm the efficiency of molecular imprinting, the response of the bare gold electrode and NIP-modified electrode to atrazine, simazine, and MCC was examined. All of these are electroactive on bare gold electrodes, and the magnitude of response to these compounds was in the order of simazine, atrazine, and MCC. In the case of the NIPmodified electrodes, the responses became smaller than those of a bare gold electrode and the order of their response was same as those of the bare electrode. In this case, the NIP limited the diffusion of atrazine, simazine, and MCC. Thus, the NIP-modified electrode did not show selectivity for atrazine (Figure 7). In the case of MIP-modified electrodes, the responses other than atrazine were smallest among these electrodes; however, that of atrazine was almost the same as for the gold electrode. This result indicated that the molecular imprinting is effective to give selectivity to atrazine in electrochemical sensing. The atrazineimprinted polymer has a specific binding site to atrazine but not to simazine or to MCC. The shape of the site does not fit compounds other than atrazine. Repeatability. Repeatability was tested with the MIP-modified electrode. Atrazine was measured from 1 to 10 µM. One calibration curve was plotted, and its slope was -2.7 µA/µM. After the first electrochemical determination of atrazine, the electrode was immersed in methanol/acetic acid to remove atrazine and product (24) Higuera, M. J.; Montoya, M. R.; Galvin, R. M.; Mellado, J. M. R. J. Electroanal. Chem. 1999, 474, 174-181.

4886

Analytical Chemistry, Vol. 75, No. 18, September 15, 2003

left in the polymer. The electrode was then immersed in LiCl for 1 h prior to the next electrochemical measurement. The second response was -2.4 µA/µM. Repeatability was sufficient to determine atrazine. Five electrodes were tested. One of them was sufficiently stable for 4 days at four determinations/ day, but two electrodes were not sufficiently stable. One electrode could be used for 3 days. At that time, the MIP had partly or thoroughly peeled off from the gold surface. A thin layer of MIP still remained on the gold electrode as observed in the atomic force microscope (data are not shown here). Therefore, allyl mercaptan was considered to be effective to keep MIP on the electrode. The adhesiveness of MIPs to the electrode surfaces should be considered. The MIPs may deform when they are exposed to solutions of different polarities. This effect is very probably stronger for thick layers. The manufacture of thin polymer layers is now under investigation. Another consideration on the adhesiveness is the structure of the electrode. In this study, we used a gold electrode deposited on a quartz crystal, which is hydrophilic. Adhesiveness was improved by coating adhesive material such as polyimide around the gold electrode. Stability of the MIP was improved at least one week or more. CONCLUSION An artificial receptor for atrazine was prepared on a gold electrode by molecular imprinting. Electrochemical reduction of atrazine was facilitated by introduction of LiCl as electrolyte into the atrazine-imprinted polymer. The MIP-modified electrode was selective to atrazine among other triazines, and it is applicable to direct atrazine sensing. NIPs did not show selective response to atrazine. In the present study, MIPs proved to be effective as a molecular-recognition element of a chemical sensor based on amperometric determination of the recognized analyte. The preparation of molecular imprinted polymers is simple and inexpensive and applicable to various molecules with different structure. The combination of electrochemical detection and molecular imprinting technique will provide simple, specific, and inexpensive sensing systems. ACKNOWLEDGMENT This work is supported in part by a grant from Ministry of Education, Culture, Sports and Technology. The authors thank Dr. Otto Doblhoff-Dier for useful comments in the preparation of this article. Received for review December 31, 2002. Accepted June 6, 2003. AC020795N