Method of evaluation of toxicity of environmental components of azov and black seas

FIELD: ecology.

SUBSTANCE: method comprises placing the fluorescent test-objects in the control and analyzed samples, irradiation with the excitation light, definition of fluorescence characteristics, by which change the toxicity of the controlled environment is assessed. Microalgae of species Scenedesmus apiculatus are used as test-objects, which are previously isolated from environmentally safe areas of the test water reservoirs.

EFFECT: use of the claimed method enables to assess quickly and accurately the toxicity of water and bottom sediments of the Azov and Black Seas.

6 tbl, 4 ex

 

The invention relates to ecology and toxicology, and can be used to assess the toxicity of water and sediments of the Azov and Black seas in the conditions of economic activity, and in emergency situations.

It is known that under the action of various environmental factors and anthropogenic pollution on aquatic ecosystems primarily changes in the photosynthetic activity of the cells of photosynthetic organisms. These changes lead to changes in all other parts of the ecosystem.

To assess the toxicity of the waters of the Azov-black sea basin used a strain of luminescent bacteria Ph. phosphoreum (Cohn) Ford (Ioualalen, Amitav. Luminous bacteria of the Black and Azov seas. Ecology of the sea. 2003. Vol. 64) (1), bacterial test "Ecolum"developed in Russia (THE 6-09-20-236-93, MSU, Moscow) (2), strains of bacteria Vibrio fischeri VKPM B-9579 (Patent RF №2346035. MCI C12N 1/20 2007) (3) and Vibrio fischeri VKPM B-9580 (Patent RF №2342434 MCI C12N 1/20) (4). These strains were isolated from water of the Black sea. Biotesting based on the sensitivity of bioluminescent bacteria to the action of toxicants present in water and sediments of marine basins. In the known methods the toxicity assessment is based on determining the change in the intensity of bioluminescence of bacteria when exposed to a current of the practical substances, present in the analyzed sample, compared with the control. However, the content of the bacterial cultures demanding in terms of cultivation, requires frequent pereselenie that leads to great effort and expense. In addition, there are known cases of loss strain of bioluminescent activity (properties) during storage.

To control the waters of the Azov-black sea basin, in addition to the bacterial cells, you can use other organisms, such as algae cells. Algae, like all autotrophs, play a vital role in the food web of the ecosystem. Violation of toxins in their physiological activity, as well as the structure of algocenoses have serious consequences for ecosystems. Research methods phytoplankton, based on the measurement of fluorescence, currently widely used in the laboratory for experimental cultures of algae, in waters, in the field. Measuring the fluorescence of phytoplankton, it is possible to calculate the concentration of chlorophyll in microalgae (gold V.M., Gajewski N.A., Tents YOU, Popelinsky VA, Scars S.A. experience in the use of fluorescence for the differential assessment of chlorophyll and plankton algae// Hydrobiol. Journe. 1986. So 22, №3) (5).

There is a method of determining the toxicity of water (AU CAS is no 1405745 MCI AC 61/00, G01N 33/18) (6), which control the change in the output intensity of the pigments in the environment under the action of toxic compounds, where as pigmentaria test object using marine red algae of the genus Callithamnion. Standard international methods of biotesting of sea water, developed under the auspices of ISO (International Standard Organization)are test systems using microalgae Phaeodactylum tricornutum and Skeletonema costatum (Water quality - Algal growth inhibition test with Skeletonema costatum and Phaeodactylum tricornutum. Draft International Standard ISO/DIS 10253.2. 1994. 12 p.) (7).

Analyses on the microalgae give a statistical advantage over many test objects, as it is possible to use a larger number of cells, requires a much smaller sample volumes and time of testing, maintenance spare cultures, due to their rare pereselenia and low demands to the cultivation conditions, does not require much effort and expense. The testing process is easy to automate.

The closest solution is selected as a prototype method for assessing the toxicity of liquid (autospid. The USSR №1515105 MCI G01N 33/18) (8)providing for the cultivation of photosynthetic test object, the excitation of the luminescence of the test object and the determination of the fluorescence characteristics change which is judged on the toxicity of the controlled fluid.

When is ispolzovanie algae to assess the toxicity of marine waters in different seas is not always achieved with the adaptation of the test object to a specific waters, that reduces the reliability of the results. In particular, when assessing the toxicity of natural waters that do not meet the natural hydrochemical composition of water in which were grown culture of the test object, the reaction of the test object can be regarded as toxic effects of the investigated water. Therefore, the search for test objects in the Azov and Black seas was aimed at the selection of microalgae, which could serve as a test-object pollutants waters of the Azov and Black seas.

The problem solved by the invention, the expansion of the number of test objects to assess the toxicity of the waters of the Azov-black sea basin, as well as increasing the accuracy of the information when assessing the toxicity of the environment. This object is achieved in that in the known method, including room fluorescent test object in the control and the sample, the irradiation of the stimulating light, the determination of the fluorescence characteristics change which is judged on the toxicity of controlled environment, according to the invention, as test objects used by the algae species Scenedesmus apiculatus, which is pre-allocated from the ecologically clean district of the studied reservoir.

Use as test object microalgae Scenedesmus apiculatus expands the number of test objects to assess toxicity to the marine the waters of the Azov-black sea basin and reduces the cost of testing.

When used as a test object indigenous algae Scenedesmus apiculatus, selected from ecologically clean areas of the Azov and Black seas as the most adapted to the environment of the studied reservoirs, will reduce errors and significantly increase the sensitivity of fluorescent biological testing.

The set of distinctive features of the described method provides the achievement of the task.

Comparison of the prototype with the claimed solution showed that the above features are distinctive, and therefore the claimed method meets the criterion of "novelty".

The method is as follows.

From clean areas of the Azov and Black seas selected water samples. Methods multiple dilutions and re-seeding of the samples emit green algae species Scenedesmus apiculatu, which is used as a test object. Suspension of microalgae is placed in the control (without toxicants) sample and analyze samples. Influence of the sample with exciting light pulses for excitation of fluorescence of the test objects. Determine the fluorescent characteristics of the test object, the modification of which is judged on the toxicity of the analyzed samples.

Example 1. From clean areas of the Azov and Black seas in may-June 2008, i.e. during the period Akti is Noah vegetation the main species of microalgae, samples were collected of water. During this period in the Azov and Black seas beetroot algae 7 departments: Cyanophyta, Chrysophyta, Bacillariophyta, Dinophyta, Cryptophyta, Chlorophyta, Euglenophyta. Methods multiple dilutions and re-seeding of selected samples was allocated 5 algological and bacteriologically pure cultures of single species of green and blue-green algae. On taxonomic facilities 3 isolates belonged to blue-green algae (Cyanophyta), and 2 strains of green algae (Chlorophyta). Blue-green algae identified as Oscillatoria laetevirens (Crouan) Gom. (=Phormidium laetevirens (Crouan et Gom.) Anagn. et. Kom.), Oscillatoria Agardhii Gom. (=Planktothrix Agardhii (Gom.) Anagn. et. Kom.) and Snowella rosea (Snow) Elenkin; green - as Oocystis borgei Snow and Scenedesmus apiculatus (W. W. et) Chod. Selected strains of algae were prepared for spectral analysis. Suspension of microalgae placed in the control (no toxins) water samples. Fluorescence spectra of solutions containing microalgae, was registered on spectrofluorophotometer RF-5301PC of Shimadzu (Japan). Using Panorama fluorescence 1.1 in scan mode (2D synchro measurement) was the analysis of the excitation spectra and luminescence and the results of their synchronization for each strain selected wavelengths with the characteristic peaks of excitement.

In the next step, we determined the sensitivity of selected cultures of microalgae to the action of a hundred the standard toxicants. An assessment of the sensitivity was performed on the relative difference in intensity of bioluminescence control and experimental samples. Suspension of microalgae were made in samples with bichromate of potassium (K2Cr2O7(the solution concentration from 0.001 to 100 mg/l), copper sulfate CuSO4(concentration of from 0.0001 to 100 mg/l) and phenol (5-1500 mg/l). Exposure times ranged from 10 to 30 minutes Response of microalgae on the impact of selected toxicants researched, exciting glow in the field maximums established for each strain of algae. It was recorded fluorescence spectra, capturing changes in light intensity at the maximum emission.

According to the results of studies of the sensitivity of all selected cultures of microalgae to the standard action of toxicants - bichromate potassium (K2Cr2O7), copper sulfate (CuSO4) and phenol were selected the most sensitive (promising for biological testing) species of microalgae Scenedesmus apiculatus and Snowella rosea.

Changing the sensitivity of selected species of microalgae when exposed to standard toxicants and the fluorescence intensity (in arbitrary units of fluorescence, his first-ever competition) microalgae Scenedesmus apiculatus and Snowella rosea in the experiment are given in table 1, 2, 3.

Table 1
The fluorescence intensity of microalgae Scenedesmus apiculatus (λwosb.220 nm, λamiss.366 nm) and Snowella rosea (λwosb.340 nm, 440 nm) in the experiment with bichromate of potash (his first-ever competition)
ControlThe concentration of toxicant K2Cr2O7(TLV 0.05 mg/l) mg/l
0,0010,010,10,51,05,010,0100,0
The fluorescence intensities of Scenedesmus apiculatus, his first-ever competition
24,520,920,319,419,519,517,214.4V0,7
The intensity of fluorescence Snowella rosea, his first-ever competition
11,311,411,512,515,318,416,515,0 2,4

td align="center"> 8,5
Table 2
The fluorescence intensity of microalgae Scenedesmus apiculatus (λwosb.220 nm, λamiss.366 nm) and Snowella rosea (λwosb.220 nm and λamiss.370 nm) in the experiment with copper sulfate (his first-ever competition)
ControlThe concentration of toxicant CuSO4mg/l
0,00010,0010,010,11,010,0100,0
The fluorescence intensities of Scenedesmus apiculatus, his first-ever competition
24,521,820,519,3of 17.515,912,21,2
The intensity of bioluminescence Snowella rosea, his first-ever competition
11,311,411,912,8the 13.414,17,9

Table 3
The fluorescence intensity of microalgae Scenedesmus apiculatus (λwosb.220 nm, λamiss.366 nm) and Snowella rosea (λwosb.340 nm, λamiss.440 nm) in the experiment with phenol (his first-ever competition)
ControlThe concentration of toxicant phenol mg/l
5,050,0100,0300700100012001500
The intensity of florescence Scenedesmus apiculatus, his first-ever competition
24,516,70,50,100000
The intensity of fluorescence Snowella rosea, his first-ever competition
11,312,914,315,1 9,27,56,76,0

From table 1 it is evident that under the influence of K2Cr2O7(0.01-10 mg/l) (MCL of 0.05 mg/l) fluorescence intensity (λwosb.220 nm, λamiss.366 nm) of the culture of Scenedesmus apiculatus decreased up to a maximum of 45-50% from the reference level (depending on exposure time and conditions fluorimetry).

For culture Snowella rosea in the presence of the same concentrations of K2Cr2O7the maximum increase in fluorescence intensity to 60% (λwosb.340 nm, λamiss.440 nm).

Higher concentrations of K2Cr2O7(at the level of 50-100 mg/l) 45-99% inhibited the fluorescence of both cultures of algae.

Table 2 shows that in solutions CuSO4(0.01-10 mg/l Scenedesmus apiculatus showed a similar sensitivity, as evidenced by the decrease in fluorescence intensity (λwosb.220 nm, λamiss.366 nm) a maximum of 50% of the control level. In a solution with a concentration of CuSO4100 mg/l was observed almost complete quenching of fluorescence suspension of Scenedesmus apiculatus.

Under the influence of low concentrations of CuSO4(0.0001-1.0 mg/l) fluorescence spectra Snowella rosea is not noticeably changed. The highest induction of luminescence (up to 25% of control, λin the b. 340 nm, λamiss.440 nm) was registered in a solution of CuSO4with a concentration of 1.0 mg/l Exposure time of the algae in the culture medium CuSO4in concentrations of 10 and 100 mg/l causes quenching of luminescence by 25 and 30%. Therefore, the sensitivity of culture Snowella rosea to CuSO4was about 2 times lower than Scenedesmus apiculatus.

From table 3 it can be seen that phenol at a concentration of 5 mg/l inhibited the fluorescence Scenedesmus apiculatus (λwosb.220 nm, λamiss.366 nm) at 10-55%, and the solution containing phenol (50 mg/l for fluorescence quenching was nearly 100%. At the same time, Snowella rosea showed low sensitivity to phenol: fluorescence intensity of 1.5 times inhibited only phenol concentrations 1000 mg/L.

Example 2. For comparative evaluation of the sensitivity of the used characteristic EU50(effective concentration) is the concentration of substance that causes 50%reduction in bioluminescence suspension of microalgae.

Using EC50(effective concentration of a toxicant that reduces the luminescence by 50%) allowed us to compare the susceptibility of algae Scenedesmus apiculatus and Snowella rosea with a sensitivity of bioluminescent bacteria E. coli RT-5, Ph. phosphoreum (Cohn) Ford and indigenous strains of bacteria Vibrio fischeri VKPM B-9579 and Vibrio fischeri VKPM B-9580 isolated from waters of the Azov and Black seas (table 4).

Table 4
The susceptibility of algae and known strains of luminous bacteria to different toxicants (inhibition of luminescence), EU50mg/l
Culture of algae and bacteria strains used as test-objectsEC50mg/l
CuSO4K2Cr2O7Phenol
Scenedesmus apiculatus10-4010-505-30
Snowella rosea20-5020-50-
Vibrio fischeri In 957911-1 .510-50125-150
Vibrio fischeri In 95802210-50125-150
Eshebo (pPLS-5)35-7 .5150>300
P. phosphoreum (Cohn) Ford4 250170
MACpx(mg/l)0.005 - in terms of C2+0.05 - substance0.001
1RF patent №2346035, 2007;2RF patent №2342434, 2007;3RF patent №79581,2001;4Malygina, Katsev, 2003.

Comparison of the sensitivity of native microalgae investigated toxic substances (table 4) showed a 2 times higher sensitivity of microalgae Scenedesmus apiculatus in accordance with the sensitivity of Snowella rosea. Therefore, for testing as the test object was selected cuts Scenedesmus apiculatus.

Comparison of the sensitivity of native microalgae Scenedesmus apiculatus average EC50investigated the toxic substances showed that Scenedesmus apiculatus more sensitive than strain Ph. phosphoreum (Cohn) Ford. In particular, in average it is about one order of magnitude more sensitive to K2Cr2O7. So, the value of EC50the microalgae Scenedesmus apiculatus the bichromate of potassium is in the range of concentrations from 10 to 50 mg/l, and Ph. phosphoreum is 250 mg/L.

The phenol value EC50for microalgae Scenedesmus apiculatus was 5-30 mg/l, which is also lower than EC50D. the I Ph. phosphoreum.

Comparison of the sensitivity of microalgae Scenedesmus apiculatus to toxic substances with sensitivity lux-strain E.coli RT-5 showed similar results, except CuSO4. Cuts Scenedesmus apiculatus more sensitive to K2Cr2O7, (~ 5 times), and phenol (approximately), but inferior in sensitivity to CuSO4(~ 4 times).

The findings suggest the potential use of microalgae Scenedesmus apiculatus to determine the toxicity of aquatic environments.

Example 3. Conducted inspection sensitivity is used as the test object algae species Scenedesmus apiculatus isolated from waters of the Black and Azov seas. From clean areas of the Azov and Black seas were selected water samples. They were placed microalgae Scenedesmus apiculatus, selected from different seas.

Studies have shown that culture of Scenedesmus apiculatus from different seas, placed in a "native" and "nonnative" environment, distinguished by the intensity of fluorescence (λwosb.220 nm, λamiss.366 nm) in the range of 12-17%, depending on the exposure conditions (table 5).

Table 5
Deviation from the monitoring the fluorescence intensity of Scenedesmus apiculatus (λwosb.220 is m, λamiss.366 nm) in the water of the Black and Azov seas (the exposure time - 10 minutes)%
No.Option waterScenedesmus apiculatus (from the water of the sea of Azov)Scenedesmus apiculatus (from waters of the Black sea)
1The Black sea water sample-17,15of 4.45
2Sample the water of the sea of Azov3,30-12,45

Thus, indigenous algae placed in "non-native" environment, showed 2-3 times larger deviation of the level of fluorescence from that of microalgae placed in the "native" environment. This deviation can distort the measurement results of the toxicity of the samples in the "non-native" environment, which points to the need for the use of indigenous microalgae (as the most adapted to the conditions of the reservoir) in the practice of biotesting of water and water extracts of sediment.

Example 4. Experiments were conducted to determine the toxicity of the components of the environment (water, sediments) of the Black sea using indigenous microalga Scenedesmus apiculatus as the test object. Were investigated 3 water samples and 3 samples of bottom sediments, selected in an area with high anthropogenic pollution (water area of the black sea port). To prepare extracts of sediments as a solvent used clear water of the Black sea, pre-filtered through a bacterial filter, in a weight ratio of 10:1 from dried at room temperature at ground level. Bottom sediment samples were shaken in an orbital shaker at 65 rpm at room temperature for one hour and then defended before the sedimentation of suspended solids. The pooled extracts were filtered through a siphon (capron network No. 76) and used in further toxicity studies.

The toxicity of water extracts of sediment was determined from the change in the fluorescence intensity of Scenedesmus apiculatus (λwosb.220 nm, λamiss.366 nm) relative to the control (pure sea water of the Black sea). The exposure time to 10 minutes While the deviation of the intensity of illumination in the test sample relative to the control (both downward and upward) less than 20% indicates the absence of toxicity, from 20 to 30% of low toxicity, from 30 to 50% - moderate toxicity, more than 50% - about acute toxicity.

The research results are summarized in table 6.

Tab the Itza 6
The results of biological testing of water samples and sediments from the contaminated waters of the Black sea on the culture of native algae Scenedesmus apiculatus (deviation from control fluorescence intensity, %; denominator - toxicity index, points)
The test sampleλwosb.220 NM, λamiss.366 nm
The exposure time, min
1030
Water 1-4,270-cent to 8.850
Water 2-to 21.771-10,670
Water 3-charged 8.520 -13,420
Sediment 1-34,492-34,862
Sediment 2-40,952-41,922
Sediment 3-52,093-48,842

According to the test results, the decrease in fluorescence intensity relative to control in water samples No. 1, 2 and 3 depending on the exposure time, were, respectively, -4.27 - -8.85%; -21.77 - -10.67%- 8.52 - 13.42%, in accordance with the above scale, characterizes water samples No. 1 and 3 as non-toxic, sample # 2, depending on the test conditions, as not toxic - slightly toxic.

The intensity of fluorescence in extracts of sediments No. 1,2 and 3 depending on the exposure time decreased relative to the control, respectively-at 34.49 - -at 34.86%, -at 40.95 - -41.92%- 52.09 - - 48.84%. According to the results of fluorimetry sediments 1 and 2 are evaluated, so as moderately toxic, sample # 3 - as acutely toxic. The results of the testing indicate moderate toxicity of bottom sediment samples No. 1 and 2 and acute toxicity - sample No. 3. In the sediment sample No. 3, the analytical data was recorded highest for the studied area of the port oil (32.6 g/kg of dry soil), ASAS (91 mg/kg), phenol (4.1 mg/kg) and polycyclic aromatic hydrocarbons (1010 mg/kg).

The test method of biotesting of water and bottom sediments taken in an area with high anthropogenic pollution (sea port) using Scenedesmus apiculatus as the test object, enabled by changes in the intensity of fluorescence (λamiss.366 nm) to establish the toxicity of water samples and bottom sediments. However, toxicity values for water samples were lower than for extracts of bottom sediments is s and in some cases correlated with the content in samples of surface-active substances, oil and phenol.

In General, high sensitivity of selected cultures of the algae tested toxicants and approbation of the method of biotesting on the basis of Scenedesmus apiculatus in the Black sea suggest a promising use of the isolated cultures of microalgae as test objects to determine the toxicity of the components of the environment marine water bodies in the complex conditions of anthropogenic pollution.

The developed method of biotesting can be used for rapid assessment of toxic substances in liquids, for example, when discharges to the environment waste (waste) water treatment and continuous monitoring of the toxicity of the environment, including emergency cases and adverse environmental situations.

The inventive method of determining toxicity differs from similar systems higher sensitivity to toxicants (registration is possible toxic effect of fluorine on the level of the MPC for water fisheries of the reservoir), rapid response (results of the analysis are recorded within one hour), inertia-free (light, exciting fluorescence, little changes of the physiological state of the test object), low cost of analysis.

Literature

1. Ioualalen, Amitav. Luminous bacteria of the Black and Azov Mor is th. Ecology of the sea. 2003. Vol. 64.

2. THE 6-09-20-236-93

3. Patent of the Russian Federation No. 2346035. MCI C12N 1/20.

4. Patent of the Russian Federation No. 2342434 MCI C12N 1/20.

5. Gold V.M., Gajewski N.A., Tents YOU, Popelinsky VA, Scars S.A. experience in the use of fluorescence for the differential assessment of chlorophyll in planktonic dorola//Hydrobiol. Journe. 1986. So 22, No. 3.

6. USSR author's certificate No. 1405745, IPC AC 61/00, G01N 33/18.

7. Water quality - Algal growth inhibition test with Skeletonema costatum and Phaeodactylum tricornutum. Draft International Standard ISO/DIS 10253.2. 1994. 12p.

8. USSR author's certificate No. 1515105 MCI G01N 33/18 (prototype).

A method of evaluating the toxicity of the components of the environment of the Azov and Black seas including room fluorescent test object in the control and the sample, the irradiation of the stimulating light, the determination of the fluorescence characteristics change which is judged on the toxicity of the controlled environment, wherein the test objects used by the algae species Scenedesmus apiculatus, which is pre-allocated from ecologically clean areas of the studied reservoirs.



 

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FIELD: agriculture.

SUBSTANCE: hydro-biological indicators - Pantle-Buck saprobity index in modification of Sladechek is measured. At the same time the hydro-chemical parameters - the pH factor, chemical oxygen demand, dissolved oxygen level and electroconductibility are measured. The composite index is calculated by the formulas. The obtained value of the composite index is compared to the data of table 1 and according to the results the ecological state of the water reservoir is determined.

EFFECT: invention enables to accelerate the determining of the ecological state of the water reservoir by hydrochemical and hydrobiological indicators.

2 tbl, 1 ex

FIELD: biotechnology.

SUBSTANCE: uniform stacking of seeds of round red radish with white tip is carried out on the filter paper in a Petri dish. 5 ml test sample of water is poured into the dish, and the liquid level in the dish must be lower than the surface of the seeds. At that the filter paper is taken of round shape with the diameter of the Petri dish with the mark on the edge, a pattern with holes for marking the places of planting seeds is placed on it. And one of the holes is oriented relative to the filter paper mark. Then the marked filter paper is placed in a Petri dish with the orientation of its mark in a northern direction on the compass, then the seeds are laid in the Petri dish on the marked places of planting on the filter paper. The water under study is poured, after germination of seeds prior to taking seedlings from the Petri dish for measuring the length of the root in each seedling, the angle of direction of its root is measured by compass as a factor of the azimuth of the root. In undergerminated seeds the azimuth of the root is taken as the absence of a quantitative value and a dash is put in the log of measurements. Subsequently, in all the seedlings in a Petri dish the length of the root is measured, and this length in the undergerminated seeds is taken as zero.

EFFECT: improvement of the method.

15 cl, 5 tbl, 1 ex

FIELD: biotechnology.

SUBSTANCE: synchronised daphnia is obtained from the most prolific clone of daphnia population, except for the laboratory population. The test samples are prepared. From the obtained daphnias the even-aged, parthenogenetic ones are selected, and concurrently the exposure is carried out to the test solution (test), in water (control), in the solution of the reference toxicant (KT+) and in solution of mutagen (Km+). The physiological condition in the original daphnia generation (F0) is recorded. The toxicity factors are determined, namely, the death rate (L0) and the fertility rate (P0). The physiological condition of daphnias of the second generation (F2) is recorded and the total mutagenic activity is determined according to the formula &=fex/fk where fex is frequency of lethal, sublethal and morphological mutations in the experiment, and a fk is frequency of lethal, sublethal and morphological mutations in the control with the subsequent comparing its value with isoeffective concentrations of reference mutagens (Km+) and toxicants (KT+) by release of limiting nuisance value and establishing the level of genotoxic activity on the basis of the results obtained.

EFFECT: improving the accuracy of the estimate.

2 dwg, 15 tbl, 4 ex

FIELD: chemistry.

SUBSTANCE: claimed invention relates to field of biophysics. Claimed are methods of determining space-time distribution of proteolytic enzyme activity in heterogeneous system, in accordance to which: provided is system in vitro, which contains sample of blood plasma, whole blood, water, lymph, colloidal solution, crystalloid solution or gel, and proteolytic enzyme or its precursor; fluorogenic, chromogenic or luminescent substrate for said enzyme id added; space distribution of signal of released substrate label is registered at specified time moments and space-time distribution of proteolytic enzyme activity is obtained by solving reverse problem of type "reaction-diffusion-convection" taking into account label binding with medium components. Also described is device for realisation of methods in accordance with claimed invention and method of diagnosing hemostasis disorders, based on their application.

EFFECT: invention can be further applied in the study of blood coagulation system and diagnostics of diseases associated with blood coagulation disorders.

22 cl, 6 dwg

FIELD: chemistry.

SUBSTANCE: invention refers to medicine, particularly to medical diagnostics, and may be used for two- and three-dimensional (tomographic) fluorescent imaging of a diagnosed object. A device comprises a fluorophore absorption band probing emitter provided with a fibre output, an emission receiver in the form of a CCD camera, an object scanning system with the emitter in a projection configuration, as well as a data processing and visualisation system. The device comprises the fluorophore emission band probing emitter provided with the fibre output, fluorophore absorption and emission band omnidirectional emitters in a reflection configuration, a second emission receiver with the fibre output in the form of a photomultiplier, the object scanning system of the photomultiplier in a projection configuration in relation to the probing emitter, as well as a scanning control unit. The data processing and visualising system is provided with original software for implementing methods for surface imaging, projection visualisation and diffuse fluorescent tomography.

EFFECT: device is characterised by simplicity and low measurement time.

2 cl, 3 dwg

FIELD: measurement equipment.

SUBSTANCE: invention relates to the field of monitoring of natural and process waters and is designed to determine partial concentrations of physical-chemical forms of uranium (VI) in aqueous solutions, which is necessary, in particular, for optimisation of the process of uranium extraction by method of underground leaching. The method consists in radiation of the volume of the investigated sample with nanosecond pulses of laser radiation in ultraviolet range and subsequent registration of dependence of intensity of a signal of mixture fluorescence on intensity of laser radiation and time of delay of receiver strobe relative to the laser pulse. The source of the laser radiation may be a AIG:Nd laser with conversion of the radiation frequency into the fourth harmonics (wave length 266 nm) with maximum energy in the pulse of at least 1 mJ. The system for registration of the fluorescence signal may be a CCD-chamber strobed by nanosecond pulses and connected to a spectral device (polychromator).

EFFECT: invention provides for increased accuracy of detection.

5 cl, 6 dwg

FIELD: agriculture.

SUBSTANCE: method relates to the field of agriculture, in particular fruit growing and selection. The method comprises the freezing of annual shoots in the dormant period in the environmental chamber. At that the evaluation of damaged shoots is carried out not visually but according to the size of the maximum quantum efficiency of photochemical reactions of the photosystem II and the relative velocity of the electron transport by the photosystem II in the cambium tissue and buds, which are determined by the microbial cell adsorption reaction fluorometer. The minimum level of fluorescence and changes in this index under the action of actinic light with density of 190 mcmol/(m2s) are recorded, and after exposure to the object of high intensity light pulse (10 000 mcmol/(m2s), 450 nm).

EFFECT: method enables to accelerate the evaluation of damage of fruit plants with frost.

2 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: group of inventions refers to laboratory diagnostics and may be used for diagnosis and monitoring of the treatment of various diseases. The method for monitoring of the treatment of the disease involves a fluorescence centre excitement of a biological fluid sample by the exposure to an emission of at least two wavelengths, and recording at least two spectra of the emission generated by the sample, respectively. The presence, degree and nature of the disease are identified by determining the spectral characteristics of the emission generated by the sample as compared to the respective reference (health) spectra and typical spectra of various diseases with the spectra compared within the range involving a laser emission dispersion line. The group of inventions also refers to a device for implementing the above method involving the lasers with various working wavelengths, fibre optic lines collected from the side of the sample into a bundle with a common tip, a spectrometer, a control unit and a computer to process the fluorescence spectra. The spectrometer comprises a collimator with removable optical light filter, a diffraction grating and a charge-coupled device matrix coupled with a signal pre-processor. The control unit regulates laser switching off/on and placing into a collimator of the optical light filter related to the switched-on laser.

EFFECT: group of inventions enables higher rate and accuracy of obtaining the analysis results.

7 cl, 1 dwg, 1 ex

FIELD: physics, optics.

SUBSTANCE: invention relates to a device for analysing luminescent biological microchips, having a sample holder and lighting means. The device includes laser sources for exciting fluorescent radiation and a fibre-optic system for distributing the laser radiation, a device for capturing the image of the sample, a filter for filtering fluorescent light of the sample and an optical system for projecting the fluorescent image of the sample on the image capturing device. The device is characterised by that the lighting means has an annular support around the periphery of which ends of the fibres of the fibre-optic system for distributing laser radiation are placed, wherein the fibre-optic system includes several bundles of optical fibres such that one bundle of fibres corresponds to one laser, wherein each bundle, on the side facing the sample when the sample is placed on the holder, is split into separate fibres, and the ends of the fibres from different lasers are arranged around the annular support with alternation and are directed towards the analysed sample when the sample is placed on the holder, at an acute angle to the axis of the annular support.

EFFECT: device increases uniformity of illuminating different portions of a biochip when illuminated with different lasers by enabling illumination of a sample with exciting light from different sides using separate lasers or any combination of lasers.

4 cl, 3 dwg

FIELD: physics.

SUBSTANCE: fluorescence detection system has an exciting radiation source and a radiation processing device (18, 20), having a line forming element (20) and a beam shaping element (18), a focusing device, a device for collecting fluorescent or phosphorescent radiation, a detector (28), a substrate (16) for holding a sample (14) and a means of scanning the exciting line. The exciting radiation is a line and is directed onto the sample at an angle larger than the critical angle between the substrate (16) and the sample (14) so that the exciting radiation undergoes total internal reflection at the substrate-sample boundary and is damped. The beam shaping element (18) is designed to shape the beam into a an annular shape (34), and the line forming element (20) is capable of shaping the annular beam, which is converted by the focusing device into an exciting line.

EFFECT: increasing the rate of measurement without losing sensitivity.

15 cl, 5 dwg

FIELD: chemistry.

SUBSTANCE: method involves adsorbing DBMBF2 or a derivative thereof on a polymer matrix containing polar groups (e.g. OH groups). Presence of pyridine vapour in air is indicated by fluorescence in the 400-500 nm region under the action of pyridine when a matrix with low fluorophore content (such that it is mainly in monomer form) is used, or by increasing fluorescence intensity in the 400-500 nm region with simultaneous decrease in intensity in the 500-600 nm region (if there is considerable content of the dimer form along with the monomer form in the matrix).

EFFECT: detecting pyridine vapour in air in 10-60 s.

3 cl, 3 ex, 7 dwg

FIELD: biotechnologies.

SUBSTANCE: proposed chimeric protein with SEQ ID NO:02 is fluorescent biosensor, built on the basis of HyPer protein and mutant of PH-domain of Btk tyrosine kinase.

EFFECT: proposed inventions allow performing simultaneous monitoring of product of hydrogen peroxide and phosphatidyl inositol-3,4,5-triphosphate in a living cell.

4 cl, 4 dwg, 3 ex

FIELD: measurement equipment.

SUBSTANCE: for classification of oil impurities on water surface, test water surface is irradiated in UV-range on excitation wave length λe, intensity of fluorescent radiation I(λ1), I(λ2), I(λ3), I(λ4) from the test water surface is recorded in four narrow spectral ranges with centres on wave lengths λ1, λ2, λ3, λ4, which have been chosen from the condition of maximum distance between classes in two-dimensional space of classification criteria and Values K1 and K2 are found for the test water surface, and belonging of oil impurity to one of the classes is estimated as per penetration of the found values K1 and K2 for the test water surface to the area corresponding to that class in two-dimensional space of classification criteria.

EFFECT: invention allows performing classification as per four groups: water with different characteristics, protein or algae in water, crude oil, heavy oil products, and light cleaned oil products.

5 dwg, 2 tbl

FIELD: medicine.

SUBSTANCE: method involves taking bone tissue fragment sample in area under examination, measuring relative laser luminescence level. The obtained values are compared to normal bone tissue characteristics. Quantitative reduction of mineral composition being found relative to reference value in normal state is diagnosed by interpreting spectral characteristics in diagnostic bandwidth of 350-550 nm.

EFFECT: high accuracy of diagnosis.

2 dwg

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