Potato glycoalkaloids detection based on conductometric sensor coupled to butyryl cholinesterase

For the construction of a biosensor sensitive to some steroidal glycoalkaloids a conductometric planar electrode as a transducer and the horse serum butyryl cholinesterase (BuChE) as a biorecognition element have been used. It lias been shown that a-solanine, a-chaconine and solanidine can be detected within the range of their concentrations from 0.2 to 100 /гМ depending on the type of steroidal alkaloid used. The detection limits were estimated to be 0.2 for chaconine and 0.5 juM for solanine I solanidine. The responses of the sensors developed were reproducible: the relative standard deviation was about 1.5 % and 5 % for intraand inter-sensor responses, respectively. Moreover, one sensor with the immobilized enzyme can be used repeatedly (for at least J00 measurements) after a simple washing by buffer and can be stored at room temperature without substantial loss in activity of the immobilized enzyme not less than 1 month. It has been shown that all the analytes investigated inhibit reversibly and competitively the horse BuChE immobilized on the transducer surface. In assays, which combined a-solanine and a-chaconine, inhibition of BuChE was not additive. A possibility to apply the biosensor developed for the quantitative detection of the total steroidal alkaloids pool in foodstuffs and some biological samples is discussed.


For the construction of a biosensor sensitive to some steroidal glycoalkaloids a conductometric planar electrode as a transducer and the horse serum butyryl cholinesterase (BuChE) as a biorecognition element have been used. It lias been shown that a-solanine, a-chaconine and solanidine can be detected within the range of their concentrations from 0.2 to 100 /гМ depending on the type of steroidal alkaloid used. The detection limits were estimated to be 0.2 for chaconine and 0.5 juM for solanine I solanidine. The responses of the sensors developed were reproducible: the relative standard deviation was about 1.5 % and 5 % for intra-and inter-sensor responses, respectively. Moreover, one sensor with the immobilized enzyme can be used repeatedly (for at least J00 measurements) after a simple washing by buffer and can be stored at room temperature without substantial loss in activity of the immobilized enzyme not less than 1 month. It has been shown that all the analytes investigated inhibit reversibly and competitively the horse BuChE immobilized on the transducer surface. In assays, which combined a-solanine and a-chaconine, inhibition of BuChE was not additive. A possibility to apply the biosensor developed for the quantitative detection of the total steroidal alkaloids pool in foodstuffs and some biological samples is discussed.
Introduction.The cultivated potato (Solanum tubero sum L.), as one of the world's major agricultural crops, is consumed daily by millions of people from diverse cultural backgrounds.Potatoes are grown in -80 % of all countries, and world-wide production stands in access of 350 million tones per annum, a figure exceeded only by wheat, maize and rice [1 ].Despite its status as a food of global importance, the potato tuber contains toxic glycoalkaloids (GA) that cause sporadic outbreaks of poisoning in humans and a lot of livestock deaths [ However, owing the large and often unpredictable variations in the GA levels, which can arise from differences in variety, locality, cultural practices and stress factors (light, disease, mechanical injury, con ditions of storage and pre-treatment, insect damage and chemicals), and the fact that so many aspects of biochemistry and toxicity of these compounds remain poorly understood, the World Health Organization has proposed that the limit should be reduced to 20-100 mg/kg.We ELISA [20] is extensively used to analyze the total GA fraction.Extraction of the alkaloids prior to assay is simple solubilisation, making the whole procedure much quicker than alternative methods.It is carried out in the wells of plastic microtitration plates.The immunoassay procedure is often extre mely specific, technically simple and does not require the services of an experienced analyst.However, the main problem of immunoassay is the high price of the biologically active material, i. e. monoclonal anti bodies and enzyme-labeled antibodies.Biosensors The alkaloids (a-chaconine, a-solanine and sola nidine) were prepared as 1 mM solutions in 5 mM acetic acid solution.
Sensor design.The planar electrodes (Fig. 1) consist of two identical pairs of platinum inter digitated electrodes on a glass substrate (thickness 0.5 mm, dimensions 5 x 40 mm).The sensitive area of each electrode pair was about 1 * 1.5 mm.These electrodes were used as transducers for conducto metric measurements.

Enzyme immobilization.
A biologically active membrane on the transducer surface was formed by a method of protein cross-linking in saturated G1A vapour [24].The mixture containing 5 % (w/v) BuChE, 5 % (w/v) BSA and 10 % (v/v) glycerol in 20 mM phosphate buffer (pH 7.5) was deposited in a drop wise way on the sensitive surface of a measuring sensitive element, while the mixture containing 10 % (w/v) BSA and 10 % (v/v) glycerol in 20 mM phosphate buffer (pH 7.5) was placed on a reference element.Then, the conductometric transducers were placed for 30 min in saturated G1A vapour.After exposure to G1A, the membranes were dried at room temperature for 30 min and before measurements the The dependence of the enzyme inhibition on the GA concentration (Fig. 2) was investigated using conductometric transducers.As can be seen in Fig. 2,  a-solanine, a It has been shown that chaconine (Fig. 2) is a more potent inhibitor of immobilized butyryl cholinesterase than the other relative compound, so lanine.Since both of them share the same aglycone, namely solanidine, it can be suggested that more strong effect of chaconine on the enzyme activity is associated with the differences in their carbohydrate component and in steric compatibility of the whole alkaloid molecule with the enzyme active site.In the assays, which combined of a-solanine and a-chaconine, inhibition of BuChE was not additive (Fig. 2

) and these results are in good agreement with those obtained by Nigg et al. [25] for inhibition of the human plasma and serum BuChE. The results ob tained (Fig. 2, curves J and 2) can be explained by the competition between the substrate and chaco nine/solanine as well as by the competition between the inhibitors, and competition was proved by expe rimental estimation of K T values for solanine, cha conine and solanidine.
An example of the methodology used for the direct detection of K } value for solanidine is depicted in Fig. 3

2 ].
Potatoes and individual alkaloids have been shown to be teratogenic; embryotoxic, and toxic [3, 4].It has been also found that GA specifically induces membrane disruptive effects on cholesterol containing membranes and are more potent in permeabilizing the outer membrane of mitochondria compared to digitonin.In vitro studies have revealed that solanine and chaconine inhibit substantially cholinesterase's activity as well [5].Solanine non-competitively inhibit calcium transport in vivo in rat duodenum.Either chaconine or solanine reduces the sodium ion active transport in frog skin [6, 7].Their high concentration may cause acute poisoning, including gastrointestinal and neurological disturbances, in man, with death being caused by the central nervous system depression.Other studies also suggest that there is an increased risk for cancers of the brain, breast, endometrium, lung, and thyroid associated with the consumption of large quantities of potatoes [8 ].An available information suggests that the sus ceptibility of humans to GA poisoning is both high and very variable: oral doses in the range of 1 -5 mg/кг body weight are marginally to severely toxic to humans whereas 3-6 mg/кг body weights can be lethal [9].Neither chaconine nor solanine levels are affected by food processing and preparation.On the contrary, baking and frying, resulting in low mois ture-containing products, considerably concentrate GA in products such as potato chips and fried peel, and the risk of possible toxicity increases greatly [10].The widely accepted safety limit for GA in tubers remains 200 mg/кг fresh weight.

Fig. 1 .
Fig. 1.General view of platinum interdigitated planar electrodes used for the development of alkaloid-specific biosensor seem to be a very promising tool to overcome most of the problems described above.A basic idea for the ISFET-based monitoring of a-solanine, a-chaconine and solanidine has been proposed by Korpan et al. [21 ], and recently optimised by Arkhypova et al. [22].Owing to simplicity of the technologies of planar electrodes production in comparison with ISFET, the objective of this study was to develop the alkaloid sensitive conductometric sensor coupled to BuChE, to investigate the main analytical characteristics of the biosensor developed and to study the mechanism of the immobilized BuChE inhibition by GA. Materials and Methods.Chemicals, Butyryl cho linesterase (pseudocholinesterase EC 3.1.1.8)from horse serum, 13 U/mg solid; butyrylcholine chloride (BuChCl); bovine serum albumin (BSA, fraction V, 98 % purity); a-chaconine, a-solanine and solanidine from potato sprouts were purchased from «Sigma» (France).Aqueous solution (25 % w/v) of glutaraldehyde (G1A) was obtained from «Sigma-Aldrich Chemie GmbH» (Germany).All other chemicals were of analytical grade and were used without any further treatment.The alkaloids (a-chaconine, a-solanine and sola nidine) were prepared as 1 mM solutions in 5 mM acetic acid solution.Sensor design.The planar electrodes (Fig.1) consist of two identical pairs of platinum inter digitated electrodes on a glass substrate (thickness 0.5 mm, dimensions 5 x 40 mm).The sensitive area of each electrode pair was about 1 * 1.5 mm.These electrodes were used as transducers for conducto metric measurements.Enzyme immobilization.A biologically active membrane on the transducer surface was formed by a method of protein cross-linking in saturated G1A vapour[24].The mixture containing 5 % (w/v) BuChE, 5 % (w/v) BSA and 10 % (v/v) glycerol in 20 mM phosphate buffer (pH 7.5) was deposited in a drop wise way on the sensitive surface of a measuring sensitive element, while the mixture containing 10 % (w/v) BSA and 10 % (v/v) glycerol in 20 mM phosphate buffer (pH 7.5) was placed on a reference element.Then, the conductometric transducers were placed for 30 min in saturated G1A vapour.After exposure to G1A, the membranes were dried at room temperature for 30 min and before measurements the

Fig. 2 .
Fig. 2. Calibration curves for the detection of solanine (/), chaconine (2) and equimolar mixture of solanine and chaconine (5) by the conductometric sensor coupled to BuChE.Measurement conditions: 10 mM K,Na-phosphate buffer, pH 7.5, room temperature and BuChCl concentration of 10 mM membranes were soaked for 20 min in a buffer solution to equilibrate the membrane system.After use, the membranes containing biologically active material were washed twice with buffer solution, dried and stored at 4 or 20 °С Measurement of enzyme activity and inhibition.Measurements were conducted in daylight at room temperature (20 °С) in a glass cell (2 ml volume) filled with K,Na-phosphate buffer, pH 7.5.The biosensor was immersed in a vigorously stirred sam ple solution.After baseline stabilization, BuChCl was added to the vessel.The differential output signal between the measuring and reference sensitive ele ments was registered with «home made» laboratory set-up, and the steady state or kinetic response of the biosensor was plotted as a function of the BuChCl concentration.The evaluation of the enzyme inhibition by GA was carried out at an excess concentration of butyrylcholine chloride and included the following steps:The biosensor was soaked in 5 mM K,Na-phosphate buffer, pH 7.5, until reaching a stable baseline output signal;Butyrylcholine chloride was added to the measu rement cell.The kinetic or steady-state output signal of the biosensor was taken as an index of the maximal catalytic activity of the immobilized enzyme, and such a value of the biosensor response was used for further evaluation of the inhibition effect of a definite GA concentration;After twice washing, the sensor output was regis tered in the buffered solution containing target ste roidal alkaloid (s) and butyrylcholine chloride as desc ribed in the previous step.The level of inhibition due