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Development and optimisation of biosensors based on pH-sensitive field effect transistors and cholinesterases for sensitive detection of solanaceous glycoalkaloids
Biosensors and Bioelectronics 18 (2003) 1047 /1053 www.elsevier.com/locate/bios
Development and optimisation of biosensors based on pH-sensitive field effect transistors and cholinesterases for sensitive detection of solanaceous glycoalkaloids Valentyna N. Arkhypova a,*, Sergei V. Dzyadevych a, Alexey P. Soldatkin a, Anna V. El’skaya a, Claude Martelet b, Nicole Jaffrezic-Renault b a
Laboratory of Biomolecular Electronics, Institute of Molecular Biology and Genetics, National Academy of Science of Ukraine, 150 Zabolotnogo Street, Kiev 03143, Ukraine b Ecole Centrale de Lyon, IFoS UMR 5621, BP 163, 69131 Ecully Cedex, France Received 12 April 2002; received in revised form 16 August 2002; accepted 20 September 2002
Abstract Highly sensitive biosensors based on pH-sensitive field effect transistors and cholinesterases for detection of solanaceous glycoalkaloids have been developed, characterised and optimised. The main analytical characteristics of the biosensors developed have been studied under different conditions and an optimal experimental protocol for glycoalkaloids determination in model solution has been proposed. Using such a biosensor and an enzyme reversible inhibition effect, the total potato glycoalkaloids content can be determined within the range of 0.2 /100 mM depending on the type of alkaloid, with lowest detection limits of 0.2 mM for a-chaconine, 0.5 mM for a-solanine and 1 mM for solanidine. The dynamic ranges for the compounds examined show that such biosensors are suitable for a quantitative detection of glycoalkaloids in real potato samples. High reproducibility, operational and storage stability of the biosensor developed have been shown. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Biosensor; pH-sensitive field effect transistor; Cholinesterases; Inhibition; Solanaceous glycoalkaloids
1. Introduction Potatoes, members of the Solanaceae plant family, serve as a major, inexpensive food source of both energy (starch) and good-quality protein. Its annual worldwide production is of about 350 million tons (FAO Production Yearbook, 1992). The US per capita potatoes consumption has been steadily increasing and is about 61 kg/year (Willard, 1993). Potatoes produce potentially toxic glycoalkaloids, both during growth and after harvest. In commercial cultivated potatoes, the primary compounds are a-solanine and a-chaconine, glycosides
* Corresponding author. Tel.: /380-44-266-07-49; fax: /380-44266-07-59. E-mail address: [email protected] (V.N. Arkhypova).
of the steroidal alkaloid solanidine. While there is some debate about their actual function in the plant, these and similar compounds have been shown to have toxic effect in humans (Ripakh and Kim, 1968; McMillan and Thompson, 1979). Glycoalkaloids appear to be more toxic for people than for animals. The toxicity may be due to their anticholinesterase activity towards the central nervous system and to cell membranes disruption affecting the digestive system and other organs. Thus, care must be taken in production of new potato varieties so that the level of glycoalkaloids did not exceed safe values. Glycoalkaloid poisoning associated with the central nervous system manifesting in such symptoms as fast and weak pulse, rapid and shallow breathing, delirium, and coma, is due to the glycoalkaloids ability to inhibit cholinesterase. The anticholinesterase activity of sola-
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naceous glycoalkaloids was first demonstrated by Pokrovskii (1956) and since has been confirmed by a number of workers both in vitro and in vivo (Orgell et al., 1958; Bushway et al., 1987; Roddick, 1989). Main solanaceous glycoalkaloids in potatoes are asolanine and a-chaconine, both triglycosides of solanidine, a steroidal alkaloid derived from cholesterol. Their molecular structures are shown on Scheme 1. Solanidine is a steroidal backbone without attached sugar moieties. Current methodology for the analysis of potato glycoalkaloids and related compounds has been extensively reviewed (Coxon, 1984; Friedman and McDonald, 1997). It includes: (1) colorimetry (Clement and Verbist, 1980); (2) thin layer chromatography (Ferreira et al., 1993); (3) gas chromatography (Herb et al., 1975); (4) high-performance liquid chromatography (Jonker et al., 1992; Crabbe and Fryer, 1980; Houben and Brunt, 1994); and (5) immunoassays (ELISA) (Morgan et al., 1983). The methods commonly used are complex and require extraction, separation and purification of samples before measurements. Several colorimetric and gas chromatographic methods include conversion of glycosides into aglycones prior to the analysis, other methods comprise the alkaloids. Besides, all these methods exploit an expensive and bulky instrumentation with high power consumption and need well-trained operators. Biosensors seem to be a very promising tool to overcome most of the problems described above. These novel analytical systems based on semiconductor structures attracted considerable attention since the innovative potential of microelectronics could be realized in new technologies adapted to the large-scale production of miniature devices such as biosensors. The biosensor research is ultimately aimed at the development of inexpensive, reliable and simple devices suitable for rapid, sensitive and selective analytical tests.
This paper describes a biosensor based on potentiometric pH-sensitive field effect transistor (pH-FET) including cholinesterases as a biorecognition element and using an enzyme inhibition phenomenon, for solanaceous glycoalkaloids determination. The ion-sensitive field effect transistors (ISFETs) were introduced by Bergveld (1970) and first used in a biosensor for penicillin detection by Caras and Janata (1980). Since then, a lot of articles have been published about biosensors based on ISFETs, especially on pHFETs: enzyme sensors (ENFET) (Karube et al., 1986; Dzyadevich et al., 1999; Mai Anh et al., 2002), cell sensors (Korpan et al., 1993), and immune sensors (Gotoh et al., 1987). The application of enzyme inhibition effect for biosensor creation has been already shown (Hart et al., 1997; Ivanov et al., 2000; Arkhypova et al., 2001), but all these sensors are based on an irreversible type of inhibition. The principle of operation of glycoalkaloid biosensor is based on following basic reactions (Scheme 2). These reactions result in proton generation, causing the pH change in membrane, which allows to use potentiometric method of measurements. Solanaceous glycoalkaloids inhibit both butyrylcholinesterase (BuChE) and acetylcholinesterase (AcChE) as shown in in vitro and in vivo studies (Orgell et al., 1958; Bushway et al., 1987; Roddick, 1989; Krasowski, 1997). The level of inhibition due to the action of glycoalkaloids can be evaluated by comparison of the biosensor responses before and after contact with a glycoalkaloids solution (Korpan et al., 2002). The main analytical characteristics of the biosensors developed were studied under different experimental conditions according to the recommended definitions and classification for electrochemical biosensors (The´venot et al., 2001).
2. Materials and methods
2.1. Materials
Scheme 1.
AcChE (EC 3.1.1.7, type VI-S: from Electric Eel) with a specific activity of 292 U/mg solid; BuChE (EC 3.1.1.8, from Horse Serum) with a specific activity of 13 U/mg solid; bovine albumin (fraction V, 98% purity), acetyl choline chloride (99% purity), butyryl choline chloride (BuChCl) (98% purity), glycoalkaloids (a-chaconine, asolanine and solanidine) from potato sprouts and glutaraldehyde (grade II, 25% aqueous solution) were purchased from Sigma-Aldrich Chemie GmbH (Steinheim, Germany). All reagents were of analytical grade and were used without any further specific treatment.
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Scheme 2.
2.2. Sensor design The ISFETs were fabricated at the Research Institute of Microdevices (Kiev, Ukraine). The potentiometric sensor chip contains identical Si3N4-ISFETs, the design and operation mode have been previously described (Frolov et al., 1993; Shul’ga et al., 1995). The ISFETs operated at a constant source current and drain-source voltage mode (Is /200 mA, Vds /1 V). The bare substrate of the sensor chip was used as a quasireference electrode. The pH-sensitivity of ISFETs was linear in the range from 2 to 12 pH with the slope of 30/ 40 mV/pH. 2.3. Enzyme immobilisation The biologically active membranes were formed by cross-linking of an enzyme with bovine albumin in a saturated glutaraldehyde vapour on the transducer surface (Dzyadevich et al., 1999; Arkhypova et al., 2001). The mixture containing 5% (w/v) enzyme, 5% (w/ v) bovine albumin, 10% (w/v) glycerol in 20 mM phosphate buffer (pH 7.4) was deposited on the sensitive surface of one transducer by the drop method, while the mixture containing 10% (w/v) bovine albumin and 10% (w/v) glycerol in 20 mM phosphate buffer (pH 7.4) was placed on the surface of the reference transducer. The use of glycerol prevents from a loss of enzyme activity during the immobilisation process, and provides better homogeneity of the membrane and better adhesion to the surface of the transducer. The sensor chip was then placed in a saturated glutaraldehyde vapour. After 30 min of exposure to glutaraldehyde, the membranes were dried at room temperature for 15 min.
stock solutions in water, and solutions of glycoalkaloids were prepared as 2 mM stock solutions in 5 mM acetic acid. Concentrations of substrates and inhibitors were adjusted by adding defined volumes of an appropriately concentrated stock solution. The differential output signal between the measuring and reference ISFET was registered using a laboratory ISFET-meter from Institute of Microtechnology (Neuchatel, Switzerland). After response measurement, the initial enzyme activity was restored by washing out the biosensor in the working buffer solution for 10/15 min. Multi-component ‘polymix’ buffer was prepared according to the following composition: 2.5 mM Tris, 2.5 mM KH2PO4, 2.5 mM citric acid, 2.5 mM sodium tetraborate, 150 mM NaCl. The pH of this buffer was adjusted by titration with either NaOH or HCl. The ‘polymix’ buffer has a stable buffer capacity over a wide range of pH including the range from 5 to 9. The influence of different pH values on the ENFET responses was studied using such ‘polymix’ buffer.
2.4. Measurements All measurements were performed in daylight at room temperature in an open glass vessel filled with a vigorously stirred 5 mM phosphate buffer solution. The solutions of substrates were prepared as 200 mM
Fig. 1. Dependence of the residual activity of the immobilised AcChE (1) and BuChE (2) on concentration of a-chaconine. The biosensor response to 1 mM substrate concentration was measured in 5 mM phosphate buffer, pH 7.4.
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3. Results and discussion The dependence of the residual activity of the immobilised BuChE and AcChE on the a-chaconine concentration is shown in Fig. 1. As depicted in this figure, BuChE biosensor is more sensitive to a-chaconine than AcChE biosensor. For other types of steroidal alkaloids such as a-solanine and solanidine the same results were obtained. In order to select optimal conditions for the glycoalkaloids determination, the responses of the BuChE biosensor to BuChCl adding without and with different glycoalkaloids in solution were measured. In Fig. 2 the calibration curve for BuChCl determination is presented along with dependencies of inhibition level of immobilised BuChE for different inhibitors. The level of inhibition was calculated as a relative decrease in the biosensor response after its contact with the inhibitor. From this figure it is clear that the best sensitivity and accuracy of measurements have been achieved for the BuChCl concentration of about 1 mM. For lower substrate concentrations, the enzyme in the membrane is in excess and involved into the substrate conversion according to the product reaction only partly (i.e. the rest of the enzyme does not participate in the enzymatic reaction). In this case, the enzyme molecules linked with inhibitor can be compensated by involving free enzyme molecules in the reaction. As a result, the experimental decrease of the biosensor response will be lower than the actual decrease of the enzyme activity due to the inhibition. This effect is typical for immobilised enzymes irrespective of either the inhibition mechanism or the system used for the detection of enzyme activity (Trevan, 1980). For high substrate concentration, both
Fig. 2. Calibration curve of potentiometric BuChE biosensor based on pH-ISFET for BuChCl determination (1), and dependence of inhibition level of immobilised BuChE on substrate concentration for 20 mM a-chaconine (2), a-solanine (3) and solanidine (4) in 5 mM phosphate buffer, pH 7.4.
substrate and inhibitor interact with the immobilised enzyme simultaneously, and the sensitivity toward the inhibitor decreases with an increase in the substrate concentration. This features only reversible mechanism of inhibition. Finally, the 1 mM concentration of BuChCl was used in further experiments. It has been also shown that inhibition does not depend on duration of the biosensor contact with glycoalkaloids. For the 1/ 30 min period of biosensor incubation with glycoalkaloids no changes in inhibition level were observed. It is well known that the choice of a buffer may influence the enzyme activity. pH-dependence of the immobilised BuChE activity toward the substrate and inhibitor was studied in a ‘polymix’ buffer solution. This complex buffer was selected in this work to avoid any influence of buffer capacity on the sensor response, as such buffer demonstrated the same buffer capacity in a broad range of pH*/from 5 to 9. Fig. 3 shows the BuChE biosensor response to the injection of 1 mM BuChCl and the enzyme inhibition level after contact with a-chaconine at different pH values. The highest response was obtained for buffer solution about pH 7.2, whereas inhibition level did not depend on solution pH in the detected pH range. These data are in good agreement with the results on cholinesterase activity toward the substrate and glycoalkaloids obtained by other authors (Imato and Ishibashi, 1995; Roddick, 1989), but differ from the results of high-pH-enhancement of disruption of membranes by glycoalkaloids (McKee, 1959; Roddick and Rijnenberg, 1986). In all following experiments an optimal pH toward the substrate was used for measurements to obtain the best resolution for further determination of inhibitors. Other important characteristics of the biosensor are the dependence of its response on the buffer concentra-
Fig. 3. Dependence of biosensor response for 1 mM BuChCl (1) and following inhibition levels of immobilised BuChE for 1 mM (2), 5 mM (3) and 25 mM (4) a-chaconine on pH. Measurements were conducted in 2.5 mM ‘polymix’ buffer.
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Fig. 4. Dependence of biosensor response for 1 mM BuChCl (1) and following inhibition levels of immobilised BuChE for 2 mM (2) and 5 mM (3) a-chaconine on buffer concentrations, pH 7.2.
Fig. 5. Dependence of biosensor response for 1 mM BuChCl (1) and following inhibition level of immobilised BuChE for 2 mM a-chaconine (2) on NaCl concentrations in 5 mM phosphate buffer, pH 7.2.
tion and ionic strength of solution. The results presented in Fig. 4 and Fig. 5 show that output signals of the potentiometric biosensor to the substrate addition are suppressed at increasing buffer and NaCl concentration. The increase in phosphate buffer concentration from 2 to 20 mM, and increase in salt concentration up to 200 mM result in an 8/9-fold decrease in the output signal. This phenomenon is well known for enzyme biosensors and can be associated with the increasing of buffer capacity and ionic strength of solution (Dzyadevich et al., 1999). At the same time, the inhibition level does not strongly depend on the buffer and NaCl concentration, unless slight increase. Therefore, the variation of buffer and NaCl concentration is not an important parameter for the glycoalkaloids determination and does not influence the inhibition level. That is why in further
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measurements the 5 mM phosphate buffer, pH 7.2, without NaCl addition was selected. There are several approaches to an evaluation of the enzyme inhibition level, which correspond to different experimental procedures. The typical response curves of biosensor versus BuChCl and a-solanine in the buffer solution were obtained due to two different protocols of measurements and then compared (Fig. 6). In the first case, first the substrate was added, and then the response was measured. After the steady-state response stabilisation the inhibitor was added, and inhibition effect was measured. In the second case, the substrate was first mixed with the inhibitor, then an appropriate volume of the mixture was added, and the response was measured. As can be seen from the figure, the final levels of signal for both cases are the same, thus either protocol can be used. It is well known that at least 95% of all glycoalkaloids in commercial potatoes are a-solanine and a-chaconine. Fig. 7 presents the calibration curves for the detection of such glycoalkaloids and its aglycone solanidine. As can be seen, the total potato glycoalkaloids can be detected within the range of 0.2 /100 mM depending on the type of alkaloid, with detection limits of 0.2 mM for achaconine, 0.5 mM for a-solanine and 1 mM for solanidine. IUPAC has recommended that the detection limit is the smallest concentration that the analyst can expect to detect with a given degree of confidence. According to this definition, the detection limit was determinate as follows: DL/3sb/S, where sb is standard deviation of the blank and S is the sensitivity (expressed as slope of the calibration curve). The dynamic ranges for the compounds examined show that such biosensors are suitable for a quantitative detection glycoalkaloids in potato samples. The biosensor practicability is often limited by its reproducibility, operational and storage stability. These characteristics were therefore investigated. Combined test of reproducibility, operational and storage stability for the biosensor developed shows very good results (Fig. 8). In this investigation the reproducibility and operational stability was analysed during 1 day or some hours, then the biosensor was stored in the working buffer solution at room temperature for a night, and after this was tested again. This procedure was repeated during several days. The responses of the BuChE biosensor based on pH-ISFET were reproducible; the relative standard deviation was about 3%. A test of operational stability demonstrated that biosensor responses remain stable with a drift of about 1% per day, when the evolution was monitored during operational conditions. Furthermore, the storage stability in a 5 mM phosphate buffer, pH 7.2 at 4 8C was quite good, the biosensor responses remaining stable for more than 3 months.
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Fig. 6. Typical response curves of BuChE biosensor for two different protocols of measurements. Measurements conditions: base line is drawn in 5 mM phosphate buffer, pH 7.2; arrows indicate the points of different aliquots of BuChCl and a-solanine adding.
4. Conclusions
Fig. 7. Calibration curves for the detection of a-chaconine (1), asolanine (2) and solanidine (3) by pH-ISFET-based biosensor. Measurements were conducted in 5 mM phosphate buffer, pH 7.2, and 1 mM BuChCl.
Fig. 8. Reproducibility, operational and storage stability of the BuChE biosensor based on pH-ISFET. Measurements were conducted in 5 mM phosphate buffer, pH 7.2, and 1 mM BuChCl.
The results presented in this work demonstrate the feasibility of butyryl cholinesterase biosensors based on pH-FETs for sensitive detection of solanaceous glycoalkaloids. The sensor proposed is rather cheap, easy in operation and requires short analysis time compared with the traditional methods of glycoalkaloids evaluation. The main analytical characteristics of the biosensors developed were studied in model solution under different conditions. In comparison with biosensors for glycoalkaloids determination, the practical application of cholinesterase biosensors for pesticide determination has significant limitations. The irreversible inhibition measurements result in the decrease in biosensor responses so that the lifetime of a biosensor is limited to 10 /20 measurements irrespective of the real stability of the immobilised enzyme. The necessity of permanent reloading or reactivation of an enzymatic layer complicates the operation of a biosensor and reduces the reproducibility of the results obtained. In the case of glycoalkaloids determination these problems were avoided because another type of enzyme inhibition was used. The glycoalkaloids biosensors developed can be a success in different fields of human activity for (i) monitoring alkaloids content in breeding lines and potential commercial varieties to avoid exceeding the alkaloids concentrations safe for consumption, (ii) choosing the appropriate cultivation conditions and post-harvest treatments to minimise the alkaloids level, and (iii) establishing and developing the alkaloids possessing insecticide, antimicrobial and fungitoxic activity. The results concerning the suitability of the biosensors developed for determination of glycoalkaloids in different potatoes will be published in the nearest future.
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Acknowledgements This work was supported by an INTAS Grant 0000151, a NATO Grant LST.CLG.977342 and PostDoc position from ‘Grand Lyon’ (to VNA).
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Imato, T., Ishibashi, N., 1995. Potentiometric butyrylcholine sensor for organo-phosphate pesticides. Biosensors Bioelectron. 10, 435 / 441. Ivanov, A.N., Evtugyn, G.A., Gyurcsa´nyi, R.E., To´th, K., Budnikov, H.C., 2000. Comparative investigation of electrochemical cholinesterase biosensors for pesticide determination. Anal. Chim. Acta 404, 55 /65. Jonker, H.H., Koops, A.J., HoogenDoorn, J.C., 1992. A rapid method for the quantification of steroidal glycoalkaloids by reversed phase HPLC. Potato Res. 35, 451 /455. Karube, I., Tamiya, E., Dicks, J.M., 1986. A microsensor for urea based on ISFET. Anal. Chim. Acta 185, 195 /200. Korpan, Y.I., Soldatkin, A.P., Starodub, N.F., El’skaya, A.V., Gonchar, M.V., Sibirny, A.A., Shul’ga, A.A., 1993. Methylotrophic yeast microbiosensor based on ion-sensitive field effect transistors for methanol and ethanol determination. Anal. Chim. Acta 371, 203 /208. Korpan, Y.I., Volotovsky, V.V., Martelet, C., Jaffrezic-Renault, N., Nazarenko, E.A., El’skaya, A.V., Soldatkin, A.P., 2002. A novel enzyme biosensor for steroidal glycoalkaloids detection based on pH-sensitive field effect transistors. Bioelectrochemistry 55, 9 /11. Krasowski, M.D., 1997. Natural inhibitors of cholinesterases: implications for adverse drug reactions. Can. J. Anaesth. 44, 525 /534. Mai Anh, T., Dzyadevych, S.V., Soldatkin, A.P., Duc Chien, N., Jaffrezic-Renault, N., Chovelon, J.-M., 2002. Development of tyrosinase biosensor based on pH-sensitive field-effect transistors for phenols determination in water solutions. Talanta 56, 627 /634. McKee, R., 1959. Factors affecting the toxicity of solanine and related alkaloids to fusarium caeruleum. J. Gen. Microbiol. 20, 686 /696. McMillan, M., Thompson, J.C., 1979. An outbreak of suspected solanine poisoning in schoolboys: examination of criteria of solanine poisoning. Quat. J. Med. 48, 227 /243. Morgan, M.R.A., McNerney, R., Coxon, D.T., Chan, H.W.-S., 1983. An enzyme-linked immunosorbent assay for total glycoalkaloids in potato tubers. J. Sci. Food Agric. 34, 593 /598. Orgell, W.H., Vaidya, K.A., Dahm, P.A., 1958. Inhibition of human plasma cholinesterase in vitro by extracts of solanaceous plants. Science 128, 1136 /1137. Pokrovskii, A.A., 1956. The effect of the alkaloids of the sprouting potato on cholinesterase. Biokhimiya 21, 705 /710. Ripakh, L.A., Kim, A., 1968. Case of mass poisoning by solanine. Sovetskaia Meditsina 22, 129 /131. Roddick, J.G., Rijnenberg, A.L., 1986. Effect of steroidal glycoalkaloids of potato on the permeability of liposome membranes. Phisiol. Plantarum. 68, 436 /440. Roddick, J.G., 1989. The acetylcholinesterase-inhibitory activity of steroidal glycoalkaloids and their aglycones. Phytochemistry 28, 2631 /2634. Shul’ga, A.A., Netchiporouk, L.I., Sandrovsky, A.K., Abalov, A.A., Frolov, O.S., Kononenko, Y.u.G., Maupas, H., Martelet, C., 1995. Operation of an ISFET with non-insulated substrate directly exposed to the solution. Sensors Actuators 30, 101 /105. The´venot, D.R., Toth, K., Durst, R.A., Wilson, G.S., 2001. Electrochemical biosensors: recommended definitions and classification. Biosensors Bioelectron. 16, 121 /131. Trevan, M.D., 1980. Immobilized Enzymes: an Introduction and Applications in Biotechnology. Wiley, Chichester. Willard, M., 1993. Potato processing: past, present and future. Am. Potato J. 70, 405 /418.
Development and optimisation of biosensors based on pH-sensitive field effect transistors and cholinesterases for sensitive detection of solanaceous glycoalkaloids Valentyna N. Arkhypova a,*, Sergei V. Dzyadevych a, Alexey P. Soldatkin a, Anna V. El’skaya a, Claude Martelet b, Nicole Jaffrezic-Renault b a
Laboratory of Biomolecular Electronics, Institute of Molecular Biology and Genetics, National Academy of Science of Ukraine, 150 Zabolotnogo Street, Kiev 03143, Ukraine b Ecole Centrale de Lyon, IFoS UMR 5621, BP 163, 69131 Ecully Cedex, France Received 12 April 2002; received in revised form 16 August 2002; accepted 20 September 2002
Abstract Highly sensitive biosensors based on pH-sensitive field effect transistors and cholinesterases for detection of solanaceous glycoalkaloids have been developed, characterised and optimised. The main analytical characteristics of the biosensors developed have been studied under different conditions and an optimal experimental protocol for glycoalkaloids determination in model solution has been proposed. Using such a biosensor and an enzyme reversible inhibition effect, the total potato glycoalkaloids content can be determined within the range of 0.2 /100 mM depending on the type of alkaloid, with lowest detection limits of 0.2 mM for a-chaconine, 0.5 mM for a-solanine and 1 mM for solanidine. The dynamic ranges for the compounds examined show that such biosensors are suitable for a quantitative detection of glycoalkaloids in real potato samples. High reproducibility, operational and storage stability of the biosensor developed have been shown. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Biosensor; pH-sensitive field effect transistor; Cholinesterases; Inhibition; Solanaceous glycoalkaloids
1. Introduction Potatoes, members of the Solanaceae plant family, serve as a major, inexpensive food source of both energy (starch) and good-quality protein. Its annual worldwide production is of about 350 million tons (FAO Production Yearbook, 1992). The US per capita potatoes consumption has been steadily increasing and is about 61 kg/year (Willard, 1993). Potatoes produce potentially toxic glycoalkaloids, both during growth and after harvest. In commercial cultivated potatoes, the primary compounds are a-solanine and a-chaconine, glycosides
* Corresponding author. Tel.: /380-44-266-07-49; fax: /380-44266-07-59. E-mail address: [email protected] (V.N. Arkhypova).
of the steroidal alkaloid solanidine. While there is some debate about their actual function in the plant, these and similar compounds have been shown to have toxic effect in humans (Ripakh and Kim, 1968; McMillan and Thompson, 1979). Glycoalkaloids appear to be more toxic for people than for animals. The toxicity may be due to their anticholinesterase activity towards the central nervous system and to cell membranes disruption affecting the digestive system and other organs. Thus, care must be taken in production of new potato varieties so that the level of glycoalkaloids did not exceed safe values. Glycoalkaloid poisoning associated with the central nervous system manifesting in such symptoms as fast and weak pulse, rapid and shallow breathing, delirium, and coma, is due to the glycoalkaloids ability to inhibit cholinesterase. The anticholinesterase activity of sola-
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naceous glycoalkaloids was first demonstrated by Pokrovskii (1956) and since has been confirmed by a number of workers both in vitro and in vivo (Orgell et al., 1958; Bushway et al., 1987; Roddick, 1989). Main solanaceous glycoalkaloids in potatoes are asolanine and a-chaconine, both triglycosides of solanidine, a steroidal alkaloid derived from cholesterol. Their molecular structures are shown on Scheme 1. Solanidine is a steroidal backbone without attached sugar moieties. Current methodology for the analysis of potato glycoalkaloids and related compounds has been extensively reviewed (Coxon, 1984; Friedman and McDonald, 1997). It includes: (1) colorimetry (Clement and Verbist, 1980); (2) thin layer chromatography (Ferreira et al., 1993); (3) gas chromatography (Herb et al., 1975); (4) high-performance liquid chromatography (Jonker et al., 1992; Crabbe and Fryer, 1980; Houben and Brunt, 1994); and (5) immunoassays (ELISA) (Morgan et al., 1983). The methods commonly used are complex and require extraction, separation and purification of samples before measurements. Several colorimetric and gas chromatographic methods include conversion of glycosides into aglycones prior to the analysis, other methods comprise the alkaloids. Besides, all these methods exploit an expensive and bulky instrumentation with high power consumption and need well-trained operators. Biosensors seem to be a very promising tool to overcome most of the problems described above. These novel analytical systems based on semiconductor structures attracted considerable attention since the innovative potential of microelectronics could be realized in new technologies adapted to the large-scale production of miniature devices such as biosensors. The biosensor research is ultimately aimed at the development of inexpensive, reliable and simple devices suitable for rapid, sensitive and selective analytical tests.
This paper describes a biosensor based on potentiometric pH-sensitive field effect transistor (pH-FET) including cholinesterases as a biorecognition element and using an enzyme inhibition phenomenon, for solanaceous glycoalkaloids determination. The ion-sensitive field effect transistors (ISFETs) were introduced by Bergveld (1970) and first used in a biosensor for penicillin detection by Caras and Janata (1980). Since then, a lot of articles have been published about biosensors based on ISFETs, especially on pHFETs: enzyme sensors (ENFET) (Karube et al., 1986; Dzyadevich et al., 1999; Mai Anh et al., 2002), cell sensors (Korpan et al., 1993), and immune sensors (Gotoh et al., 1987). The application of enzyme inhibition effect for biosensor creation has been already shown (Hart et al., 1997; Ivanov et al., 2000; Arkhypova et al., 2001), but all these sensors are based on an irreversible type of inhibition. The principle of operation of glycoalkaloid biosensor is based on following basic reactions (Scheme 2). These reactions result in proton generation, causing the pH change in membrane, which allows to use potentiometric method of measurements. Solanaceous glycoalkaloids inhibit both butyrylcholinesterase (BuChE) and acetylcholinesterase (AcChE) as shown in in vitro and in vivo studies (Orgell et al., 1958; Bushway et al., 1987; Roddick, 1989; Krasowski, 1997). The level of inhibition due to the action of glycoalkaloids can be evaluated by comparison of the biosensor responses before and after contact with a glycoalkaloids solution (Korpan et al., 2002). The main analytical characteristics of the biosensors developed were studied under different experimental conditions according to the recommended definitions and classification for electrochemical biosensors (The´venot et al., 2001).
2. Materials and methods
2.1. Materials
Scheme 1.
AcChE (EC 3.1.1.7, type VI-S: from Electric Eel) with a specific activity of 292 U/mg solid; BuChE (EC 3.1.1.8, from Horse Serum) with a specific activity of 13 U/mg solid; bovine albumin (fraction V, 98% purity), acetyl choline chloride (99% purity), butyryl choline chloride (BuChCl) (98% purity), glycoalkaloids (a-chaconine, asolanine and solanidine) from potato sprouts and glutaraldehyde (grade II, 25% aqueous solution) were purchased from Sigma-Aldrich Chemie GmbH (Steinheim, Germany). All reagents were of analytical grade and were used without any further specific treatment.
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Scheme 2.
2.2. Sensor design The ISFETs were fabricated at the Research Institute of Microdevices (Kiev, Ukraine). The potentiometric sensor chip contains identical Si3N4-ISFETs, the design and operation mode have been previously described (Frolov et al., 1993; Shul’ga et al., 1995). The ISFETs operated at a constant source current and drain-source voltage mode (Is /200 mA, Vds /1 V). The bare substrate of the sensor chip was used as a quasireference electrode. The pH-sensitivity of ISFETs was linear in the range from 2 to 12 pH with the slope of 30/ 40 mV/pH. 2.3. Enzyme immobilisation The biologically active membranes were formed by cross-linking of an enzyme with bovine albumin in a saturated glutaraldehyde vapour on the transducer surface (Dzyadevich et al., 1999; Arkhypova et al., 2001). The mixture containing 5% (w/v) enzyme, 5% (w/ v) bovine albumin, 10% (w/v) glycerol in 20 mM phosphate buffer (pH 7.4) was deposited on the sensitive surface of one transducer by the drop method, while the mixture containing 10% (w/v) bovine albumin and 10% (w/v) glycerol in 20 mM phosphate buffer (pH 7.4) was placed on the surface of the reference transducer. The use of glycerol prevents from a loss of enzyme activity during the immobilisation process, and provides better homogeneity of the membrane and better adhesion to the surface of the transducer. The sensor chip was then placed in a saturated glutaraldehyde vapour. After 30 min of exposure to glutaraldehyde, the membranes were dried at room temperature for 15 min.
stock solutions in water, and solutions of glycoalkaloids were prepared as 2 mM stock solutions in 5 mM acetic acid. Concentrations of substrates and inhibitors were adjusted by adding defined volumes of an appropriately concentrated stock solution. The differential output signal between the measuring and reference ISFET was registered using a laboratory ISFET-meter from Institute of Microtechnology (Neuchatel, Switzerland). After response measurement, the initial enzyme activity was restored by washing out the biosensor in the working buffer solution for 10/15 min. Multi-component ‘polymix’ buffer was prepared according to the following composition: 2.5 mM Tris, 2.5 mM KH2PO4, 2.5 mM citric acid, 2.5 mM sodium tetraborate, 150 mM NaCl. The pH of this buffer was adjusted by titration with either NaOH or HCl. The ‘polymix’ buffer has a stable buffer capacity over a wide range of pH including the range from 5 to 9. The influence of different pH values on the ENFET responses was studied using such ‘polymix’ buffer.
2.4. Measurements All measurements were performed in daylight at room temperature in an open glass vessel filled with a vigorously stirred 5 mM phosphate buffer solution. The solutions of substrates were prepared as 200 mM
Fig. 1. Dependence of the residual activity of the immobilised AcChE (1) and BuChE (2) on concentration of a-chaconine. The biosensor response to 1 mM substrate concentration was measured in 5 mM phosphate buffer, pH 7.4.
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3. Results and discussion The dependence of the residual activity of the immobilised BuChE and AcChE on the a-chaconine concentration is shown in Fig. 1. As depicted in this figure, BuChE biosensor is more sensitive to a-chaconine than AcChE biosensor. For other types of steroidal alkaloids such as a-solanine and solanidine the same results were obtained. In order to select optimal conditions for the glycoalkaloids determination, the responses of the BuChE biosensor to BuChCl adding without and with different glycoalkaloids in solution were measured. In Fig. 2 the calibration curve for BuChCl determination is presented along with dependencies of inhibition level of immobilised BuChE for different inhibitors. The level of inhibition was calculated as a relative decrease in the biosensor response after its contact with the inhibitor. From this figure it is clear that the best sensitivity and accuracy of measurements have been achieved for the BuChCl concentration of about 1 mM. For lower substrate concentrations, the enzyme in the membrane is in excess and involved into the substrate conversion according to the product reaction only partly (i.e. the rest of the enzyme does not participate in the enzymatic reaction). In this case, the enzyme molecules linked with inhibitor can be compensated by involving free enzyme molecules in the reaction. As a result, the experimental decrease of the biosensor response will be lower than the actual decrease of the enzyme activity due to the inhibition. This effect is typical for immobilised enzymes irrespective of either the inhibition mechanism or the system used for the detection of enzyme activity (Trevan, 1980). For high substrate concentration, both
Fig. 2. Calibration curve of potentiometric BuChE biosensor based on pH-ISFET for BuChCl determination (1), and dependence of inhibition level of immobilised BuChE on substrate concentration for 20 mM a-chaconine (2), a-solanine (3) and solanidine (4) in 5 mM phosphate buffer, pH 7.4.
substrate and inhibitor interact with the immobilised enzyme simultaneously, and the sensitivity toward the inhibitor decreases with an increase in the substrate concentration. This features only reversible mechanism of inhibition. Finally, the 1 mM concentration of BuChCl was used in further experiments. It has been also shown that inhibition does not depend on duration of the biosensor contact with glycoalkaloids. For the 1/ 30 min period of biosensor incubation with glycoalkaloids no changes in inhibition level were observed. It is well known that the choice of a buffer may influence the enzyme activity. pH-dependence of the immobilised BuChE activity toward the substrate and inhibitor was studied in a ‘polymix’ buffer solution. This complex buffer was selected in this work to avoid any influence of buffer capacity on the sensor response, as such buffer demonstrated the same buffer capacity in a broad range of pH*/from 5 to 9. Fig. 3 shows the BuChE biosensor response to the injection of 1 mM BuChCl and the enzyme inhibition level after contact with a-chaconine at different pH values. The highest response was obtained for buffer solution about pH 7.2, whereas inhibition level did not depend on solution pH in the detected pH range. These data are in good agreement with the results on cholinesterase activity toward the substrate and glycoalkaloids obtained by other authors (Imato and Ishibashi, 1995; Roddick, 1989), but differ from the results of high-pH-enhancement of disruption of membranes by glycoalkaloids (McKee, 1959; Roddick and Rijnenberg, 1986). In all following experiments an optimal pH toward the substrate was used for measurements to obtain the best resolution for further determination of inhibitors. Other important characteristics of the biosensor are the dependence of its response on the buffer concentra-
Fig. 3. Dependence of biosensor response for 1 mM BuChCl (1) and following inhibition levels of immobilised BuChE for 1 mM (2), 5 mM (3) and 25 mM (4) a-chaconine on pH. Measurements were conducted in 2.5 mM ‘polymix’ buffer.
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Fig. 4. Dependence of biosensor response for 1 mM BuChCl (1) and following inhibition levels of immobilised BuChE for 2 mM (2) and 5 mM (3) a-chaconine on buffer concentrations, pH 7.2.
Fig. 5. Dependence of biosensor response for 1 mM BuChCl (1) and following inhibition level of immobilised BuChE for 2 mM a-chaconine (2) on NaCl concentrations in 5 mM phosphate buffer, pH 7.2.
tion and ionic strength of solution. The results presented in Fig. 4 and Fig. 5 show that output signals of the potentiometric biosensor to the substrate addition are suppressed at increasing buffer and NaCl concentration. The increase in phosphate buffer concentration from 2 to 20 mM, and increase in salt concentration up to 200 mM result in an 8/9-fold decrease in the output signal. This phenomenon is well known for enzyme biosensors and can be associated with the increasing of buffer capacity and ionic strength of solution (Dzyadevich et al., 1999). At the same time, the inhibition level does not strongly depend on the buffer and NaCl concentration, unless slight increase. Therefore, the variation of buffer and NaCl concentration is not an important parameter for the glycoalkaloids determination and does not influence the inhibition level. That is why in further
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measurements the 5 mM phosphate buffer, pH 7.2, without NaCl addition was selected. There are several approaches to an evaluation of the enzyme inhibition level, which correspond to different experimental procedures. The typical response curves of biosensor versus BuChCl and a-solanine in the buffer solution were obtained due to two different protocols of measurements and then compared (Fig. 6). In the first case, first the substrate was added, and then the response was measured. After the steady-state response stabilisation the inhibitor was added, and inhibition effect was measured. In the second case, the substrate was first mixed with the inhibitor, then an appropriate volume of the mixture was added, and the response was measured. As can be seen from the figure, the final levels of signal for both cases are the same, thus either protocol can be used. It is well known that at least 95% of all glycoalkaloids in commercial potatoes are a-solanine and a-chaconine. Fig. 7 presents the calibration curves for the detection of such glycoalkaloids and its aglycone solanidine. As can be seen, the total potato glycoalkaloids can be detected within the range of 0.2 /100 mM depending on the type of alkaloid, with detection limits of 0.2 mM for achaconine, 0.5 mM for a-solanine and 1 mM for solanidine. IUPAC has recommended that the detection limit is the smallest concentration that the analyst can expect to detect with a given degree of confidence. According to this definition, the detection limit was determinate as follows: DL/3sb/S, where sb is standard deviation of the blank and S is the sensitivity (expressed as slope of the calibration curve). The dynamic ranges for the compounds examined show that such biosensors are suitable for a quantitative detection glycoalkaloids in potato samples. The biosensor practicability is often limited by its reproducibility, operational and storage stability. These characteristics were therefore investigated. Combined test of reproducibility, operational and storage stability for the biosensor developed shows very good results (Fig. 8). In this investigation the reproducibility and operational stability was analysed during 1 day or some hours, then the biosensor was stored in the working buffer solution at room temperature for a night, and after this was tested again. This procedure was repeated during several days. The responses of the BuChE biosensor based on pH-ISFET were reproducible; the relative standard deviation was about 3%. A test of operational stability demonstrated that biosensor responses remain stable with a drift of about 1% per day, when the evolution was monitored during operational conditions. Furthermore, the storage stability in a 5 mM phosphate buffer, pH 7.2 at 4 8C was quite good, the biosensor responses remaining stable for more than 3 months.
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Fig. 6. Typical response curves of BuChE biosensor for two different protocols of measurements. Measurements conditions: base line is drawn in 5 mM phosphate buffer, pH 7.2; arrows indicate the points of different aliquots of BuChCl and a-solanine adding.
4. Conclusions
Fig. 7. Calibration curves for the detection of a-chaconine (1), asolanine (2) and solanidine (3) by pH-ISFET-based biosensor. Measurements were conducted in 5 mM phosphate buffer, pH 7.2, and 1 mM BuChCl.
Fig. 8. Reproducibility, operational and storage stability of the BuChE biosensor based on pH-ISFET. Measurements were conducted in 5 mM phosphate buffer, pH 7.2, and 1 mM BuChCl.
The results presented in this work demonstrate the feasibility of butyryl cholinesterase biosensors based on pH-FETs for sensitive detection of solanaceous glycoalkaloids. The sensor proposed is rather cheap, easy in operation and requires short analysis time compared with the traditional methods of glycoalkaloids evaluation. The main analytical characteristics of the biosensors developed were studied in model solution under different conditions. In comparison with biosensors for glycoalkaloids determination, the practical application of cholinesterase biosensors for pesticide determination has significant limitations. The irreversible inhibition measurements result in the decrease in biosensor responses so that the lifetime of a biosensor is limited to 10 /20 measurements irrespective of the real stability of the immobilised enzyme. The necessity of permanent reloading or reactivation of an enzymatic layer complicates the operation of a biosensor and reduces the reproducibility of the results obtained. In the case of glycoalkaloids determination these problems were avoided because another type of enzyme inhibition was used. The glycoalkaloids biosensors developed can be a success in different fields of human activity for (i) monitoring alkaloids content in breeding lines and potential commercial varieties to avoid exceeding the alkaloids concentrations safe for consumption, (ii) choosing the appropriate cultivation conditions and post-harvest treatments to minimise the alkaloids level, and (iii) establishing and developing the alkaloids possessing insecticide, antimicrobial and fungitoxic activity. The results concerning the suitability of the biosensors developed for determination of glycoalkaloids in different potatoes will be published in the nearest future.
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Acknowledgements This work was supported by an INTAS Grant 0000151, a NATO Grant LST.CLG.977342 and PostDoc position from ‘Grand Lyon’ (to VNA).
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