Molecularly imprinted polymer (biomimetic) based potentiometric sensor for atrazine

Sensors and Actuators B 123 (2007) 65–70

Molecularly imprinted polymer (biomimetic) based potentiometric sensor for atrazine K. Prasad a , K.P. Prathish a , J. Mary Gladis a , G.R.K. Naidu b , T. Prasada Rao a,∗ a

b

Regional Research Laboratory (CSIR), Trivandrum 695 019, India Department of Environmental Sciences, S.V. University, Tirupati 517 502, India

Received 15 May 2006; received in revised form 4 July 2006; accepted 25 July 2006 Available online 30 August 2006

Abstract A biomimetic potentiometric sensor was developed by dispersing the atrazine imprinted polymer particles in di-n-octyl phthalate plasticizer and then embedding in polyvinyl chloride matrix. The sensor responds to atrazine in the pH range 2.5–3.0 over a wide working range of 0.0001–10 mM with a detection limit of 0.5 ␮M (0.1 ppm). This sensor has a reasonably faster response time of ∼2 min and high selectivity with respect to other important classes of pesticides and herbicides. The utility of the sensor was successfully tested for field monitoring of atrazine in ground waters by spiking known amounts of atrazine. © 2006 Elsevier B.V. All rights reserved. Keywords: Molecularly imprinted polymer particles; Atrazine; Potentiometric sensor; Ground waters

1. Introduction In the past few years, herbicides and pesticides have been widely employed in order to increase yields and improve the quality of agricultural production. These compounds are indispensable in the modern and profitable agricultural scenario but they are also a major source of contamination of the natural environment besides their merits. Hence, they are allowed at a certain level in the real environment for the safety, necessitating methodologies to make the regular protocol for control of these pollutants [1]. Atrazine is a typical candidate of great importance in the family of herbicides with better efficiency for control of weed and have been applied in a large scale with a great deal of consumption. Yet atrazine is suspected as one of the endocrine disrupters, can cause multiplicate types of cancers and is considered to have the abilities such as interrupting regular hormone function, cause birth defects, reproductive tumours and weight loss in mother and embryos [2,3]. In view of its persistence and solubility, atrazine and its metabolites enters natural waters and can cause serious problem for environmental safety and human health.

∗

Corresponding author. Tel.: +91 471 2515317; fax: +91 471 2491712. E-mail address: [email protected] (T.P. Rao).

0925-4005/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2006.07.022

The design and development of portable field devices such as sensors rather than laboratory-based instruments in monitoring atrazine at trace levels is prime challenge to analytical chemists. In view of the lack of selectivity of the conventional chemical sensors, molecularly imprinted polymer (MIP) recognition element based sensors are gaining wide acceptance now-a-days in the scientific community [4]. Consequently, MIP based conductometric [5,6], amperometric [7] and thickness-shear mode acoustic [8] sensors have been reported for the determination of atrazine. To our knowledge [9], MIP based potentiometric sensor has not been developed for atrazine in particular and pesticides, in general. In this paper, we describe a potentiometric sensor for atrazine present in spiked groundwaters by dispersing atrazine MIP particles in di-n-octyl phthalate (DOP) and embedding in polyvinylchloride (PVC) matrix and forming a membrane. 2. Experimental 2.1. Reagents and materials Atrazine, simazine, 2,4-D, 2,4,5-T, phorate, parathion and dichlorovos were obtained from SUPELCO, USA. Stock standard solutions (0.01 M) of atrazine were prepared by dissolving 0.2157 g of atrazine in 1.0 ml of concentrated hydrochloric acid

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adjusted the pH to 2.7 ± 0.2 with dilute NaOH after the addition of 10 ml of 1 M monochloroacetic acid buffer and made upto 100 ml with deionized water. Methacrylic acid (MAA), ethylene glycol dimethacrylate (EGDMA), 2,2 -azobisisobutyronitrle (AIBN), di-n-octyl phthallate (DOP), 2-nitrophenyloctyl ether (NPOE), bis(2-ethylhexyl) sebacate (BEHS), tris(2-ethylhexyl) phosphate (TEHP), sodium tetraphenylborate (NaTPB), oleic acid (OA) and high molecular mass poly(vinyl chloride) (PVC) were obtained from Aldrich (Milwauke, USA). The solutions of 1.0 × 10−2 to 1.0 × 10−8 M were prepared by aqueous dilution of a definite volume from the stock standard solution. All other chemicals were of analytical grade reagents. Deionized water was used throughout. 2.2. Preparation of molecularly imprinted (MIP) or non-imprinted (NIP) polymer particles The synthesis of atrazine-imprinted polymer was similar to the one reported by Turiel et al. [10] for propazine. Briefly, atrazine (1 mmol) and methacrylic acid (4 mmol) were taken in 50 ml round bottom flask and the mixture was left in contact for 5 min for pre-arrangement. Subsequently, EGDMA (20 mmol), AIBN (2 mmol) and 5 ml of toluene were added. The mixture was purged with N2 for 5 min and the flask was sealed under this atmosphere. It was then placed in an oil bath at 80 ◦ C to start the polymerization process. After 12 h, the obtained polymers were ground and sieved, and the particles with sizes between 50 and 105 ␮m were collected. Atrazine and unpolymerized monomer were removed by soxhlet extraction with 100 ml of acetic acid for 1 h and 100 ml of chloroform for 2 h. Then, the particles were suspended in acetone and settled for 4 h. The sedimented particles were discarded and those not sedimented were collected by centrifugation. The particles collected were suspended in acetone again and settled for 4 h, followed by centrifugation. This procedure was repeated for four times. The resulting MIP particles were dried to constant weight under vacuum at 60 ◦ C and were used in following experiments [8]. NIP particles were similarly prepared without the addition of atrazine. 2.3. Fabrication of the sensor The PVC membrane sensors were fabricated by following the general procedure. Ninety milligrams of PVC and 25 mg of NaTPB (or OA) were dissolved in 2.5 ml of tetrahydrofuran (THF). Forty-five milligrams of MIP or NIP particles were dispersed in 0.2 ml of DOP (NPOE/BEHS/TEHP) and were added to the above solution, homogenized and then poured in a Teflon mould of 21 mm of internal diameter. The THF was allowed to evaporate at room temperature. PVC based polymer membranes were obtained with thickness of ∼0.45 mm. A blank membrane was also prepared in a similar manner, maintaining the same composition without MIP or NIP particles. The membranes were glued to one end of a pyrex glass tube with Araldite. The tube was then filled with 10−3 M of atrazine internal filling solution. The sensor was stored in air when not in use.

2.4. Analytical procedure The sensor was conditioned in 25 ml of 0.1 M monochloroacetic acid buffer (pH 2.7) for 0.5 h. The pH of the test solution (50 ml) was maintained at 2.7 ± 0.2 after the addition of 5 ml of 1.0 M monochloroacetic acid buffer. The potentials of the test solution were measured with the following cell assembly for different concentrations of atrazine in the range 1.0 × 10−8 to 1 × 10−2 M. The response of the sensor was examined by measuring the electromotive force (EMF) of the following electrochemical cell: Ag|AgCl|1.0 × 10−3 M atrazine|PVC membrane|sample solution|KCl (saturated)|HgCl2 |Hg. The EMF was plotted as a function of the logarithm of atrazine concentration. 2.5. Analysis of ground water samples Ground water samples spiked with known amounts of atrazine were analysed using the above-fabricated MIP based atrazine potentiometric sensor by following the analytical procedure mentioned above. 3. Results and discussion Molecularly imprinted polymer particles prepared with dysprosium(III) ion template have been dispersed in NPOE and embedded in PVC selectively recognizes dysprosium(III) ion over several other alkali, alkaline earth and transition metal ions in the concentration range 8 × 10−6 to 1.0 × 10−1 M with a detection limit of 2 × 10−6 M [11]. In this paper, the suitability of molecularly imprinted polymer particles dispersed in plasticizer and embedded in PVC to recognize atrazine has been examined. 3.1. Effect of membrane composition Literature reports on conventional potentiometric sensors for inorganics depends on various features of membrane such as the properties of the plasticizer, nature and amount of ion recognizing material used [11,12]. Thus, different aspects of the membrane preparation using atrazine MIP particles were optimized on similar lines. 3.1.1. Nature of plasticizer Addition of appropriate plasticizer leads to optimum physical properties and ensures high mobility of the atrazinium ions in the membrane. These solvent mediators strongly influence the working concentration range of potentiometric sensors. As it is well known that the plasticizers improve the electrochemical properties of conventional potentiometric sensors, the effect of different plasticizers on the performance of MIP based sensors was first investigated. Fig. 1 shows the potential response obtained for MIP based sensors with different plasticizers DOP, NPOE, BEHS and TEHP. From the figure, it is seen that the membrane with DOP and BEHS offered a linear response of 56.0 ± 0.1 mV over the range 1.0 × 10−4 to 1 × 10−2 M. On the other hand, there is no linear response range in case of

K. Prasad et al. / Sensors and Actuators B 123 (2007) 65–70

Fig. 1. Potential response of MIP based atrazine potentiometric sensor fabricated with different plasticizers.

NPOE and TEHP. Furthermore, the sensor with DOP provides better response down to 1.0 × 10−7 M with detection limit of 5 × 10−7 M or 0.1 ppm compared to BEHS (as there is no response to atrazine concentration below 10−5 M). This observation of better performance of DOP plasticized atrazine sensors is analogous to the report by Ganjali et al. [13] for lanthanum ion. It was noticed that MIP based membranes were found to be brittle in the absence of plasticizer and sensor performance could not be checked. 3.1.2. Effect of addition of lipophilic salt/ionic additives There are several reports on the effect of addition of lipophilic salts or ionic additives upon the characteristics of conventional potentiometric sensors [14] as they reduce the anionic interference and lower the electrical resistance of the membranes. The effect of addition of OA or NaTPB or none on the performance of MIP based sensor for atrazine was examined (Table 1). The addition of NaTPB to MIP based membrane responds to atrazine in the range 1.0 × 10−7 to 1.0 × 10−2 M in comparison to 1.0 × 10−5 to 1.0 × 10−2 M (OA) and a 1.0 × 10−6 to 1.0 × 10−2 (none). However, the response in the latter case is non-linear presumably due to the anion interference. These twin observations of better working concentration range and uniform slope in the case of NaTPB added membrane were in tune with the reports by Singh and Saxena [14] and Prasad et al. [11].

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Fig. 2. Effect of pH on the response of MIP based atrazine potentiometric sensor.

3.1.3. Effect of MIP particles to PVC ratio The ratio of PVC to MIP particles was found to play a key role in the sensor performance since the weight of MIP particles determines the number of binding sites available for selective rebinding of atrazine. The results in Table 1 show that the membrane having the weight of PVC to MIP particles in the ratio 1:0.5 gives the best performance. In the case of membranes with 1:0.25 ratio, the total number of binding sites available for rebinding of atrazine are relatively lower for the membrane to respond effectively. On the other hand, during the preparation of membranes with 1:1 ratio, the MIP particles are dispersed non-uniformly resulting in poor performance. 3.2. Effect of pH of test solution The effect of pH of test solution on the performance of MIP based atrazine sensor was studied by varying the pH in the range 1.0–3.0. The results obtained are shown in Fig. 2 from which it is clear that the optimum pH for better and identical response characteristics is 2.5–3.0. Hence, the pH of the test solution was adjusted to 2.7 ± 0.2 after the addition of 5 ml of 1.0 M monochloroacetic acid buffer. 3.3. Dynamic response time Dynamic response is yet another factor that measures the sensing ability of the sensor. The response time was recorded

Table 1 Optimization of membrane ingredients during fabrication of MIP based atrazine potentiometric sensor Weight of PVC (g)

0.09 0.09 0.09 0.09 0.09

DOP (ml)

0.2 0.2 0.2 0.2 0.2

Weight of lipophilic salt (g) OA

NaTPB

– – – 0.025 –

0.025 0.025 0.025 – –

Atrazine MIP particles (g)

Working concentration range (M)

0.045 0.023 0.090 0.045 0.045

1.0 × 10−7 1.0 × 10−7 1.0 × 10−5 1.0 × 10−5 1.0 × 10−6

to 1.0 × 10−2 to 1.0 × 10−2 to 1.0 × 10−2 to 1.0 × 10−2 to 1.0 × 10−2

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Fig. 3. Dynamic response of MIP based atrazine potentiometric sensor for stepwise concentration change of atrazine (A) 1.0 × 10−6 M, (B) 1.0 × 10−5 M, (C)1.0 × 10−4 M, (D) 1.0 × 10−3 M and (E) 1.0 × 10−2 M.

by changing the atrazine concentration in test solution over a concentration range of 1.0 × 10−6 to 1.0 × 10−2 M (see Fig. 3). The actual potential versus time traces for MIP particles based sensor reaches equilibrium response in a time of about 2 min. To evaluate the reversibility of atrazine sensor, a similar procedure in the opposite direction was adopted. The measurements performed in the sequence of high to low concentration (from 1.0 × 10−2 to 1.0 × 10−6 M) indicate that the MIP based sensor was reversible (see Fig. 3) analogous to conventional chemical sensors. 3.4. Sensitivity and detection limit The potential responses of the MIP, non-imprinted and blank membrane sensors fabricated under the optimal conditions arrived above was studied and the results obtained are shown in Fig. 4. As seen from the figure, the plot obtained for

Fig. 5. Potentiometric response signal of the MIP or NIP based atrazine potentiometric sensor to atrazine and other pesticides/herbicides for a decade change of concentration from 1.0 × 10−6 to 1.0 × 10−5 M. (1 → atrazine, 2 → parathion, 3 → 2,4-D, 4 → phorate, 5 → dichlorovos, 6 → 2,4,5-T, 7 → simazine).

the MIP based sensor offers a working range of 1.0 × 10−7 to 1.0 × 10−2 M with a linear response of 56.0 ± 0.1 mV/decade in the range 1.0 × 10−4 to 1.0 × 10−2 M. The limit of detection was 5 × 10−7 M or 0.1 ppm. In contrast, the non-imprinted and blank membranes gave Nernstian response only for 1 decade concentration change. i.e. 1.0 × 10−3 to 1.0 × 10−2 M. Better response characteristics of MIP based sensor over non-imprinted polymer membrane in all the concentrations is attributed to the “Imprinting Effect”. 3.5. Sensor selectivity Several common pesticides and herbicides including simazine were tested using the MIP and NIP based atrazine sensors in order to determine the degree of interference from each. The potentiometric response results from the exposure to interferences individually were recorded and the results obtained are shown in Fig. 5. The chemicals that are most likely to interfere gave no false positive readings, indicating that the both MIP and NIP sensors have good selectivity for atrazine excepting simazine. The interference from simazine was reported by Luo et al. [8] who described MIP based thickness-shear mode acoustic sensor for atrazine. Quite interestingly, atrazine sensor based on MIP modified gold electrode senses simazine better than atrazine [7]. These findings can be explained by the similar size and shape of these two triazine herbicides. On the other hand, other compounds that do not interfere belonging to organophosphorus, phenoxy acetic acid pesticides or herbicides respectively. However, the higher E value for atrazine with MIP over NIP sensor and its lower value for MIP compared to NIP sensor for other interferents clearly indicate the “Imprinting Effect”. 3.6. Stability and reusability

Fig. 4. Potential response of atrazine imprinted, non-imprinted and blank membrane potentiometric sensors with respect to atrazine concentration.

The important criteria required for any sensing device in addition to sensitivity and selectivity is stability and reusability. The above developed MIP based atrazine sensor was found to be stable (deviation less than 1.0 mV for 5 × 10−6 M of atrazine)

K. Prasad et al. / Sensors and Actuators B 123 (2007) 65–70 Table 2 Analysis of ground water (45 ml of sample + 5 ml of 1 M monochloroacetic acid) Sample

Ground water

a

Concentration of atrazine (␮g/ml) Added

Founda

– 0.10 0.20 1.0 2.0

– 0.10 ± 0.01 0.19 ± 0.01 1.00 ± 0.05 1.95 ± 0.05

Recovery (%)

– 100 95 100 97.5

Average of three determinations.

for 3 months and can be reused for more than 30 times without any loss in sensing ability. Furthermore, the sensor does not require any conditioning, i.e. no need for soaking in conditioning solutions. 3.7. Analytical application It is clear from the selectivity studies that several pesticides and herbicides which are likely to be present in agricultural waters do not have any deleterious effect on atrazine sensor performance. So, it was decided to analyse the ground water samples by spiking known amounts of atrazine. The results obtained are shown in Table 2 from which it is clear that the MIP based atrazine sensor can reliably be used for monitoring the natural waters, which, if found, can alert the authorities for appropriate control measures. 4. Conclusions In conclusion, this report provides a new strategy to construct a potentiometric biomimetic sensor for direct, rapid and highly selective detection of atrazine. The strategy involves the dispersion of atrazine imprinted MIP particles in di-n-octylphthalate and embedded in polyvinylchloride matrix and cast as membrane after dissolving the latter in tetrahydrofuran. The stability, reusability and dynamic response time are analogous to conventional chemical sensors. The highlights of the developed atrazine sensor is the high degree of selectivity and its portable nature enabling field studies. Acknowledgements One of us (J.M.G.) is grateful to CSIR, New Delhi for the award of Research Associateship. We (T.P.R. and K.P.) are thankful to STED, Government of Kerala for sponsoring the project on development of MIP based sensors for pesticides. References [1] Q. Zhou, W. Wang, J. Xiao, J. Wang, G. Liu, Q. Shi, G. Guo, Comparison of the enrichment efficiency of multiwalled carbon nanotubes, C18 silica and activated carbon as the adsorbents for solid phase extraction of atrazine and simazine in water samples, Microchim. Acta 152 (2006) 215–224. [2] T. Gebel, S. Kevekordes, K. Pav, R. Edenharder, H. Dunkelberg, In vivo genotoxicity of selected herbicides in the mouse bone-marrow micronucleus test, Arch. Toxicol. 71 (1997) 193–197.

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[3] J. Kniewald, M. Jakominic, A. Tomljenovic, B. Simic, P. Romac, P. Vranesic, Z. Kniewald, Disorders of male rat reproductive tract under the influence of atrazine, J. Appl. Toxicol. 20 (2000) 61–68. [4] C. Alexander, H.S. Andersson, L.L. Andersson, R.J. Ansell, N. Kirsch, I.A. Nicholls, J. O’Mahony, M.J. Whitcombe, Molecular imprinting science and technology: a survey of the literature for the years upto and including 2003, J. Mol. Recognit. 19 (2006) 106–180. [5] S.A. Piletsky, E.V. Piletskaya, A.V. Elgersma, K. Yano, I. Karube, Yu.P. Parhometz, A.V. El’skaya, Atrazine sensing by molecularly imprinted membranes, Biosens. Bioelectron. 10 (1995) 959–964. [6] T.A. Sergeyeva, S.A. Piletsky, T.L. Panasyuk, A.V. El’skaya, A.A. Brovko, E.A. Slinchenko, L.M. Sergeeva, Conductimetric sensor for atrazine detection based on molecularly imprinted polymer membranes, Analyst 124 (1999) 331–334. [7] R. Shoji, T. Takeuchi, I. Kubo, Atrazine sensor based on molecularly imprinted polymer-modified gold electrode, Anal. Chem. 75 (2003) 4882–4886. [8] Ch. Luo, M. Liu, Y. Mo, J. Qu, Y. Feng, Thickness shear mode acoustic sensor for atrazine using molecularly imprinted polymer as recognition element, Anal. Chim. Acta 428 (2001) 143–148. [9] T. Prasada Rao, K. Prasad, J.M. Gladis, Biomimetric sensors for toxic pesticides and inorganics based on optoelectronic/electrochemical transducers—an overview. Talanta (communicated). [10] E. Turiel, A. Martin-Esteban, P. Fernandez, C. Perez-Conde, C. Camara, Molecular recognition in a propazine-imprinted polymer and its application to the determination of triazines in environmental samples, Anal. Chem. 73 (2001) 5133–5141. [11] K. Prasad, R. Kala, T. Prasada Rao, G.R.K. Naidu, Ion imprinted polymer based ion-selective electrode for the trace determination of dysprosium(III) ions, Anal. Chim. Acta 506 (2006) 69–74. [12] M. Shamsipur, M. Yousefi, M. Hosseini, M.R. Ganjali, Lanthanum(III) PVC membrane electrodes based on 1,3,5-trithiacyclohexane, Anal. Chem. 74 (2002) 5538–5543. [13] M.R. Ganjali, A. Daftari, M. Rezapour, T. Puorsaberi, S. Haghgoo, Gliclazide as novel carrier in construction of PVC-based La(III)-selective membrane sensor, Talanta 59 (2003) 613–619. [14] A.K. Singh, P. Saxena, A highly selective thallium(I) electrode based on a thia substituted macrocyclic ionophore, Talanta 66 (2005) 993–998.

Biographies Mr. K. Prasad received MSc degree in environmental sciences at the Sri Venkateshwara University, Tirupati in the year 2003. His research interests are in the field of solid phase extraction, coprecipitation, chemical sensors, imprinted polymers, organic and inorganic pollutants. Mr. K.P. Prathish is a PhD scholar in the Inorganic Materials Group, Regional Research Laboratory, CSIR, Trivandrum. He has received his MSc in analytical chemistry from Kerala University in the year 2004. His research interests are in the field of sensors, imprinted polymers, chemical warfare agents, pesticides. Dr. (Mrs.) J. Mary Gladis has received her MSc degree in 1998 from M.S. University, Thirunelveli and joined as research scholar in Regional Research Laboratory (CSIR) after clearing CSIR, UGC-NET examination conducted by CSIR, New Delhi. She was awarded PhD degree in chemistry in the year 2004 from University of Kerala, Trivandrum. She was conferred with “Young Scientist Award” by Kerala State Council for Science, Technology & Environment during its 16th Kerala Science Congress in the year 2004. Presently, she is working as CSIR Research Associate in RRL, Trivandrum. She has to her credit one US and 2 Indian patents, 8 reviews and 20 papers in international refereed SCI journals. Prof. G.R.K. Naidu has 30 years of teaching and research experience. His research interests include trace metal analysis in biological and environmental samples, solid phase extraction, liquid–liquid extraction, imprinted polymer based sensors for toxic inorganics and pesticides. He has published 75 papers in reputed journals and guided 9 students for PhD and 15 students for Mphil degree. He is a member of many national and international scientific bodies. He visited Germany, Japan, Turkey, Belgium and UK as visiting scientist. He

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has collaborated extensively with reputed national and international scientific institutes. He is currently heading the Department of Environmental Sciences, SV. University, Tirupati. Dr. T. Prasada Rao received his PhD degree in chemistry in 1981 from Indian Institute of Technology, Chennai, after his postgraduation in analytical chemistry from Andhra University. He worked as scientist in different CSIR laboratories of India such as IICT, Hyderabad; CECRI, Karaikudi, and RRL, Trivandrum and currently is Deputy Director and scientist-in-charge, Inorganic Materials Group

of RRL. Recipient of Prof. T.L. Ramachar award and Andhra University Medal in 1987 and 1994, respectively. Has to his credit 1 US patent, 6 Indian patents and published 175 research papers including 23 review articles in SCI journals. Authored one chapter each in three books and contributed an article to Encyclopedia of Analytical Science (2nd edition). Eight students were awarded PhD degrees under his guidance. His current research interests are solid phase extraction, flow injection analysis, molecular/ion imprinted polymers and biomimetic sensors.