Results and discussion


Usually different living organisms (Crustacea, fish, algae, fungi, some vegetables and others) are used to control total toxicity of environmental materials. An International Standard exists as the basis of the determination of some indexes of Daphnia immobilization (ISO 6341:1996(E)). Unfortunately, it is a very routine procedure. Other approaches which are used in practical applications are based on control of oxygen consumption by micro-organisms or determination of their luminescence.

2.1.1. Method with the use of Daphnia as the sensitive object

We propose a principal new approach based on the determination of the chemiluminescence (ChL) level of a live Daphnia medium. The differences of measuring cell signals before and after introducing Daphnia into the solution to be analyzed were recorded. In the experiments, Daphnia magna St. (Cladocera) was used, which was kept in the medium according to the International Standard rules (ISO 6341:1996(E)). In preliminary experiments, it was shown that only 1-5 Daphnia are sufficient for these experiments (Ivashkevich et al., 2002). The excited ChL of the medium was recorded in the presence of luminol and H2O2. The optimal concentrations of the above numerated chemicals were preliminary established in the special experiments (Ivashkevich et al., 2002). 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.

Depending on source of toxic substances we have obtained deviations of ChL values from the initial levels, which were commensurate with the intensity of the toxic effect. 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 using the biosensor based on the determination of ChL level of the live Daphnia medium. It was found that the generally accepted method allows use of just 0.1 mg/l of potassium biochromate as the minimum level. At the same time, the sensitivity of the proposed biosensor approach was almost two orders higher (Levkovetz et al., 2002). It is necessary to mention that the overall time of analysis made a big difference in both cases (about 24 h and 30 min for the generally accepted and biosensor methods, respectively).

The sensitivity of Daphnia to mycotoxins T2 and patulin was demonstrated by Gojster et al. (2003) and Pilipenko et al. (2007). Diapason of the measurements of T2 mycotoxin by the generally accepted method was in the range of concentration of 0.01-0.1 mg/l. At the same time, the biosensor method had a range from 0.001 to 1 mg/l. As for patulin, it was possible to make quantitative determinations by the biosensor method in the range of 0.001-1 mg/l.

2.1.2. Method with the use of bioluminescent bacteria as the sensitive entity

In the investigations, Photobacterium phosphoreum K3 (IMB B-7071), Vibrio fischeri F1 (IMB B-7070) and Vibrio fischeri Sh1 purified from the Black Sea and the Sea of Azov were used. The level of bioluminescence (BL) was measured by the developed devices. The level of toxicity was presented as the concentration which caused a 50% decrease of the intensity of BL (EC50). For all cases of signal measurements, the value of EC50 oscillated in the range of 7-19 mg/l depending on the time of incubation of the bacteria in the T2 mycotoxin solution. It is necessary to emphasise that the sensitivity of V. fischeri F1 to micotoxin T1 is much higher in comparison with the sensitivity of Ph.phosphoreum Sq3 (Katzev et al., 2003).

Increasing patulin concentration from 0.63 to 40 mg/l caused sufficient decrease in the BL intensity at the influence on Ph.phosphoreum Sq3 during 12-60 min. The value of EC50 for patulin was in the range 0.63-1.25 mg/l (Katzev et al., 2003). The dose-effect of patulin at the low concentration (as low as 1 mg/l) may be confidently registered in the case of three repeated measurements for each point. If this methodological approach is followed in the analysis, the toxic effect of patulin to bioluminescent bacteria may be revealed at concentrations of less than 0.15 mg/l. Moreover, extending time of influence up to 90 min, the toxic effect of patulin increased and value of EC50 was in the range 0.15-0.63 mg/l. Decreasing the pH of the medium to the lower physiological limit (5-5.5), the sensitivity increased up to one order. The value of EC50 has analogy with the semi-lethal dose established for animals and it correlates with other indexes of toxicity (cytotoxicity, irritation of mucouses, etc.) (Elnabarawy et al., 1988). It is necessary to mention that the intestinal barrier in animals is destroyed at patulin concentrations of about 1 mg/l (Manfoud et al., 2002). Taking this fact into consideration, the above indicated data testify that the proposed biosensor analysis with the use of bioluminescent bacteria may be effective for screening samples of water, juice, foods and other environmental substances.

In 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), it was revealed that the most of the investigated substances are inhibitors of this process. At first, the cationic and anionic SAS had similar kinetics of inhibition. Second, nonionic SAS, have an additional stage in which the inhibition is absent or some activation of bioluminescence is observed. Therefore, for revealing the toxicity of this group of SAS, it is necessary to incubate these substances with bacteria for a long time.


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 (Starodub and Starodub, 2000).

In these experiments, the simazine conjugates and antiserum to simazine were obtained according to Yazynina et al. (1999) and presented by Professor B. Dzantiev of the A.N. Bach Institute of Biochemistry, Moscow, Russia. Conjugates of 2,4-D with proteins and enzymes were obtained with the help of 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%. The antiserum to 2,4-D did not have a cross-reaction with simazine.

The principles of the design and operation of biosensors were presented by Starodub et al. (1999). Specific antibodies to herbicides were immobilised through the staphylococcal protein A. The analysis was performed using the sequential saturation method where antibodies are left unbound after their exposure to the native herbicide in the investigated sample, then have interacted with the labelled herbicide. The sensitivity of the EIS structures based sensor to simazine, when the HRP-conjugates were used, was approximately 5 ^g/l. The linear plot of the sensor response lay in the range of the concentrations from 5 to 150 ^g/l. This sensitivity of the EIS structures based sensor towards both herbicides was lower than is needed in practice. We tried to elucidate the main reasons for such a situation. One of them may be connected with difficulties to record 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 the HRP label to the GOD one and obtained a sensitivity of the analysis approximately five times higher. The linear plots for simazine and 2,4-D were in the range of 1.0-150 and 0.25-150 ^g/l, respectively (Starodub and Starodub, 1999b; Starodub et al., 2000).

An immune biosensor based on the EIS structures attracts attention because of the simple analytical procedure and possibility of undertaking multi-parametrical control of the environment. For repeated analyses, 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 offers the possibility of carrying out analysis of eight to ten samples simultaneously. It is suitable for a wide screening of not only herbicides but also other types of toxicants. Other types of biosensors may be used for the verification of the analytical results, for example, based on the ISFETs, the sensitivity of which in the determination of the above mentioned herbicides is at the level of 0.1 ^g/l or less (up to 0.05 ^g/l) which corresponds to practice demands (Starodub and Starodub, 1999a, 2001; Starodub et al., 2000). We believe that the sensitivity of the EIS structures based immune sensor can be increased still further. One of the possible ways of doing this could be the development of special suitable membranes. It is necessary to provide a very high density of the immobilised 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 prospective material for such membranes (Shirshov et al., 1997; Rebrijev et al., 2002). 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 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 are very cheap and they can be stored for a long time in a refrigerator.

Since a number of 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 other ones (urease) with the thiol groups react with HMI, there is the possibility of simultaneous determination of these classes of toxic elements (Starodub et al., 1998; Rebrijev and Starodub, 2001).

The sensitivity of HMI and PhOrPe determination depends essentially on the incubation time of the enzyme membranes in the environment of these analytes. Two different approaches were tested: (1) registration of the sensor output signal in the mixture of a substrate and analytes; (2) separation of the inhibition reaction from the subsequent measurement of the residual enzyme activity. In the latter case, the threshold sensitivity of toxin analysis was about ten 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 lay within the range from 10-4 to 10-7 M, depending on the type of the metal used. The range of linear detection covered two to three 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 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. The activity of GOD depends on the presence of HMI for concentrations above 10-4 M. GOD was used as the reference enzyme which has a minimal reaction in respect of both types of groups of toxins.


For this purpose, we apply SPR, TRIE and calorimetric based biosensors.

2.3.1. Analysis by SPR and TIRE based optical immune biosensors

The principles of construction of SPR biosensor and the main algorithm of analysis with its help were described by Starodub et al. (1997, 1999).

As a rule, we have analysed in detail three main variants of approaches: (a) specific antibodies from an antiserum were immobilised on the gold surface of an SPR transducer through the intermediate layer from Staphylococcal protein A or some lectin, and free analyte was in the solution to be analysed (direct method of analysis); (b) conjugate (NP, simazine, 2,4-D or T2 myco-toxine with some protein - BSA, or STI, or Ova) was directly immobilised on the gold surface of the SPR transducer, and free analyte with an appropriate antiserum was in solution (competitive approach with the immobilized conjugate); (c) the specific antibodies from the antiserum was immobilised as in "a", and free analyte and its conjugate with some protein were in the solution to be analysed (competitive method with the immobilized antibodies); and (d) immobilised and oriented antibodies as in "c" react with free analyte and then with the appropriate conjugate (approach to saturation of active binding sites on the surface). It was observed that orientation of the antibodies on the surface is more effective with the help of protein A than with the use of lectins. Maybe, this is connected with the possibility of presence of some carbohydrates not only in the Fc-fragment of the antibodies but also in the Fab-fragments.

It was found that the sensitivity of 2,4-D analysis by the direct method is about 5-10 ^g/l, which is not high. Such a low level of sensitivity with the direct method of analysis is observed in case of the determination of other low weight substances, for example, T2 mycotoxin and NPh. Methods "b" to "d" above were much more sensitive. A biosensor based on the TIRE allows us to identify mycotoxin T2 up to 0.15 ng/ml (Starodub and Starodub, 1999; Starodub et al., 2006). Both optical immune biosensors can provide the sensitivity of analysis which is needed for practice. The overall time of analysis is about 5-10 min, if the transducer surface is prepared beforehand. It is necessary to mention that the immune biosensor based on the SPR is simpler then the TIRE biosensor. In addition, to former may be developed as a portable device.

2.3.2. Analysis by calorimetric immune biosensor

We will demonstrate the efficiency of an immune biosensor for the determination of low molecular substances using the results obtained in the experiments with NPh. For successful development of the calorimetrical biosensor, it was necessary to first set the optimal concentration of antiserum (for example, antiserum to NPh). For this purpose, 150 ^l of antiserum in different concentrations was brought in a measuring cell and incubated for 15 min to establish a baseline (for this time the temperature in a barn was set at an optimum level). Then, 50 ^l solution of NPh in concentrations of 1, 5 and 10 ^g/ml were brought into the cell. Thus, it was set that the optimum concentration of antiserum was about 5 mg of protein in 1 ml.

For the determination of NPh in solutions with the help of a thermal biosensor, it was necessary to construct a corresponding calibration curve. For this purpose, 150 ^l of antiserum (in a concentration of 5 ^g of protein in 1 ml) was brought into a measuring cell and then 50 ^l of NPh in a range of concentration from 0.5 up to 10 ^g/ml was pumped into the measuring cell. This demonstrated the opportunity of "direct" detection of NPh by a calorimetrical biosensor with the sensitivity of about 1 ^g/ml. The overall time of analysis is about 20-30 min.

Certainly, the sensitivity of the determination of NPh by thermal immune biosensor is much less than in the case of application of the SPR or TIRE biosensor, but it is necessary to mention the simplicity of carrying out the measurement. Maybe, a thermal biosensor could be used for the screening of toxic elements in environmental materials with subsequent verification of the results of analysis by optical immune biosensors.


The creation of cheap and effective technology for the removal of toxic pollutants from water is an urgent need in modern environmental protection. The first and most important problem is modifying and changing technology, aiming towards energy savings and reaching the minimal emission levels in the hydrosphere. This problem can be resolved by an optimal combination of chemical-technological methods with biological ones (Klimenko et al., 2002). Treatment expenses depend on the degree of purification needed. There are certain purification limits determined by economy, under which the enterprise becomes non-profitable. The role of a combination of natural biodegradation processes with chemical-technical methods in this context is most important. The toxicity of pollutants entering the environment and their transformation as a result of waste-water treatment must to be taken into account during technology creation. Recent experiences indicate that the efficiency for the purification of waste-water from toxic xenobiotics can be improved by combining adsorption and oxidation methods with biological methods. However, effective automatic control of water treatment is needed for the optimization of the clean-up process. Biosensors offer unique possibilities of obtaining cheap, fast and sensitive control units that can be incorporated as sensors for water toxicity control and regulation at different stages of the treatment process (Goncharuk et al., 2007; Klimenko et al., 2007). In this report, the results about the application of optical and other types of biosensors for feedback control of water purification process will be presented.

The technology was accomplished by successive pre-photooxidation steps (O3 and UV-irradiation), followed by simultaneous biological degradation in a biosorber with activated carbon (AC) with immobilized degradable bacteria. From the start, we proposed using a Daphnia chemiluminescent test for the express determination of total toxicity during water purification. It was established that this biotest is very sensitive to the presence of toxic pollutants, such as nonylphenolethoxylates, for example. Quantitatively, their control is possible at concentrations of about 20 mg/l and less. It was demonstrated that: (a) the optimal regime of photoozonation may be achieved if the concentration of the initial substances decreased by 50% from the initial; (b) the use of activated carbon after photo-ozonation caused a sharp decrease in general toxicity of the treated water due to adsorption of some organic radicals and semi-decay of pollutants. The stability of the biosorber work with AC is provided by simultaneous biodegradation of the AC during solution filtration through the AC layer. Special methods were developed to increase the efficiency of AC biodegradation to 95-98% from its initial capacity. To provide stability of biosorber operation, special requirements were developed on the sorbents, first as regards their operating conditions, and second as regards their porosity. The pilot scale system aimed at the complete treatment process based on a joint action of physical-chemical and biochemical processes with biosensor control has been developed and assembled. The general scheme of this system is presented below (Fig. 1).

For the control of total toxicity of the initial water and for the determination of individual toxic substances (for example of NphEO), it is necessary to use the chemilminescent and bioluminescent tests and one of the immune biosensors, based on electrolyte semiconductor structures or surface plasmon resonance (SPR). The thermal cell biosensors with Sacchromices cerevisia and bioluminescent test with Vibrio fischeri as the sensitive biological elements were non-effective at the low concentrations of such toxic subsrances as detergents. It is the same situation in the intermediate stage of water purification, after its treatment by ozone and UV-radiation. At the final stage of water purification, it is more suitable to use a Daphnia chemi-luminescent biosensor and immune biosensor based on ion-sensitive field effect transistors (ISFETs), as this is more sensitive in comparison with the other immune biosensors.

Torg Gard Clutch

Figure 1. Overall scheme of pilot scale system with biosensor feedback control for water purification from surfactants. Where: 1-compressor, 2-ozone generator; 3-electronic manometer; 4-manometer; 5-gas rotameter; 6-water trap; 7-back valve; 8-reactor for catalytic photoozonation; 9-UV-lamp of low pressure; 10-photometric gas cuvette; 11-ozone trap (KI solution); 12-gas clock; 13-energy source for UV-lamp of low pressure; 14-computer; 15, 16-tanks, containing surfactant solution; 17, 18-water rotameters; 19, 20-peristaltic pumps; 21-bioreactor; 22-AC-active carbon; 23-AC with immobilized microorganisms; 24-biosensors for initial solution; 25-biosensors for solution after photoozonation; 26-biosensors for purified water

Figure 1. Overall scheme of pilot scale system with biosensor feedback control for water purification from surfactants. Where: 1-compressor, 2-ozone generator; 3-electronic manometer; 4-manometer; 5-gas rotameter; 6-water trap; 7-back valve; 8-reactor for catalytic photoozonation; 9-UV-lamp of low pressure; 10-photometric gas cuvette; 11-ozone trap (KI solution); 12-gas clock; 13-energy source for UV-lamp of low pressure; 14-computer; 15, 16-tanks, containing surfactant solution; 17, 18-water rotameters; 19, 20-peristaltic pumps; 21-bioreactor; 22-AC-active carbon; 23-AC with immobilized microorganisms; 24-biosensors for initial solution; 25-biosensors for solution after photoozonation; 26-biosensors for purified water

During the process of treatment, the following different integral parameters of water quality were analyzed: chemical oxygen demand, permanganate oxidizability, biological oxygen demand, total organic carbon, residual concentration of ozone, dissolved peroxides, total toxicity and concentration of individual toxic elements. The technology is suitable for the textile industry (band textiles, silken, knitted, artificial technical fabric and cotton factories as well as wool-spinning mills), and for water quality control in galvanizing and oil extraction processes. The using of this technology leads to recycling of the processing water within an industry. The results might be used for wastewater purification from toxic pollutants (surfactants, phenols, pesticides, oil products) and production of drinking water from polluted sources of water supply.


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