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Immunosensor for the determination of the herbicide simazine based on an ion-selective field-effect transistor
Analytica Chimica Acta 424 (2000) 37–43
Immunosensor for the determination of the herbicide simazine based on an ion-selective field-effect transistor N.F. Starodub a,∗ , B.B. Dzantiev b , V.M. Starodub a , A.V. Zherdev b a
Department of Biochemistry and Sensoric System, A.V. Palladin’s Institute of Biochemistry of Ukrainian National Academy of Sciences, 9 Leontovicha Str., 252030 Kyiv, Ukraine b A.N. Bach Institute of Biochemistry, Russian Academy of Sciences, Leninsky prospect 33, 117071 Moscow, Russia
Received 23 March 2000; received in revised form 15 August 2000; accepted 15 August 2000
Abstract An immunosensor based on the ion-selective field effect transistor (ISFET) was developed for the express determination of herbicide simazine. Polyclonal antibodies against simazine were attached to the ISFET gate via staphylococcal protein A. Two methods of simazine detection were used — (i) competitive immune assay when native (detected) and peroxidase-labelled simazine molecules competed for binding with antibodies on the ISFET surface, and (ii) sequential saturation of antibodies, left unbound after their exposure to native simazine in investigated sample, with labelled simazine. The catalytic activity of bound peroxidase was measured in the presence of ascorbic acid and H2 O2 . The limit of simazine detection by competitive immune assay was 1.25 ng ml−1 , the linearity was observed in the range of 5–175 ng ml−1 . Sequential saturation of the antibodies led to the growth of the assay sensitivity up to 0.65 ng ml−1 . Acidic treatment of the ISFETs allowed using them several times without loss of the signal amplitude. The analysis had rapid kinetics, its overall time including duration of all preparation stages was around 50 min. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Immunosensor; ISFET; Herbicide; Simazine; Competitive immunoassay
1. Introduction Intensive and non-controlled agricultural application of herbicides and other classes of pesticides led to their wide spread in the environment. Due to pesticides extreme toxicity and their ability to induce different physiological disorders in humans and animals [1], the content of these substances in soil, water and food should be controlled. Biosensors are effective de∗ Corresponding author. Tel.: +380-44-229-4743; fax: +380-44-229-6365 E-mail address: [email protected] (N.F. Starodub).
vices for such analyses. Different types of biosensors were proposed for pesticide determination both for group-specific screening of toxic agents [2–5] and for control of individual compounds [6–13]. Application of immunochemical recognition allows reaching high specificity of biosensors [8,12]. Therefore, the combination of biosensor approach with antigen–antibody reaction seems to be rather suitable and promising since it can provide sensitive, fast, simple and inexpensive analyses. To fulfil all practical demands, optimal instrumental devices and biochemical protocols should be chosen. Silicon transducers, like ion-selective field effect transistors (ISFETs), are extremely perspective for bioassays. They can be manufactured by common
0003-2670/00/$ – see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 3 - 2 6 7 0 ( 0 0 ) 0 1 1 4 3 - 0
38
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technologies. Moreover, they combine measurements and preliminary processing of signals and also have other advantages [14,15]. Herbicide simazine (2-chloro-4,6-di(N-ethylamino)1,3,5-triazine) is one of most widely used compounds in agricultural practice of different countries [1]. Therefore, it was chosen as a model antigen for our investigations. The presented study was undertaken to apply the ISFET technique for simazine immunodetection.
2. Experimental 2.1. Materials 1 - Ethyl - 3(3 - dimethylaminopropyl)carbodiimide, dimethylformamide (both from ICN), N-hydroxysuccinimide (Sigma), 2,4,6-trinitrobenzensulfonic acid (CalBiochem), Triton X-100 (Serva), Tween-20, glutaraldehyde and ascorbic acid (Sigma) and 2,20 -azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS, Boehringer Mannheim) were used in the experiments. Purified preparations of simazine, its 2-ethylcarboxy- and 2-propylcarboxy- derivatives were generously provided by Dr. S.A. Eremin, Moscow State University, Russia. Bovine serum albumin (BSA, Minsk Institute of Epidemiology and Microbiology, Belorussia), hemocyanin from Paralithodes camtschatica (HC, generously provided by Dr. I.Yu. Sakharov, Moscow State University, Russia), soybean trypsin inhibitor (STI, Reanal), ovalbumin (OA, Serva) and horseradish peroxidase (HRP, RZ = A403 /A280 = 3.0, Biolar, Latvia) were used for syntheses of simazine–protein conjugates. 2.2. Syntheses of simazine–protein conjugates, obtaining of antiserum and analysis of its specificity The simazine–protein conjugates and the antiserum specific to simazine were obtained by immunisation of Chinchilla rabbits according to procedure described in [16]. Specificity of the antiserum was tested using closiest structural analogues of simazine, namely atrazine, propazine, promethrine, terbutylazine, amethrine, terbutrine, asiprotrine, cyanazine, semerone, methoprotrine, and deethylatrazine. The
antisera cross-reacted with atrazine (89%), terbutylazine (80%), and propazine (10%). Other analytes demonstrated cross-reaction in the range 0.7–6.2%. The antisera didn’t interact reliably with pesticides from other structural classes (such as pyrethroids or derivatives of phenoxyacetic acid); the cross-reactions for those analytes were less than 0.1%. 2.3. Preparation of the ISFET-based immunosensors Chips used in the experiments contained two ISFETs and a reference electrode. They were manufactured by the “Microprocessor” plant (Kyiv, Ukraine). pH sensitivity of the ISFETs was 52 mV per pH unit. The first (reference) ISFET was covered with the BSA, and the second (working) ISFET with the anti-simazine antiserum or firstly by the staphylococcal protein A (Sigma) and then by the antiserum. For attaching biomolecules to the ISFET surface glutaraldehyde (GA, Sigma) was used as cross-linking agent. It formed covalent bonds [17] between the silicon nitride surface of the ISFET gate and proteins being immobilised. The detailed protocol of the immobilisation was the following. The ISFET surface was cleaned by its sequential immersion into the mixture of potassium dichromate and sulphuric acid (10 s), water (vigorous washing), acetone, ethanol and PB (10 mM Na-phosphate buffer, pH 7.3, containing 100 mM NaCl). Then, by drop coating the 2.5% water solution of the GA was placed on the prepared surface. After 90 min at room temperature (25◦ C) the GA was placed again according to the same procedure. Then after 90 min from the second GA deposition, the chip was washed by distilled water. After this, BSA (20 g ml−1 in PB), or non-diluted antiserum, or protein A (20 g ml−1 in the PB) was placed on the gate surface of the ISFET and kept during 1 h at humid conditions. The treatment of the chip with the GA was carried out at the room temperature. At last, the chip was kept overnight at 4◦ C, and then, after vigorous washing with the PB, was immersed into the glycine solution (10 mg ml−1 in the PB and at the room temperature) in order to block non-reacted groups of the GA. After 30 min at the room temperature the ISFET was washed with the PB. When the protein A was immobilised, a step of the specific antibody binding was added. The antisimazine non-diluted antiserum was placed on the
N.F. Starodub et al. / Analytica Chimica Acta 424 (2000) 37–43
ISFET with the preliminary adsorbed protein A. The chip was incubated for 1 h in humid conditions and then washed with the PB. The prepared chips were stored in dry state at 4◦ C. 2.4. ISFET-based immunodetection of simazine Antigen–antibody interactions between immobilised antibodies and simazine were allowed in the PB. The level of the signal was recorded after the chip was immersed for 30 min into the solution of the HRP-labelled simazine. For the competitive assay the chip was immersed for 30 min into the solution containing both native and labelled simazine (Choice of reactants’ concentrations is described in the Section 3). After those incubations the chip was washed with the PhB and immersed into the HRP substrate solution. The measurements of the HRP activity were carried out in the working buffer (WB) containing 10 mM tris-HCl, 100 mM NaCl and 15 mM ascorbic acid (Sigma) as it was described in [18]. pH of the WB was 7.5. The hydrogen peroxide was added to the measuring cell (volume = 1 ml). The concentrations of above mentioned components of the WB were chosen according to the previous paper [18]. The concentration of H2 O2 was 5 or 10 mM. The signals (dV/dt) of the ISFETs were registered by electronic device (constructed at the Radiophysics Department of the Kiev National University named after Taras Shevchenko) providing signal amplification and its processing on the basis of a custom-made computer program. All measurements were undertaken at the room temperature. After every assay the chip was treated with the 0.1 M HCl (5 min) and carefully washed with the PhB.
3. Results and discussion The proposed immunosensor technique is based on the detection of the pH shift on the ISFET gate during HRP catalytic reaction. The substrate conversion causes a local basic pH shift, because dehydroascorbic acid formed is a more neutral compound compared to ascorbic acid [18]. Tests of pH shifts without native simazine molecules indicated that simazine–HRP conjugate (0.1 g ml−1 ,
39
by HRP) cannot be detected in the proposed system when antiserum has been directly attached to the ISFET gate. It was due to presence of a big concentration of non-IgG type proteins in antiserum which competed for aldehyde groups on the transducer surface. The signal was registered only when antibodies were immobilised via protein A. In this case IgG molecules specifically bound with protein A, and concentration of simazine specific IgG on the transducer surface sharply decreased. As can be seen from the Fig. 1, the pH shift occurred during 40–60 s after addition of H2 O2 . The type of sensor response is determined by the absence of electrolyte retaining membrane on the transducer surface. The enzyme was directly immobilised on the gate of surface of the ISFET via crosslinking with GA (see Section 2.3 of Section 2). We varied concentrations of the HRP–simazine conjugate in the range 0.1–0.6 g ml−1 (by the HRP). It was found that the maximal sensor output (about 95 mV) corresponded to 0.20 g ml−1 of the conjugate. Under these conditions binding sites of specific immunoglobulins were saturated. It should be noted that the sensor output depends on quantity of antigenbinding sites on the ISFET surface. Therefore, oriented immobilisation of antibodies via staphylococcal protein A (without GA treatment of the antibodies) is an effective way to reach higher signals. The concentration of the HRP-labelled simazine during the competitive detection of the simazine was equal to 0.25 g ml−1 (by the HRP). Change of the ISFET potential of the immunosensor as a function of the simazine concentration is presented in Fig. 2 (mean values from 8 to 10 measurements). Reliable decrease of the sensor signal was observed down to 1.25 ng ml−1 of simazine in the analysed mixture. The linearity of signal decrease was observed in the range of simazine concentrations from 5 to 175 ng ml−1 ; in this range the potential of the ISFET gate varied from 10 to 74 mV. The standard deviation was on average about 5%. It is necessary to mention a high reproducibility of results between different measurents by individual ISFETs. To achieve such reproducibility ISFETs obtained from the same silicone plate were used. Moreover, both the electrical characteristics of these transducers and the procedure for biological membrane preparation were standardised. In the latter
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Fig. 1. Typical response of the sensor at the addition of hydrogen peroxide. The ISFET potential is recorded as a function of time.
case, it was very important to fulfil exactly the protocol of cleaning and activation of gate surface as well as to provide constant humid conditions during 1 h of interaction of protein A with the activated groups of the gate surface. As a rule, all these processes were
fulfilled by the group’s technology, and no less than ten ISFETs were prepared simultaneously. Since ISFETs are very sensitive to different factors (such as buffer capacity, ionic strength, pH level, light irradiation, presence of ions affecting the charge of
Fig. 2. Change of the ISFET potential of the immunosensor as a function of simazine concentration (the competitive scheme).
N.F. Starodub et al. / Analytica Chimica Acta 424 (2000) 37–43
41
Fig. 3. Non-specific sensor response at the addition of hydrogen peroxide and WB. The ISFET potential is recorded as a function of time.
the gate surface, etc.) [19–21], their non-specific responses to addition of the WB or hydrogen peroxide were analysed. For this purpose a differential scheme was used: the first ISFET contained antibodies immobilised via protein A, the second had BSA immobilised via GA. It was shown (Fig. 3) that the WB did not influence the sensor output, while hydrogen peroxide in the concentrations mentioned above (10 mM) initiated small (<10 mV) responses. H2 O2 caused acidic pH shift in contrast to the basic changes during determination of the HRP activity. Thus neither WB nor H2 O2 contributed to the sensor’s basic pH shifts during the simazine recording. The study of optimal concentration of hydrogen peroxide indicated that the sensor output was about 15–20% higher for 10 mM of H2 O2 than for 5 mM of H2 O2 . We have not investigated the correlation between hydrogen peroxide concentration and level of the sensor signal yet. This will be task for our further research in this area, since we think that the optimisation of this step may improve the sensitivity of the proposed analysis. The overall time of the assay including the duration of all preparation stages was 50 min. The limiting stage of the analysis is the competition between the native and the HRP-labelled simazine for binding with the antibodies (30 min). We performed preliminary
experiments in which the duration of this stage was reduced to 10 min while the other assay conditions remained unchanged. In this case the sensor showed the sensitivity about 6.50 ng ml−1 which was more than five times lower than the sensitivity of the assay performed according to the procedures described above. Therefore, reduction of the assay time may lead to the decrease of its sensitivity. According to our preliminary investigations (data not shown) the sensitivity of the sensor can be increased by decrease of the WB capacity since according to existing data [21] the buffer capacity of the analysed sample has a significant influence on the sensor response. Another approach used to increase the sensitivity was so called saturation protocol of immunoassay [22]. The chip with immobilised antibodies was incubated with native simazine in investigated sample and then in order to saturate unbound antibodies with the HRP-labelled simazine (0.25 g ml−1 , by HRP). Both stages lasted 20 min. This approach allowed reaching sensitivity 0.65 ng ml−1 and linearity in the range 1.25–185 ng ml−1 (Fig. 4, mean values from 10 to 12 measurements). Destruction of antigen–antibody bonds by treatment of the chips with 0.1 M HCl for 5 min made it possible to reuse the chips for two to three measurements
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Fig. 4. Change of the ISFET potential of the immunosensor as a function of simazine concentration (the sequentional saturation scheme).
without signal decrease. Between the measurements chips were stored in dry state at 4◦ C. Before every new assay they were washed with PhB for 2 h. The proposed analytical system may be compared with other immunochemical approaches based on the ISFETs. Schasfoort et al. [23] immobilised antigens or antibodies on a porous membrane covering the ISFET gate. In this work stepwise change of electrolyte concentration produced a transport of ions through the membrane–protein layer resulting in a transient membrane potential, which was measured by the ISFET. The transport was determined by charge density in the protein layer, which changes upon formation of antigen–antibody complexes. The ISFET allowed detection of 10−6 M of antibodies. Sakai et al. [24] constructed a sensor by immobilising of human immunoglobulin G (IgG) on the ISFET surface. The assay involves a competitive binding of urease-labelled anti-IgG and IgG to be determined by membrane-bound IgG followed by electrochemical determination of the catalytic activity of bound urease. The detection limit of this sensor was 10 g ml−1 of IgG and the assay time 60 min. Comparison of these approaches with the sensor presented in this paper suggests that proposed variant has higher sensitivity and sometimes shorter assay time.
4. Conclusion A new ISFET-based immunosensor for the determination of herbicide simazine was created. Two schemes of the immunoassay were applied — competitive immune analysis and so called sequential saturation of antibodies. The activity of bound peroxidase was measured by basic pH shift of ascorbic acid solution after addition of hydrogene peroxide. The overall time of analysis was about 50 min. When the investigations were carried out according to the first scheme, the sensor allowed detecting simazine at concentrations up to 1.25 ng ml−1 . Linearity was observed in the range 5–175 ng ml−1 . Sequential saturation of antibodies, left unbound after their exposure to native simazine in investigated sample, with labelled simazine allowed to increase the sensitivity of the immune sensor to 0.65 ng ml−1 with the linearity in the range 1.25–185 ng ml−1 .
Acknowledgements This work was made possible by the Award PL979112 of the INCO-Copernicus Foundation. The authors are grateful to E.V. Yazynina from A.N. Bach
N.F. Starodub et al. / Analytica Chimica Acta 424 (2000) 37–43
Institute of Biochemistry (Moscow), who prepared and characterised simazine–protein conjugates. [9]
References [1] M. Mazzullo, R. Mesirca, M. Paolini, G. Cantelli-Forti, P. Perocco, P. Ciaccia, S. Grilli, J. Environ. Pathol. Toxicol. Oncol. 16 (1997) 231. [2] N.F. Starodub, Yu.M. Shirshov, V.M. Starodub, A.L. Kukla, M.I. Kanjuk, A.V. Prokopovich, R. Merker, Electrochem. Soc. Proc. 97-19 (1997) 799. [3] N.F. Starodub, Yu.M. Shirshov, W. Torbicz, N.I. Kanjuk, V.M. Starodub, A.L. Kukla, in: D.P. Nikolelis et al. (Eds.), Biosensors for Direct Monitoring of Environmental Pollutants in Field, Kluwer Academic Publishers, Dordrecht, 1998, p. 209. [4] N.F. Starodub, M.I. Kanjuk, A.L. Kukla, Y.M. Shirshov, Anal. Chim. Acta 385 (1999) 461. [5] N.F. Starodub, A.L. Kukla, M.I. Kanjuk, Y.M. Shirshov, in: Proceedings of the IXth International Trade Fair and Conference on Sensors, Transducers and Systems, Nurnberg, Germany, 1999, Vol. 2, p. 105. [6] G. Jeanty, J.-L. Marty, Biosens. Bioelectron. 13 (1998) 213. [7] N. Mionetto, J.-L. Marty, I. Karube, Biosens. Bioelectron. 9 (1994) 463. [8] L. Campanella, C. Colapicchioni, G. Favero, M.P. Sammartino, M. Tomassetti, in: Proceedings of the VIIIth International
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Conference for Solid State Sensors and Actuators and Eurosensors IX, Stockholm, Sweden, 1995, p. 481. D.M. Ivnitskii, J. Rishpon, Biosens. Bioelectron. 9 (1994) 569. P. Skladal, T. Kalab, Anal. Chim. Acta 316 (1995) 73. T. Imato, N. Ishibashi, Biosens. Bioelectron. 10 (1995) 435. R. Rouillon, M. Sole, R. Carpentier, J.-L. Marty, Sens. Actuators B 26–27 (1995) 477. T. Kalab, P. Skladal, Electroanalysis 9 (1997) 293. P. Bergveld, Sens. Actuators 1 (1981) 17. P. Bergveld, Lecture Notes of ICB Seminars Biomeasurements, Electrochemical Measurements of Biochemical Quantities, Warsaw, Poland, 1995, p. 60. E.V. Yazynina, A.V. Zherdev, B.B. Dzantiev, V.A. Izumrudov, S.J. Gee, B.D. Hammock, Anal. Chem. 71 (1999) 3538. D.L. Harame, L.J. Bousse, J.D. Shott, J.D. Meindl, IEE Trans. Electron Dev. ED-34 (1987) 1700. V. Volotovsky, N. Kim, Biosens. Bioelectron. 13 (1998) 1029. M.J. Eddowes, Sens. Actuators 11 (1987) 265. S.D. Caras, D. Petelenz, J. Janata, Anal. Chem. 57 (1985) 1920. M.J. Eddowes, Sens. Actuators 7 (1985) 97. T.T. Ngo, H.M. Lenhoff, Enzyme-Mediated Immunoassay, Plenum Press, New York, 1985, p. 446. R.B.M. Schasfoort, P. Bergveld, J. Bomer, R.P.H. Kooyman, Sens. Actuators 17 (1989) 531. H. Sakai, N.I. Kaneki, H. Hara, Anal. Chim. Acta 230 (1990) 189.
Immunosensor for the determination of the herbicide simazine based on an ion-selective field-effect transistor N.F. Starodub a,∗ , B.B. Dzantiev b , V.M. Starodub a , A.V. Zherdev b a
Department of Biochemistry and Sensoric System, A.V. Palladin’s Institute of Biochemistry of Ukrainian National Academy of Sciences, 9 Leontovicha Str., 252030 Kyiv, Ukraine b A.N. Bach Institute of Biochemistry, Russian Academy of Sciences, Leninsky prospect 33, 117071 Moscow, Russia
Received 23 March 2000; received in revised form 15 August 2000; accepted 15 August 2000
Abstract An immunosensor based on the ion-selective field effect transistor (ISFET) was developed for the express determination of herbicide simazine. Polyclonal antibodies against simazine were attached to the ISFET gate via staphylococcal protein A. Two methods of simazine detection were used — (i) competitive immune assay when native (detected) and peroxidase-labelled simazine molecules competed for binding with antibodies on the ISFET surface, and (ii) sequential saturation of antibodies, left unbound after their exposure to native simazine in investigated sample, with labelled simazine. The catalytic activity of bound peroxidase was measured in the presence of ascorbic acid and H2 O2 . The limit of simazine detection by competitive immune assay was 1.25 ng ml−1 , the linearity was observed in the range of 5–175 ng ml−1 . Sequential saturation of the antibodies led to the growth of the assay sensitivity up to 0.65 ng ml−1 . Acidic treatment of the ISFETs allowed using them several times without loss of the signal amplitude. The analysis had rapid kinetics, its overall time including duration of all preparation stages was around 50 min. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Immunosensor; ISFET; Herbicide; Simazine; Competitive immunoassay
1. Introduction Intensive and non-controlled agricultural application of herbicides and other classes of pesticides led to their wide spread in the environment. Due to pesticides extreme toxicity and their ability to induce different physiological disorders in humans and animals [1], the content of these substances in soil, water and food should be controlled. Biosensors are effective de∗ Corresponding author. Tel.: +380-44-229-4743; fax: +380-44-229-6365 E-mail address: [email protected] (N.F. Starodub).
vices for such analyses. Different types of biosensors were proposed for pesticide determination both for group-specific screening of toxic agents [2–5] and for control of individual compounds [6–13]. Application of immunochemical recognition allows reaching high specificity of biosensors [8,12]. Therefore, the combination of biosensor approach with antigen–antibody reaction seems to be rather suitable and promising since it can provide sensitive, fast, simple and inexpensive analyses. To fulfil all practical demands, optimal instrumental devices and biochemical protocols should be chosen. Silicon transducers, like ion-selective field effect transistors (ISFETs), are extremely perspective for bioassays. They can be manufactured by common
0003-2670/00/$ – see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 3 - 2 6 7 0 ( 0 0 ) 0 1 1 4 3 - 0
38
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technologies. Moreover, they combine measurements and preliminary processing of signals and also have other advantages [14,15]. Herbicide simazine (2-chloro-4,6-di(N-ethylamino)1,3,5-triazine) is one of most widely used compounds in agricultural practice of different countries [1]. Therefore, it was chosen as a model antigen for our investigations. The presented study was undertaken to apply the ISFET technique for simazine immunodetection.
2. Experimental 2.1. Materials 1 - Ethyl - 3(3 - dimethylaminopropyl)carbodiimide, dimethylformamide (both from ICN), N-hydroxysuccinimide (Sigma), 2,4,6-trinitrobenzensulfonic acid (CalBiochem), Triton X-100 (Serva), Tween-20, glutaraldehyde and ascorbic acid (Sigma) and 2,20 -azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS, Boehringer Mannheim) were used in the experiments. Purified preparations of simazine, its 2-ethylcarboxy- and 2-propylcarboxy- derivatives were generously provided by Dr. S.A. Eremin, Moscow State University, Russia. Bovine serum albumin (BSA, Minsk Institute of Epidemiology and Microbiology, Belorussia), hemocyanin from Paralithodes camtschatica (HC, generously provided by Dr. I.Yu. Sakharov, Moscow State University, Russia), soybean trypsin inhibitor (STI, Reanal), ovalbumin (OA, Serva) and horseradish peroxidase (HRP, RZ = A403 /A280 = 3.0, Biolar, Latvia) were used for syntheses of simazine–protein conjugates. 2.2. Syntheses of simazine–protein conjugates, obtaining of antiserum and analysis of its specificity The simazine–protein conjugates and the antiserum specific to simazine were obtained by immunisation of Chinchilla rabbits according to procedure described in [16]. Specificity of the antiserum was tested using closiest structural analogues of simazine, namely atrazine, propazine, promethrine, terbutylazine, amethrine, terbutrine, asiprotrine, cyanazine, semerone, methoprotrine, and deethylatrazine. The
antisera cross-reacted with atrazine (89%), terbutylazine (80%), and propazine (10%). Other analytes demonstrated cross-reaction in the range 0.7–6.2%. The antisera didn’t interact reliably with pesticides from other structural classes (such as pyrethroids or derivatives of phenoxyacetic acid); the cross-reactions for those analytes were less than 0.1%. 2.3. Preparation of the ISFET-based immunosensors Chips used in the experiments contained two ISFETs and a reference electrode. They were manufactured by the “Microprocessor” plant (Kyiv, Ukraine). pH sensitivity of the ISFETs was 52 mV per pH unit. The first (reference) ISFET was covered with the BSA, and the second (working) ISFET with the anti-simazine antiserum or firstly by the staphylococcal protein A (Sigma) and then by the antiserum. For attaching biomolecules to the ISFET surface glutaraldehyde (GA, Sigma) was used as cross-linking agent. It formed covalent bonds [17] between the silicon nitride surface of the ISFET gate and proteins being immobilised. The detailed protocol of the immobilisation was the following. The ISFET surface was cleaned by its sequential immersion into the mixture of potassium dichromate and sulphuric acid (10 s), water (vigorous washing), acetone, ethanol and PB (10 mM Na-phosphate buffer, pH 7.3, containing 100 mM NaCl). Then, by drop coating the 2.5% water solution of the GA was placed on the prepared surface. After 90 min at room temperature (25◦ C) the GA was placed again according to the same procedure. Then after 90 min from the second GA deposition, the chip was washed by distilled water. After this, BSA (20 g ml−1 in PB), or non-diluted antiserum, or protein A (20 g ml−1 in the PB) was placed on the gate surface of the ISFET and kept during 1 h at humid conditions. The treatment of the chip with the GA was carried out at the room temperature. At last, the chip was kept overnight at 4◦ C, and then, after vigorous washing with the PB, was immersed into the glycine solution (10 mg ml−1 in the PB and at the room temperature) in order to block non-reacted groups of the GA. After 30 min at the room temperature the ISFET was washed with the PB. When the protein A was immobilised, a step of the specific antibody binding was added. The antisimazine non-diluted antiserum was placed on the
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ISFET with the preliminary adsorbed protein A. The chip was incubated for 1 h in humid conditions and then washed with the PB. The prepared chips were stored in dry state at 4◦ C. 2.4. ISFET-based immunodetection of simazine Antigen–antibody interactions between immobilised antibodies and simazine were allowed in the PB. The level of the signal was recorded after the chip was immersed for 30 min into the solution of the HRP-labelled simazine. For the competitive assay the chip was immersed for 30 min into the solution containing both native and labelled simazine (Choice of reactants’ concentrations is described in the Section 3). After those incubations the chip was washed with the PhB and immersed into the HRP substrate solution. The measurements of the HRP activity were carried out in the working buffer (WB) containing 10 mM tris-HCl, 100 mM NaCl and 15 mM ascorbic acid (Sigma) as it was described in [18]. pH of the WB was 7.5. The hydrogen peroxide was added to the measuring cell (volume = 1 ml). The concentrations of above mentioned components of the WB were chosen according to the previous paper [18]. The concentration of H2 O2 was 5 or 10 mM. The signals (dV/dt) of the ISFETs were registered by electronic device (constructed at the Radiophysics Department of the Kiev National University named after Taras Shevchenko) providing signal amplification and its processing on the basis of a custom-made computer program. All measurements were undertaken at the room temperature. After every assay the chip was treated with the 0.1 M HCl (5 min) and carefully washed with the PhB.
3. Results and discussion The proposed immunosensor technique is based on the detection of the pH shift on the ISFET gate during HRP catalytic reaction. The substrate conversion causes a local basic pH shift, because dehydroascorbic acid formed is a more neutral compound compared to ascorbic acid [18]. Tests of pH shifts without native simazine molecules indicated that simazine–HRP conjugate (0.1 g ml−1 ,
39
by HRP) cannot be detected in the proposed system when antiserum has been directly attached to the ISFET gate. It was due to presence of a big concentration of non-IgG type proteins in antiserum which competed for aldehyde groups on the transducer surface. The signal was registered only when antibodies were immobilised via protein A. In this case IgG molecules specifically bound with protein A, and concentration of simazine specific IgG on the transducer surface sharply decreased. As can be seen from the Fig. 1, the pH shift occurred during 40–60 s after addition of H2 O2 . The type of sensor response is determined by the absence of electrolyte retaining membrane on the transducer surface. The enzyme was directly immobilised on the gate of surface of the ISFET via crosslinking with GA (see Section 2.3 of Section 2). We varied concentrations of the HRP–simazine conjugate in the range 0.1–0.6 g ml−1 (by the HRP). It was found that the maximal sensor output (about 95 mV) corresponded to 0.20 g ml−1 of the conjugate. Under these conditions binding sites of specific immunoglobulins were saturated. It should be noted that the sensor output depends on quantity of antigenbinding sites on the ISFET surface. Therefore, oriented immobilisation of antibodies via staphylococcal protein A (without GA treatment of the antibodies) is an effective way to reach higher signals. The concentration of the HRP-labelled simazine during the competitive detection of the simazine was equal to 0.25 g ml−1 (by the HRP). Change of the ISFET potential of the immunosensor as a function of the simazine concentration is presented in Fig. 2 (mean values from 8 to 10 measurements). Reliable decrease of the sensor signal was observed down to 1.25 ng ml−1 of simazine in the analysed mixture. The linearity of signal decrease was observed in the range of simazine concentrations from 5 to 175 ng ml−1 ; in this range the potential of the ISFET gate varied from 10 to 74 mV. The standard deviation was on average about 5%. It is necessary to mention a high reproducibility of results between different measurents by individual ISFETs. To achieve such reproducibility ISFETs obtained from the same silicone plate were used. Moreover, both the electrical characteristics of these transducers and the procedure for biological membrane preparation were standardised. In the latter
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Fig. 1. Typical response of the sensor at the addition of hydrogen peroxide. The ISFET potential is recorded as a function of time.
case, it was very important to fulfil exactly the protocol of cleaning and activation of gate surface as well as to provide constant humid conditions during 1 h of interaction of protein A with the activated groups of the gate surface. As a rule, all these processes were
fulfilled by the group’s technology, and no less than ten ISFETs were prepared simultaneously. Since ISFETs are very sensitive to different factors (such as buffer capacity, ionic strength, pH level, light irradiation, presence of ions affecting the charge of
Fig. 2. Change of the ISFET potential of the immunosensor as a function of simazine concentration (the competitive scheme).
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Fig. 3. Non-specific sensor response at the addition of hydrogen peroxide and WB. The ISFET potential is recorded as a function of time.
the gate surface, etc.) [19–21], their non-specific responses to addition of the WB or hydrogen peroxide were analysed. For this purpose a differential scheme was used: the first ISFET contained antibodies immobilised via protein A, the second had BSA immobilised via GA. It was shown (Fig. 3) that the WB did not influence the sensor output, while hydrogen peroxide in the concentrations mentioned above (10 mM) initiated small (<10 mV) responses. H2 O2 caused acidic pH shift in contrast to the basic changes during determination of the HRP activity. Thus neither WB nor H2 O2 contributed to the sensor’s basic pH shifts during the simazine recording. The study of optimal concentration of hydrogen peroxide indicated that the sensor output was about 15–20% higher for 10 mM of H2 O2 than for 5 mM of H2 O2 . We have not investigated the correlation between hydrogen peroxide concentration and level of the sensor signal yet. This will be task for our further research in this area, since we think that the optimisation of this step may improve the sensitivity of the proposed analysis. The overall time of the assay including the duration of all preparation stages was 50 min. The limiting stage of the analysis is the competition between the native and the HRP-labelled simazine for binding with the antibodies (30 min). We performed preliminary
experiments in which the duration of this stage was reduced to 10 min while the other assay conditions remained unchanged. In this case the sensor showed the sensitivity about 6.50 ng ml−1 which was more than five times lower than the sensitivity of the assay performed according to the procedures described above. Therefore, reduction of the assay time may lead to the decrease of its sensitivity. According to our preliminary investigations (data not shown) the sensitivity of the sensor can be increased by decrease of the WB capacity since according to existing data [21] the buffer capacity of the analysed sample has a significant influence on the sensor response. Another approach used to increase the sensitivity was so called saturation protocol of immunoassay [22]. The chip with immobilised antibodies was incubated with native simazine in investigated sample and then in order to saturate unbound antibodies with the HRP-labelled simazine (0.25 g ml−1 , by HRP). Both stages lasted 20 min. This approach allowed reaching sensitivity 0.65 ng ml−1 and linearity in the range 1.25–185 ng ml−1 (Fig. 4, mean values from 10 to 12 measurements). Destruction of antigen–antibody bonds by treatment of the chips with 0.1 M HCl for 5 min made it possible to reuse the chips for two to three measurements
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Fig. 4. Change of the ISFET potential of the immunosensor as a function of simazine concentration (the sequentional saturation scheme).
without signal decrease. Between the measurements chips were stored in dry state at 4◦ C. Before every new assay they were washed with PhB for 2 h. The proposed analytical system may be compared with other immunochemical approaches based on the ISFETs. Schasfoort et al. [23] immobilised antigens or antibodies on a porous membrane covering the ISFET gate. In this work stepwise change of electrolyte concentration produced a transport of ions through the membrane–protein layer resulting in a transient membrane potential, which was measured by the ISFET. The transport was determined by charge density in the protein layer, which changes upon formation of antigen–antibody complexes. The ISFET allowed detection of 10−6 M of antibodies. Sakai et al. [24] constructed a sensor by immobilising of human immunoglobulin G (IgG) on the ISFET surface. The assay involves a competitive binding of urease-labelled anti-IgG and IgG to be determined by membrane-bound IgG followed by electrochemical determination of the catalytic activity of bound urease. The detection limit of this sensor was 10 g ml−1 of IgG and the assay time 60 min. Comparison of these approaches with the sensor presented in this paper suggests that proposed variant has higher sensitivity and sometimes shorter assay time.
4. Conclusion A new ISFET-based immunosensor for the determination of herbicide simazine was created. Two schemes of the immunoassay were applied — competitive immune analysis and so called sequential saturation of antibodies. The activity of bound peroxidase was measured by basic pH shift of ascorbic acid solution after addition of hydrogene peroxide. The overall time of analysis was about 50 min. When the investigations were carried out according to the first scheme, the sensor allowed detecting simazine at concentrations up to 1.25 ng ml−1 . Linearity was observed in the range 5–175 ng ml−1 . Sequential saturation of antibodies, left unbound after their exposure to native simazine in investigated sample, with labelled simazine allowed to increase the sensitivity of the immune sensor to 0.65 ng ml−1 with the linearity in the range 1.25–185 ng ml−1 .
Acknowledgements This work was made possible by the Award PL979112 of the INCO-Copernicus Foundation. The authors are grateful to E.V. Yazynina from A.N. Bach
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Institute of Biochemistry (Moscow), who prepared and characterised simazine–protein conjugates. [9]
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