A new molecularly imprinted polymer (MIP)-based electrochemical sensor for monitoring 2,4,6-trinitrotoluene (TNT) in natural waters and soil samples

Biosensors and Bioelectronics 25 (2010) 1166–1172

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A new molecularly imprinted polymer (MIP)-based electrochemical sensor for monitoring 2,4,6-trinitrotoluene (TNT) in natural waters and soil samples Taher Alizadeh a,∗ , Mashaalah Zare b , Mohamad Reza Ganjali b,c , Parviz Norouzi b,c , Babak Tavana b a b c

Department of Applied Chemistry, Faculty of Science, University of Mohaghegh Ardabili, Ardabil, Iran Center of Excellence in Electrochemistry, Faculty of Chemistry, University of Tehran, Tehran, Iran Medical Nanotechnology Research Centre, Tehran University of Medical Sciences, Tehran, Iran

a r t i c l e

i n f o

Article history: Received 14 August 2009 Received in revised form 29 September 2009 Accepted 5 October 2009 Available online 12 October 2009 Keywords: 2,4,6-Trinitrotoluene Molecularly imprinted polymer Voltammetric sensor Carbon paste

a b s t r a c t A high selective voltammetric sensor for 2,4,6-trinitrotoluene (TNT) was introduced. TNT selective MIP and non-imprinted polymer (NIP) were synthesized and then used for carbon paste (CP) electrode preparation. The MIP, incorporated in the carbon paste electrode, functioned as selectively recognition element and pre-concentrator agent for TNT determination. The prepared electrode was used for TNT measurement by the three steps procedure, including analyte extraction in the electrode, electrode washing and electrochemical measurement of TNT. The MIP-CP electrode showed very high recognition ability in comparison to NIP-CP. It was shown that electrode washing after TNT extraction led to enhanced selectivity. The response of square wave voltammetry for TNT determination by proposed electrode was higher than that of differential pulse voltammetry. Some parameters affecting sensor response were optimized and then a calibration curve plotted. A dynamic linear range of 5 × 10−9 to 1 × 10−6 mol l−1 was obtained. The detection limit of the sensor was calculated equal to 1.5 × 10−9 mol l−1 . This sensor was used successfully for TNT determination in different water and soil samples. © 2009 Elsevier B.V. All rights reserved.

1. Introduction Due to health, ecological and security risks caused by long- and short-term exposure to explosive compounds, there is considerable interest in their measurements in environmental samples. Extensive efforts have been devoted to the development of innovative and effective sensors, capable of monitoring explosives rapidly in the low concentration and complex matrixes. Electrochemical sensors are among the widely used devices for TNT determination because of inherent sensitivity of these methods (Saravanan et al., 2006; Zimmermann and Broekaert, 2005; Agu et al., 2005; Wang and Thongngamdee, 2003). In order to enhance the sensitivity and also selectivity, the use of chemically modified electrodes is a common option (Wang et al., 2004; Hrapovic et al., 2006; Wang and Pumera, 2006; Shi et al., 2007). However, the moderate selectivity of these sensors can be considered as a main problem in this case. The use of electrochemical biosensors is the proper way to overcome the moderate selectivity problem of the electrochemical sensors based on the chemically modified electrodes (Naal et al., 2002). Immunosensor is another class of biosensors and it involves the use of antibodies as biosensing element. Reaction takes place between a target analyte and a specific antibody. These

∗ Corresponding author. E-mail address: [email protected] (T. Alizadeh). 0956-5663/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2009.10.003

immunosensors can be used for the detection of explosive compounds. For instance, the use of a membrane-based continuous flow displacement immunoassay for detection of nanomolar quantities of explosives has been reported (Rabbany et al., 1998). Although biological receptors have specific molecular affinity, have been widely used in diagnostic bioassays and chemo/biosensors, they are often produced via complex protocols with a high cost, and require specific handling conditions because of their poor stability. Also, the natural receptors for many detected analytes do not exist (Whitcombe et al., 2000; Wulff, 2002; Haupt and Mosbach, 2000; Ye and Haupt, 2004). Thus, there has been a strong driving force in synthesizing artificial recognition receptors. Molecular imprinting is one of the most efficient strategies that offers a synthetic route to artificial recognition systems by a template polymerization technique (Mosbach, 2006; Tao et al., 2006; Sun et al., 2004; Piletskaya et al., 2005; Huang et al., 2004; Hall et al., 2006). The synthesis technique is simple and cheap, and the resultant MIP materials exhibit; high selectivity, excellent mechanical strength, durability to heat, acid and base conditions and better engineering possibility than biological counterparts (Ye and Haupt, 2004). Moreover, the introduction of synthetic design into molecular imprinting strategy can even make a host element suitable for the analyte, for which the natural receptor does not exist. These characteristics allow MIP materials as recognition elements to be used in a wide range of fields (Andersson et al., 1990; Spivak, 2005).

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Although the main applications continue to be in selective separation, MIP-based sensors for the detection of active molecules, pharmaceuticals and environmental pollutants, are perhaps the most challenging and have attracted considerable interest in recent years (Piletsky and Turner, 2002; Holthoff and Bright, 2007a,b; Stephenson and Shimizu, 2007). The sensors with MIP recognition element can identify and quantify the target species by converting the analyte-MIP binding event into a physically readable signal. Some chemical sensors, designed for TNT determination based on the MIP technology, can be found in the literatures. A chemical sensor, with capability to detect TNT at ppb quantities, has been developed to detect 2,4,6-trinitrotoluene utilizing planar integrated optical waveguide (IOW) attenuated total reflection spectrometry (Walker et al., 2007). In that work submicron thick films of organically modified sol–gel polymers, containing imprinted selective sites for TNT molecules, were deposited on the waveguide surface as the sensing layer. Binding of TNT and subsequent conversion to the anion, results in the attenuation of light, propagating through the waveguide and creating a spectrophotometric sensing device. In the other work TNT was measured by the quartz crystal microbalances (QCM) sensor coated with thin layer of MIP containing TNT selective sites (Bunte et al., 2007). Recently, another electrochemical sensor for the detection of TNT with enhanced sensitivities by imprinting of structure-like picric acid as substituting templates in the composite film of Au nanoparticles and conductive polymers has been reported. The ppt level detection limit and high selectivity was reported for this sensor (Riskin et al., 2008). These works are interesting particularly with respect to the reported low detection limit and high selectivity. However, complex instrumentation requirement and complicated sensor preparation protocol together with high cost of the methods are the main disadvantages of these works. In this work, a new, cheap and simple method was applied for design and preparation of the high selective and sensitive electrochemical sensor for TNT determination at sub-ppb quantities (DL ∼ 0.34 ppb). The MIP having recognition sites for TNT was used as a recognition element, in the carbon paste electrode. This biomimetic modifier functioned as selectivity increasing and preconcentrator agent for TNT determination. The prepared electrode was used for TNT determination by the three steps procedure, including analyte extraction in the electrode, electrode washing and electrochemical measurement of TNT. It was found that the washing step had the main effect on the selectivity improvement of the sensor by removing the weakly absorbed interferences from the electrode without considerable effect on the sensitivity of the sensor. The optimized sensor was used successfully for TNT determination in water and soil samples.

2. Experimental 2.1. Instruments and reagents Electrochemical data were obtained with a three-electrode system using a potentiostat/galvanostat model PGSTAT302, Metrohm. The differently prepared MIP or NIP involved sensors were used as a working electrode. A platinum wire and an Ag/AgCl electrode were used as the counter and reference electrodes, respectively. Methacrylic acid (MAA), obtained from Sigma–Aldrich (Munich, Germany), was purified by passing them through a short column of neutral alumina, followed by distillation under reduced pressure. Ethylene glycol dimethacrylate (EDMA), obtained from Fluka (Buchs, Switzerland), was distilled under reduced pressure in the presence of hydroquinone inhibitor and stored at 4 ◦ C

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until use. 2,4,6-trinitrotoluene, para-nitrophenol, aniline, phenol, nitrobenzene, n-eicosane and 2, 2 -azobisisobutyronitrile (AIBN) were supplied by Sigma–Aldrich (Munich, Germany), and used as received. Graphite powder was purchased from Fluka (Buchs, Switzerland). Other chemicals were of analytical grade and were purchased from Merck (Darmstadt, Germany). 2.2. Molecularly imprinted polymers preparation In order to produce a molecularly imprinted polymer template molecule (1 mmol), methacrylic acid (4 mmmol) and 50 ml of dry chlorofom were placed into a 100 ml round-bottomed flask and the mixture was left in contact for 10 min. Subsequently, EDMA (24 mmol) and AIBN (0.2 mmol) were added. The flask was sealed and the mixture was purged with nitrogen for 15 min. Polymerization took place in a water bath at 60 ◦ C for 24 h. The final polymer was simply powdered and the template was removed by Soxhlet extraction with methanol for 48 h. The complete removal of template from the polymer was traced by the square wave voltammetry method. The non-imprinted polymer (NIP) was prepared similar to the MIP, except that the template was not present in the polymerization media. In order to obtain finer and smaller MIP particles, the obtained powder was sequentially immersed three times in acetonitrile, and the supernatant portions were collected for final use. 2.3. Preparation of the sensors For construction of the sensor (MIP-CP or NIP-CP), 0.05 g graphite was homogenized in a mortar with 0.01 g of powdered 2,4,6-trinitrotoluene MIP or NIP for 10 min. Subsequently, neicosane, 0.03 g was melted in a dish in a water bath heated at 45–50 ◦ C. The graphite/MIP blend was then added to the melted n-eicosane and mixed with a stainless steel spatula. The final paste was used to fill a hole (2.00 mm in diameter, 3 mm in depth) at the end of an electrode body previously heated at 45 ◦ C. After cooling at room temperature, the excess of solidified material was removed with the aid of sand paper. The electrode can be reused after each experiment by moving the electrode surface on a paper in order to rub out a thin layer of the electrode surface. 2.4. TNT measurement in real samples In order to determine the TNT in the real samples, the sensor was immersed into the spiked solution with pH 4.5, adjusted by acetate buffer solution (0.15 mol l−1 ). After incubation for 10 min the sensor was washed by emplacing it in the water/acetonitrile (97:3) solution for 15 s, then, the electrode was transferred to the electrochemical cell containing 10 ml of HCl solution (0.07 mol l−1 ). The pre-potential of −1.0 V was applied to the electrode for 30 s. Finally, the square wave voltammograms in the potential range of 0.0–1.0 V with SW potential amplitude of 50 mV and frequency of 150 Hz were recorded and the current peak was used for final determination. 3. Results and discussion 3.1. Cyclic voltammetry behavior of TNT Electrochemical behavior of TNT in different mediums and various electrodes has been studied and reported (Schmelling et al., 1996; Plambeck, 1982; You et al., 1997; Wang et al., 1998). However, the cyclic voltammetry of TNT was investigated by using pure carbon paste electrodes having no MIP or NIP. The obtained voltammogram is shown in Fig. 1. As can be seen, three distinct reductions

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Fig. 1. Electrochemical behavior of TNT in the carbon paste electrode when the potential scanned from 1.0 to −1.0 V and vice versa; potential scan rate = 0.03 V s−1 , pH = 1.1, TNT = 8 × 10−5 mol l−1 .

are observed during the decreasing potential sweep, likely corresponding to the formation of hydroxylamine from the sequential reduction of the three NO2 groups. Conversely, the increasing sweep only indicates one oxidation peak at the positive potential. The oxidation peak may correspond to further transformation of the moieties formed during the decreasing potential sweep. Thus it is related to the oxidation of hydroxylamine (Hilmi et al., 1999). According to a report (Hilmi et al., 1999) when the potential was maintained for a few minutes at the potentials corresponding to the three observed reductions peak, the peak obtained at positive potential (oxidation peak) increased with increasing reduction time. This phenomenon was also observed in the present work. This observation indicates that by increasing the reduction time in a negative potential it can be possible to increase the peak height of oxidation peak. This fact can be used for TNT sensor sensitivity increasing if the oxidation peak is applied for sensor as determination potential. Besides, it can be seen that the oxidation peak of TNT at carbon paste electrode is partly higher in comparison to the three other reduction peaks of TNT. Therefore, in the further investigation in the case of both cyclic voltammetry and square wave voltammetry, at first the proper negative pre-potential was applied to the MIP-CP or NIP-CP, converting the –NO2 groups of TNT to the –NHOH groups, and then the potential sweep was carried out in the positive potential range. 3.2. MIP-CP and NIP-CP electrodes responses to TNT and washing effect In order to study the TNT recognition ability of MIP, the MIPCP, NIP-CP and CP electrodes were prepared and inserted into the TNT containing solutions. After 7 min the electrodes were removed from the TNT solution and cyclic voltammetry was carried out. The obtained results are shown as voltammograms, relating to the oxidation peak of the reduced product of TNT which was obtained after applying the pre-potential of −1.0 V for 10 s to each electrode (Fig. 2). As can be seen, the CV signal of MIP-CP electrode (voltammogram a) is higher than that of the NIP-CP electrodes (voltammogram c). This indicates that the MIP in the carbon paste electrode can intensively uptake TNT from the aqueous solution in comparison to the NIP-CP. For further evaluation of MIP, the electrodes were

Fig. 2. Recorded TNT responses from different electrodes immersed in the TNT solution (8 × 10−6 mol l−1 ) (voltammogram of a, c and e). The effect of electrode washing on the related cyclic voltammetry response (scanned from −0.2 V to 1.0 V ad vice versa) after removing the electrodes from the TNT solution (voltammogram b, d and f). TNT extraction conditions; pH = 5 (acetate buffer: 0.15 mol l−1 ), extraction time = 7 min, washing time = 15 s. electrochemical cell solution pH = 1.3, potential scanning rate = 0.03 V s−1 , pre-potential = −1.0 V, pre-potential applying time = 10 s.

inserted into the washing solution for a short time (15 s) just after they were removed from the TNT solution. The obtained results are shown as the voltammograms (b) and (d) in Fig. 2. As it is clear, the electrodes washing after TNT extraction does not noticeably affect the TNT signal in the MIP-CP. However, at the same time, the response of the NIP-CP electrode decreases considerably by washing. According to the voltammograms of (e) and (f) in Fig. 2 (right), the response of CP electrode disappears after washing the electrode. These results indicate that MIP-CP electrode has more affinity towards TNT in comparison to the NIP-CP and CP electrodes. The washing process can remove the weakly and nonspecifically absorbed TNT molecules from the electrode surface, the state which is dominant in the case of NIP-CP and CP. However, TNT molecules which are incorporated in the selective sites of MIP are not removed easily by the washing process. This behavior of MIP-CP can be used for selectivity enhancement of MIP-CP by a simple washing step. The constructed MIP particles during the non-covalent approach usually contain selective sites with various affinities for the template. Some of them are cavities with matchable sizes to the template molecule. These are template recognition sites, constructed with regular and perfect shape in the polymerization period, and thus have more affinity for TNT. TNT molecules located in the mentioned cavities are tightly absorbed to the MIP, and thus the washing of MIP-CP electrode, do not remove TNT easily from the electrode (Caro et al., 2002). The cavities with incomplete or irregular shape, and also the non-selective binding sites cannot absorb TNT molecules so tightly. The portion of TNT molecules absorbed by such mentioned binding sites can be removed from MIP-CP electrodes by the washing process.

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Fig. 3. Comparison of square wave voltammetry and differential pulse voltammetry for TNT determination by proposed sensor after applying pre-potential of −0.8 V for 20 s to the electrode. TNT extraction conditions; pH = 5, extraction time: 7 min, washing time: 10 s. electrochemical cell solution pH: 1.3, pre-potential = −0.8 V, pre-potential applying time = 20 s.

3.3. Electrochemical method selection In order to achieve a high sensitive sensor, the selection of a proper electrochemical technique is most important. Thus, we tested different voltammetric methods such as differential pulse voltammetry and square wave voltammetry, known as high sensitive methods, in the same conditions of extraction and washing. The obtained results of these experiments (Fig. 3) showed that the response of square wave voltammetry for TNT is considerably better than that of differential pulse voltammetry. Thus, this method was selected as a main electrochemical method for TNT determination by the prepared sensor. 3.4. Optimization of parameters affecting TNT determination The optimization process for the designed sensor was divided into three sections including optimization of carbon paste composition, extraction parameters and electrochemical determination conditions. 3.4.1. MIP-CP composition optimization In order to find the best composition for MIP-CP electrodes, the amount of different ingredients of the electrode including MIP, carbon and n-eicosane was changed in the fixed conditions of extraction and voltammetric determination, and the obtained responses were used for conclusion. The MIP-CP electrode was prepared with a fixed amount of carbon and n-eicosane, and different amounts of MIP. The resulted electrode at each case was used for TNT extraction and determination. The obtained results are presented in Fig. 4(I). It is clear that the maximum response for the prepared sensor appears in the MIP amount of 0.025 g. Higher amounts of MIP in the MIP-CP electrode can increase the sensor response because of providing more recognition sites on the electrode surface as it is evident in the corresponding curve. However, enhancement of the MIP amount, more than a threshold amount, leads to a decrease in the prepared sensor response, probably because of electrode surface conductivity decreasing. Similar experiments were also carried out in order to investigate the effect of carbon and neicosane amounts on the prepared electrode response for TNT, by variation of these parameters amounts in the MIP-CP electrode, followed by recording the obtained results. These results are shown in Fig. 4(II) and (III). From the corresponding curves, the optimum

amount of carbon and n-eicosane were found to be, 0.035 and 0.025 g for carbon and n-eicosane amounts, respectively. At first, the increasing the carbon content of the MIP-CP electrode leads to an increase in the electrode response, but, after a definite point, further carbon content increasing results in lowering the corresponding signal. The first increase in response can be related to electron transferring capability enhancement of the electrode in the presence of higher carbon content. Furthermore, the electrode response decreasing with the carbon content enhancement, can be attributed to the fact that the more carbon amount of on the electrode surface leads to decrease the MIP content of electrode surface. The optimum amount of n-eicosane is required for MIP-CP electrode preparation. Presence of higher amounts of binder (n-eicosane) in the MIP-CP electrode leads to a decrease in electrode response, because of electrode surface conductivity decreasing. 3.4.2. Washing effect As described previously, the washing of electrode led to remove the weakly adsorbed species on the electrode surface. This fact can be used for sensor selectivity improvement and omitting the interference effects, if proper washing conditions is applied. It was found that neutral water containing small amount of organic solvent decrease the NIP-CP signal, whereas the NIP-CP signal did not affected considerably by the mentioned mixture. Different organic solvent such as ethanol, methanol, acetone and acetonitrile were added to the water (2% in water) and tested as washing solution. In the case of acetonitrile the response difference between MIP-CP and NIP-CP was higher than that for other tested solvent. Thus acetonitrile was selected as organic solvent type to mix with water. The difference between the responses of MIP-CP and NIP-CP in the similar conditions of extraction and determination was increased by increasing the organic solvent content of washing solution from 0 to 3% and after that it remained constant. However, presence of acetonitrile higher than 5% declined the physical properties of the electrode surface probably because of swelling effect of organic solvent on the MIP particles presented in the carbon paste. Thus 3% acetonitrile was chosen in this case. The time of electrode immersion in the washing solution was also investigated. It was found that the response difference between MIP-CP and NIP-CP increase till 15 s and afterwards the aimed sig-

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Fig. 4. Optimization of carbon paste electrode compositions; variation of electrode response for TNT with changing of (I) MIP, (II) carbon and (III) n-eicosane amounts of MIP-CP electrode.

nal reached to partly steady state. Thus 15 s was chosen as optimal washing time. 3.4.3. TNT extraction conditions optimization The pH of TNT solution as a commonly considering parameter was noticed and its effect on the TNT extraction in the electrode was studied. For this purpose, the prepared electrodes were inserted into the TNT solutions with various pH values where they were incubated for 7 min at the constant stirring rate. After the mentioned time, the electrode was removed from the solution and immersed into the solution of the electrochemical cell. The results of this experiment are shown in Fig. 5(I). In the pH range of 2–5 the TNT related electrochemical signal can be observed, and thus the TNT extraction amount is relatively high. It seems that no considerable variation in extraction of TNT is resulted by pH changing in this range of a pH higher than 6, the extraction amount tends to decrease. According to these results, the pH of 4.5 fixed with acetate buffer (0.15 mol l−1 ), was chosen as an optimum pH for TNT extraction in the electrode. In order to optimize the stirring rate in the extraction period, TNT was extracted in the prepared MIP-CP electrodes at various stirring rates, whereas the other extraction parameters such as time, pH and TNT extraction were the same and constant. The obtained results showing the TNT related square wave voltammetry signal variation against the stirring rates are presented in Fig. 5(II). As can be seen, the greater the stirring rate, the higher the electrode response is for TNT. This indicates the high effect of the stirring rate on the TNT extraction in the MIP-CP electrode. The growth in TNT voltammetric response with the stirring rate increasing continues up to 400 rpm. However, further enhancement in stirring rate does not affect considerably the TNT extraction. Therefore, the stirring rate of 500 rpm was chosen as optima for this variation.

Extraction time was another main parameter which was examined. For this aim the prepared electrodes were inserted into the TNT solutions for various times. Afterwards the electrodes were removed from the solutions followed by square wave voltammetry. The obtained results are shown in Fig. 5(III). According to this figure the increasing of extraction time leads to an intensive increase in the TNT extraction amount in the electrode until about 10 min. After, the response increasing rate with time enhancement is not so considerable. In order to decrease the TNT analyzing time, as much as possible, the time of 10 min was selected for the extraction. 3.4.4. Optimization of square wave voltammetry parameters In the case of square wave voltammetry measurement, the main important parameters which could be optimized were; pH of solution of electrochemical cell, applied pre-potential amount, time of exerted pre-potential and also frequency of used potential in square wave voltammetry. Fig. 5(IV) shows that the anodic peak current is dependent on the pH. The anodic peak current decreased with increasing pH. Thus, in order to obtain high electrochemical responses of the sensor, the acidic pH of about 1.1 was fixed by using HCl (0.07 M) solution. The effect of pre-potential magnitude and its exertion time was investigated. It was found that the pre-potential of −1.0 V is appropriate, and applying higher value pre-potential to the electrode did not further increase the response amount. The optimization of pre-potential applying time is demonstrated in Fig. 5(V), where the peak current increases with increasing the pre-potential. Applying time up to about 30 s where it attains plateau, thus the optimum amount of 30 s was considered for this parameter. The effect of square wave frequency on the final response of the sensor was examined. The obtained results are shown in Fig. 5(VI). As can be deduced the frequency of 150 Hz is the best option in this case. The peak current initially increased

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Fig. 5. Optimization of different conditions affecting the TNT extraction and determination in the MIP-CP electrode; variation of electrode response for TNT with changing of (I) TNT solution pH, (II) stirring rate, (III) electrode incubation time, (IV) pH of electrochemical solution, (V) pre-potential applying time and (VI) frequency of square wave potential.

with increase in SW amplitude and reached a maximum at around 50 mV. 3.5. Analytical characterization After the optimization and establishment of the determination method for the prepared MIP-CP sensor, various ions and molecules were examined with respect to their interference with the determination of TNT. The tolerance limit was established as the maximum concentration of foreign species that caused a relative error of 5% in the analytical signal It is worth noticing that the values of the current response used for the calibration curve are actually the absolute values of the oxidative peak current, observed after electrode incubation in different concentrations of TNT solution, and cathodic pre-potential of −1.0 V applied to the electrode. For 70 nM of TNT, the results showing the interference levels of some species are given in Table 1. It can be seen that the developed sensor do not affected by presence of different organic and inorganic species in the samples even in their concentrations considerably higher than that of TNT. The prepared and optimized MIP-CP sensor response against TNT concentration variation was checked. The calibration graph (shown in Fig. 6) of the prepared sensor showed

a linear relationship over TNT concentration in the range of 5 × 10−9 to 5.0 × 10−6 mol l−1 with a detection limit of 1.5 × 10−9 mol l−1 (S/N = 3). Each point of the calibration graph is the average of three replications. 3.6. Determination of TNT in water and soil samples The analytical usefulness of the prepared electrochemical sensor for determination of TNT was demonstrated by applying it to the determination of TNT in water and soil samples. The absence of TNT was first verified in the non-spiked samples simply, by applying the proposed method to the non-spiked samples and observing no TNT related response. Water samples of tap water and ground water were spiked with TNT. For analysis of soil samples, approximately

Table 1 Interference levels for some tested species in the determination of TNT by developed sensor. Species

Interference level

K+ , Ca2+ , Cl− , SO4 2− , Co2+ , Ni2+ Fe2+ , Zn2+ , Cu2+ Pb2+ , Hg2+ Phenol Aniline para-Nitrophenol Benzoic acid Nitrobenzene

<650 550 <500 100 100 60 <50 65

Fig. 6. Calibration curve obtained for developed sensors at optimized conditions (inset: linear range of calibration curve).

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Table 2 Determination of TNT in different water and soil samples. Sample

Spiked [TNT]/1 × 10−9 mol l−1

Founda [TNT]/1 × 10−9 mol l−1

Recovery (%)

RSD (%)

Tap water Ground water Soil

100 100 200b

98.1 97.23 193.8b

98.1 97.2 96.9

1.9 2.7 5.3

a b

Number of sample assayed = 5. (␮g g−1 ).

Table 3 Comparison of proposed technique with two other previously developed method for determination of TNT in different water and soil samples. Sample

Spiked [TNT]/1 × 10−6 mol l−1

Determined by Proposed methoda [TNT]/1 × 10−6 mol l−1

Reference methoda [TNT]/1 × 10−6 mol l−1

Tap water

10.0 20.0

9.5(±0.23) 20.4 (±0.57)

9.7(±0.33) 21.0 (±0.37)

Ground water

10.0 15.0

10.33 (±0.30) 14.23 (±0.41)

9.43 (±0.21) 14.23 (±0.35)

200.0b 100.0b

201.13 (±9.85) 102.63 (± 5.25)

202.33 (±6.85) 98.67 (±3.35)

Soil a b

Number of sample assayed = 5. (␮g g−1 ).

1 g soil, previously spiked with TNT at the 200 ␮g g−1 levels, was accurately weighed and introduced into a 30 ml centrifuge tube. Next, 2 ml acetonitrile was added and the mixture was shaken for 30 min. After centrifugation at 3500 rpm for 20 min, the liquid was filtered through a Nylon syringe filter. A 10 ␮l aliquot of this extract was diluted to 10 ml with acetate buffer solution of pH 4.5. The electrode was immersed into the resultant solution for 10 min followed by inserting the electrode in the washing solution for 15 s. Finally the electrode was emplaced in the electrochemical cell containing HCl (0.07 M) and the SW voltammetry response was recorded for determination purpose according to the calibration curve, previously described. Table 2 summarizes the results obtained for the three samples analysed. As can be seen, the recoveries achieved are acceptable for all the samples tested, the confidence intervals being calculated for a significance level of 0.05. Table 3 shows the results obtained from analysis of different samples by using the introduced work and other voltammetric method (Saravanan et al., 2006) as a reference method in order to statistically verify the performance of the sensor for TNT determination. Those obtained by MIP-CP method and reference voltammetric method were statistically compared by the paired t-test. This test revealed the absence of significant differences between the results obtained by both methods (at 95% confidence level). There was thus no evidence of the presence of systematic error in the results. 4. Conclusion Very high selective square wave voltammetry sensor for TNT determination at low concentration was proposed. The MIP functioned as both pre-concentrator and high selective recognition element in the carbon paste structure. Washing of the MIP-CP electrode after TNT extraction led to enhance the selectivity without considerable loss in sensitivity and detection limit of the sensor. The proposed sensor was used successfully for TNT determination in real samples. References Agu, L., Montenegro, D.V., Seden, P.Y., Pingarron, J.M., 2005. Anal. Bioanal. Chem. 382, 381–387.

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