Liquid photopolymerizable compositions as immobilized matrix of biosensors.

Series of liquid photopolymerizable compositions (LPhPC) based on oligouretanemetacrylate (OUM-1000T and OUM-2000T) and oligocarbonatemetacrylate (OCM-2), monomethacrylic ether of ethylene glycol and vinylpyrrolidone (VP) were tested. It was shown that the LPhPC, which contained VP (as basic hydrophylic matrix), OCM-2 (cross-linking agent) and OUM-2000T (to increase adsorption of polymer) was the most optimal. The blend contained 3 g/100 ml of enzyme. ISFET based biosensors for analysis of glucose and urea had the following characteristics: linear response in the range of concentrations 0.1-10 mmol/l, 0.05-20 mmol/l, angle of slope of concentration curve--30 mV/pC, 38 mV/pC, and response time of approximately 10-15, 5-10 min, correspondingly. The value of Km for immobilized urease and beta-glucose oxidase (GOD) achieved 0.85 and 3.1 mmol/l, respectively. It was established that under immobilization conditions at 20 degrees C the residual activity of GOD was about 35% from the initial level, the residual activity of horseradish peroxidase (HRP) and urease was 42% and 20%, respectively. In case of an immobilization of GOD at -50 degrees C its residual activity reached almost 50% from the initial level. It was investigated how different sources of UV radiation and different substances (including specific and non-specific substrates) influenced stability of the enzymes in the LPhPC and in the prepared membrane at storage. Dynamics of changes of enzyme activity at the process of photo immobilization was characterized, and requirements for enzyme maximal storage were selected. The proposed LPhPC may be prepared in advance since enzymes do not lose their activity during 2 months. Therefore, two processes, i.e. manufacturing of a transducer and preparation of a biological membrane on its surface, can be combined in one. In order to achieve this, approaches of modern electronics, such as for example photolithography, can be used. The developed LPhPC is homogenous, non-active to biological substances, permeable for the analyzed sample, can be prepared using a simple immobilization procedure, and has a defined hydrophobic-hydrophilic balance and sufficient level of adhesion to transducer surfaces. These all cover the requirements to modern biosensors.

Registration of T-2 mycotoxin with total internal reflection ellipsometry and QCM impedance methods.

A sensitive optical method of total internal reflection ellipsometry (TIRE) in conjunction with immune assay approach was exploited for the registration of T-2 mycotoxin in a wide range of concentrations from 100 microg/ml down to 0.15 ng/ml. Association constants of 1.4x10(6) and 1.9x10(7)mol(-1)s for poly- and monoclonal T-2 antibodies, respectively, were evaluated from TIRE kinetic measurements. According to TIRE data fitting, binding of T-2 molecules to antibodies (at saturation) has resulted in the increase in adsorbed layer thickness of 4-5 nm. The QCM impedance measurements data showed anomalously large mass increase and film softening, most likely, due to the binding of large T-2 aggregates to antibodies.

Thermal biosensor for detecting nonylphenol in the environment.

The main goal of the research was the development of thermal immune biosensor for highly sensitive and specific determination of nonylphenol (NPh), based on measuring the heat released as a result of the interaction between hapten and specific antibodies. As it was shown previously, in case of SPR based immune biosensor a number of algorithms of analysis was realized, including "competitive" (with the sensitivity on the level of about 7-10 ng/ml), "direct" (10 ng/ml) ways, and the so called algorithm "to saturation" (about 2-5 ng/ml). The time of analysis by immune SPR biosensor is about 10 min (on the previously prepared transducer surface, including immobilization of sensitive structures). The developed thermal biosensor provides direct detection of NPh with the sensitivity of about 1 microg/ml and the overall time of analysis of about 20-30 min. In spite of a lower sensitivity of the thermal biosensor, it is less sensitive to admixtures in real samples and simpler in use than the biosensor based on SPR and, consequently, the thermal biosensor is more applicable in the field conditions.

Antibody immobilisation on the metal and silicon surfaces. The use of self-assembled layers and specific receptors.

The use of Staphylococcal protein A and lectins as intermediate immobilising agents allows operators to orient antibodies (Ab) towards the solution due to the presence of a specific binding sites of immunoglobulin (Ig) molecules. Antibodies of different species of animals have unequal affinities to individual lectins. The effective thickness of immobilised Ab's depends on the type of substrates used and increases in the following sequence: bare gold or silicon surface, the surface treated with self-assembled polyelectrolytes (PESA) or with protein A or some lectins deposited on the preliminary formed polyelectrolyte layer. The glycolysated protein of jp51 may be selectively immobilised from the mixture of retroviral proteins (p24 and jp51), if it is necessary to distinguish infected animals from preliminarily immunised ones by means of a vaccine based on p24 protein. It was shown that the use of Staphylococcal protein A, instead of some lectins as intermediate layer for the Ab immobilisation, does not lead to a more sensitive determination of such low-weight toxins as 2,4-dichlorophenoxyacetic acid (2,4-D). The above-mentioned results were obtained with surface plasmon resonance (SPR) technique.

[Immunoenzyme analysis of non-ionic surfactants in water].

The approaches for high sensitive and specific determination of nonylphenol (NP) with the help of immune enzymatic (ELISA) method have been developed. The process of preparation of conjugates of NP with proteins, antiserum obtaining, purification of immunoglobulin (Ig) fractions and study of specificity of the obtained antibodies were described in detail. It was shown that the antiserum and total Ig fraction do not differ in respect of specificity and binding abilities. The developed algorithm of ELISA method fulfillment is able to provide NP revealing with the sensitivity in the range of 20-50 ng/ml and working controlled concentrations--from 20-50 to 1000 ng/ml.

[Structuring of recognizing elements of biosensors. Genetic constructs for the directed immobilization and combination of recognizing and signal generating structures]. / Strukturirovanie uznaiushchikh élementov biosensornykh datchikov. Geneticheskie konstruktsii dlia napravlennoi immobilizatsii i sovmeshcheniia uznaiushchikh i generiruiushchikh signal struktur.

Data about the genetic-engineering approaches for providing of directed immobilisation of biological molecules on the transducer surface and for indirect revealing the formed specific complexes are analysed in this review. Creation of nanostructures on the transducer surface and a possibility to do directed transfer of biological molecules from one surface on the other one are considered as well.

[Effect of mycotoxin T-2 on bioluminescence intensity of bacteria]. / Vliianie mikotoksina T-2 na intensivnost' bioliuminestsentsii bakterii.

The acute and chronic toxicity of T-2 was studied by bioluminescent method with the use of two strains of luminous bacteria--P. phosphorum Sq3 u V. fischeri F1 as biological objects. It was shown that in acute experiments after 10 min incubation of bacteria in the presence of T-2 the bioluminescence inhibition on the 50% level was observed at the toxin concentration equal to 12 mg/mL. In chronic experiments such a level of bioluminescence inhibition was registered after 16 hours incubation at the toxin concentration of 18 mg/mL. T-2 toxicity was also investigated in the presence of different serum albumin concentrations. It decreases with the increase of albumin concentration at the short term of incubation (5 min) of the mixture to be analyzed. In case of the longer term of incubation (up to 30 min) of this mixture T-2 toxicity was restored. Probably, it is a result of destruction of protein-toxin complex, which is, evidently, reversible and may be characterized by some index. It is necessary to emphasize that the sensitivity of T-2 analysis increases under the decrease of pH value up to lower bacterial physiological level, i.e. to 5-5.5. The revealed abilities of T-2 toxin effect on the intensity of bacterial bioluminescence may be used under the development of instrumental analytical approach on the basis of biosensor technology for testing this toxin in the environment. Taking into account the analysis simplicity and rapidity, such analytical device may have a perspective for wide practical application.

[Directed immobilization of recognizing elements of biosensor transducers]. / Orientirovannaia immobilizatsiia uznaiushchikh élementov biosensornykh datchikov.

Data about the directed immobilisation of recognising structures on metal and silicon surfaces of biosensor transducers are analyzed in the review. Attention is paid to such approaches as pretreatment of surface by silanes, thiol compounds as well as to formation of polyelectrolyte-based layers.

[Effect of surface-active substances on bioluminescence intensity of bacteria]. / Vliianie poverkhnostno-aktivnykh veshchestv na intensivnost' liuminestsentsii bakterii.

The study of sensitivity of luminous bacteria isolated from the Black and Azov seas to surfactants from various classes was carried out. It was shown that cationic surfactants had a strong inhibition effect on bacterial luminescence in contrast to anionic and in particular nonionic surfactants. To increase the luminous bacteria sensitivity to the action of OP-10 (nonionic surfactant) and ABS (anionic surfactant), which are widely used in industry, several approaches have been developed. They include modulation of bacterial sensitivity by the additives of cationic substances, use of luminous bacteria at a logarithmic stage of growth, realization of biotesting at low pH = 5.5. The use of these approaches allows to lower effective concentrations of OP-10 and ABS, which caused a decrease of bioluminescence by 50%, 3-200 times and opens perspectives for the use of the bioluminescent method to study these surfactants toxicity on the principle of biosensorics.

[Express control of toxic substances and pathogenic microorganisms. Immune analysis and immune sensors]. / Ekspressnyi kontrol' toksicheskikh veshchestv i patogennykh mikroorganizmov. Immunnyi analiz i immunnye sensory.

In the review the overall characteristics of biological warfare components, which represent danger to people in the case of their application by military or terrorist groups are discussed. The main part of the review is devoted to modern approaches of antibody obtaining and, in particular, preparation of specific recombinant immunoglobulins as well as to different immune chemical methods of determination of individual toxins and pathogenic microorganisms. A special attention is paid to existing data about the development of rapid, selective, high sensitive, simple and fully automated instrumental methods on the basis of biosensor technology which is designed for the control of components of biological warfare in environmental objects. Additionally industrially manufactured biosensors and their characteristics are given and analyzed.

[Liquid photo-polymerized compositions as an immobilized matrix of biosensors]. / Zhidkie fotopolimerizuiushchiesia kompozitsii v kachestve immobilizatsionnogo matriksa.

Series of liquid photopolimerized compositions based on oligourethanemetacrylate (OUM-1000T and OUM-2000T) and oligocarbonatemethacrylate (OCM-2), butilmethacrylate, methacrylic that acid, monomethacrylic ether of ethylene glycol and vinylpirrolidone (VP) were tested. It was shown the optimal variant of enzyme sensor development was a composition containing VP (a basic hydrophylic matrix), OCM-2 (crosslinked components) and OUM-2000T (crosslinked and increasing adsorption of polymer component). The blend contains 3% of enzyme. The obtained biosensors as based on immobilized beta-glucose oxidase and ureases have the following charachteristics: the linear response in the range of the concentration 0.1-10 mM, 0.05-20 mM, angle of slope of curve 30 mV/pC, 38 mV/pC, and response time 10-15, 5-10 mines, respectively. The maximal response of urease sensor was in the diapazon of pH 6.0-6.5. The increase of NaCl concentration in the solution to 300 mM caused reduction of sensor response. Under this concentration the was latter equal to half of initial response. Further increase of NaCl concentration (to 500 mM) doesn't lead to further response reduction. K(m) was calculated and it was shown, that amount of immobilized urease and beta-glucose oxidase in photopolymer material was equal 0.85 and 3.1 mM respectively.

[Chemiluminescence of daphnia cultivating medium and optimization of conditions for its determination]. / Khemiliuminestsentsiia sredy kul'tivirovaniia dafnii i optimizatsiia uslovii ee opredeleniia.

The article deals with the optimization of conditions for the chemiluminescence determination. The Daphnia habitat was shown to have no spontaneous chemiluminescence. This was revealed using hydrogen peroxide and luminol, the optimal concentrations of which were 23 and 1.6 x 10(-2) mmol/L. p-Iodphenol at low concentrations (4 x 10(-5)-2 x 10(-3) mmol/L) did not render its effect chemiluminescence signal while at high concentrations (4 x 10(-2) mmol/L) an inhibition of chemiluminescence was observed. To obtain the needed intensity of chemiluminescence no more than 5 daphnia persons is required to incubate in volume of 10 mL of sample for analyzing. The intensity of chemiluminescence of daphnia cultivating medium and the sensitivity of this organism to potassium chromate increased at the temperature increasing from 24 to 32 degrees C. Daphnia cultivating medium can be preserved in refrigerator for several hours without lost of chemiluminescence signal.

[Optimization of the conditions of immobilization of enzymes in a photopolymeric membrane]. / Optimizatsiia uslovii immobilizatsii fermentov v fotopolimernoi membrane.

The residual activity of enzymes immobilized in the membrane on the basis on 1-vinyl-2-pyrrolidinone as photopolymerizable composition is studied. It is established, that under conditions of the immobilization at 20 degrees C the residual activity glucoseoxidase is about 35% from a initial level, horseredish peroxidase and urease from Jeack beans--42% and 20%, respectively. In case of an immobilization of beta-glucoseoxidase -50 degrees C it reaches almost 50% from a initial level. It was investigated the influence of different sources of UV-radiation and different substances on stability of the enzymes in the composition and in the immobilization matrix at storage. Dynamic of changes of enzyme activity at the photoimmobilization was characterized, and also the requirements for providing of its maximal storage was selected.

[Analysis of specific nucleotide sequences. DNA biosensors]. / Analiz spetsificheskikh nukleotidnykh posledovatel'nostei. DNK-biosensory.

Information about common molecular-biological approaches for the determination of the specific nucleotide sequences in genetic materials was given in the review. Main attention was paid to consideration of the ways for DNA biosensor creation. The information about the types of such biosensors was presented in detail and characteristics of the developed devices were cited. Separately the question about the use of the instrumental analytical approaches for the identification of genetic materials of individual pathogenic microorganisms was viewed.

[Electrochemical sensor based on photopolymeric membranes for determining urea]. / Elektrokhimicheskii sensor na osnove fotopolimernykh membran dlia opredeleniia mocheviny.

A new electrochemical enzymatic sensor based on the ion selective field effect transistors (ISFETs) and photocurable membrane was developed for the determination of urea. For the immobilization of urease on the gate surface of the ISFET a simple method, involving the use of liquid photocurable compositions on the basis of vinylpirollidone, oligouretanemetacrylate and oligocarbonatemetacrylate, was applied. The linearange of the response of the developed electrochemical sensor lies in the range 0.05-20 mM. The latter corresponds to the claims of the medical practice. The overall time of the analysis is 5-10 min. The effects of the buffer concentration and its pH as well as temperature and presence of ammonia ions in the measuring medium on the amplitude of the sensor response were estimated. The duration of sensor work is as shortest 40 days. The proposed sensor on the basis of the ISFET is promising for the express analysis of the level urea in blood, while the developed method of membrane preparation with entrapped enzyme can be combined with the integral technology of producing of the biosensors semiconductor transducers.

[Photopolymers as an immobilized matrix in biosensing]. / Fotopolimery kak immobilizatsionnyi matriks v biosensorike.

Biosensors are perspective devices for analysis of substances in chemistry, biological chemistry, medicine, biotechnology and also for environment status monitoring. Their advantages are compact size, short time of analysis, display high response and simplicity in usage. The working characteristics of biosensors often depend on efficacy of a biological stuff immobilization on the surface of transducer. In this context there is a need for development of immobilization methods capable to provide for execution of the following demands: 1) compatibility of this process with technology of building transducer; 2) simplicity in fulfillment, cheapness and expressness at manufacture of biomembranes; 3) ability to provide the maximal safety of a biological stuff activity and its high adhesion to a surface of transducer; 4) reproducibility at serial application and capacity of standardizing. Usage of photocrosslinked and photopolymerized compound at the immobilization of a biological stuff allows to provide execution of the listed above demands. The present review is devoted to features of application of the given class of compound at building of biosensors.

Immobilization of biocomponents for immune optical sensors.

Immobilisation of both human immunoglobulin(IgG) and antiimmunoglobulin (anti-IgG) was performed by means of polyelectrolyte self-assembly. This technique was compared with direct immobilisation of the immune components on bare gold and their covalent binding via glutaraldehyde as a bifunctional reagent. Additionally, the immune components were properly oriented during their immobilisation by using a predeposited layer of the protein A. Methods of the surface plasmon resonance (SPR) and planar interferometry were employed for monitoring the immobilisation as well as specific immune reaction. It was shown that in case of the use of polyelectrolyte self-assembly it is possible to achieve the sensitivity of the analysis up to 30 ng/ml for SPR and up to 1 ng/ml for planar interferometer based immune sensors.

[A new electrometric approach to studying specific biomolecular complexes]. / Novyi élektrometricheskii podkhod k issledovaniiu spetsificheskikh kompleksov biomolekul.

In the article the results of the influence of some types of biological molecules and their specific immune complexes on the volt-amperic characteristics of surface-barrier contact structures with the super thick metal film are presented. Moreover, the possibility to develop on this basis a simple instrumental method for separate registration of initial components and products of their interaction is discussed. It was shown that the volt-amperic characteristics of surface-barrier structures of Ni-Si changed at the deposition of myoglobin and its specific monoclonal antibodies. These structures more essentially reacted to the formed presence of specific immune complex and, in particular, to the direct formation of this complex on the surface at the subsequent use of the initial immune components in comparison with their separate presence. For all investigated types of biological molecules and their specific complexes we found optimal thickness of metal film when changes of volt-amperic characteristics of system achieved maximum level. It was concluded that this electrometrical method was suitable for the express and separation free registration of specific immune complex.

[Morpho-functional characteristics of hemoglobinopathies]. / Morfo-funktsional'n'ie kharakteristiki Ieritrona.

The approaches to the problems of primary diagnostics of the hereditary hematoglobinopathias caused by unbalanced synthesis of alpha and beta chains, as well as modern state of screening of hereditary thalassemic hematoglobinopathias are considered. The result of the available information analysis takes into account numerous peculiarities of this problem, including economic ones. Experience of an estimation of the displays of unbalanced synthesis of globin chains concerning the morphology of red blood cells is presented in the generalized form. The latter is a very important for preliminary diagnostics the hematoglobinopathias because, for the first, it may be realized at primary units of public health system service, for the second, it allows to narrow to an acceptable level the group of persons whose blood requires more complex and expensive researches.

Development of the myoglobin sensor based on the surface plasmon resonance.

Bioaffinic sensor based on the surface plasmon resonance (SPR) with golden layer as a transducer was developed. This sensor was applied for control of antigen-antibodies (Ag-Ab) interactions. For modification of transducer surface the spontaneously organized molecular assemblies of two types of mercaptan were formed. It was shown that the created bioaffinic sensor allowed to detect minimal concentration of antibodies, about 1 ng/ml, to the human heart myoglobin, which preliminary was immobilized on the sensitive surface. The particularities of the dynamics of protein interaction were observed. Duration of incubation with the biospecific component was no more than 5 minutes. After rupturing of specific binding it was possible to obtain initial signal again during 7-9 cycles.