Лазер-индуцированная флуоресцентная спектроскопия в диагностике тканевой гипоксии (обзор)

The aim of review is to discuss the results of studies on diagnosis of tissue hypoxia by laser-induced spectroscopy, as well as to identify promising trends and prospects of this technique for its further application in experimental and clinical medicine.The review presents the findings of studies of the fluorescence intensity of endogenous fluorophore molecules (reduced nicotinamide adenine dinucleotide, oxidized flavin adenine dinucleotide) as markers of ischemic injury of internal organs (brain, heart, liver, kidneys, etc.). The principles of fluorescence laser-induced spectroscopy in vivo are discussed. The historical aspects of the subject are also covered. The results of the use of the technique in experimental and clinical studies of tissue hypoxia and ischemia are shown. Difficulties in interpreting the intensity values of autofluorescent signal of the studied molecules are revealed. It was noted that the tissue autofluorescence in a long-term anoxia remains unknown, and there are no structured ideas about the impact of exogenous and endogenous factors on autofluorescence intensity.In conclusion, the use of laser-induced fluorescence spectroscopy to diagnose tissue ischemia is a promising area of experimental and clinical medicine, which still has various unresolved issues, despite a large number of studies in this domain.Цель обзора — анализ опыта диагностики тканевой гипоксии методом лазер-индуцированной спектроскопии, а также выявление перспективных направлений и потенциальных возможностей данного метода для дальнейшего его применения в экспериментальной и клинической медицине.В обзоре представили данные исследований интенсивности флуоресценции эндогенных молекул-флуорофоров (никотинадениндинуклеотида восстановленного, флавинадениндинуклеотида окисленного) как маркеров ишемического повреждения внутренних органов (мозга, сердца, печени, почек и др.). Рассмотрели принципы метода флуоресцентной лазер-индуцированной спектроскопии in vivo. Уделили внимание историческим аспектам темы. Показали результаты применения метода в экспериментальных и клинических исследованиях тканевой гипоксии и ишемии. Выявили трудности в интерпретации значений интенсивности аутофлуоресцентного сигнала исследуемых молекул. Отметили, что неизвестным остается период сохранения способности ткани к аутофлуоресценции в условиях длительной аноксии, отсутствуют систематизированные представления о влиянии экзогенных и эндогенных факторов на интенсивность аутофлуоресценции.Сделали вывод, что применение метода лазер-индуцированной флуоресцентной спектроскопии с целью диагностики тканевой ишемии является перспективным направлением в экспериментальной и клинической медицине, не исчерпавшим себя, и оставляющим ряд нерешенных вопросов, несмотря на большое количество исследований в данной области

Determination of pharmaceuticals based on indole alkaloids with amperometric DNA-sensors and enzyme immunoassay test-system

Bioaffine methods were developed for determining indole-containing alkaloids ajmaline and vincristine. These methods are based on the proposed amperometric DNA-sensors and on immunoenzyme test-system with a spectrophotometric indication of the analytical signal. The complexing between ajmaline and immobilized native DNA (n-IDNA) allows effective preliminary concentration from test solutions on the biosensor. The time of analysis is 25-30 min, limit of detection (LOD) for ajmaline is 1.0×10-10 M. The test-system utilized the immunological reaction of ajmaline with its antibodies and the enzyme marker, horseradish peroxidase, LOD is 1.5×10-9 M. Anti-cancer vincristine interaction with immobilized renatured DNA (r-IDNA) was studied and determination was carried out using an amperometric DNA-sensor. Bioaffine membrane drug concentration and reactivation of the sensor was performed. The affinity binding constant Kbind for vincristine-r-IDNA complex calculated by Scatchard's method was found to be high enough [(5.0±0.4)×105 ll mol] confirming high specificity of the complexing with r-IDNA. The duration of the assay is 40 min. The developed method is characterized with an LOD of 1.1×10-9 M, with the lack of the need for long sample processing. The pharmaceuticals were determined by those methods in model solutions of blood serum and in tablets and solutions for injections. © Taylor & Francis Group, LLC

Amperometric biosensor based on denatured DNA for the study of heavy metals complexing with DNA and their determination in biological, water and food samples

Amperometric biosensor (BS) has been elaborated based on the stationary mercury-film electrode (SMFE) with silver support and cellulose nitrate (CN) membrane containing immobilized single-stranded DNA (ssIDNA). The sorption isotherms and ssDNA-heavy metal binding constants have been obtained with the BS. According to these data, the chosen heavy metals form the following series of binding strength with ssIDNA: Pb(II)>Fe(III)>Cd(II). It has been found that upon the competitive adsorption, there exists practically simultaneous sorption of different ions at ssIDNA containing membrane. The method of the determination of heavy metals based on preconcentration of metal ions on the BS followed by the destruction of DNA-metal complexes with ethylenediamine tetraacetate (EDTA) and voltammogram recording has been proposed. The lower limits of detectable contents are 1.0×10-10, 1.0×10 -9 and 1.0×10-7 mol l-1 for Pb(II), Cd(II) and Fe(III), respectively. Heavy metals have been assayed in natural and drinking water, milk and blood serum samples even under simultaneous presence with a selectivity factor of 1:10. The effect of matrix components has been estimated. © 2004 Elsevier B.V. All rights reserved

Electrochemical biosensors based on nucleic acids and their use in bioaffinity assays for determining DNA and and its effectors

The analytical capabilities of electrochemical biosensors based on nucleic acids are systematized. Immobilization methods that retain the biological activity of nucleic acids and provide an opportunity to use them as multipurpose analytical reagents are described. The use of the above sensors in bioaffinity assays for determining DNA and its effectors in biochemical analysis and environmental monitoring and for determining the nucleotide composition of DNA is demonstrated in many examples. © Pleiades Publishing, Inc., 2006

Analytical possibilities of amperometric choline esterase biosensors in continuous-flow analysis

A model set-up of a simple and convenient continuous-flow analyzer (enzyme reactor) that includes an amperometric choline esterase biosensor for the determination of potential contaminants of the environment is developed. Various modifications of the biosensors developed are used for determination of toxicants in a flow within the concentration range from 1 × 10-3 to n × 10-13 M over 16-36 h. ©1996 Plenum Publishing Corporation

An enzyme amperometric sensor for toxicant determination

An enzyme sensor with amperometric recording of the analytic signal for determination of a number of cholinesterase (ChE) inhibitors (P-, Cl-containing pesticides, heavy metals) has been developed. It consists of a mercury film-covered silver electrode and ChE immobilized in the nitrocellulose film in the presence of glutaraldehyde. It is shown that the enzyme electrode can be used as a component of an enzyme reactor for the determination of ChE inhibitors and activators under flow conditions. © 1991

Bioaffine methods for determining ajmaline using an amperometric DNA-sensor and an immunoenzyme spectrophotometric test system

Bioaffine methods are developed for determining indole-containing alkaloid ajmaline, which has a cytostatic effect and is used as a cardiac drug. These methods are based on the proposed amperometric DNA-sensor and on immunoenzyme test system with the spectrophotometric indication of the analytical signal. The complex formation between ajmaline and immobilized native DNA allows ajmaline to be efficiently preconcentrated on the biosensor from test solutions. Optimum conditions for preconcentrating ajmaline and those for reactivating the biosensor for its repeated use are found. The time of analysis is 25-30 min, the determination limit for ajmaline is 3.0 × 10-10 M (RSD = 33%). In the test system, the immunological reaction of ajmaline with its antibodies and the enzyme marker, horseradish peroxidase, are used. The determination limit is 4.0 × 10-9 M (RSD = 33%). Ajmaline is determined by the two methods in model solutions of blood serum and in tablets and solutions for injections. © 2009 Pleiades Publishing, Ltd

Amperometric DNA biosensor for the determination of auto-antibodies using DNA interaction with Pt(II) complex

A method of denatured DNA immobilisation on cellulose nitrate film has been developed. A modified film of uniform and stable surface has been used as a bio-sensitive part of amperometric DNA biosensor based on the stationary mercury-film covered silver electrode. The biosensor has been used to devise a new variant of solid-phase immunoassay of auto-antibodies (Ab) in blood serum without separation of components. The content of auto-Ab was monitored by measuring the currents of catalytic hydrogen evolution (with potentials of -1.2 and -1.4V) resulting from the complexing of Pt(II) with DNA or auto-Ab respectively. The determination has been performed within a wide concentration area of 5.0×10-10 to 7.0×10-8M. The limit of detection is 3.0×10-10M. The affinity constants for the immunoreaction of DNA-antibodies have been found to be 1.25×10 9 and 2.50×108M-1, which confirms the specificity of the interaction. The protocol of the immunoassay has been proposed and the procedure of diagnosing Aleutian mink disease (AMD) has been described here. © 2003 Elsevier B.V. All rights reserved

Amperometric sensors based on immobilised enzymes

The state, problems, and prospects of the development of one of the new fields of analytical chemistry — "Reagent-free" analytical methods — are described and surveyed using as an example enzyme-based amperometric sensors. Questions associated with the design of biosensors and the characteristics of their functioning under various conditions are examined. The applied aspects of the use of enzyme electrodes of this type in various fields of science and the national economy are noted. The bibliography includes. © 1991 IOP Publishing Ltd