Nanosized materials have shown a relevant potential for practical application in a broad number of research fields, in industrial production and in everyday life. However, these substances acquire new properties and therefore may be biologically very active. This raise questions their potential toxic effects on living organisms. In some cases the nanosized materials or nano-composites possess distinct positive properties in enhancing the adaptation of plants in unfavorable conditions and in decreasing the negative effect of some chemical substances. The information about the positive and negative effects of nano-materials as well as the data concerned to the innovative approaches used by authors for the rapid assessment of the total toxicity with the exploitation of bacteria, Daphnia and plants are given. In last case a special attention is paid to the control of natural bioluminescence and chemoluminescence of living medium of organisms, the energy of the seed germination and the efficiency of the photosynthetic apparatus in growing plants by the estimation of chlorophyll fluorescence by the special “Floratest” biosensor. Three specific clases of nano-materials are analysed: a) nano-particles ZnO, Ag2O, FeOx, TiO2 and others, b) colloidal suspension of the same compounds, and c) nanostructured layered clay materials (acid saponites and Nb-containing saponite clays). The next features are analyzed: the biocidal activity (for nanoparticles), the improvement of the nutrition of plants on calcareous soils (for colloidal structures), the activity and performances as heterogeneous catalysts (for Nb-containing saponites, as selective oxidation catalysts for toxic organosulfur compounds into non-noxious products). The chemical and physical characterization of the nanosized materials described here was studied by different spectrophotometric and microscopic techniques, including AFM and SEM.
It is presented the experimental results about the investigations of the efficiency of the structured nano-pourous silicon (sNPS) application as transducer in the immune biosensors designed for the control of retroviral bovine leucosis (RBL) and the determination of the level such mycotoxins as T2 and patulin among environmental objects. Today, there is an arsenal of the traditional immunological methods that allow for the biochemical diagnostics of the above diseases and control of toxins but they are deeply routine and can not provide the requirements of practice for express analysis, its low cost and simplicity. Early to provide practical demands we developed immune biosensors based on SPR, TIRE and thermistors. To find more simple variant of the assay we studied the efficiency sNPS as trasducer in immune biosensor. The registration of the specific signals was made by measuremets of level of chemiluminescence (ChL) or photocurrent. The sensitivity of biosensor for both variants of the specific signal registration at the determination of T2 and patulin was about 10-20 ng/ml. Sensitivity analysis of RBL by this immune biosensors exceeds traditionally used approaches including the ELISA-method too. The optimal serum dilution of blood at the screening leukemia should be no less than 1:100, or even 1:500. The immune biosensor may be applied too for express screening leucosis through analysis of milk. In this case the optimal serum dilution of milk should be about 1:20. The total time of analysis including all steps (immobilization of specific Ab or antigens on the transducer surface and measurements) was about 40 min and it may be a sharp decline if the above mentione sensitive elements will be immobilized preliminary measurements. It is concluded that the proposed type of transducer for immune biosensor is effective for analysis of mycotoxins in screening regime.
In last time gardens are often at the dangerous of viruses and bacteria infections. To preserve not only the coming
harvest, but, in generally, to provide stability and growing horticultures the development of new generation of the
analytical techniques for remote express screening vegetative state arrays and direct control of the appropriate infection
if appearance of its maybe expected on the basis of previous surveys are very actually and important. For continuous
monitoring we propose the application of the complex of the optical analytical devices as “Floratest” and “Plasmatest”
(both produced in Ukraine) which is able to control step by step general situation with vegetable state and verify
concrete situation with infection. General screening is accomplished on the control of the intensity of chlorophyll
induction (IChF), namely, registration of so called Kautsky curve which testifies about physiological mechanisms of
energy generation, accumulation and effective ways of its realization in cells. The measuring may be done by direct
way on the number of individual vegetables and remote screening of massive with transferring registered signal direct
in the laboratory. Next step of control connected with the application of the surface plasmon resonance (SPR) based
immune biosensor which is able to determine concrete bacteria (for example, Erwinia amilovora) with the limit
detection about 0.2 μg/ml, the overall time of the analysis within 30 min (5 min of the duration of one measurement).
The traditional ELISA-method showed the sensitivity to this pathogen about 0.5 μg/ml, overall time of the analysis
several hours and obligatory using additional expensive reagents.
The real manifestations of biowarfare were only during local military conflicts. In previous times the danger with this
factor forms through activation of terrorist organizations. To prevent non-desirable consequences and to preserve the
lives and health of people it is necessary to provide constant control by the application of complex of the new generation
of the instrumental devices based on the principles of biosensorics which allow rapid revealing step by step: total toxicity
of environmental objects, presence of appropriate groups and specific chemical substances among them. It is
demonstrated simple and rapid estimation of the total toxicity through the control of the intensity of chlorophyll
fluorescence (IChF) by the direct or remote ways by the device “Floratest” (Ukraine). There is possible on the basis of
IChF curve of growing plants from some territory during appropriate period to reveal appearance of toxic substances in
this area. Their presence in more local cases may be done by using bioluminescent bacteria (pure Ukrainian strains)
or/and controlling short term Daphnia living medium by developed portable chemiluminometer. For the revealing of
some groups of toxic elements it is recommended the cerium oxide ISFETs based enzymatic biosensors. The last and
devices based on SPR (“Plasmotest”, Ukraine), porous silicon (with the registration of biospecific interaction
macromolecules by luminescence or electro conductivity) and some nano-metal oxides were realized in immune
biosensors at the determination of content of number of mycotoxins, some microorganisms (Salmonella spp.) and
diagnostics of viral disease (retroviral leucosis). We present the main characteristics of the above mentioned devices and
give confirmation that all the analysis meets practice demands. Overall time of analysis is in range 10 min and it is very
simple and may be realized in field conditions.
The results of studies about the effects of colloidal solutions of Fe and Zn on the photosynthetic activity of plants of yellow lupine affected by carbonate chlorosis are given. It is shown that the impression of plants by carbonate chlorosis causes a decrease in the efficiency of photosystem II and in result of that the affected plants lag in a weight. Processing plants by the colloidal solutions of iron and zinc creates conditions for improvement of function of the photosynthetic apparatus of plants.
The purpose of the present study was to determine the effectiveness of the chlorophyll fluorescence method induction for the estimation of the functional state of the photosynthetic activity of plants. The study was done using the "Floratest" prototype portable device. It was found that the representative plants in three ecological zones have a significant difference of indexes, what, through the changes of the chlorophyll fluorescence induction, reflects the processes of energy transformation at the early stages of photosynthesis. It was stated that value of Kpl plateau in the Kautsky curve can be used as a test for the early selection of the horse chestnut forms, resistant to the influence of the complex environmental factors. The results in bean leaves indicated that the fluorescent indices are sensitive to the effects of such stressors as acidification, salinity, dehydration.
The effect of silver and zinc oxide nanoparticles in combination with alginate on bioluminescent Photobacterium leiognathi Sh1 bacteria was investigated. Silver nanoparticles were found to be more toxic than zinc oxide nanoparticles on bioluminescent bacteria. The nanoparticles and their ions released results in the same effect, however, it was absent in combination with alginate. The effective inhibiting concentration (EC50) for silver nanoparticles was found about 0.3 – 0.4 μg mL-1, which was up to two times larger then for zinc oxide nanoparticles. The absence of sodium chloride in the tested media prevented the formation of colloidal particles of larger size and the effective inhibition concentrations of metal derivatives were lower than in the presence of sodium chloride.
The main modern approaches which were realized at the development of new generation of biosensors intended for application in field of diagnostics, food quality control and environmental monitoring are presented. The main attention was paid to creation of the multi-parametrical and multi-functional enzymatic and immune biosensors which were realized for the complex diagnostics of diabetes, autoimmune state and for the control of process of sugar production. The label-free bioaffine devices based on the nano-porouse silicon (NPS) with the registration of specific formed signal by chemiluminescence (ChL) and photoresistivity and intended for the determination mycotoxins and diagnostics of retroviral bovine leukemia (RBL) are analyzed too. Improving of ion sensitive field effect transistors (ISFETs) through changing silicon nitride on the cerium oxide is discussed as perspective approach in case of micotoxins and Salmonella control. In the conclusion the possibility to replace biological sensitive elements by artificial ones is considered.
In this work we have designed high-specific biosensors for Salmonella typhimurium detection based on the surface plasmon resonance (SPR) and total internal reflection ellipsometry (TIRE). It has been demonstrated high selectivity and sensitivity of analysis. As a registering part for our experiments the Spreeta (USA) and “Plasmonotest” (Ukraine) with flowing cell have been applied among of SPR device. Previous researches confirmed an efficiency of SPR biosensors using for detecting of specific antigen-antibody interactions therefore this type of reactions with some previous preparations of surface binding layer was used as reactive part. It has been defined that in case with Spreeta sensitivity was on the level 103 – 107 cells/ml. Another biosensor based on the SPR has shown the sensitivity within 101 – 106 cells/ml. Maximal sensitivity was on the level of several cells in 10 ml (up to the fact that less than 5 cells) which has been obtained using the biosensor based on TIRE.
It was compared the efficiency of patulin control at the application of such optical biosensors which were based on the surface plasmon resonance (SPR) and nano-porous silicon (sNPS). In last case the intensity of the immune reaction was registered by measuring level of chemiluminescence (ChL) or photocurrent of nPS. The sensitivity of this mycotoxin determination by first type of immune biosensor was 0.05-10 mg/L Approximately the same sensitivity as well as the overall time analysis were demonstrated by the immune biosensor based on the nPS too. Nevertheless, the last type of biosensor was simpler in technical aspect and the cost of analysis was cheapest. That is why, it was recommend the nPS based immune biosensor for wide screening application and SPR one for some additional control or verification of preliminary obtained results. In this article a special attention was given to condition of sample preparation for analysis, in particular, micotoxin extraction from potao and some juices. Moreover, it was compared the efficiency of the above mentioned immune biosensors with such traditional approach of mycotoxin determination as the ELISA-method. In the result of investigation and discussion of obtained data it was concluded that both type of the immune biosensors are able to fulfill modern practice demand in respect sensitivity, rapidity, simplicity and cheapness of analysis.
New type of the multi-immune sensor was elaborated. It is based on electrolyte-insulator-semiconductors structures and intended for determination of such herbicides as simazine, atrazine and 2,4-D. The specific antibodies were immobilized on nitrocellulose disks, which were placed in measuring cells. The analysis was fulfilled by sequential saturation of antibodies, left unbound after their exposure to native herbicide in investigated sample, with labelled herbicide. If horse radish peroxidase (HRP) was used as label the sensitivity of this multi-immune sensor was about 5 and 1.25 (mu) g/L for simazine and 2,4-D, respectively. At the changing of HRP by (beta) -glucose oxidase the sensitivity of analysis of these herbicides increased approximately in 5 times. The linear plots of the registered concentrations were in the range of 1,0-150,0 and 0,25-150,0 ng/mL for simazine and 2,4-D respectively. It was recommended to use the developed immune sensor for wide screening of herbicides in environment. The ways for increasing of its sensitivity were proposed.
A new sensitive method based on bioinduced chemiluminescence and Daphnia as a test object is proposed for the evaluation of general toxicity of environment. The method was tested at the determination of biological toxicity of the solution of potassium chromate and at the control of water toxicity of some rivers of Ukraine. Special attention was paid to the optimization of conditions for the chemiluminescence determination. The medium of Daphnia staying was shown to have no spontaneous chemiluminescence. This was revealed using hydrogen peroxide and luminol, the optimal concentrations of which were 23 and 1.6x10-2 mmol/L. p-Iodphenol at low concentrations (4x10-5- 2x10-3 mmol/L) did not effect chemiluminescence signal but at high concentrations (4x10-2 mmol/L) an inhibition of chemiluminescence was observed. To obtain the needed intensity of chemiluminescence it is necessary to incubate no more than 5 Daphnia persons in volume of 10 mL of sample to be analyzed. The intensity of chemiluminescence of Daphnia staying medium and the sensitivity of this organism to potassium chromate increased at the temperature increasing from 24 to 32 degree(s)C. Medium of Daphnia staying can be preserved in refrigerator for several hours without loss of chemiluminescence signal.
System for determination of glucose in aqueous solution based on silicon nitride ISFET has been created and tested. Glucose oxidase deposited onto the gate of ISFET provided high sensitivity towards presence of glucose in analyzed solution. Concentration dependence has been studied. It was found that the detection limit was less than 0.1 mMol/l and response was linear with a slope amounting to 55 - 57 mV/decade.
Immobilization of human immunoglobuline (IgG) (AG) and goat on human IGG antibodies (AB) as well as AB/AG specific reaction were studied with planar polarization interferometry (PPI). In this novel method, polarized laser beam was coupled into the planar waveguide made on silicon wafer and consisted of 20nm Si3N4 layer sandwiched between two 1.5 micrometers SiO2 layers with the sensing window etched in the top SIO2 layer. One of the immune components was deposited by means of polyelectrolyte self- assembly on top of the Si3N3 layer within the sensing window, P-component of the polarized light is sensitive to adsorption, while s-component serves as a reference. Thus the outcoming light intensity depends on the phase shift between s- and p-components. Different sequences of immobilization of the immune components were studied with both surface plasmon resonance (SPR) and PPI methods. It was shown that predeposition of a monolayer of protein A, which is believed to affect the orientation of the immune components, causes an additional increase in the sensitivity. PPI method allowed us to improve substantially the sensitivity towards AB/AG reaction as compared to traditional SPR method. Particularly, of specific binding of 3ng/ml AG was registered.
Composite films containing enzyme and indicator molecules were produced by means of polyelectrolyte self-assembly. These membranes provide two functions: (i) molecular recognition of the substratum by respective enzyme, and (ii) optrode transducing, when the products o the substratum decomposition affect optical spectra of indicator molecules. Apart from direct registration of enzyme reactions, inhibition reactions can also be monitored with this method. Particularly, heavy metal salts and phosphor organic pesticides acting as inhibitors for Urease and Cholinesterase, respectively, were registered. Composite PESA films were deposited onto glass slides and consisted of several layers of poly(alylamine) hydrochloride (PAA) alternated with indicator molecules, either Cyclo-tetra- chromotropylene or Thymol Blue, both containing SO3- Na+ groups. Then a few layers of PAA/enzyme were deposited on top. A typical structure of the samples was (PAA/Indicator)n/(PAA/Enzyme)m/PAA with n equals 1-5. The obtained films were characterized with UV-visible absorption spectroscopy. The effect of the substrate decomposition on the UV-vis spectra of respective indicator molecules was studied. The inhibition of enzymes Urease and Cholinesterase by heavy metal ions and phosphor organic pesticide, respectively was found. The results obtained show the prospects towards development of optical enzyme sensor arrays.
In the paper the results about changes of the current- voltage characteristics of the surface-barrier structures (SBS) after the deposition of myoglobin and its specific monoclonal antibodies are presented. It was shown that the SBS structures with ultra-thin films or with ultra-thin layer of the porous silicon are sensitive to the presence of the protein or its specific immune complex on their surfaces. The response of the system depended on the conditions of the specific complex formation. The characteristic optimum of the thickness of Ni film or the layer of the porous silicon was revealed when the changes of the I-V characteristics had a maximum level.
The problem of jaw-face traumatism is actual in surgical stomatology and fracture of lower jaw make up about 90% from all types of traumas. Number of approaches that include non- specific stimulation of immune system by intravenous laser irradiation were used to raise efficacy of treatment of above indicated fractures. But the application of laser beam irradiation must be controlled by independent biochemical and immunological methods. In particular it is considered the necessity of preliminary analysis of immune system state to determine dose loading of this factor. In dependence on immune system activity the laser beams can promote treatment or reduction of its efficiency. In this report the experimental data about necessity of such control in case of usage of laser beam irradiation are demonstrated. The levels of thymic serum activity (TSA), inhibitors TSA (ITSA) and sub-fractional distribution of substances in blood serum were analyzed in two groups of patients with the fracture of lower jaw: (1) with ordinary treatment and (2) with additional inclusion of number of procedures of laser irradiation. The total quantity of investigated patients was 16. It was shown that: (1) at the irradiation of patients with the normal original TSA level in blood plasma: (a) TSA decreased; (b) ITSA appeared; (c) laser-correlation spectrums changed that indicated about increase of level of substance with less translation mobility and (d) the overall term of recovery increased for 1 day; (2) when the original TSA was lower, above mentioned parameters changed into opposite direction as it is indicated in point 1. So, low intensive laser radiation can have both positive and negative effect in dependent on original organism state.
At the creation and application of biosensors appeared a number of problems which are: 1) optimization of process connected with stabilization of the structure of biomolecules at the integration with the transducers to preserve their maximum activity and 2) search of approaches to accomplish repeated analysis of substances which are irreversible inhibitors of activity of above mentioned molecules. In this article the results obtained in time of solving of these problems at the usage of enzymes as sensitive bilayer of biosensors are analyzed. For stabilization of the structure of such enzymes as:(beta) - glucose oxydase, urease, cholinesterases during their immobilization the following approaches were examined: 1) usage of one or combination of chemical substances: protein, saccharose, ethylendiamine tetraacetic acid (EDTA), glycerol, ditiotrie-tole (DTT) and specific substrates or their homologues; 2) variation of covalent crosslinking methods including usage of bifunctional reagents in aqueous and vaporous phases; 3) change of time of the influence of this reagent. Optimization of these parameters can allow to preserve about 70-80 percent of initial enzyme activity at the usage of such bifunctional reagent as glutaraldehyde. For repeated analysis of phosphoroganic pesticides and heavy metal ions which are irreversible inhibitors of enzymes the following approaches were applied: 1) treatment of enzyme membrane by special reactivating substances ; 2) usage of easily replaceable enzymatic membrane. It was shown that the last way is more preferable, particularly if alginate gel or nitrocellulose is used for direct enzyme immobilization or preparation of separated biomembrane respectively. Standard deviation of sensor responses for different membrane castings did not exceed 10 percent. At the same time this parameter changed more strongly after even one use of reactivating reagents.
The photoluminescence of the porous silicon obtained by special procedure with the usage of the chemical and laser beam treatment of silicon crystal was investigated in water, buffer and solution containing sodium chloride. It was demonstrated that the intensity of the photoluminescence did not practically change at the above mentioned conditions as well as after antigen or antibody immobilization on the porous silicon surface. But this parameter of the photoluminescence dramatically decreased in case of specific immune complex formation on the silicon surface. The level of the photoluminescence extinguishing depended on duration and intensity of immune reaction. It is proposed to use discovered effect for creation of the immunosensors based on the direct registration of immunocomplex formation.
The main scheme of the measuring cell as well as the sensor device, the analysis conditions, and the results of the quantitative determination of several antigens are presented. Special attention is paid to the immobilization of one of the immunocomponents on the fiber optic surface and three approaches were investigated: (1) covalent binding of the immunocomponent with the help of glutaraldehyde to the surface preliminary treated by aminopropilethoxysilane, (2) direct binding of the immunocomponent to silicon oxide surface by BrCN, and (3) usage of a special membrane closely connected to the fiber optics. The investigations showed that the level of immobilized biomolecules may be achieved almost two fold more by the second approach in comparison with the first one. Good results can also be received by the usage of the special membrane. The sensitivity of the fiber optic immunosensors intended for the determination of the antigens selected was comparable with that made by the enzyme-linked immunosorbent assay (ELISA) method. But in all cases the rate of analyses by immunosensors was much higher than that of the ELISA method, which is the. main advantage of such types of immunosensors.
The main scheme of the cell for measuring as well as itself sensor device, the analyses condition and the results of quantitative determination of several antigens such as estradiol-17, chorionic gonadotropin, antibodies to influenza virus, total IgG, and cell of Salmonella Typhimurium are presented. Special attention is paid to immobilization of one of the immunocomponents on the fiber optic surface.
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