Biotests

Technical data. Industrial Biotest Labs, Northbrook, lU., 1970. 

It is established by biotesting that complexation and adsor ption ar e the most important processes promoting transformation of metal compounds in biologically and the chemically inactive forms and essential decrease their toxicity. The kinetics data have shown the maximal decrease in toxicity was observed in natural water where the complexation occurred with participation of both DOM and added HS. 

By results of biotesting sewage dumped in a reservoir the payment for toxie dump pays off. More often in toxieologieal experiments as test-objeet ai e used Daphnia. Toxieity of water of eity reservoirs of Astrakhan during the different periods 2004 by use Daphnia magna is investigated. Results of reseai ehes are shown in the table. 

Paper Biotests of Animal Tissue Cholinesterase Preparations 128... 

Methods of Photometric Analysis of Ache-Biotests Image 139... 

Fig-1 The main scheme of AChE biotests technology (AChE - acetylcholinesterase PVA - polyvinil alcohol)... 

Principle Biosensors consist of paper matrixes and tissue enzyme preparations, often the pure enzyme AChE or AChE-containing cells. As seen in Fig.l, main scheme of the preparation technology and procedure includes (i) the preparation of same kinds of biotests-biosensors, which are paper matrixes impregnated with tissue preparation of AChE and covered by polymer film. (ii) biochemical reactions of the AChE activity with and without inhibitors tested and (iii) the photometric analysis of the samples for quantitative estimation of the biochemical reactions. 

Matrixes Three types of paper filters (i). Filtrak, Germany, the filter Number 388 [(0.025) - soft, wide pores] filters No. 90 (0.15) and No. 90 (0.25) - dense, narrow pores) (ii). chromatographic papers FN-5, FN-11 (Germany) the kapron membrane (pore size 0.2 microns) and (iii). the membrane Vladipor MFA-MA No. 6 (Kazan industrial associations Tasma )] were applied to matrixes for biotests. The circles (6 -10 mm dia) are cut out from the matrixes materials by special instrument. The most stable results are received with use of matrixes of chromatographic papers FN-5, FN-11. 

Procedure Some details of AChE-biotests preparation are shown in Fig. 2. 

The preparation of AChE-biotest includes the following stages, (i). the preparation of homogenate from tissue with high cholinesterase activity (electrical organ acetone powder). Homogenate are preparated in phosphate... 

Fig. 2 The scheme of preparation of AChE-biotests (details in text)...

Materials required AChE preparations, commercial acetylcholinesterase, horse (blood) serum preparation, human (blood) serum preparation, or matrix biotests with these immobilized enzyme (see section 15.2.1)... 

Procedure Cholinesterase activity in analyzed tissue or the matrix (biotest with immobilized AChE) is determined in the incubation media [consisting of substrate ATCh - 34 mmol maleate buffer 0.1 M, pH = 6.0- 6.5 ml sodium citrate 0.1 M - 0.5 ml CuS045H20 0.03M -1.0 ml distilled H20 (or inhibitor in variant with toxin analyzed) -1.0 ml potassium ferricyanide 0.005 M -1 ml.] Volume of incubation media in one test - 400 mcl. As a blank (control sample), a treatment of the exposure without the substrate is used. If inhibitory effects of allelochemical (or any toxin) are analyzed, before the substrate addition the sample was preliminary exposed to allelochemical inhibitor. Two methods for the AChE-biotests may be recommended (i) in microcells ( stationary conditions ) and (ii) in flowing columns-reactors ( dynamic conditions ). 

In cells of 0.5 volume ml at the special incubation plate, the biotests (on one in a cell) are located. In each cell, 400 mcl of the incubation medium is added (see Section 3.1) air is squeezed out with thin glass stick from under the biotest and the incubation begins at room temperature. After the incubation each cell with the biotest is washed by distilled water (3 x 200 ml) and the reaction is stopped by adding 96 % ethyl alcohol (100 ml per cell). After 15 min, alcohol is removed from the cells and the biotests are dried at room temperature for 5-10 h (or at 50°C for 1-2 h). 

II. The incubation of AChE-biotests in the flowing columns-reactors... 

AChE-biotests are located in a special column-reactor connected with the peristaltic pump and the incubation is done in flowing conditions. For this purpose special flowing, termistatic column-reactor is used (described in Supplement 2). 

The reactions of AChE-biotests results in the red-brown product (Hettchet pigment). The reaction of AChE-biotests on inhibitors can be estimated visually. The residual activity of AChE in biotests after the action of different concentrations of eserine (physostigmine) and proserine (neostigmine) is seen in Fig. 3. 

Fig. 3 AchE-biotests after normal colour reaction and after action of inhibitors...

The quantitative estimation of biotests reaction can be expressed in terms of optical density (D) or in terms of factor of reflection coefficient (Kref) ... 

The significance of biotests D, measured in the air or clean matrixes is used in the microplate photometers. 

In the photometric analysis, a calibration of samples is made with the 5-30 ml of the water solutions of Congo red dye (Congo red Ind., Beijing Huagonchang) pH 5.6. This dye has the absorbance maximum 500 nm. Before the measurements, the tests are dried up on air at 50°C for 60 min. The absorbance of Congo red is also used as a blank for the absorbance measurements of AchE-biotests on the matrixes. 

Experiment 3. Estimation of residual enzyme activity in the biotest To calculate the residual activity of AChE-biotest after AChE inhibitors action on the biotest, three measurements will be done (i) measurement Dn of the biotests after incubation with substrate, (ii) measurement Dw/s after incubation without substrate and (iii) measurement Ding after incubation with substrate and inhibitor. The amount of residual activity (A) will be calculated in percentage with the following formula ... 

For example, Table 1 shows the results of the calculation D (Experiment 2) and residual activity A (Experiment 3) in AChE-biotests (Fig. 2) after the action of the ezerine and prozerine. 

Table 1 Results of photometric analysis of AChE-biotests (see Fig.3)...

Protocols of use First register the image brightness of paper matrixes in plates (plate 1) then again register the second image of the plate with biotests (plate 2) (see suppl. 3). The value of D was calculated by the standard formula ... 

Materials required The cholinesterase preparation (biotests or simple extracts from biological tissues), the reagents for a reaction medium choosen (see section 3.2.), alkaloid physostigmine salycilate and its derivative neostigmine ("Sigma"). 

For the calculation of biotests optical density and computer analysis see 1.3.1 Experiment No. 3. 

Photometric measurements of colouring in the AChE-biotests after biochemical hydrolysis of acetylthiocholine may be made by using any... 

The use of the 96-cells plate and microplate photometers represents a convenient and fast way of quantitative photometric analysis of reactions of the chemical tests and biotests made on the basis of paper materials. The firm microplate photometers are supplied with the necessary software and systems of scanning a plate which carry out not only one-wave, but also the multiwave photometric analysis, that will enable us to increase the accuracy of the analysis. 

The measurement of optical density in AChE-biotests after analytical procedure can be done on special microplate photometer, for example, MicroReader 4 (Hyperion Inc., USA). The measurement of optical density was at X = 490 nm. For installation of microplate photometer parameters, the software of the device is used. According to our data, the best results were found with the use of modified 96-cells plate (Budantsev and Budantseva, 2005). They differ from a standard plastic plate by absence of cells bottom. 

Indirect smeasurement of optical parameters AChE-biotests (TV-computer analysis)... 

---