_____________________________Nickolay F. Starodub et al �

InvIted revIew

abstract

Received for publication: Feb. 19, 2008. Revised: May 07, 2008.

rEZUMat

1 Institute of Biochemistry of National Academy of Sciences of Ukraine, Kiev, Ukraine, 2 Academy of Food Technologies, Odessa, Ukraine

Correspondence to:Nickolay F. Starodub, Institute of Biochemistry of National Academy of Sciences of Ukraine, 9 Leontovicha Str., 03087 Kiev, Ukraine, Tel: +380-44-279 47 43.Email: nikstarodub@yahoo.com

INtrODUctION

Despite the 1�72 Biological Weapons Convention, it appears worldwide that some nations are continuing to develop and produce biological warfare agents (BWA).

MYcOtOXINs aND OtHEr LOW WEIGHt tOXINs as INstrUMENt OF bIOtErrOrIsts: EXPrEss INstrUMENtaL cONtrOL aND sOME WaYs tO DEcONtaMINatE POLLUtED ENVIrONMENtaL ObJEcts

nickolay F. Starodub1, Ljudmila n. Pilipenko2, Inna v. Pilipenko2, Antonina v. egorova2

Objective: The instrumental analytical devices based on the principles of biosensorics and developed in Institute of Biochemistry of Ukraine are presented in this review. Material and methods: The main attention is given to chemical substances related to endocrine disrupting factors. Results: Our investigations in field of biosensors include: 1) Control of the total toxicity by the use of the bioluminescent bacteria with the optical signal registration and estimation of the intensity of Daphnia exometabolites by the chemiluminescent way; 2) Revealing the groups of toxic elements by the electrochemical- and thermo-biosensors with the sensitive elements presented by the special micro organisms and enzymes; 3) Determination of the concrete toxic elements by the electrochemical and optical biosensors based on enzymes and immune components as selective structures on one side and different types of transducers (surface plasmon resonance, porous silicon, electrolyte-insulator structures) on the other side. Conclusions: The developed biosensors respond to the practical demands within respect to: sensitivity, selectivity, simplicity of analysis, cost of analysis, and fulfillment of time of analysis. Problems that should be solved in the near future are: improving stability of biological materials and the development of effective procedure for their integration in biosensors simultaneously with transducer production; replacement of natural biological materials by artificial selective receptors.Key Words: low molecular weight toxins, mycotoxins, bioterrorism, express control

Obiectiv: Dispozitivele analitice instrumentale bazate pe principiile biosenzoricit\]ii, dezvoltate n Institutul de Biochimie din Ucraina sunt examinate n aceast\ trecere n revist\. Material [i metode: Principalele aspecte examinate se refer\ la substan]ele chimice legate de factorii agresivi endocrini. Rezultate: Investiga]iile noastre n domeniul biosenzorilor includ: 1) Controlul toxicit\]ii totale prin utilizarea bacteriilor bioluminiscente cu nregistrarea optic\ a semnalului [i estimarea intensit\]ii exometaboli]ilor de Daphnia prin chimioluminiscen]\; 2) Eviden]ierea de grupuri de elemente toxice prin biosenzori electrochimici [i termici cu elementele sensibile prezentate de anumite microorganisme [i enzime; 3) Determinarea elementelor toxice concrete prin biosenzori electrochimici [i optici baza]i pe enzime [i componente immune ca structuri selective, pe de o parte, [i prin diferite tipuri de transduceri (rezonan]\ plasmonic\ de suprafa]\, silicon poros, structuri de izolare electrolitic\), pe de alt\ parte. Concluzii: Biosenzorii dezvolta]i r\spund cerin]elor practice n privin]a: sensibilit\]ii, selectivit\]ii, simplicit\]ii analitice, costului [i duratei analizei. Problemele care ar trebui rezolvate n viitorul apropiat sunt: ameliorarea stabilit\]ii materialelor biologice [i dezvoltarea de procedee eficiente de integrare a lor n biosenzori simultan cu producerea de transduceri; nlocuirea materialelor biologice naturale cu receptori artificiali selectivi.Cuvinte cheie: toxine cu greutate molecular\ mic\, micotoxine, bioterorism

Today, the problem of environment protection from BWA, that are used not only for solving local conflicts, but also for psychological terror aiming to arouse panic among people of a specific country and to create permanent terror in the word, is very current. Some information on countries working at the program of biological weapons has been given earlier.1 The investigations undertaken by this program were directed on the induction of acute diseases among people and animals, destruction of sex status, prevention of antibiotic effects and others.

BWA are divided into three main groups: toxins (the so-called mid-spectrum agents), viruses, and bacteria. Toxins are biochemicals of various origin

_____________________________10 TMJ 2008, Vol. 58, No. 1 - 2

and chemical structure. Mycotoxins may serve as one of characteristic example of BWA. These substances are represented by a large group including more than 300 individual toxins produced by different fungi strains.2 T2, aflatoxins, zearalenone, patulin and others cause a great interest since they are widespread and characterized by a high level of toxicity.

T2 mycotoxin has a more toxic effect (400 times more) than mustard gas and lewisite. It is well-known that mycotoxin T2 was packed into rockets, bombs, cisterns, some explosive cylinders, hand-grenades that were used in Laos and Afghanistan as yellow rain.3,4 This mycotoxin may be prepared in a very simple way. Both circumstances (simplicity to obtain and high level of toxicity) form a very serious problem since these toxic elements may be used as instrument for bioterrorists.

It is necessary to mention that, generally, the use of toxins by terrorists, compared to others groups of BWA, is the most probable since viruses and bacteria present big danger not only for intended victims, but also for executors of the terrorist act.

The provision of appropriate services by simple, very selective and sensitive methods for express revealing of components BWA in environment is a very important approach among others directed to prevent serious consequences. Unfortunately, the analytical methodologies for the analysis of mycotoxins as well as other low molecular weight toxins include such instrumental analysis as high-performance liquid or gas chromatography with mass spectroscopy or liquid chromatography with mass spectroscopy.

As these methods are laborious and costly, the development of innovative approaches, such as immune analysis and particular chemo- and biosensors, is very urgent.5-10

In this review, the main attention will be paid to some mycotoxins and other chemical substances related to a group named endocrine disrupting factors, for example, heavy metal ions (HMI), nonylphenol, surface active substances (SAS) and some pesticides.

At first, we will present the proposed instrumental analytical methods based on the biosensorics principles and intended for the express revealing of the above-numerated toxins in environmental objects. This aspect will be analyzed from two points of view: estimation of total toxicity level at the screening analysis of environmental objects and then determination of the concentration of individual toxins in these objects.

MatErIaLs aND MEtHODs

For the analysis of the instrumental analytical device used for the control of total toxicity of environmental objects and for the determination of concentration of individual substances in these objects the main attention will be paid to the modern approaches based on the principles of biosensorics.

At first, we will analyze the efficiency of the developed biosensor for work with the standard solution. Then the obtained results will be compared with those found by the application of biosensors in real conditions. The type of biosensors, their design particularities and the details of analysis procedure will be presented in the appropriate section of this article.

Directions of our activity in the field of biosensors reflect three aspects: fundamental research, creation of working prototypes and development of some elements of technology. Fundamental aspect includes: selection of types of transducer (physical surface) and physical-chemical signals for the registration of the interaction of biological molecules as well as choosing the sensitive biological material and development of effective methods for the oriented immobilization of this material. Working prototypes of biosensors are created for application in the area of human and veterinary medicine, ecology and biotechnology. After examination of the biosensor work in real conditions, some elements regarding the technology of biosensor production are described.

The main biological and physical elements used by us in the development of biosensors as well as the principles of registration of signal generated after interaction of the sensitive biological material with the analyzed substance are shown in Table 1.

Our investigations in field of development of biosensors and their application include the several steps: 1) control of the total toxicity of environmental objects by the use of bioluminescent bacteria with the optical signal registration and the determination of the intensity of Daphnia exometabolites by the chemiluminescence, 2) demonstration of the toxic elements by the electrochemical and thermo-metrical biosensors with the sensitive elements presented by special microorganisms and enzymes; 3) determination of the concrete toxic elements by the electrochemical and optical biosensors based on the enzyme and immune components as selective structures, on one side, and the different types of transducers including surface plasmon resonance, porous silicon, electrolyte-insulator structures, on the other side.

_____________________________Nickolay F. Starodub et al 11

rEsULts

Instrumental analytical approaches for deter-mination of the total toxicity of environmental objects

Different living organisms (Crustacea, fish, algae, fungi, some vegetables and others) are commonly used to control total toxicity of environmental objects. International standard exists based on the determination of some indexes of Daphnia immobilization.11

Unfortunately, it is a very routine procedure. Other approaches with practical application are based on the control of oxygen consumption by microorganisms or determination of their luminescence.

Method using Daphnia as sensitive objectWe propose a principal new approach based on

the determination of the chemiluminescence (ChL) level of Daphnia living medium. The differences of measuring cell signals before and after introducing Daphnia in the solution to be analyzed were registered. In our experiments, we used Daphnia Magna Straus (Cladocera), which was kept in a culture medium according to the international standard protocols.11 In preliminary experiments it was shown that one to five Daphnia’s only are sufficient for experiments.12 The exited ChL of the medium was registered in the presence of luminol and hydrogen peroxide. Luminol, p-iodphenol and hydrogen peroxide were added to the medium for the enhancement of this ChL. The optimal concentrations of the above-numerated chemicals were preliminary established in the special experiments.12

Stationary, semi-portable and portable devices supplied by optrods, high sensitive photomultiplier, or photo resistors were created for the determination of the intensity of ChL. (Fig. 1) Depending on the source of toxic substances, we have obtained deviations of ChL values from the initial levels, which were in accordance with the intensity of toxic effect. To compare obtained results, the standard method with immobilization of Daphnia was performed.

Potassium biochromate was used as the standard chemical solution and its toxicity was checked by the generally accepted method (according to the index of Daphnia immobilization) and biosensor based on the determination of level of ChL Daphnia living medium. The obtained results are presented in Figure 2. It was demonstrated that the generally accepted method allows to reveal 0.1 mg/L of potassium biochromate as minimal level. At the same time, the sensitivity of the proposed biosensor approach was almost two-order higher.13 It is necessary to mention that the overall time of analysis differed significantly in both cases (about 24 h and 30 min for the generally accepted and biosensor methods, respectively).

Sensitivity of Daphnia to mycotoxins of T2 and patulin was demonstrated before and is shown in Figure 3.13 Diapason of the measurements of T2 mycotoxin was in the range of 0.01 to 0.1mg/L. At the same time, when the biosensor method was used, this range was from 0.001 to 1 mg/L. Moreover, the level of sensitivity did not depend on time of Daphnia incubation in solution to be analyzed. As far as patulin is concerned, there was possibility to quantitatively determine it by the biosensor method in ranges from 0.001 to 1 mg/L.

Table 1. Main components and the principles of signal measurements used for the development of biosensors.

––

_____________________________12 TMJ 2008, Vol. 58, No. 1 - 2

Figure 1. Analytical devices for luminescence registration. A. Optrode based optical biosensor, where: 1-9 - fiber optics with immobilized cells, photodiod, system for signal registration, computer, glass container, sample, syringe for reagent introducing, pressure compensator and support, respectively. B. Stationery luminometer supplied by photomultiplier. C. Portable luminometer, where: 1, 2 - electronic block and reactor box, respectively.

Method using bioluminescent bacteria as sensitive object

In the investigations Photobacterium phosphoreum K3 (IMB B-7071), Vibrio fischeri F1 (IMB B-7070) and Vibrio fischeri Sh1 purified from Black Sea and Sea of Azov were used. The level of bioluminescence (BL) was measured by the devices which is shown in Figure 1. The samples contained 0.8 ml of the tested substance in 2.5% solution of NaCl, 0.1 ml of 0.5 M phosphate (pH 7.0) or phosphate citrate (pH 5.5) buffers and 0.2 ml of bioluminescent bacterial suspension including 5x105 cells/ml. Alternately, bioluminescent bacteria (105 cells) were immobilized in sepharose gel (about 0.1 ml) deposited at the end of fiber optics.

Figure 2. The estimation of biological activity of Daphnia (as sensitive structure) after staying into solution of potassium bichromate by: generally accepted (A) and biosensor (B) methods.

Figure 3. T2 (A, B) and patulin (C) determination diapasons by the generally accepted (A) and biosensor (B, C) methods.

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Fig. 2. The estimation of biological activity of Daphnia (as sensitive structure) after staying into

solution of potassium bichromate by: generally accepted (A) and biosensor (B) methods.

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solution of potassium bichromate by: generally accepted (A) and biosensor (B) methods.

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_____________________________Nickolay F. Starodub et al 13

In both cases, the BL intensity (I) was registered through 30-120 min. The level of toxicity was presented as the concentration, which caused 50% decrease of the intensity of BL (EC50). As it can be seen in Figure 4, for all cases of signal measurements the value of EC50 oscillated in the range of 7 to 1� mg/L, depending on the time of bacteria incubation with the T2 mycotoxin solution. It is necessary to underline that the sensitivity of V. fischeri F1 to mycotoxin T1 is much higher in comparison with the sensitivity of Ph. phosphoreum Sq3.14

Figure 4. Concentration of T2 mycotoxin caused half decreasing of BL of V. fischeri F1 at different time of exposition. 1-3: 10, 20 and 30 min of exposition in solution to be analysed, respectively.

Figure 5. Changes of BL intensity of Ph. phosphoreum at the effect of different concentration of patulin during 90 min, where: 1-4: 0; 0.15; 0.63; 1.25 mg/L, respectively.

The increase of patulin concentration from 0.63 to 40 mg/L has caused sufficient decrease of the BL intensity incubated with Ph. phosphoreum Sq3 for 12 to 60 minutes. The value of EC50 for

patulin was between 0.63 and 1.25 mg/L.15 The dose-effect of patulin at the low concentration (as low as 1 mg/L) may be confidently registered in case of three repeated measurements for each point. If this algorithm of analysis is followed, the toxic effect of patulin to bioluminescent bacteria may be revealed at a concentration below 0.15 mg/L. (Fig 5)

Moreover, when the time of incubation was prolonged (up to �0 min) the toxic effect of patulin increased and the value of EC50 was in between 0.15 and 0.63mg/L. The decrease of medium pH to the lower physiological limit (5-5.5) resulted in a sensitivity increase up to one order. The value of EC50 has analogy with semi lethal dose established for animal and it correlates with other indexes of toxicity (cytotoxicity, irritation of mucous membranes, etc.).16

It is necessary to mention that the intestinal barrier in animals is destroyed at a patulin concentration of about 1 mg/L.17 Taking into consideration of this fact, the above data testify that the proposed biosensor analysis with the use of bioluminescent bacteria may be effective for the screening of samples of water, juice, foods and other environmental objects.

The study of the influence of different types of SAS on the intensity of bioluminescence of bacteria (Ph. phosphoreum K3 (IMB B-7071), V. fischeri F1 (IMB B-7070) and V. fischeri Sh1) has revealed that the main part of the investigated substances function as inhibitors of this process. The analysis of kinetics of inhibition displayed a number of peculiarities. (Fig. 6) At first, the cationic and anionic SAS had the similar kinetics of inhibition. Secondly, nonionic SAS have an additional stage during which the inhibition is absent or some activation of bioluminescence is observed. Therefore, for revealing toxicity of this group of SAS it is necessary to incubate these substances with bacteria a long time.18

Figure 6. Kinetics of bacterial bioluminescence inhibition by different types of SAS. Δ – non-ionic; ◊ - anionic and □ – cationic SAS.

Fig. 4. Concentration of T2 mycotoxin caused half decreasing of BL of V. fischeri F1 at different

time of exposition. 1-3 – 10, 20 and 30 min of exposition in solution to be analysed,

respectively.

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Fig. 5. Changes of BL intensity of Ph.phosphoreum at the effect of different concentration of

patulin during 90 min, where: 1-4 – 0; 0.15; 0.63 1.25 mg/L, respectively.

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Fig. 6. Kinetics of bacterial bioluminescence inhibition by different types of SAS.

– non-ionic; - anionic and – cationic SAS.

_____________________________14 TMJ 2008, Vol. 58, No. 1 - 2

Biosensor determination of groups of toxic elements

To determine group-specific toxic substances, for example, phosphororganics, chlororganics, cyanides and others we have developed a multi-biosensor based on electrolyte-insulator-semiconductor (EIS) structures.1� The overall view of such biosensor, with an electronic part developed in Institute of Semiconductor Physics of National Academy of Sciences of Ukraine, is given in Figure 7. One of these biosensors is closed and the reference electrode is situated nearby. Another biosensor is open and has two independent channels, each with five measuring cells. The biosensor is operated by a special software.

Figure 7. EIS-structures based biosensor.

In these experiments, the simazine conjugates and antiserum to simazine were obtained according to recommendations from Prof. B. Dzantiev (A.N. Bach Institute of Biochemistry, Moscow, Russia).20

Сonjugates of 2,4-D with proteins and enzymes were obtained with the help of the Fenton’s reagent. The antiserum to simazine cross reacted with atrazine (89%), terbutylazine (80%), and propazine (10%). Other analytes demonstrated cross-reaction in the range of 0.7-6.2%. Antiserum to 2,4-D did not have any cross-reaction with simazine. The principles of design and work of biosensors were presented by the author in another paper.21 Specific antibodies to herbicides were immobilized through the staphylococcal protein A. The analysis was performed using the sequential saturation method, when antibodies left unbound after their exposure to native herbicide in investigated sample, then have interacted with the labeled herbicide. The basic sensitivity of the EIS structures sensor to simazine, when the horse radish peroxidase (HRP)-conjugates were used, was approximately 5 µg/L. The linear plot of the sensor response lied in the range of the concentrations from 5 to 150 µg/L. This sensitivity of the EIS structures based sensor towards both herbicides was lower than it is requested

in practice. We tried to elucidate the main reasons for such a situation. One of them may be connected with difficulties to register sensor output due to the formation of air bulbs, which appear as a result of high activity of the HRP. Use of high concentrations of ascorbic acid may be another reason for the lower sensitivity of this sensor. We changed HRP to the glucose oxidase (GOD) and obtained a sensitivity of the analysis approximately 5 times higher. The linear plots for simazine and 2,4-dichlorophenoxyacetic acid (2,4-D) were in the range of 1.0-150 and 0.25-150 µg/L, respectively.22,23

Immune biosensor based on the EIS structures attracts attention because of the simple procedure of the analysis and existing possibility to fulfill multi-parametrical control of the environment. For the repeated analysis, replaceable membranes are very suitable. The overall time of the analysis is about 40 min. Therefore, the EIS structures based immune sensor may be used for wide screening of the environment for the presence of herbicides. It gives possibility to carry out analysis of 8-10 samples simultaneously. It is suitable for a wide screening of not only herbicides but also other types of toxicants. For the verification of the results of analysis others types of biosensors may be used, for example those based on the ISFETs, with a sensitivity for the determination of the above mentioned herbicides is at the level of 0.1µg/L and less (up to 0.05 µg/L) and corresponds to practice demands.22-24

We believe that the sensitivity of the EIS structures based immune sensor can be increased further. One way to do this could be the development of special suitable membranes. It is necessary to provide a very high density of the immobilized specific antibodies on the membrane surface. Moreover, it would be very effective if these antibodies were immobilized not only on the membrane surface but also in its large-scale pores, which would be accessible for large molecules of conjugates of herbicides with enzymes. In our opinion, synthetic biologically compatible polymers, which can be prepared in a simple way with different levels of density and porosity, can serve as a perspective material for such membranes.25,26 Of course, to increase the sensitivity of the analysis, it would also be very efficient to use monoclonal antibodies with a high level of affinity to analytes, to choose enzyme labels with a high turnover of the activity, and to provide preservation of the enzyme activity during preparation of the conjugate. If the membranes were prepared in advance, the duration of the analysis may be shortened up to 10 min. Membranes are simple to prepare, they

_____________________________Nickolay F. Starodub et al 15

are very cheap and can be stored for a long time in refrigerator.

Since the enzymes which have serine residuum in the active centre (first of all butyrylcholine esterase – BChE, acetylcholine esterase – AChE and total choline esterase – ChE) are very sensitive to phosphororganic pesticides (PhOrPe) and others (urease) with the thiol groups react with HMI, there is a possibility to simultaneous determine these classes of toxic elements.27,28

The sensitivity of HMI and PhOrPe determination essentially depends on the incubation time of enzyme membranes in environment containing these analytes. Two different approaches were tested: (1) registration of the sensor output signal in the mixture of a substrate and an analytes, (2) separation of the inhibition reaction from the following measurement of the residual enzyme activity. In the latter, the threshold sensitivity of toxin analysis was about 10 times higher. The time of incubation was chosen experimentally and it was 15 min. The concentration of HMI that could be determined by the urease channel of the sensor array lied within the range from 10-4 to 10-7 M, depending on the type of the metal used. The range of linear detection covered 2-3 orders of the concentration change.

The effects of both pesticides are very similar. The limit of detection of pesticides indicated above was 10-7 M. The range of the linear response was from 10-5 to 10-7 M. At the same time, the sensitivity of BChE to HMI was substantially lower than that of urease. The maximum sensitivity of BChE to HMI was for concentrations of more than 10-4 M. Activity of GOD depends on the presence of HMI for concentrations above 10-4 M. The diagram of the responses of channels with different enzymes (BChE, AChE, urease, ChE and GOD) is presented in Figure 8. GOD was used as reference enzyme that has a minimal reaction in respect of both types of groups of toxins.

Biosensor determination of individual toxinsFor this purpose, we use SPR, TRIE and

calorimetric based biosensors.

Analysis by SPR and TIRE based optical immune biosensors

The overall view of this biosensor is shown in Figure �. Its measuring part was developed in the Institute of Semiconductor Physics of National Academy of Sciences of Ukraine. The principles of construction of SPR biosensor and main algorithm of analysis with its help were described elsewhere.2�,30

Figure 8. Time diagram of the array operation of five different enzyme reactions. The mixture contained: 5x10-4 mol/l of Co2+- ions (point-lines); 10-5 mol/l of DVDP (dashed lines).

Figure 9. SPR based immune biosensor.

For experimental purposes we chose the optimal conditions for performing the analysis. As a rule, we have analyzed in detail three main variants of approaches:

a). Specific antibodies from antiserum were immobilized on the gold surface of SPR transducer through intermediate layer from Staphylococcal protein A or some lectin and free analyte was in solution to be analyzed (direct way of analysis);

b). Conjugate of nonylphenol, or simazine, or 2,4-D or T2 mycotoxin with some protein – bovine serum albumin, or soybean tripsin inhibitor, or ovalbumin was directly immobilized on gold surface of SPR transducer and free analyte with appropriate antiserum were in solution (competitive way with the immobilized conjugate);

c). The specific antibodies from antiserum were immobilized as in “a” and free analyte and its conjugate with some protein were in solution to be analyzed (competitive way with the immobilized antibodies);

d). Immobilized and oriented as in “c” antibodies react with free analyte and then with appropriate conjugate (approach with tsaturation of active binding sites on the surface).

Fig. 8. Time diagram of the array operation of five different enzyme reactions. The

mixture contained: 5.10-4 mol/l of Co2+- ions (point-lines); 10-5 mol/l of

DVDP (dashed lines).

_____________________________16 TMJ 2008, Vol. 58, No. 1 - 2

It was shown that orientation antibodies on the surface is more effective with the help of protein A in comparison with the use of lectins. Maybe it is connected with the possibility of presence of some carbohydrates not only in Fc-fragment of antibodies but alos in Fab-fragments.

It is known that the sensitivity of 2,4-D analysis by direct method is about 5-10 µg/L, which is not high. Such low level of sensitivity using direct analysis is observed in case of the determination of other low weight substances, for example: T2 mycotoxin, nonylphenol. Much more sensitive are the methods “b” to “d”. (Fig. 10). Biosensor based on the TIRE allows to us reveal mycotoxin T2 up to 0.15 ng/ml.8,31

Figure 10. Calibration curves for the determination of nonylphenol by SPR based immune biosensor by direct (1) and competitive (2) ways.

Both optical immune biosensors can provide the method sensitivity needed for practice. Overall time of analysis is about 5-10 minutes if transducer surface is preliminary prepared. It is necessary to mention that the immune biosensor based on the SPR is simpler then TIRE biosensor. Additionally, the latter can be realized as portable device.

Analysis by calorimetric immune biosensorThis biosensor was designed in two different

forms: as micro calorimeter and thermistor-based device. We will demonstrate the efficiency of immune biosensor for the determination of low molecular substances by the results obtained in the experiments with nonylphenol.

For a successful development of the calorimetrical biosensor, it was necessary at first to set the optimal concentration of antiserum (for example antiserum to nonylphenol). For this purpose 150 µl antiserum in different concentrations was brought in a measuring cell and incubated for 15 minutes to establish a baseline (for this the temperature in the barn was set at an

optimum level). Then 50 µl solution of nonylphenol in concentrations of 1; 5 and 10 µg/ml were brought into the cell. Thus, it was determined that the optimum concentration of antiserum was about 5 mg of protein in 1 ml.

For the assay of nonylphenol in solutions with the help of thermal biosensor, it was necessary to build-up a corresponding calibration curve. For this purpose, 150 µl of antiserum ( concentration: 5 µg of protein in 1 ml) was brought into a measuring cell; then 50 µl of nonylphenol in a range of concentration from 0.5 up to 10 µg/ml was pumped into the measuring cell. (Fig. 11) Thus, we demonstrated the possibility of “direct” detection of nonylphenol by calorimetrical biosensor with the sensitivity about 1 µg/ml. The overall time of analysis is about 20-30 min.

Figure 11. "Direct" detection of nonylphenol by the calorimetrical biosensor.

Certainly, the determination of nonylphenol by thermal immune biosensor is much less sensitive than in case of application of SPR or TIRE biosensor, but it is necessary to mention the simplicity of the method. One possibility would be that the thermal biosensor is used for the screening of toxic elements in environment, followed by verification of the results using optical immune biosensors.

DIscUssION

The analysis of the above presented results confirms that the biosensor currently developed are able to provide control of total level of toxicity and sensitive revealing of individual toxicants according to the modern practice demands. It is necessary to underline that the protection of environment from contamination by different toxic elements demands a great attention and global decisions, as it was recently done in case of ozone problem. Approximately one hundred years ago around 300 stations of passive control of ozone level in atmosphere were described, but only 20 years ago real interest in this problem was shown, resulting in a number of decisions at the

Fig. 10. Calibration curves for the determination of nonylphenol by SPR based immune

biosensor by direct (1) and competitive (2) ways.

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_____________________________Nickolay F. Starodub et al 17

governamental level.32 The negative conditions sharply increase the appearance of genetic anomalies and intensify their phenotypic expression.33-35 Of special concern is the contamination of water resources. In this situation, the problem of monitoring the wastewater and control of activities of bioterrorists become priorities. The main principles of such monitoring are the result on intensive research and include: colorimetric analysis in combination with membrane technology, spectroscopy with the application of photodiode massive, fluorescent analysis and biosensor control.36

Certainly, the role of biosensors in the control of environmental objects increases with every year, as scientist strive to improve their technical and analytical characteristics and to move near to the most complete fulfillment of the practice demands. This may be achieved in case of the development of the multi-parametrical systems, which will be able to analyze several samples and to determine a wide range of different substances. This may be achieved by the application of the hybrid organic and non-organic sensor technologies, as well as by the use of the thin layer technology.37,38 Chemical modeling of the selective biological sites for the binding of analytes is a very important research direction for the elaboration of new-generation instrumental analytical devices.3�,40

They may allow the avoidance of problems with the operational stability of sensors and the obtaining of an uninterrupted work regime. So, it was shown that the revealing of number of OChS (dichlorpropylene, chloroform, toluene, simazine, 2,4-D, T-2 mycotoxin and others) in water sources may be done by using of calixarenes on the quartz resonator or gold surface of SPR.3�,40

Certainly, it is necessary to develop new, more effective membranes, which should be stable during long term of biosensor work in different mediums.41

Unfortunately, today it is not possible to appreciate the advantages of some types of biosensors versus others regarding the efficiency of control of some individual toxins but it will be done in the near future. The improving abilities of biosensors, in particular concerning their sensitivity, selectivity and optimization of the assay method, gives the possibility to achieve such level which is inhered in traditional instrumental approaches and ELISA-method. In any case, biosensor analysis is more rapid and simple.

Once the all above-mentioned problems are solved, the ecological services will be provided with reliable analytical instruments. Nevertheless, even today, a positive answer may be given to the question which was entered in the title of article published in

journal of “Biosensors” more than 20 years ago: “Can biosensors help to protect drinking water?”42

cONcLUsIONs

1. The developed prototypes of biosensors respond to the practical demands with respect to:

- Sensitivity;- Selectivity;- Simplicity of analysis; - Cost of analysis;- Assay duration.2. Further improvement of some developed

prototypes of biosensors may help to speed up their production.

3. Problems that need a solution in the near future are:

- Improving stability of biological materials and the development of effective procedure for their integration in biosensors simultaneously with transducer surface production;

- Replacement of natural biological materials by artificial selective receptors;

- Certification of the perspective biosensor prototypes for the performance of specific analysis in practice.

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