The metal complexes of the porphyrin-ketones-sensitive element for the optical determination of oxygen in a liquid or gaseous medium and the method for determining the oxygen

 

(57) Abstract:

Use: in analytical chemistry, veterinary science, medicine, biotechnology and food industry for the optical determination of oxygen. The inventive platinum and palladium complexes of new substituted porphyrin-ketones. The sensing element for the optical determination of oxygen in a liquid or gaseous medium in the form of molded polystyrene products distributed in it platinum or palladievye complexes of new substituted porphyrin-ketones as a phosphorescent dye. The sensitive element is made in the form of a film thickness of 1-20 microns. The film can be fixed on a solid substrate or the optical element. Molded product may contain a layer of oxygen-dependent enzyme. The method for determining the oxygen through the sensor in contact with the analyzed sample and linked via an optical fiber with a phosphorescence detector with optimal sensitivity 700-850 nm. The phosphorescence of the sensing element excite the led in the field of absorption of the dye and register it in pulse mode with a time resolution with variables in the Oia oxygen are calculated according to the intensity and/or lifetime of the phosphorescence from onexperience the nature of the decay of phosphorescence. Platinum and palladium complexes of new substituted porphyrin-ketones have quantum yields and lifetimes of phosphorescence, close to the porphyrin complexes, and intense absorption band in the region 560-620 nm. The shift of the visible absorption band is 50-60 nm in comparison with metal. Phosphorescence is shifted to the long wavelength region of about 100 nm. This allows you to simplify the process of optical oxygen detection in liquid or gaseous environment and improves the accuracy of determination.10 Il.

The invention relates to the field of analytical chemistry and technology and can be used in medical diagnostics, clinical medicine, biotechnology, food industry, veterinary medicine, environmental studies.

In cases where the task is to determine or continuous monitoring of biologically active compounds, the most effective is the use of biosensors.

Molecular oxygen is one of the most important objects of analysis and at the same time, the substrate of many enzymatic reactions. It is also effective tusitala luminescence dyes. The quenching efficiency of luminescence which is registered stewing. If successful, the selection luminescense dye matrix for it and the corresponding elements of optoelectronics you can create effective optical oxygen sensors, capable of exact quantification of oxygen. The corresponding device may be implemented using a compact, simple and cheap (fiber) optical equipment [1]

Optical detection of oxygen can be carried out by measuring the luminescence intensity or lifetime of the luminescence of the dye. The latter approach is preferred since it does not depend on the concentration of the dye, its change in time and the fluctuations of the operating characteristics of the components of the optical detection system [2]

Currently, the literature describes several approaches to the creation of optical oxygen sensors, and enzyme biosensors based on them. As the active oxygen-sensitive element are commonly used polymer compositions on the basis of luminescent dyes, which are permeable to molecular oxygen dissolved in the analyzed system. It runs as an oxygen membrane, which is applied or attached to the end of obtainig items proposed to use a polymer composition based on polyaromatic dyes (for example, pyrene or decacyclene [3]), and also on the basis of the fluorescent complexes of ruthenium (Ru(bpy)3, Ru(phen)3 and others) [4] However, polyaromatic dyes require UV excitation, which is difficult is implemented on fiber-optic equipment and LEDs. In addition, the short duration of the luminescence of these dyes complicates the creation of sensors based on the principle of measuring lifetimes. This is due to the performance of semiconductor electronics (LEDs and photodiodes), which is usually about 1-2 microseconds. Complexes of ruthenium, as a rule, possess a complex coordination chemistry and photochemistry and poorly soluble in the polymers commonly used to obtain the oxygen-sensitive compositions. This leads to practical difficulties in creating a simple, compact and cheap devices.

Closest to the invention are phosphorescent dyes sensitive elements on their basis and method of determining oxygen described in the patent [5] is the use of phosphorescent dyes, porphyrin nature of Pt - and Pd-complexes of porphyrins. These dyes have absorption bands in the visible region of the spectrum and have an intense FOS is assured of oxygen sensors and biosensors based on these compounds, the following problems occur. First, the platinum complexes of porphyrins, which are most convenient for the detection of oxygen in the physiological concentration range of About 20% in the air or 10-250 mm in water), poorly compatible with existing semiconductor optoelectronics, mainly with light sources. Green and dark green LEDs (on the basis of the GaP, with a narrow emission spectrum and a maximum at 557 and 567 nm, respectively) are ineffective for excitation of luminescence Pt-porphyrins (they have a narrow absorption band with a maximum at 535 nm). At the same time, the blue light-emitting diodes (based on SiC with a wide range of emission maxima in the region of 480 nm) have substantially lower energy output than LEDs in the visible spectrum [7] moreover, the integral of the overlap of their emission and absorption Pt-porphyrins small.

Palladium complexes of porphyrins are more convenient spectral characteristics and can be worked green LEDs. However, in normal conditions (air saturation) their luminescence hundreds of times extinguished by oxygen due to too much time of life, which is of the order of 1 millisecond. This makes them unsuitable for measuring oxygen in physiological diporphyrin for optical detection of oxygen also occurs a number of difficulties. These include, in particular, that the kinetics of luminescence described in the patent [5] polymer compositions based on metalloporphyrins (PVC skin, polymethylmethacrylate with plasticizers as matrices) has mnogochislennyi character. This significantly complicates the calibration, the experimental data and the determination of oxygen concentrations by luminescence and can affect the measurement accuracy. Low photochemical stability of porphyrin compounds also limits their application in optical sensors.

Not currently describes the creation and use of fiber-optic devices for detection of oxygen-based semiconductor electronics and phosphorescent porphyrin dyes, and enzyme biosensors.

The present invention is the synthesis of new fluorescent dyes that are derived metal complexes of porphyrins, mainly Pt(II)- and Pd(II)-complexes, which are promising, in particular for use in fiber-optic oxygen sensors.

Another aim of the invention is to develop a sensitive elements on the basis of these new dyes, mainly on armentia membranes on the basis of the above oxygen-sensitive compositions and oxygen-dependent enzymes. Called sensitive elements are used for optical detection of oxygen and/or biologically active compounds (metabolites) in samples.

Finally, the aim of the invention is to develop a new method of determining the oxygen, which is based on the use of the above-described phosphorescent dyes and sensitive elements.

The invention and implementation are as follows.

Synthesis of new fluorescent dyes is carried out by directed chemical modification of the porphyrin macrocycle, which is held so that, referring to the minimum extent the ability of the corresponding metal complexes, intensively to phosphoresce at room temperature, change in this spectral characteristics of phosphorescence. Derivatives of porphyrins are obtained by selective oxidation of a specific fragment of the porphyrin macrocycle, which leads to change as a result of electronic spectra and some physical characteristics. The process of chemical modification is ultimately in obtaining new compounds, porphyrin-ketones (or oxo-chlorines), and then to get the sledge class of compounds, they differ in the structure, optical and physical properties from the corresponding porphyrin compounds and related complexes of chlorins, dihydrochloride, etc., [8]).

The structure of the complexes of porphyrin-ketones (I) and related complexes of porphyrins (II) and chlorine (III) below.

The scheme of synthesis of metal complexes of the porphyrin ketone shown below.

Pt - and Pd-complexes of porphyrin-ketones also intensively phosphorescent at room temperature, quantum yields and lifetimes of phosphorescence are close to the corresponding porphyrin complexes. They have an intense absorption band (excitation) in the field of 580-620 nm, which is 50-60 nm longer wavelengths compared with the corresponding metalloporphyrins. Spectrum of phosphorescence are also offset in the long wavelength region of more than 100 nm. In particular, Pt-complexes of porphyrin-ketones have long wavelength absorption maximum 591 nm and the maximum phosphorescence at 758 nm. Thanks to these properties, they are well compatible with a semiconductor visible light sources (for example, yellow LEDs having the optimum emission at 586 nm) and can be effectively used for detection of oxygen Onov shifted respectively by 10 and 30 nm in the red region compared with platinum complexes and also effectively excited yellow or orange and red LEDs.

New connections are formally partially oxidized derivatives of porphyrins, in addition to a better spectral characteristics, compares favorably to the known high resistance to photo - and chemical oxidation. The relevant experimental data are given in the supplemental materials.

As noted above, the long-wave shift of the electronic spectra of the obtained compounds is achieved by reducing the aromaticity macrocyclic metal complex patterns. A similar effect is also known for fluorescent chlorin, bacteriochlorin and their metal complexes (M. Gouterman. The Porphyrins/ed. D. Dolphin, 1979, v. 3, pp.1-165). However, these structures, which formally can be considered as recovered porphyrins are highly susceptible to photooxidation and unpromising for similar applications. High instability to photo - and chemical oxidation have also porphyrin-diodes and their metal complexes of intermediate compounds for the synthesis of porphyrin-ketones (see scheme synthesis).

The metal complexes of various porphyrin-ketones derived from the corresponding porphyrins and differ only in the lateral substituents porphyrin core in 1-8 provisions have ACLs present invention.

On the basis of the above metal complexes-porphyrin-ketones, mainly hydrophobic arbitrary, and polystyrene developed y the oxygen-sensitive composition (film). New compositions have several advantages compared to the previously described compositions of Pd - and Pt-porphyrins. In particular, their luminescence is described ownexperience law decay, which greatly simplifies the measurement procedure and the processing of the experimental data when registering oxygen measuring principle the lifetime of the luminescence. They have a fast and reversible response to changing oxygen concentrations. These polymeric compositions are intended for use in fiber-optic oxygen sensors, and enzyme biosensors based on them as the oxygen-sensitive elements (membranes or coatings). The composition may contain small additions of plasticizers to improve their mechanical and adhesive properties. Membrane and/or coatings on the basis of the above compositions allow you to precisely and quantitatively to determine the oxygen content on the basis of a measurement of the intensity and/or lifetime of the luminescence.

Enzyme membranes get rodnymi membranes. For example, the enzyme glucose oxidase (GO), which catalyzes the reaction:

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quantitative evaluation of the content of the substrate can lead indirectly by the level of oxygen consumption.

The enzyme membrane is prepared by immobilization of the enzyme or enzyme system, one of the substrates which is oxygen, directly to the above-described oxygen-sensitive polymeric compositions [1,2] thus Obtained membrane is sensitive to the corresponding second substrate of the enzyme. With the introduction of the substrate in saturated air of the analyzed solution, this membrane gives luminescent response (fire luminescence due to the removal of quenching by oxygen), which is proportional to the concentration of the substrate. Membrane with immobilized glucose oxidase, lackadasical, ethanolamides, cholesterol oxidase, arcasoy, etc. can be used as active elements in the respective devices (biosensors) instead of the above-described oxygen membranes to determine, respectively, glucose, lactate, ethanol, cholesterol, uric acid, etc.

New method of determination of oxygen based on the use of the above-described sensitive optoelectronics and electronic circuits microsecond temporal logic, which allow the measurement of the intensity and/or lifetime of the luminescence of the above compositions.

The invention is illustrated by the following examples.

Example 1. Synthesis of Pt-octaethylporphyrin-ketone (hydrophobic, water-insoluble dye).

(a) 100 mg octaethylporphyrin (EIA) is dissolved in 30 ml of chloroform. Add 200 mg OsO4and 3 drops of pyridine. Survive 24 hours in the dark and then passed through the solution a current of hydrogen sulphide within 10 minutes the Precipitate of sulphide of osmium filtered. The solution is evaporated in vacuum to dryness. The remainder chromatographic on a column of silica gel in the system chloroform/ether 95/5, Collect the main fraction of CIS-diol of octaethylporphyrin, evaporated, and crystallized from chloroform/methanol. A yield of about 40%

b) 40 mg of CIS-diol of octaethylporphyrin dissolved in 10 ml of concentrated sulfuric acid and incubated for 10 minutes at room temperature. The mixture is then poured on fine ice, neutralized aqueous ammonia. The precipitate is filtered off, dried, dissolved in 5 ml of chloroform and chromatographic on a column of silica gel in chloroform. Collect the main fraction of octaethylporphyrin-ketone. Crystallized from chloroform/methanol. A yield of about 90%

in) 30 is 3 hours. The mixture is then evaporated to dryness. The residue is boiled with 50 ml of chloroform for 15 minutes, the resulting solution was evaporated to dryness. The remainder chromatographic on a column of silica gel in the system chloroform/acetone 95/5. Collect the main fraction of the platinum complex octaethylporphyrin-ketone (Pt-EIA-ketone). Evaporated, to obtain 18 mg of the basic substance. A yield of about 60%

Elemental analysis data for Pt-octaethylporphyrin-ketone. Found, C - 57,99; H 5,90; N 7,50. C36H44N4OPt. Calculated C - 58,07; H 5,96; N 7,53.

Example 2. Getting Pd-octaethylporphyrin-ketone. 30 mg octadecylamine (see example 1) dissolved in 10 ml of dimethylformamide, add a solution of 100 mg PdCl2in 10 ml of dimethylformamide. Heated at 110oC for 30 minutes. Cooled to 70-80oWith add dropwise 5 ml of water, cooled. The precipitation was filtered and crystallized from chloroform/methanol. Getting 27 mg of palladium complex octaethylporphyrin-ketone. A yield of about 90%

Elemental analysis data for Pd octaethylporphyrin-ketone. Found - 65,89; H 6,69; N 8,50. C36H44>N4OPd. Calculated C - 65,99; H 6,77; N 8,55.

Example 3. Synthesis of Pt-coproporphyrin I-ketone (water soluble dye) and tetraethyl ester.

(a) 100 mg tetraethyl ester to aetiologie ester coproporphyrin I yield 30%

b) 30 mg of CIS-diol is dissolved in 10 ml of concentrated sulfuric acid at 0oC under stirring, and incubated for 15 minutes. The solution is poured onto 100 g of ice, neutralized with ammonia. The precipitate is filtered, dried, chromatographic. A yield of about 75% tetraethyl ester coproporphyrin I-ketone (tee KP-ketone).

in) 20 mg tea KP-ketone dissolved in 5 ml of benzonitrile add 150 mg of K2PtCl6and boil 1 hour. The solution is evaporated to dryness. The residue is extracted with chloroform. Chromatographic on a column of silica gel in the system chloroform/ether 10/1. The output of the platinum complex of the tetraethyl ester of coproporphyrin I-ketone 75%

Elemental analysis data for Pd coproporphyrin I-ketone tea. Found, C To 59.51; H By 5.87; N 6,27. C44H52N4Pd. Calculated C - 59,56; H 5,91; N Of 6.31.

g) 15 mg tetraethyl ester Pd-coproporphyrin I-ketone dissolved in 10 ml of dioxane, was added 100 mg of KOH in 1 ml of water. Heated at 70oC for 4 hours. Add 50 ml of water, neutralized to pH 5 with hydrochloric acid. The precipitate is filtered off and dried. The output of the Pt-coproporphyrin I-ketone 100%

Elemental analysis data for Pd coproporphyrin I-ketone. Found, C - 55,71; H 4,63; N 7,20. C36H36N4O9Pd. Calculated C 55,78; H To 4.68; N, 7.23 Percent.

2identical to the complexes octaethylporphyrin-ketone (see example 2).

Complexes Ethiopian-ketone (R1= R3= R5= R7=CH3, R2=R4=R6=R8=CH2CH3) fully synthesized similarly to the corresponding complexes of octaethylporphyrin (see examples 1, 2 of the description).

Synthesis of Pd and Pt complexes of coproporphyrin III-ketone and tetraethyl esters. Coproporphyrin III (R1= R3= R5= R8= CH3, R2=R4R6R7CH2CH2COOCH2CH3oxidize OsO4followed by incubation with sulfuric acid and purified analogously to example 1. In this view of the asymmetric structure of the source porphyrin is obtained a mixture of the four major isomers of coproporphyrin-ketone, which spectral indistinguishable (i.e., identical) is used to obtain Pt and Pd complexes, the method is completely the same as those described in examples 2, 3.

Example 5. Luminescent properties of metal complexes of the porphyrin-ketones. Corrected excitation spectra and unadjusted emission spectra (phosphorescence) shot on luminescence spectrometer LS-50 (Perkin Elmer, UK) for dilute solutions of dyes (0.1-1.0 mm) to fit the h-ketone shown in Fig.1. Spectra similar Pd-derivatives are shown in Fig.2. As you can see, chemical modification leads to a significant long-wave mixing in the spectra of excitation and emission of new dyes.

Spectra of excitation and emission of various metal complexes of derivatives of porphyrin-ketones (for example Pt-EIA-ketone, Pt-coproporphyrin I-ketone and tetraethyl ester Pt-coproporphyrin I-ketone) is almost identical to the above described (Fig.1, 2).

Comparative properties of complexes of porphyrin-ketones listed in the table.

The life time of the phosphorescence Pt-octaethylporphyrin-ketone and Pd-octaethylporphyrin in obeskislorozhennaja solutions (micellar 1% solution of Triton X-100 containing 10 mg/ml sodium sulfite, pH 7.0) at room temperature for 60 and 450 microseconds, respectively (25oC). For the corresponding metalloporphyrins these values are of the order of 95 and 1100 microseconds. For derivatives of various patterns temporal characteristics are also similar.

Example 6. Preparation and properties of polymer compositions based on PT-porphyrin-ketones.

10 mg Pt-EIA-ketone dissolved in 1 ml of chloroform. The resulting solution is mixed with 10 ml of 5% solution of polystyrene To obtain the oxygen-sensitive coatings and membranes the solution of the polymer composition is applied in a thin layer on the horizontal surface of the optical element (glass plate, transparent polyester film or the end face of the optical fiber), and dried in air for 1-12 hours. Thus obtained elements coated with a film of a polymer composition with a thickness of 1-20 microns have satisfactory mechanical properties and can be used for optical detection of oxygen in the water and gas phases. A high concentration of dye in the film provides effective absorption of the exciting light (the optical density at a wavelength of 595 nm is 0.1-2 units) and high levels of fluorescent signal.

Figure 3 shows the decay kinetics of the luminescence of the polymer compositions of Pt - and Pd-octaethylporphyrin-ketones in polystyrene and their linearization by ownexperience law. The nature of the dependency does not change under different oxygen concentrations and the change in the concentration of dye in the polymer in the range of 0.1-50 mm last. The lifetimes of luminescence dyes in the composition in the absence of tusitala (oxygen) are 63,0 ISS and 450 μs for Pt - and Pd-complex, respectively, at 25oC.

Figure 4 shows the calibration curve for the determination of oxygen (air pressure) in the gas phase using polymeric oxygen-Cosi (microseconds), and in linearized form in the coordinates of the stern-story (to/t [Q]). The amplitude changes of the life time for obeskislorozhennaja and saturated air environments is about 4 times at 25oC. Changes in luminescence intensity and character similar to the changes the lifetime of the luminescence, indicating a truly dynamic type quenching of the luminescence of dyes in polymer compositions.

Similarly receive polymeric compositions based on Pt-, Pd-complexes tetraethyl ester coproporphyrin I-ketone and other hydrophobic porphyrin-ketones. Fluorescent and oxygen-sensitive properties of the obtained compositions similar to the above.

Content luminescense dye in the polymer composition is dictated by practical considerations. The lower limit is due to the sensitivity of the detecting system, since the magnitude of the fluorescence signal is directly associated with the concentration of the dye. The upper limit is due, on the one hand, the solubility of the dye in the polymer, and on the other, the efficiency of absorption of the exciting light in this composition. If the absorption efficiency is close to 100% of the almost all exciting snotty, for the system Pt-octaethylporphyrin-ketone-polystyrene specified in the description of the investigated range of dye concentrations of 0.1-50 mm, which corresponds to the weight range listed then in the formula: upper limit is 5% (V/V). At higher ratio by evaporation of the solvent (toluene), the dye in the polymer film aggregates and/or precipitates, and the specific fluorescence signal is not growing. At 5% weight content of the dye absorption efficiency of the exciting light 3-micron film of the composition is about 50% which is close to theoretical limit and quite satisfying practical tasks.

Comparison of photostability of complexes of porphyrins and porphyrin-ketones was carried out as follows. By the same methods (see above) were made oxygen-sensors based on pt-EIA and Pt-EIA-ketone, which were then subjected to intense irradiation with polychromatic light in the same conditions. After 18 hours the residual fluorescence signal (intensity) were respectively 34% and 95% i.e. photochemical stability of the oxygen-sensitive membrane on the basis of Pt-EIA-ketone about 10 times better than for similar based on Pt-EIA. These are the ptx2">

Example 7. Determination of oxygen with the use of polymer compositions based on platinum complexes of porphyrin-ketones.

This was constructed a prototype fiber-optic oxygen sensor, shown in General form in Fig.5. Its main component - submersible active element, which is an oxygen-sensitive membrane on the basis of the above-described polymer compositions, mounted on the end of a bifurcated fiber optic bundle. Harness ensures efficient optical connection between the oxygen membrane and an optoelectronic detector.

Fluorescent detector optimized for measuring the luminescence of the above compositions. It uses a pulse measurement mode long luminescence, which is based on the coherent modulation of the intensity of the light source (led) and a photodetector (photodiode). This mode allows you to define the light signals after a certain time (delay time) after a short flash of light source. The time delay may be variable, its value is comparable to the decay time of the luminescence of the composition. This allows you to define multiple point kinetics Zay to calculate thus, the lifetime of the specific signal (luminescence).

A block diagram of an optical oxygen sensor, which includes an optoelectronic fluorescence detector and an oxygen membrane-based Pt-octaethylporphyrin-ketone, is shown in Fig.6. It includes:

luminestra membrane on the basis of Pt-complex porphyrin-ketone;

(fiber)optical node (forked light harness);

semiconductor pulsed light source (led or laser) with optimum issue in the field of 550-650 nm;

the photodetector (photodiode) with optimum sensitivity in the field of 700-850 nm;

the electrical circuit of the modulation of the intensity of the light source;

the electrical circuit of the modulation voltage on the photodetector;

the electrical circuit of the two modulation schemes, providing their feedback and variable time delay;

the unit preamplification and/or amplification of an electrical signal from the photodiode;

block processing signal and the output signal (analog and/or digital);

power supply DC.

The device operates as follows.

Oxygen membrane (disc 8 mm diameter, mounted on a common end razdvoenie (fiber)optical communication with a detector of luminescence. Optical channel: led membrane photodiode is also equipped with optical filters for effective discrimination of the exciting light and phosphorescence. The led provides the excitation of the luminescence of the composition in the region of absorption of the dye, the photodiode check the phosphorescence emitted by the dye in the corresponding spectral region. The electrical signal from the photodiode is scheme preamplification and amplification, and optionally converted from analog to digital. Modulation schemes operating in the coherent mode with a fundamental frequency of about 1 kHz is required for work measurement and reference signals. The working signal from the photodiode is measured after a certain time after the decay of the led (the delay time), which is comparable with the duration of the luminescence of the dye: the range of 10-100 microseconds. When in measurement mode lifetimes work signal is measured at several values of time delay. The reference signal is measured when the delay time is significantly longer than the length of luminescence: the order of 300-1000 microseconds. The integration time of a single signal (gate account) is comparable to the decay time of the dye and is of the order of 100 microseconds. Lying is the new life of the luminescence shown in Fig.7. Schema processing and output of information with regard to the magnitude of the reference signal to calculate the integral of specific signals (in units of luminescence intensity or lifetimes) and the corresponding oxygen content in the analyzed environment. The device provides for initial calibration for oxygen of at least two points and periodic pagkalinawan.

The above-described device allows to measure the intensity and/or lifetime of the luminescence of the above oxygen of membranes or coatings placed on the end of a bifurcated optical fiber, and the results of these measurements to calculate the oxygen content in the analyzed sample.

Figure 8 shows a typical curve of the luminescence response of the film to the change of oxygen concentration in the system (aqueous solution). 95% of the response is of the order of 10 seconds, including the time of transfer of the active element from a solution saturated with air in obeskislorozhennuju solution.

Example 8. Obtaining sensitive elements using enzyme-oxidase.

a) a sensor element for determining glucose.

A polymer composition was prepared and applied onto the polyester bases, and there is oxidase. It is as follows. 50 mg of the enzyme dissolved in 1 ml of distilled water and add a solution of glutaraldehyde to a final concentration of 0.2% (V/V). The resulting solution is applied on a horizontal surface oxygen membrane, evenly distributing it on an area about the size of 25 cm2and dried with dry air (1-3 hours). Thus obtained glucose oxydase membrane stored at +4oC in dry form or in phosphate buffer, pH 7.0 with 0.1% of sodium azide.

For detection of glucose using the above-described oxygen sensor (see example 6), an active element which has the above-described glucose oxydase membrane, and not the oxygen membrane. The measurements were carried out in 0.05 M phosphate buffer. Fiber optic active element sequentially immersed in solutions with different concentration of glucose (in the intervals washed with pure buffer) and is controlled by changing the intensity of luminescence. The characteristic curve of the luminescence response of fiber-optic glucose biosensor submersible type shown in Fig.9. The ultimate response in the sample of glucose (fixed signal) after stabilization of fluorescent signal (characteristic time 2-hivnet or lifetime) determine the corresponding glucose in the sample, using the previously obtained calibration.

b) a sensor element for determining cholesterol.

Enzymatic membrane-based oxygen-sensitive composition pt-EIA-ketone-polystyrene and cholesteroloxidaze (Pseudomonas fluorescens) was obtained similarly to the method of example 7. To 0.2 ml of the enzyme (3 mg/ml, 100 U/ml) was added glutaric aldehyde at a final concentration of 0.2% was applied to the surface of the oxygen membrane (area 10 cm2), and incubated 1 hour at room temperature (do not dry sweep). Then the membrane was washed and stored in buffer at 4oC.

Figure 10 shows the response of the enzyme membrane cholesterol, 3 mg/ml Conditions: 0.05 M K-phosphate buffer, pH 7.0, 10 mg/ml cholate sodium, 23oC. measurement procedure analogous to example 7.

Thus, the patent describes a new class of phosphorescent dyes with long-wave spectral characteristics and high photochemical stability, which are promising for practical applications. In particular, sensors, developed on their basis, as well as the method for determining the oxygen allow precise quantitative detection of oxygen and spectrum vajneishie pictures

Fig. 1. Spectra of excitation and emission spectra (B) Pt-octaethylporphyrin (____) and Pt-octaethylporphyrin-ketone Conditions: 1 μm solution of dye in obeskislorozhennuju micellar aqueous solution (1% Triton X-100, 10 mg/ml sodium sulfite, pH 7), 25oC.

Fig. 2. Spectra of excitation and emission spectra (B) Pd-octaethylporphyrin (____) and Pd-octaethylporphyrin-ketone Conditions: 1 μm solution of dye in obeskislorozhennuju micellar aqueous solution (1% Triton X-100, 10 mg/ml sodium sulfite, pH 7), 25oC.

Fig. 3. The kinetics of decay of phosphorescence compositions of Pt-octaethylporphyrin (a) and Pd-octaethylporphyrin-ketone (B) and polystyrene and their linearization by ownexperience law attenuation. Conditions: 25oC, the ratio of polymer/dye 100/1.

Fig.4. The calibration curve for the determination of oxygen (air pressure) in the gas phase with the composition Pt-octaethylporphyrin-ketone - polystyrene. Curve 1 in the coordinates (tq, P), curve 2 linearization in the coordinates of the stern-story (to/t, P). Conditions are similar to Fig.3.

Figure 5. The principle of operation of fiber-optic oxygen (bio)sensor.

Fig. 6. The block diagram of the opto-luminescent detector kiltie led; 5, the modulation scheme of the photodiode; 6 timing diagram and task delay time; 7 preamp and/or amp; 8 processor for processing and displaying information.

Fig. 7. The scheme of the modulation voltage to the light source and the photodetector used in optoelectronic detector for measuring the intensity and/or lifetime of the luminescence of the polymer compositions.

Fig.8. Stability integral fluorescent signal (intensity), and the response curve of fiber-optic oxygen sensor to the change of oxygen concentration in the analyzed aqueous solution (B). Changes in the intensity (and thus the lifetime of the luminescence) on the curve B in the region of 40 sec reflects the transfer process of the active element from the saturated air solution in obeskislorozhennuju environment.

Fig. 9. Response submersible fiber optic glucose biosensor membrane type at various concentrations of glucose.

Fig. 10. Response submersible enzyme membrane-based oxygen-sensitive polymeric composition and cholesteroloxidaze on the presence of cholesterol (3 mg/ml) in the analyzed sample.

1. The metal complexes of the porphyrin-ketones of General formula

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M Pt2+or Pd2+,

as phosphorescent dyes.

2. The metal complexes of the porphyrin-ketones under item 1, characterized in that the substituents R1-R8means ethyl.

3. The metal complexes of the porphyrin-ketones under item 1, characterized in that the substituents R1, R3, R5and R7mean methyl, and R2, R4, R6and R8mean CH2CH2COOH.

4. The metal complexes of the porphyrin-ketones under item 1, characterized in that the substituents R1, R3, R5and R7mean methyl, and R2, R4, R6and R8mean CH2CH2SOON3.

5. The sensing element for the optical determination of oxygen in a liquid or gaseous medium, representing a molded product of kislorodopronitsaemaya polymer distributed therein phosphorescent dye, characterized in that it is made in the form of articles molded from polystyrene, containing a dye metalcomplex porphyrin-ketones of General formula

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where R1-R8is hydrogen, lower alkyl or-CH2CH2R9;

R9hydrogen or alkyl;

M Pt2+or Pd2+.

7. The element at PP.5 and 6, characterized in that it is made in the form of a film thickness of 1-20 microns.

8. The element at PP.5 and 7, characterized in that it is made in the form of a film, fixed on a solid substrate or the optical element.

9. The element at PP.5 and 8, characterized in that the molded article further comprises a layer of oxygendependent enzyme.

10. The method for determining the oxygen involving the use of a sensitive element on the basis permeable to oxygen polymer distributed therein phosphorescent dye in contact with the analyzed sample and linked via an optical fiber with a phosphorescence detector, excitation of phosphorescence sensitive element led in the field of absorption of the dye, the registration of the phosphorescence and the calculation of oxygen concentration on the intensity and/or lifetime of phosphorescence, characterized in that the polymer used as the polystyrene, as a phosphorescent dye metal complexes of General formula

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where R1-R8are radicals from the group of hydrogen, lower alkyl or CH2-CH2-COO-R9;

R9hydrogen or alkyl;

11. The method according to p. 10, characterized in that the phosphorescent dye use Pt-octaethylporphyrin-ketone, which is characterized by the optimal value of phosphorescence at the wavelengths of excitation and phosphorescence 591 and 790 nm, respectively, and the excitation carry out the yellow led CaAs, characterized by the optimal value of the emission at 586 nm.

 

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The invention relates to medicine, in particular, clinical Oncology, and can be used in medical examination of the population to identify patients with malignant diseases, regardless of the stage and localization process, as well as for examination of cancer patients with suspicion of recurrence

The invention relates to medicine, in particular to the treatment of bronchopulmonary diseases
The invention relates to medical instrumental diagnostics, in particular to ultrasonic methods in the clinic of internal diseases and endocrinology

The invention relates to the field of cardiology, may be used in the treatment of patients with stable angina various functional classes

The sensor ozone // 2055347
The invention relates to okonometrie, in particular to sensitive elements of the ozone sensor designed for the analysis of ozone in the atmosphere

The invention relates to biophysical methods of analysis, designed to determine the concentrations CN-ions in solution and can be used to control the content of the cyanide ion in the technological cycle of gold extraction factories and other enterprises, as well as in their wastewater

The invention relates to techniques for luminescence analysis and can be used mainly to study the luminescence of biological membranes (lipid and protein parts)

The invention relates to a plasma methods of obtaining excited particles and their analysis in the gas stream

The invention relates to chemiluminescent analysis
The invention relates to analytical chemistry, to methods for determination of metal ions in solutions, and can be used in the development of optical sensors, giving a sensitive, rapid response to changing concentrations of uranium, lead, mercury in solutions

The invention relates to techniques for analytical control of the substance and can be used in mining, glass industry, with exploration for rapid determination of trace mineral in industrial raw quartz

The invention relates to analytical chemistry, and in particular to methods qualitative determination of trihexyphenidyl
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