Dyes based on dipyrromethene boron difluoride with biphotonic absorption and their use

FIELD: physical analytical methods.

SUBSTANCE: invention relates to bioanalytical methods involving dye-labeled indicators. Bioanalytical method without separation is provided directed to measure analyte obtained from biological liquid or suspension, wherein are used: analyte microparticles as first biospecific reagent; and second biospecific reagent labeled with biphotonic fluorescent dye based on dipyrromethene boron difluoride containing at least one water solubility imparting group selected from ammonium salt and sulfonic or carboxylic acid alkali metal salt and at least one chemically active group selected from carboxylic acid, reactive carboxylic acid ester, carboxylic acid anhydride, maleimide, amine and isothiocyanate. In the method of invention, laser is focused onto reaction suspension and biphotonically excited fluorescence from individual microparticles (randomly flowing or oriented by pressure provided by emission of exciting laser through focal volume of laser beam).

EFFECT: increased efficiency of bioanalyses.

5 cl, 5 dwg, 5 tbl, 25 ex

 

The scope of the invention

The present invention relates to a dye-based diferida dipyrromethene and to the use of these dyes under two-photon excitation. In addition, the present invention relates to the use of dyes on the basis of diferida dipyrromethene and their conjugates in bioanalytical assays based on two-photon excitation.

Background of the invention

Publications and other materials used in this description to illustrate the prerequisites of the invention, and in particular documents containing further details on the practical implementation, are included in this description by reference.

Dyes based diferida dipyrromethene

Dyes based diferida dipyrromethene were first described Treibs and Krauzer, Liebigs Ann.Chem. 718 (1968) 208, and Wories H.J. et al., Recl.Trav.Chim.Pays-Bas 104 (1985) 288. Since then, the dye-based diferida dipyrromethene found various applications. Dyes based diferida dipyrromethene exhibit strong fluorescence in the visible region of the spectrum. Dyes based diferida dipyrromethene have the following common characteristics: high quantum yield, a sharp absorption bands and emission and high absorption coefficient. Currently, commercially available wide times kobresia dyes on the basis of diferida dipyrromethene (Haugland R.P., Handbook of Fluorescent Probes and Research Chemicals, 6thed, Molecular Probes, Eugene, OR, 1996). The synthesis and characterization of fluorescence of these dyes have been described in publications and patents. US 4916711, US 5189029, US 5446157 and EP 0361936 in the name of Morgan and Boyer describe the application of dyes on the basis of diferida dipyrromethene in photodynamic therapy (PDT) and for receiving laser radiation. According to Morgan and Boyer, dye-based diferida dipyrromethene have low triplet-triplet absorption, together with a low threshold of action of the laser makes it possible to obtain a laser beam of higher intensity than conventional laser dyes. Moreover, high photochemical stability of these dyes provides a reduced decomposition of the dye.

Fluorescent dyes are widely used in the methods of fluorescence microscopy as an indicator labels for containment of biological structures, methods, fluorescent immunoassay for the quantitative determination of analytical samples for flow cytometrical analysis of cells and for many other purposes. Typically, these fluorescent dyes are attached to biomolecules via a covalent bond. For such tagging these dyes need a functional group which can interact with the other functional group is Oh in biomolecule. Traditionally used reactive functional groups include reactive esters of carboxylic acids, anhydrides of acids, hydrazines and isothiocyanates. US 4774339 describes the use of dyes on the basis of diferida dipyrromethene as fluorescent labels. In accordance with US 4774339 possessing fluorescent properties of the dye-based diferida dipyrromethene not have the sensitivity to solvent or pH. These dyes also have a narrow absorption band and emission, high quantum yield and high light fastness.

The main chromophore (I) dye-based diferida dipyrromethene has a peak absorption and emission at 500 nm.

The main chromophore of the dye-based diferida dipyrromethene

The wavelengths of the absorption spectrum and emission of dyes on the basis of diferida dipyrromethene can usually be changed by changing the substituents of the chromophore. The lengthening of the chain π-electronic conjugation leads to the shift of the bands of the emission and absorption of radiation to large wavelengths. The lengthening of the chain π-e-mate will also affect the light fastness, solubility and quantum yield of fluorescence. US 5274113, US 5451663 and WO 93/09185 describe the agents of tagging on the basis of diferida dipyrromethene that have my peak absorption is Oia between 525 nm and 650 nm. This shift in the wavelengths of absorption and emission was achieved by adding an unsaturated organic group in the chromophore. These patents describe the use of aryl, heteroaryl and alkenylphenol deputies for chain elongation π-e-mate. US 5248782 describes the dyes on the basis of heteroaryl-substituted diferida dipyrromethene and US 5187288 describes the dyes on the basis of the ethynyl-substituted diferida dipyrromethene, which have a peak absorption between 550 and 670 nm. The shift in the wavelengths of absorption and emission in most cases accompanied by an increase of the absorption coefficient and light fastness. The lengthening of the chain π-e-mate is also described by J. Chen et al., J.Org.Chem., 65 (2000) 2900. Chen J. et al. describe the dyes on the basis of aryl-substituted diferida dipyrromethene, which have a peak absorption between 620 and 660 nm and the fluorescence emission between 630 and 680 nm. US 5433896 describes the dyes on the basis of diferida dipyrromethene containing condensed aryl substituents. Such dyes are based diferida dipyrromethene have peak absorption and emission between 600 nm and 740 nm. The molar absorption coefficient of the dye-based diferida dipyrromethene often above 100000 cm-1M-1.

Worries H.J. et al., Recl.Trav.Chim.Pays-Bas 104 (1985) 28 describe how the introduction of sulfosalicylate in the dye-based diferida dipyrromethene higher, thus, the hydrophilicity of these dyes. They report on mono - and desulfurating dye-based diferida dipyrromethene, which have a peak absorption between 495 and 491 nm and peak emission between 515 and 510 nm.

Dyes based diferida dipyrromethene found various applications as fluorescent labels. Synthesis and diversity of these dyes described in publications and patents mentioned above. However, most of these dyes have the disadvantage, as their inherent hydrophobicity, which limits their applicability for fluorescent labeling of biomolecules. This is especially true of dyes on the basis of diferida dipyrromethene that contain aryl substituents.

Dyes based diferida dipyrromethene can be used not only as fluorescent markers, but also as laser dyes as probes in photodynamic therapy for the treatment of tumors. These dyes have also found application in the dyeing of microparticles (US 5723218) and as a medium for optical registration (US 6060606).

Two-photon excitation

In 1931, Maria Geppert-Meyer Ann.Phys. 9 (1931, 273) put forward the idea that the molecule can simultaneously absorb two photons. This phenomenon for a long time did not find practical application, up until not become available sources intensive the CSOs laser light. Two-photon excitation is created, when the focus of the source of intense light, the density of photons per unit volume and per unit time is high enough to ensure that two photons simultaneously absorbed the same chromophore. The absorbed energy is the sum of the energy of the two photons. The possibility of two-photon excitation depends on the square of the density of photon flux. The absorption of two photons, thus, is a nonlinear process of second order. As a result of simultaneous absorption of two photons by a single chromophore get the chromophore in the excited state. Relaxation of this excited state is possible due to the emission of a photon of higher energy than photons of the illuminating laser. In this regard, the method, which includes two-photon excitation and subsequent radiative relaxation, called two-photon fluorescence. Two-photon fluorescence usually has spectral characteristics similar to the induced one-photon fluorescence of the same chromophore (Xu C. and W.W. Webb, J.Opt.Soc.Am.B, 13 (1996) 481). Molecules that can exist in two simultaneously absorbed photons and the relaxation of the excited state which is accompanied by the emission of fluorescence, in the context of this invention are referred to as two-photon f is uorescent dyes.

One of the main features of two-photon excitation is that the excitation occurs only in a clearly defined three-dimensional (3D) region of the focal point. As a result of such features get high three-dimensional spatial localization of the generated fluorescence emission. Due to the nonlinear nature of the excitation, generates minimal background fluorescence outside the focal point, around the sample and optical devices. Another key feature of two-photon excitation is that the illumination and emission take place in a significantly different range of wavelengths. As a consequence, the penetration of multiple distributed light in the detector channel can be easily weaken, using filters pass low frequencies (decrease of at least 10 orders of magnitude). Because of the excited volume is very small, two-photon excitation is most suitable for monitoring small-volume samples and patterns.

Characteristic features of two-photon excitation, low background and a small amount of excitation suggest that two-photon excitation is best suited for applications where the detection of fluorescence from small volumes. In fact, the volume of two-photon excitation is femtoliter range the area or even lower depending on the optical parameters of the system. In such small volumes excitation at any given point in time there is only a small number of fluorescent molecules. One of the characteristic features relating to the methods of two-photon excitation is low, the effective absorption cross section of the fluorophores. Another feature is that two-photon excitation is usually carried out using pulsed lasers, with intervals between pulses from traditional lasers more than the lifetime of the fluorophores, which leads to reduced velocity excitation. These features together with a small amount of excitation give a relatively low signal level. Another problem concerns the intensity of strong lighting, which can lead to unexpected effects. These effects can be divided into two different categories: the cumulative effect of multiple pulses (energy storage) and the effect of one pulse (direct nonlinear absorption of the second or higher order). The cumulative effect of multiple pulses is of particular importance when using pulsed lasers with high pulse repetition frequency (lasers emitting pulses with sub-picosecond intervals of the repetition frequency of about 100 MHz). When such a pulse repetition rate possible accumulation of EN is rgii to stable triplet state of the dye molecules in a long time. Triplet States are usually not radiative, and thus, the accumulation of energy to these States reduces the output of two-photon fluorescence. The efficiency of two-photon excitation (Ex2ph) proportional to the maximum power (Pp) and pulse width (τutilaser light (Ex2ph∝Pp2uti). In accordance with this, the use of pulsed lasers with high power and moderate repetition rate of the pulses (picosecond-nanosecond pulsed lasers with repetition rate in kHz) leads to high efficiency of two-photon excitation. In this context, high efficiency two-photon excitation means that almost all of the dye molecules in the focal point of the laser beam excited by one laser pulse. Figure 1 shows the two-photon excitation of dye molecules depending on light intensity. In the ideal case, the curve of the dependence of emission from the light intensity coincides with the curve of dependence of two-photon excitation from the light intensity. This usually happens when the light intensity lower intensity required to achieve saturation (A, 1), and two-photon-excited fluorescence, so about what atom, is a quadratic dependence on light intensity. At the level of the plateau (Figure 1) or near (In, 1) (when to get the maximum two-photon-induced fluorescence using a laser with high power two-photon excitation) the effect of one pulse becomes especially important. In this case, the absorption in the excited state and stimulated emission can reduce the yield of two-photon fluorescence and cause deviations from the quadratic dependence of the emission intensity lighting. Depending on the sample, the appearance of such effects may limit the maximum usable light intensity.

Erlich J.E. et al. Opt.Lett. 22 (1997) 1843, J.W. Baur et al., Chem. Mater. 11 (1999) 2899 and Kim O.-K. et al. Chem.Mater. 12 (2000) 284 reported that the coefficients of two-photon absorption can vary greatly depending on the measurement conditions such as the intensity and duration of the pulse laser beam. It was noted that when using very intense laser light to measure the effective cross-section of two-photon absorption or release of a two-photon fluorescence, can play the role of some linear processes. The phenomenon of excited state absorption, as has been suggested, is the cause of the extinction of some fluorophores in conditions of two-photon excitation Fischer A. et al., Appl.Opt. 34 (1995) 1989).

Xu C. And W.W. Webb, J.Opt.Soc.Am. B, 13 (1996) 481 reported on the effective two-photon absorption cross sections of different fluorescent dyes, including dyes based diferida dipyrromethene described Wories H.J. et al., Recl.Trav.Chim.Pays-Bas 104 (1985) 288. Investigated Xu and Webb dye-based diferida dipyrromethene has a maximum of one-photon absorption at 495 nm and maximum emission at 515 nm. Spectra of two-photon excitation were recorded in the range between 690 and 1050 nm. In this study, the light source was a laser Ti:Sapphire with a nominal pulse duration of 80 femtoseconds. According to Xu and Webb effective cross section of two-photon absorption dye-based diferida dipyrromethene roughly an order of magnitude lower than that of Rhodamine C. Xu and Webb did not report any observations concerning the quenching of Rhodamine With high intensity laser light or any deviations from the quadratic dependence of the emission intensity lighting. However, the quenching of Rhodamine reported previously, Fischer A. et al., Appl. Opt. 34 (1995) 1989. According to Fischer, A. et al. the effect of damping in two-photon excitation leads to a lower three-dimensional resolution in two-photon microscopy.

Bioanalytical applications of two-photon-excited fluorescence.

Fluorescence finds various applications to the tion in the field of bioanalysis. In the last three decades in practice includes such applications, such as immunoassays, DNA analysis-hybridization and analyses of receptor binding using fluorescence as a detection method. In these analyses the use of reaction specific Biogradska when determining the amount of analyte in the sample. The amount of analyte can be determined by monitoring the fluorescence signal, which depends on the amount of analyte bound peroxidase. These analyses can also be based on monitoring changes in the fluorescence characteristics when specific binding reactions. This change characteristics of the fluorescence can be either a change in fluorescence intensity, a change in the wavelength of emission, the time change decomposition or change in the polarization of fluorescence.

The immunoassays are widely used in clinical diagnostics for the determination of certain diseases or physiological States. The immunoassays can be divided into two categories of analyses of different types: competitive and non-competitive assays. In the competitive method, the labeled antigen (second biospecific reagent) competes with the analyte for binding with a limited number of antibodies (primary biospecific reagent). The concentration of the analyte can be calculated from the ratio of labeled antigen, related what happened with the antibody, or on the basis of the proportions of the free fraction of labeled antigen. In non-competitive method (immunodeficiency method) analyte associated with excessive number of binding antibodies (primary biospecific reagent). Excessive amounts of labeled antibodies (second biospecific reagent) bound to another site of the analyte. The amount of analyte can be determined on the basis of the fraction of labeled antibody, contacting the analyte. Analysis methods can be divided into heterogeneous and homogeneous (without a separation) methods. Separation of bound and free fractions is necessary in heterogeneous assays, but not in homogeneous assays [Miyai K., Principles and Practice of Immunoassay, (ed. Price C.P. and D.J. Newman) Stockton Press, New York 1991, 246 and Hemmila I.A., Applications of Fluorescence in Immunoassays, (ed. Winefordner J.D.) John Wiley & Sons, New York 1991].

One of the first reports on analytical applications of two-photon excitation, was published Sepaniak et al., Anal.Chem., 49 (1977) 1554. They discussed the possibility of using two-photon excitation fluorescence for HPLC detection. Demonstrated low background and the simplicity of the system.

The applicability of two-photon fluorescence excitation in laser scanning microscopy was first demonstrated by Denk et al., Science, 248 (1990) 73. They used laser dye synchronized modes, ensure the pot is to femtosecond pulses with a frequency of emission of 80 MHz. Parish Ti-sapphire lasers has facilitated the implementation of two-photon excitation in a standard laser scanning fluorescent microscope. Two-photon excitation fluorescence has been widely discussed in the literature in the last decade. It was reported in many studies related to methods of imaging using two-photon excitation of fluorescence. The development of this technology led to the industrial manufacturing systems two-photon laser scanning microscopes.

Lakowicz et al., J.Biomolec.Screening 4 (1999) 355 showed that time-dependent damping of the intensity of DAPI (4,6-diamidino-2-phenylindol)associated with DNA and calcium-dependent fluorophores can be measured using two-photon fluorescence excitation. Lakowicz et al. reported use of multiphoton excitation for use in screening high-throughput. They showed that the induced two-photon fluorescence of Fluorescein can be easily measured in advance tablets high density.

Bioanalytical applications of two-photon-excited fluorescence, which are described in the literature, for the most part relate to methods of imaging using two-photon microscopy (Denk W. et al. US 5034613, W. Denk et al., Science 248 (1990) 73). Using two-photon fluorescence excitation in laser scanning of the respective microscopy provides a characteristic three-dimensional spatial resolution without the use of pin holes, what is necessary for confocal microscopy. With a simple optical designs microscopy with two-photon excitation provides a three-dimensional spatial resolution comparable with the results of conventional confocal microscopy with single-photon excitation. The disadvantage of the methods single-photon excitation is the need for expensive lasers capable of generating a strong ultrashort pulses with high repetition rate.

Recent developments cheaper laser technology is very promising in terms of potential applications of the technology of two-photon fluorescence excitation in routine bioanalytical tasks (Hannien P. et al., Nat.Biotechnol. 18 (2000) 548; Soini J.T. et al. Single Mol. 1 (2000) 203; WO 98/25143 and WO 99/63344). In accordance with WO 98/25143 and WO 99/63344 instead of expensive lasers are synchronized modes for two-photon excitation it is possible to use a laser diode excitation with microchips with passive Q-gate. Such lasers are monobloc, small in size, simple in construction and cheap. WO 98/25143 and WO 99/63344 describe the use of two-photon excitation in bioanalytical method. This bioanalytical method can be used for analysis of analytes in solution or in biological suspensions, and use of microparticles as the light is awaydays on bioreactive solid phase, with which is associated the main biospecific reagent. In this bioanalytical method uses the second biospecific reagent labeled with two-photon fluorescent dye. In accordance with WO 98/25143 and WO 99/63344, the contacting of the particles with the analyte and the second biospecific reagent in the volume of the reaction mass and the use of fluorescence detection based on two-photon excitation makes it possible method of analysis, without separation. The amount of analyte associated with the main biospecific reagent and particles, determined by using the detection signal of two-photon-excited fluorescence coming from the labeled second biospecific reagent. Labeled second biospecific reagent may contact either the analyte (non-competitive immunodeficiency method), or with the main biospecific reagent (competitive method). The main and second biospecific reagents are biologically active molecules such as haptens, biologically active ligands, drugs, peptides, oligonucleotides, nucleotides, nucleic acids, polypeptides, proteins, antibodies or antibody fragments. In accordance with WO 98/25143 and WO 99/63344, a laser with high efficiency two-photon excitation focus in the reaction suspension and two-photon-vozbuzhdeno the fluorescence measured from individual microparticles during their flow through the focal volume of the laser beam. Alternatively, the microparticles are captured for the period of detection of fluorescence optical layer near the laser beam. The trapping of particles at the focal point of the laser beam based on the optical pressure exerted on the particle illuminating laser. In accordance with WO 98/25143 optical trapping increases the duration of passage of the particles at the focal point of the laser beam and reduces the dead time of the measurement. In accordance with WO 98/25143 optical trapping requires a relatively high average power laser and power lighting. In this case, the average power of the laser determines the collection efficiency. The repetition frequency of pulses from a laser with microchips relatively low and, thus, it is necessary to use a high pulse energy and, consequently, a high maximum power of the illuminating laser to produce the average power of the laser is sufficient for optical trapping. The use of high maximum power, and laser with high efficiency two-photon excitation can lead to lower output of the two-photon-excited fluorescence and may also reduce the signal:background analyses without separation due to the effect of single (individual) pulse. However, in practice, to obtain the maximum signal is La fluorescence, often it is reasonable to use the laser as possible with the highest efficiency of two-photon excitation.

PURPOSE AND BRIEF description of the INVENTION

The aim of the present invention is the provision of improved bioanalytical methods without separation for measurement of the analyte from a biological fluid or suspension.

The present invention relates to a bioanalytical method without separation for measuring analyte in a biological fluid or suspension comprising microparticles as binding on bioreactive solid phase, the second biospecific reagent labeled with two-photon fluorescent dye, the laser focus in the reaction suspension, the measurement of two-photon-excited fluorescence from individual microparticles with arbitrary for, or directed pressure of the exciting laser radiation through the focal volume of the laser beam. Two-photon fluorescent dye has the structure (II):

Any at least one of the groups R1, R2, R3, R4, R5, R6or R7represents a substituted or unsubstituted phenyl, thienyl, pyrrolidinyl, fornillo, oxazolidinyl, isoxazolyl, oxadiazolyl, imidazolidinyl, benzoxazolyl, benzothiazolyl, benzimidazo the ilen, benzofuranyl, indolenine, coupled atenilol, dianilino or trainingnow group, and at least one of the groups R1, R2, R3, R4, R5, R6or R7is substituted for chemically active groups, which can be used for covalent binding with other molecules, and at least one of the groups R1, R2, R3, R4, R5, R6or R7is substituted for the receiving group, which imparts water-solubility, and the rest of the group R1, R2, R3, R4, R5, R6or R7each independently selected from the group comprising hydrogen, halogen, alkyl, cyano, carboxy, each of which optionally may be substituted; or a group R1, R2, R3, R5, R6or R7represent a substituted or unsubstituted alkyl group, R4represents hydrogen or substituted or unsubstituted alkyl, and at least one of the groups R1, R2, R3, R4, R5, R6or R7is substituted for chemically active groups, which can be used for selective covalent binding with other molecules, and at least one of the groups R1, R2, R3, R4, R5, R6or R7is C is displaced to obtain group imparts water-solubility.

BRIEF DESCRIPTION of DRAWINGS

Figure 1 presents a graph of the dependence of two-photon excitation from the light intensity.

On Figa presents dyes based on alkyl-substituted diferida dipyrromethene.

On Fig.2b and 2C presents the dyes on the basis of heteroaryl-substituted diferida dipyrromethene.

On Fig.2d presents dyes based on aryl-substituted diferida dipyrromethene.

On File and 2f presents the dyes on the basis of phenylethynyl-substituted diferida dipyrromethene.

On Figg and 2h presents the dyes on the basis of thienyl-substituted diferida dipyrromethene.

Figure 3 presents the linker connection.

Figure 4 presents a graph of the dependence of two-photon fluorescence signal from the analyte concentration (AFP) in the immunoassay.

Figure 5 presents the optical scheme of the device used for measurement of two-photon fluorescence.

DETAILED description of the INVENTION

Used in this application, the terms mean the following:

"Two-photon fluorescence" is a process involving the simultaneous absorption of two photons of the same chromophore and the subsequent radiative relaxation of the excited state.

"Two-photon fluorescent dye - molecules that can vozbujdat the Xia two simultaneously absorbed photons and the relaxation of the excited state is accompanied by emission of fluorescence.

"The cumulative effect of multiple pulses" effect, resulting from the accumulation of excitation energy to a long-term stable triplet state. This effect causes a decrease of the fluorescence yield when using sub-picosecond lasers with pulse repetition frequency of about 100 MHz for two-photon excitation. This effect causes a deviation from the quadratic dependence of two-photon fluorescence from light intensity to achieve saturation of the dye.

"The effect of the separate pulse" effect, which is a direct result of the nonlinear absorption of second or higher order. This effect causes a decrease of the fluorescence yield when using lasers with high power pulse (Pp) and the nominal pulse duration is in the range of picoseconds to nanoseconds (τuti) with a repetition frequency in kHz for two-photon excitation. This effect causes a deviation from the quadratic dependence of two-photon fluorescence from light intensity to achieve saturation of the dye.

"The efficiency of two-photon excitation" is the number of excitations per unit of time. The efficiency of two-photon excitation (Ex2ph) proportional to the maximum power (Pp) and the duration of the pulse is a (τ utilaser light (Ex2ph∝Pp2uti).

"Laser with high efficiency two-photon excitation" laser capable of two-photon excitation of fluorescent dye close to saturation (C. 1).

"The signal:background ratio between the signal obtained from the dye on the surface of the particles, and the signal obtained from the dye in solution.

"Light intensity" - the maximum laser power per unit cross-sectional area of the beam.

"Average power" - the energy of the laser beam emitted per second.

"Microparticle" - spherical or non-spherical solid object that can be used as a solid-phase carrier for chemical or biochemical particles (molecules). It is usually assumed that the microparticles have a diameter of 0.01-100 μm and may comprise a polymer, glass, silicon dioxide or other substance.

The present invention relates to fluorophores with two-photon absorption with high outputs of two-photon-excited fluorescence and demonstrates extremely low damping under the action of a separate pulse even in conditions of high-intensity laser light. The present invention offers a two-photon fluorescent dyes that can be used in bioanalytical methods for the Ah without separation, based on the use of laser with high efficiency two-photon excitation. It was found that the dyes on the basis of diferida dipyrromethene and conjugates of these dyes, which is the object of the present invention provide excellent outputs two-photon-excited fluorescence. Two-photon fluorescent dyes based diferida dipyrromethene and conjugates of these dyes, which is the purpose of the present invention are ideally suited for bioanalytical systems based microparticles, because the dyes on the basis of diferida dipyrromethene and conjugates of these dyes exhibit exceptionally low damping under the action of a separate pulse even at high average power laser is required for optical trapping of microparticles. The present invention offers a new two-photon fluorescent dyes based diferida dipyrromethene, which are particularly suitable for labelling biomolecules. Such two-photon fluorescent dyes based diferida dipyrromethene are inherently hydrophilic and soluble in aqueous solutions. The solubility of these dyes in aqueous solutions makes them ideal for use in bioanalytical methods.

Figure 1 ol is dstable a graph of the dependence of two-photon excitation from the light intensity. At low intensities of illumination (A) excitation is quadratic dependence on light intensity. To obtain the maximum two-photon-excited fluorescence dye should be excited close to saturation (In). On the plateau of saturation (C) almost all of the dye molecules become excited. On Figa presents dyes based on alkyl-substituted diferida dipyrromethene described in examples 1-3 as compounds 1-3. On Fig.2b and 2C presents the dyes on the basis of heteroaryl-substituted diferida dipyrromethene described in examples 4-9 as connections 4-9. X+is a cationic counterion. On Fig.2d presents the dyes on the basis of phenyl-substituted diferida dipyrromethene described in examples 10 and 11 as compounds 10 and 11. X+is a cationic counterion. On File and 2f presents the dyes on the basis of phenylethynyl-substituted diferida dipyrromethene described in examples 12 and 13 as connections 12 and 13. X+is a cationic counterion. On Figg and 2h presents the dyes on the basis of thienyl-substituted diferida dipyrromethene with amino, arylamino, maleimide and isothiocyanates reactive groups described in examples 14-17 as compounds 14-17. X+is a cationic counterion. On Phill linker compound 18, which is used to increase the solubility of dyes in aqueous solutions. X+is a cationic counterion. Figure 4 presents a graph of the dependence of two-photon fluorescence signal from the analyte concentration (AFP) immunoassay based on two-photon fluorescence, as described in example 21. The AFP concentration 0 ng/ml are presented as concentration of 0.1 ng/ml due to the logarithmic scale used in figure 4. Figure 5 presents the optical scheme of the device used for measurement of two-photon fluorescence. The Nd:YAG laser with microchips with passive Q-gate 1 is used as the light source for two-photon-excited fluorescence. The laser wavelength is 1064 nm, pulse duration of 1 NS and a repetition rate of 20 kHz pulses. Naturally diverging beam of laser light is directed via a dichroic mirror 2 via the optical scanner 3 to the inlet of the lens 4. The lighting at the entry hole is filling (Gaussian) about 70% full of holes. Then, the objective lens focuses the illuminating light in a special cell 5 having a thin optical bottom (thickness 0.2 mm). Average optical output power reaching the sample is 50 mW. The focal volume is about 1 femtolitr. The signal of two-photon-excited fluoresc is ncie within 540-700 nm going from full hole lens 4 and directed through the dichroic mirror 2 and narrowband optical filter 6 to the photomultiplier tube 7. When measuring particles is measured as a light two-photon-excited fluorescence and back scattered (reflected) by the laser light particles. Dichroic mirror 2 and the window 8 2%beam splitting reject back-scattered light to the hole of small diameter 9, which makes the detection of scattered signal confocal. The detector for scattered signal is a GaAs photodiode 10 to the detection of near-infrared region of the spectrum. Particles are constantly monitored, using three-dimensional beam scanner 3. Hu-Scanner is an optical-mechanical and is so close to the inlet of the lens as possible. Because of the partial light entrance aperture of the lens deflection at full articulation Hu scanners does not cause any significant cutting beam by the edge of the hole. When the amplitude of the back scattered signal exceeds a pre-set threshold level, it indicates the appearance of a particle near the focal volume. And the x - and y-scanning mirror stop. Optical pressure caused by the illuminating laser beam, detects a particle and directs it through the center of focus of the laser beam. The fluorescence signal is measured when the particle is in focus, i.e. the back scattered signal exceeds a predefined threshold. In addition to the to Hu-scanning, exercise also and z-scan, slowly moving the lens in an axial direction. The amplitude of such movement is 150 μm, and this movement does not stop for particle measurement. Despite the slow z-scan, the particles remain in focus under the action of optical safety forces. Two-photon fluorescent dyes, which are used in combination with lasers with high efficiency two-photon excitation, it is necessary to choose so that the fluorophore had minimum repayment under the action of an individual pulse. This provides the excitation density of fluorophores close to saturation and, thus, maximizing the signal-to two-photon fluorescence. The right choice of fluorescent dye also makes possible the application of optical trapping of microparticles in bioanalytical studies, which are based on two-photon excitation and the use of microparticles as a solid carrier, as described in WO 98/25143 And WO 99/63344. When using lasers with high repetition rate of pulses occurs, the singlet-triplet hybridization also becomes an important parameter for the selection of a suitable fluorophore. Using fluorophores that have a low rate of singlet-triplet hybridization, it is possible to avoid the accumulation of energy to non-radiation is telego triplet state. Two-photon fluorescent dyes based diferida dipyrromethene, which is the object of the present invention meet these two basic requirements. Moreover, the location of the absorption bands and emission of two-photon fluorescent dyes based diferida dipyrromethene can be adjusted by a suitable substitution of the main chromophore. This quality is essential in regulating properties of the chromophore, suitable for a single laser. Quantum yield of fluorescence of the dyes on the basis of diferida dipyrromethene is usually more than 70%. High quantum yield with low speed damping under the action of a separate pulse makes it possible for strong two-photon-excited fluorescence when used as a light source of the laser with high efficiency two-photon excitation. The outputs of the two-photon-excited fluorescence of dyes on the basis of diferida dipyrromethene of the present invention are surprisingly high. In contrast to previously published data [Xu C. and W.W. Webb, J.Opt.Soc.Am.B, 13 (1996) 481] it was found that the outputs of the two-photon-excited fluorescence of dyes on the basis of diferida dipyrromethene even higher than the output of the two-photon-excited fluorescence of Rhodamine C. moreover, mi is kalinoe damping of the two-photon fluorescent dyes based diferida dipyrromethene under the action of a separate pulse ensures the operation of the laser with high efficiency two-photon excitation in as the light source without substantial loss of the three-dimensional spatial resolution of the system. Two-photon fluorescent dyes based diferida dipyrromethene also can be modified by suitable substitution of the side chain, so that they can be used both in aqueous and in organic media.

The present invention relates to bioanalytical methods described in Hannien P. et al., Nat.Biotechnol. 18 (2000) 548; Soini J.T. et al. Single Mol. 1 (2000) 203; WO 98/25143 and WO 99/63344. The present invention offers a two-photon fluorescent dyes, in combination with the analytical method described in (Hannien P. et al., Nat.Biotechnol. 18 (2000) 548; Soini J.T. et al. Single Mol. 1 (2000) 203; WO 98/25143 and WO 99/63344), ensures high accuracy of the analysis. Two-photon fluorescent dyes based diferida dipyrromethene and conjugates of these dyes, which is the object of the present invention, can be used in bioanalytical method based on the use of laser with high efficiency two-photon excitation without any substantial reduction of the signal:background.

In accordance with the present invention the second biospecific reagent mark two-photon fluorescent dye-based diferida dipyrromethene. Second biospecific reagent is a biologically act the main molecule, such as a hapten, a biologically active ligand, drug, peptide, oligonucleotide, nucleotide, nucleic acid, polypeptide, protein, antibody or antibody fragment. Secondary biospecific reagent labeled with two-photon fluorescent dye-based diferida dipyrromethene, you may contact either the analyte (competitive analysis), or with primary biospecific reagent (analysis of competitive binding). The amount of analyte associated with the primary biospecific reagent, determine the detection signal of two-photon-excited fluorescence coming from the biospecific secondary reagent labeled with two-photon fluorescent dye-based diferida dipyrromethene.

Optical trapping (described in WO 98/25143), which increases the duration (passing) of a particle at the focal point of the laser beam and reduces the dead time of the measurement, requires a relatively high average power laser. High average power, low frequency pulses leads to high pulse energy, which can lead to the quenching of fluorescence under the influence of the individual pulse. In accordance with the present invention, two-photon fluorescent dyes based diferida dipyrromethene ideal for system bioanalytic the definition definitions using microparticles. These dyes exhibit exceptionally low damping under the action of an individual pulse, even at high average power laser is required for optical trapping of microparticles.

Two-photon fluorescent dyes based diferida dipyrromethene and conjugates of these dyes, which is the object of the present invention can also be used in bioanalytical method of analysis, without separation, based on the tracking of two-photon-excited fluorescence of unbound biospecific reagent. The main biospecific reagent is bound or microparticles, or on the walls of the cuvette. Second biospecific reagent labeled with two-photon fluorescent dye-based diferida dipyrromethene, you may contact either the analyte (competitive analysis), or with primary biospecific reagent (analysis of competitive binding). The amount of the analyte, who contacted the microparticles or on the walls of the cuvette through the primary biospecific reagent is determined on the basis of the signal of two-photon-excited fluorescence coming from the unbound second biospecific reagent labeled with two-photon fluorescent dye-based diferida dipyrromethene.

Two-photon fluorescent dyes on onomatopoeia dipyrromethene, who are the object of the present invention have the General structure (II):

In this structure, R1, R2, R3, R4, R5, R6or R7selected from substituents including hydrogen, halogen, alkyl, cycloalkyl, alkenyl, arylalkyl, aryl, alkylaryl, heteroaryl, acyl, alkoxy, cyano, carboxy, amino, hydroxyl, alkoxycarbonyl, nitro, alkylamino, dialkylamino, sulfo, separately or in combination. In addition, the substituents in the chromophore can be further modified to obtain a reactive functional group. Reactive groups that can be used for selective covalent binding with other molecules, include, but are not limited to, derivatives of carboxylic acids, reactive esters of carboxylic acids, anhydrides of carboxylic acids, maleimide, sulphonylchloride, sulfanilamide, hydrazines, amines, alcohols, arylazide, isocyanates, aldehydes, halogenated, triazine or isothiocyanates.

The substituents that can be used to shift the emission and absorption to higher wavelengths, are essential for the regulation of the properties of the major chromophore suitable for a particular laser. For example, in two-photon excitation using Nd:YAG lasers at a wavelength of 1064 the m band emission of fluorescence must be greater than 532 nm (the sum of the energy of the two 1064 nm photons). A convenient way to shift the absorption bands and emission to large wavelengths is a lengthening chain π-e-mate. This can be accomplished, for example, by adding unsaturated organic groups in the main chromophore. In accordance with this preferred compounds of the present invention have a structure where at least one of the substituents R1, R2, R3, R4, R5, R6or R7selected from the group comprising phenyl, thienyl, pyrrolyl, furanyl, oxazolyl, isoxazolyl, oxadiazolyl, imidazolyl, benzoxazolyl, benzothiazolyl, benzimidazolyl, benzofuranyl, indolyl, conjugated ethynyl, dienyl, trienyl, with any of the above substituents is optionally substituted by one or more substituents. Optional substituents are preferably selected from the group including halogen, hydroxy, alkoxy, cyano, nitro, carboxy, amino, alkylamino, dialkylamino, sulfo and linear or branched alkyl, preferably containing 1-10 carbon atoms, which may contain heteroatoms, substituted heteroatoms or containing heteroatoms side chains. The other substituents R1, R2, R3, R4, R5, R6or R7each independently selected from the group comprising hydrogen, halogen, ALCO is si, cyano, carboxy, alkoxycarbonyl, nitro, alkylamino, dialkylamino, sulfo and linear or branched alkyl, preferably containing 1-10 carbon atoms, which may contain heteroatoms, substituted heteroatoms or containing heteroatoms side chains. The most preferred compounds include those in which R1is substituted or unsubstituted phenyl, teinila, Petroliam or phenylethenyl; R2, R3and R4represent hydrogen; R5, R6and R7each independently be either hydrogen or an alkyl or substituted alkyl.

The preferred compound of the present invention may also have a structure where at least one of the substituents R1, R2, R3, R4, R5, R6or R7is a-YZ, where Y is the connecting link, and Z represents a reactive group that can be used for covalent binding of the chromophore with other molecules, such as biomolecules. Glue-Y - represents either a covalent bond or C1-C20linear or branched alkylenes, Allenova, alkalinous, Aracinovo group, or a combination of these groups. Glue-Y can also contain heteroatoms, substituted heteroatoms or contains gets rotoma side chain or cyclic residues. Glue-Y may also include, or consist of, the remains of the polymers, preferably residues of polymers, such as polypeptides, polysaccharides, polynucleotides, polyesters, etc. the Rest of the substituents R1, R2, R3, R4, R5, R6or R7each independently selected from the group comprising hydrogen, halogen, alkyl, cycloalkyl, alkenyl, alkynylaryl, aryl, arylalkyl, heteroaryl, acyl, alkoxy, cyano, carboxy, amino, hydroxyl, alkoxycarbonyl, nitro, alkylamino, dialkylamino, sulfo, each of which is optionally substituted. Preferred substituents defined above.

Dyes based diferida dipyrromethene generally soluble in organic solvents. But, in particular, dye-based diferida dipyrromethene containing aryl substituents are hydrophobic in nature and insoluble in aqueous solutions. However, the solubility in water is usually required for bioanalytical applications. The hydrophilicity and solubility of the fluorescent label in aqueous solutions is often important to reduce nonspecific binding, preservation svetopisemskih properties of the label in the labeled biomolecule and biological properties of labeled biomolecules. Solubility in water can be achieved by the introduction of suitable groups which impart water is rastvorimosti, the main chromophore. The group, which imparts water-solubility, preferably include ammonium salts or alkali metal sulfonic or carboxylic acids, ammonium or hydroxyl group. Solubility in water is also achieved by the introduction of a peptide or carbohydrate fragment to the dye.

Accordingly, preferred compounds of the present invention have a structure where the absorption bands and emission are chosen appropriately, the connection comprises a reactive side chain, which can be used for covalent binding of the compounds with other molecules, and the connection also includes a group which increases the solubility in aqueous solutions. Accordingly, preferred compounds of the present invention have a structure where at least one of the groups R1, R2, R3, R4, R5, R6or R7represents a substituted or unsubstituted phenyl, thienyl, pyrrolyl, furanyl, oxazolyl, isoxazolyl, oxadiazolyl, imidazolyl, benzoxazolyl, benzothiazolyl, benzimidazolyl, benzofuranyl, phenylethenyl or indolyl; and at least one of the groups R1, R2, R3, R4, R5, R6or R7is substituted for chemically active groups, which can be used for selecti the aqueous covalent binding with other molecules and/or for further chemical modifications; and at least one of the groups R1, R2, R3, R4, R5, R6or R7is substituted for the receiving group, which imparts water-solubility. Accordingly, the most preferred compounds have the structure where R1represents a substituted or unsubstituted phenyl, thienyl, pyrrolyl or phenylethenyl, R2, R3, R4and R5each independently be either hydrogen or alkyl, R6or R7Deputy, which most preferably has the formula:

where X+represents a cation, and the remaining substituent R6or R7is either hydrogen or alkyl. Preferred compounds of the present invention have a structure where groups of R1, R2, R3, R5, R6or R7represent a substituted or unsubstituted alkyl group, R4is either hydrogen, or substituted or unsubstituted alkyl; and at least one of the groups R1, R2, R3, R4,R5, R6or R7is substituted for chemically active groups, which can be used for selective covalent binding with other molecules and/or for further chemical modifications; and, hence, is her least one of the groups R1, R2, R3, R4, R5, R6or R7is substituted for the receiving group, which imparts water-solubility. Preferred substituents defined above. Examples of two-photon fluorescent dyes based diferida dipyrromethene, which are soluble in water and, therefore, particularly suitable for labelling biomolecules presented in examples 3 and 8-17. These two-photon fluorescent dyes based diferida dipyrromethene are water soluble and have very low damping under the action of an individual pulse, even when illuminated by laser two-photon excitation.

Two-photon fluorescent dyes based diferida dipyrromethene of the present invention can be connected with biospecific molecules with obtaining a two-photon fluorescent conjugates. Biospecific molecule is a biologically active molecule, such as a hapten, a biologically active ligand, drug, peptide, oligonucleotide, nucleotide, nucleic acid, polypeptide, protein, antibody or antibody fragment. Two-photon fluorescent conjugates can be used in bioanalytical systems, where the specific signal obtained through two-photon-excited fluorescence ukazannoj the conjugate. Two-photon fluorescent dyes based diferida dipyrromethene and conjugates of the present invention can also be used for staining of cells and tissues.

Following non-limiting examples are intended to further illustrate the invention. In the examples below, these compounds (compound 1-18) disclosed on Figa-2h and 3.

Example 1

3,3',5,5'-tetramethyl-4,4'-carboxymethyl-2,2'-dipyrromethene (1)

2-Etoxycarbonyl-3,5-dimethyl-4-methoxycarbonylmethylene (1.0 g, 3.35 mmol) was dissolved in formic acid (3 ml) was added Hydrobromic acid (48%, 3 ml). The reaction mixture was stirred at 100°C for 2.5 hours. After cooling the mixture to room temperature it was left overnight at room temperature. The product crystallized from the reaction mixture in the form of small orange needle-like crystals. The reaction mixture was filtered and the crystalline product was washed with water. The product, compound 1, was dried in a vacuum desiccator. The output amounted to 677 mg (84%). Additional amount (43 mg) of product was obtained from the filtrate after 2 days of maturation at +4°C. the Total yield amounted to 720 mg (86%).

2-Etoxycarbonyl-3,5-dimethyl-4-methoxycarbonylmethylene received as described by E. Bullock et al., J.Chem.Soc. (1958) 1430.

Example 2

4,4-debtor-1,3,5,7-tetramethyl-4-Bora-3A,4 is-diaza-s-indocin-2,6-dipropionate acid (2)

Dipyrromethene (compound 1) (500 mg, 1,17 mmol) suspended in chloroform (20 ml) was added triethylamine (4.3 ml, 31 mmol). Source dipyrromethene was dissolved immediately after the addition of triethylamine (4.3 ml, 31 mmol). Added diethyl ether complex·the boron TRIFLUORIDE (5 ml, 31 mmol). The reaction mixture was stirred at room temperature for 2.5 hours. The reaction mixture was diluted with chloroform (50 ml), washed with dilute HCl (5%, 30 ml) and water (30 ml). Added a small amount of ethanol (5%) for efficient extraction. The chloroform phase is evaporated to dryness on a rotary evaporator and the product (compound 2) was besieged from a mixture of ethanol/water (7 days at +4about(C) obtaining a brown-orange powder. The product, compound 2, after drying in a vacuum dessicator was 420 mg (92%).

1H NMR (JEOL JNM-LA400, DMSO-d6, 400MHz, δ ppm): 2,19 (s, 6H, 2-CH3), to 2.29 (t, 4H, 2x-CH2-), of 2.38 (s, 6H, 2-CH3), to 2.57 (t, 4H, 2x-CH2), 7,53 (s, 1H, Ar-CH=).

Example 3

Succinimidyl ether 4,4,-debtor-1,3,5,7-tetramethyl-6-carboxyethyl-4-Bora-3A,4A-diaza-s-indocin-2-propionic acid (3)

4,4-Debtor-1,3,5,7-tetramethyl-4-Bora-3A,4A-diaza-s-indocin-2,6-dipropionate acid (compound 2) (102 mg, 0.26 mmol) was dissolved in N,N-dimethylformamide (2.5 ml, anhydrous). Was added N-hydroxysuccinimide (90 mg, 0.78 mmol) and N,N (54 mg, 0.26 mmol). The reaction mixture was stirred for 24 hours at room temperature. N,N-Dimethyformamide evaporated (5 mbar, 40-50° (C) and the product was purified column chromatography using silica gel as stationary phase and a mixture of dichloromethane:acetic acid (100:8:1, V/V/V) as eluent. The fractions containing the desired monosyllabically ester (compound 3)were combined and evaporated to dryness. The residue was dissolved in a small amount of dichloromethane. Added petroleum ether to precipitate the product. The solution was filtered and the precipitated product (compound 3) was obtained as an orange solid. Yield 50 mg (39%).

1H NMR (JEOL JNM-LA400, DMSO-d6, 400MHz, δ ppm): of 2.21 (s, 3H, CH3), 2,22 (s, 3H, CH3), a 2.36 (t, 2H, -CH2-), is 2.40 (s, 3H, CH3), is 2.41 (s, 3H, CH3), at 2.59 (t, 2H, -CH2-), of 2.72 (t, 2H, -CH2-), 2,80 (s, 4H, 2-CH2-), 2,85 (t, 2H, -CH2-), 7,60 (s, 1H, Ar-CH=). UV-VIS (Ocean Optics SD2000, MeOH):λmax=523 nm (ε=91000).

Example 4

Methyl ether of 4,4,-debtor-5-(2-thienyl)-1,3-dimethyl-4-Bora-3A,4A-diaza-s-indocin-2-propionic acid (4)

2-Formyl-5-(2-thienyl)pyrrole (150 mg, 0.84 mmol) and 2,4-dimethyl-3-carboxyethylpyrrole (140 mg, 0.84 mmol) was dissolved in a mixture of dichloromethane:methanol (10:1, 30 ml). Was added phosphorus oxychloride (77 μl, 0.84 mmol) and the solution was stirred at room temperature for 18 hours. P is a promotional mixture was evaporated to dryness and the residue was dissolved in dichloromethane (150 ml). Was added N-ethyl-N,N-Diisopropylamine (of 1.44 ml, 8.4 mmol) and diethyl ether complex·the boron TRIFLUORIDE (of 1.06 ml, 8.4 mmol). Immediately after adding diethyl ether complex·the boron TRIFLUORIDE observed a strong orange fluorescence. The reaction mixture was washed with water, dried with sodium sulfate and evaporated to dryness. The crude product was purified column chromatography using silica gel as stationary phase and dichloromethane as eluent. The fractions containing the desired product were evaporated to dryness, getting 304 mg (93%) of methyl ester of 4,4-debtor-5-(2-thienyl)-1,3-dimethyl-4-Bora-3A,4A-diaza-s-indocin-2-propionic acid (compound 4).

1H NMR (Bruker AM200, CDCl3, 200MHz, δ ppm): of 2.21 (s, 3H, Ar-CH3), 2,47 (t, 2H, -CH2-), at 2.59 (s, 3H, Ar-CH3), is 2.74 (t, 2H, -CH2-), 3,68 (s, 3H, -SOON3), 6,70 (d, 1H, ArH), 6,91 (d, 1H, ArH), 7,05 (s, 1H, Ar-CH=), to 7.15 (d, 1H, ArH), 7,40 (d, 1H, ArH), of 8.04 (d, 1H, ArH). FAB-MS: MH+389. UV-VIS (Ocean Optics SD2000, MeOH): λmax=560 nm (ε=82000).

2-(2-Thienyl)pyrrole was obtained according to C.G. Kruse et al, Heterocycles 26 (1985) 3141 and subjected to formirovanie obtaining 2-formyl-5-(2-thienyl)pyrrole, in accordance with E. Bullock et al. J.Chem.Soc. (1963) 2326.

2,4-Dimethyl-3-carboxyethylpyrrole received the following way: 2-etoxycarbonyl-3,5-dimethyl-4-methoxycarbonylmethylene (1.5 g, 5.9 mmol) and potassium hydroxide (6.6 g) was dissolved in ethylene glycol (50 m is). The reaction mixture is boiled under reflux (190aboutC) for 3 hours in nitrogen atmosphere. Was added water (10 ml) and the mixture is boiled under reflux for 2 hours. The cooled reaction mixture was diluted with water (200 ml), acidified with concentrated hydrochloric acid and was extracted with diethyl ether. The organic extracts were combined, dried with sodium sulfate and evaporated to dryness. The product was purified column chromatography using silica gel as stationary phase and a mixture of dichloromethane:methanol (10:1, V/V) as eluent. The fractions containing the product were combined and evaporated to dryness to obtain 720 mg (72%) of 2,4-dimethyl-3-carboxyethylpyrrole.

Example 5

4,4-Debtor-5-(2-thienyl)-1,3-dimethyl-4-Bora-3A,4A-diaza-s-indocin-2-propionic acid (5)

Methyl ester of 4,4-debtor-5-(2-thienyl)-1,3-dimethyl-4-Bora-3A,4A-diaza-s-indocin-2-propionic acid (compound 4) (107 mg, 0.27 mmol) was dissolved in a mixture of tetrahydrofuran:water (3:2, 50 ml). Added phosphoric acid (85%, 3 ml) and the mixture is boiled under reflux for 5 days. The reaction mixture was diluted with water and the product was extracted with dichloromethane. The organic phase was dried with sodium sulfate and evaporated to dryness. The product was purified column chromatography using silica gel as stationary phase and a mixture of di is Loretan:methanol (10:1, V/V) as eluent. The fractions containing the desired product were combined and evaporated to dryness. The product was led from a mixture of dichloromethane:petroleum ether to obtain 68 mg (66%) of 4,4-debtor-5-(2-thienyl)-1,3-dimethyl-4-Bora-3A,4A-diaza-s-indocin-2-propionic acid (compound 5).

1H NMR (Broker AM200, DMSO-d6, 200MHz, δ ppm): 2,22 (s, 3H, Ar-CH3), is 2.40 (t, 2H, -CH2-), of 2.51 (s, 3H, Ar-CH3), 2,63 (t, 2H, -CH2-), to 6.80 (d, 1H, ArH), 7,11 (d, 1H, ArH), 7,19 (DD, 1H, ArH), of 7.69 (s, 1H, Ar-CH=), 7,73 (d, 1H, ArH), 7,88 (d, 1H, ArH).

Example 6

Succinimidyl ether 4,4-debtor-5-(2-thienyl)-1,3-dimethyl-4-Bora-3A,4A-diaza-s-indocin-2-propionic acid (6)

4,4-Debtor-5-(2-thienyl)-1,3-dimethyl-4-Bora-3A,4A-diaza-s-indocin-2-propionic acid (compound 5) (57 mg, 0.15 mmol) was dissolved in anhydrous N,N-dimethylformamide (3 ml). Was added N,N'-dicyclohexylcarbodiimide (47 mg, 0.23 mmol) and N-hydroxysuccinimide (52 mg, 0.45 mmol) and the mixture was stirred at room temperature for 20 hours. The reaction mixture was diluted with dichloromethane and precipitated N,N'-dicyclohexylmethane was filtered. The filtrate was evaporated to dryness and the product was purified column chromatography using silica as stationary phase and a mixture of dichloromethane:acetone:acetic acid (100:8:1, V/V/V) as eluent. The fractions containing the desired product were combined and evaporated to dryness. The product was dried the vacuum desiccator to obtain 35 mg (75%) Succinimidyl ether 4,4-debtor-5-(2-thienyl)-1,3-dimethyl-4-Bora-3A,4A-diaza-s-indocin-2-propionic acid (compound 6).

1H NMR (Bruker AM200, CDCl3, 200MHz, δ ppm): of 2.23 (s, 3H, Ar-CH3), 2,61 (s, 3H, Ar-CH3), 2,74-2,82 (2t, 4H, 2-CH2-), of 2.86 (s, 4H, -CH2-) 6,72 (d, 1H, ArH), 6,93 (d, 1H, ArH), was 7.08 (s, 1H, Ar-CH=), to 7.15 (DD, 1H, ArH), 7,41 (d, 1H, ArH), of 8.06 (d, 1H. ArH).

Example 7

Glu-Tau-derived 4,4-debtor-5-(2-thienyl)-1,3-dimethyl-4-Bora-3A,4A-diaza-s-indocin-2-propionic acid (7)

Succinimidyl ether 4,4-debtor-5-(2-thienyl)-1,3-dimethyl-4-Bora-3A,4A-diaza-s-indocin-2-propionic acid (compound 6) (35 mg, 0,074 mmol) and Glu-Tau-linker (compound 18) (19 mg, 0,074 mmol) was dissolved in anhydrous N,N-dimethylformamide (1 ml). Added anhydrous triethylamine (31 μl, 0.22 mmol) and the reaction mixture was stirred at room temperature for 30 minutes the Reaction mixture was evaporated to dryness under reduced pressure. The crude product was used without further purification.

MS (Voyager DE-PRO MALDI-TOF, PerSeptive Biosystems, matrix α-cyano-4-cinnamic acid, negative mode): Calculated 609 (M-1), found 609 (M-1).

Example 8

Glu-Tau-operations derivative of 4,4-debtor-5-(2-thienyl)-1,3-dimethyl-4-Bora-3A,4A-diaza-s-indocin-2-propionic acid (8)

Glu-Tau-derived 4,4-debtor-5-(2-thienyl)-1,3-dimethyl-4-Bora-3A,4A-diaza-s-indocin-2-propionic acid (compound 7) (0,074 mmol) was dissolved in anhydrous N,N-dimethylformamide (2 ml). Was added N,N'-dicyclohexylcarbodiimide (46 mg, 0.22 mmol) and N-hydroxysuccinimide (26 mg, 0.22 mmol)and the mixture was stirred at room temperature for 48 hours. The precipitated N,N'-dicyclohexylmethane was filtered and the filtrate was evaporated to dryness. The product was dried in a vacuum dessicator and used without further purification.

MS (Voyager DE-PRO MALDI-TOF, PerSeptive Biosystems, matrix α-cyano-4-cinnamic acid, negative mode): Calculated 706 (M-1), found 706 (M-1).

Example 9

Glu-Tau-operations derivative of 4,4-debtor-5-(2-pyrrolyl)-1,3-dimethyl-4-Bora-3A,4A-diaza-s-indocin-2-propionic acid (9)

Glu-Tau-operations derivative of 4,4-debtor-5-(2-pyrrolyl)-1,3-dimethyl-4-Bora-3A,4A-diaza-s-indocin-2-propionic acid (compound 9) was obtained by a method described in examples 7 and 8, using as starting compound 4,4-debtor-5-(2-pyrrolyl)-1,3-dimethyl-4-Bora-3A,4A-diaza-s-indocin-2-propionic acid.

MS (Voyager DE-PRO MALDI-TOF, PerSeptive Biosystems, matrix α-cyano-4-cinnamic acid, negative mode): Calculated 689 (M-1), found 689 (M-1).

Example 10

Glu-Tau-operations derivative of 4,4-debtor-5-phenyl-1,3-dimethyl-4-Bora-3A,4A-diaza-s-indocin-2-propionic acid (10)

Glu-Tau-operations derivative of 4,4-debtor-5-phenyl-1,3-dimethyl-4-Bora-3A,4A-diaza-s-indocin-2-propionic acid, compound 10, was obtained by a method described in examples 7 and 8, using as starting compound Succinimidyl ether 4,4-debtor-5-phenyl-1,3-dimethyl-4-Bora-3A,4A-diaza-s-indocin-2-propionic acid.

MS (Voyger DE-PRO, MALDI TOF, PerSeptive Biosystems, matrix α-cyano-4-cinnamic acid, negative mode): Calculated 700 (M-1), found 700 (M-1).

Example 11

Glu-Tau-operations derivative of 4,4-debtor-5-(4-methoxyphenyl)-1,3-dimethyl-4-Bora-3A,4A-diaza-s-indocin-2-propionic acid (11)

Glu-Tau-operations derivative of 4,4-debtor-5-(4-methoxyphenyl)-1,3-dimethyl-4-Bora-3A,4A-diaza-s-indocin-2-propionic acid, compound 11, was obtained by a method described in examples 7 and 8, using as starting compound Succinimidyl ether 4,4-debtor-5-(4-methoxyphenyl)-1,3-dimethyl-4-Bora-3A,4A-diaza-s-indocin-2-propionic acid.

MS (Voyager DE-PRO MALDI-TOF, PerSeptive Biosystems, matrix α-cyano-4-cinnamic acid, negative mode): Calculated 730 (M-1), found 730 (M-1).

Example 12

Glu-Tau-operations derivative of 4,4-debtor-5-styryl-1,3-dimethyl-4-Bora-3A,4A-diaza-s-indocin-2-propionic acid (12)

Glu-Tau-operations derivative of 4,4-debtor-5-styryl-1,3-dimethyl-4-Bora-3A,4A-diaza-s-indocin-2-propionic acid, compound 12, was obtained by a method described in examples 7 and 8, using as starting compound Succinimidyl ether 4,4-debtor-5-styryl-1,3-dimethyl-4-Bora-3A,4A-diaza-s-indocin-2-propionic acid.

MS (Voyager DE-PRO MALDI-TOF, PerSeptive Biosystems, matrix α-cyano-4-cinnamic acid, negative mode): Calculated 726 (M-1), found 726 (M-1).

Example 13

Glu-Tasksengine derivative of 4,4-debtor-5-(4-methoxystyrene)-1,3-dimethyl-4-Bora-3A,4A-diaza-s-indocin-2-propionic acid (13)Glu-Tau-operations derivative of 4,4-debtor-5-(4-methoxystyrene)-1,3-dimethyl-4-Bora-3A,4A-diaza-s-indocin-2-propionic acid, compound 13, was obtained by a method described in examples 7 and 8, using as starting compound Succinimidyl ether 4,4-debtor-5-(4-methoxystyrene)-1,3-dimethyl-4-Bora-3A,4A-diaza-s-indocin-2-propionic acid.

MS (Voyager DE-PRO MALDI-TOF, PerSeptive Biosystems, matrix α-cyano-4-cinnamic acid, negative mode): Calculated 756 (M-1), found 756 (M-1).

Example 14

Glu-Tau-amino derivatives of 4,4-debtor-5-(2-thienyl)-1,3-dimethyl-4-Bora-3A,4A-diaza-s-indocin-2-propionic acid (14)

Glu-Tau-operations derivative of 4,4-debtor-5-(2-thienyl)-1,3-dimethyl-4-Bora-3A,4A-diaza-s-indocin-2-propionic acid (compound 8) (0,062 mmol) was dissolved in anhydrous N,N-dimethylformamide (3 ml). Was added triethylamine (26 μl, 0.185 mmol) and Ethylenediamine (42 μl, of 0.62 mmol) and the mixture was stirred at room temperature for 30 minutes the Reaction mixture was evaporated to dryness and the product was besieged from a mixture of dichloromethane-carbon tetrachloride. The product, compound 14, was dried in a vacuum dessicator and used without further purification.

MS (Voyager DE-PRO MALDI-TOF, PerSeptive Biosystems, matrix α-cyano-4-cinnamic acid, negative mode): Calculated 651 (M-1), found 651 (M-1).

Example 15

Glu-Tau-relaminarization 4,4-debtor-5-(2-thienyl)-1,3-dimethyl-4-Bora-3A,4A-diaza-s-indocin-2-propionic acid (15)

Glu-Tau-operations derivative of 4,4-debtor-5-(2-thienyl)-1,3-dimethyl-Bora-3A,4A-diaza-s-indocin-2-propionic acid (compound 8) (0,074 mmol) was dissolved in anhydrous N,N-dimethylformamide (3 ml). Was added triethylamine (31 μl, 0.22 mmol) and 4-(2-amino-ethyl)aniline (15 mg, 0.11 mmol) and the mixture was stirred at room temperature for 3 hours. The reaction mixture was evaporated to dryness and the product was besieged from a mixture of dichloromethane/carbon tetrachloride. The product, compound 15 was dried in a vacuum dessicator and used without further purification.

MS (Voyager DE-PRO MALDI-TOF, PerSeptive Biosystems, matrix α-cyano-4-cinnamic acid, negative mode): Calculated 727 (M-1), found 727 (M-1).

Example 16

Glu-Tau-isothiocyanatobenzene 4,4-debtor-5-(2-thienyl)-1,3-dimethyl-4-Bora-3A,4A-diaza-s-indocin-2-propionic acid (16)

Glu-Tau-relaminarization 4,4-debtor-5-(2-thienyl)-1,3-dimethyl-4-Bora-3A,4A-diaza-s-indocin-2-propionic acid (compound 15) (0,048 mmol) was dissolved in a mixture of acetone (9 ml) and NaHCO3(9 ml, aq., the feast upon.). Added thiophosgene (183 μl, 2.4 mmol) and the mixture was stirred at room temperature for 1.5 hour. The reaction mixture was diluted with water (50 ml) and dichloromethane (50 ml). The aqueous phase was twice washed with dichloromethane (40 ml). The product containing aqueous phase was extracted with phenol (2·20 ml). The product containing phenol phase was washed with water (40 ml) and diluted with diethyl ether (200 ml). Phase phenol/diethyl ether was extracted with water (4·30 ml) and the combined aqueous phase is twice washed with diethyl ether (30 ml). The product, sod is Rashi aqueous phase, was evaporated to dryness and the product was besieged from a mixture of dichloromethane-carbon tetrachloride. The product, compound 16, was dried and stored in a vacuum desiccator.

MS (Voyager DE-PRO MALDI-TOF, PerSeptive Biosystems, matrix α-cyano-4-cinnamic acid, negative mode): Calculated 769 (M-1), found 769 (M-1).

Example 17

Glu-Tau-maleimide derivative of 4,4-debtor-5-(2-thienyl)-1,3-dimethyl-4-Bora-3A,4A-diaza-s-indocin-2-propionic acid (17)

Glu-Tau-amino derivatives of 4,4-debtor-5-(2-thienyl)-1,3-dimethyl-4-Bora-3A,4A-diaza-s-indocin-2-propionic acid (compound 14) (0.015 mmol) was dissolved in anhydrous N,N-dimethylformamide (3 ml). Was added triethylamine (2,1 μl, 0.015 mmol) and N-Succinimidyl 4-maleimidomethyl (3.2 mg, at 0.020 mmol) and the mixture was stirred at room temperature for 2 hours. The reaction mixture was evaporated to dryness and the product was besieged from a mixture of dichloromethane-carbon tetrachloride. The product, compound 17 was again dried and stored in a vacuum desiccator.

MS (Voyager DE-PRO MALDI-TOF, PerSeptive Biosystems, matrix α-cyano-4-cinnamic acid, negative mode): Calculated 816 (M-1), found 816 (M-1).

Example 18

Glu-Tau-linker (18)

A derivative of glutamic acid, BOC-Glu(OtBu)-OSu (Nova Biochem, 500 mg, 1.25 mmol) was dissolved in anhydrous N,N-dimethylformamide (5 ml). In a solution of triethylamine (1.20 ml, the rate of 8.75 mmol) in water (10 ml) was dissolved taurine (782 mg, of 6.25 mmol). A solution of taurine was added to the solution Glu(OtBu)-OSu and the reaction mixture was stirred at room temperature for 30 minutes Added ethanol precipitated taurine was filtered and the filtrate was evaporated to dryness. The residue was dissolved in triperoxonane acid (2 ml) and stirred at room temperature for 2 hours. The reaction mixture was evaporated to dryness and the residue was dissolved in a mixture of dichloromethane:methanol. The product (compound 18) was besieged in the form of a white solid with a slow evaporation of methanol on a rotary evaporator. Desired Glu-Tau-linker (compound 18) was obtained with the yield 64% (204 mg).

MS (ZABSpec-oaTOF, Fisons Instruments, glycerol matrix): 255 (M+1), found 255 (M+1).

Example 19

The measurements of two-photon fluorescence of the selected two-photon fluorescent dyes using a laser with high efficiency two-photon excitation

The outputs of the two-photon fluorescence was measured using the experimental device is shown schematically in Figure 5. Rhodamine (Eastman Kodak) was chosen as a standard for comparison because of its well-characterized fluorescent properties. Three dye-based diferida dipyrromethene with names BODIPY 530/550, BODIPY 558/568, and BODIPY 564/576, as well as the R-phycoerythrin (R-PE) were purchased from a company Molecula Probes. All organic dyes were dissolved in N,N-dimethylformamide and diluted with absolute ethanol to a concentration of 100 nm. The original solution of R-PE was diluted sauverny the phosphate salt solution (50/150 nm, pH 7.4). The results are presented in table 1. The results are normalized relative to the signal of Rhodamine (100). As can be seen from table 1, the outputs of fluorescence dyes on the basis of diferida dipyrromethene of the same order as the fluorescence yield of Rhodamine C. High outputs of fluorescence dyes on the basis of diferida dipyrromethene make them an attractive alternative to dyes of the type of Rhodamine s R-phycoerythrin has the highest fluorescence yield in the table of dyes. However, the applicability of R-D is limited due to the large size. The complexity of the selective activation of R-D also limits its use as a label.

Table 1

The outputs of the two-photon fluorescence of the selected two-photon fluorescent dyes
Fluorescent dyeThe normalized output of the two-photon fluorescence [a.u.]
dyes based diferida dipyrromethene
Connection 318
Compound 8219
BODIPY 530/55066
BODIPY 558/568101
BODIPY 564/576128
Rhodamine100
-RE 618

Example 20

Tagging mouse IgG anti-AFP two-photon fluorescent dye-based diferida dipyrromethene 3

To a solution of 1.5 mg (9.3 mmol) of mouse IgG anti-AFP (clone A) in 400 μl of phosphate buffered saline (10/150 nm, pH 7.4) was added a 20-fold excess of compound 3 in anhydrous N,N-dimethylformamide (25 μl, conc.=7.5 mm). Added 40 μl of NaHCO3(1M, aq.) and the mixture is incubated at room temperature for 2 hours. The product was purified on a gel filtration column (NAP-5 (Amersham Pharmacia Biotach, Uppsala, Sweden), using as eluent phosphate buffered saline (50/150, 10 mm NaN3, pH 7.4). Mobile orange fraction was rapidly collected and the degree of labeling was determined spectrophotometrically. Received the degree of labeling 5 fluorophores for protein.

Example 21

Comparison of R-PE, Alexa 532 (Molecular Probes) and dye-based diferida dipyrromethene (3) immunoassay using particles

The analysis model is immunodeficiency analysis without separation, uses of 3.1 μm amino-modified polystyrene microparticles (Amino Modified Microspheres PA05N, Bangs Laboratories, Inc., Fishers, IN, USA) as a solid phase. Fragments of mouse monoclonal anti-IgG Fab' (clone) covalently bound with microparticles using heterobifunctional ε-maleimido prolactinemia linker. The original suspension anti-AFP-coated microparticles were diluted with buffer to analyze TRIS pH 8.0 to a concentration of particles of 0.05%. Mouse monoclonal IgG anti-AFP (clone A) was labelled with dye-based diferida dipyrromethene (compound 3), Alexa 532 (Molecular Probes, Eugene, Oregon) or R-PE (Molecular Probes, Eugene, Oregon) and used as an indicator. The indicators were diluted with a buffer for analysis until the final concentration was 1.58 nm (compound 3), of 1.45 nm (Alexa 532) and 0.78 nm (R-RE, Molecular Probes). 5 μl of the suspension of microparticles and 10 μl of the indicator was mixed with 10 ál of the standard substances Alpha-1-fetoprotein (X0900, Dako A/S, Denmark). The reaction mixture was incubated for 2 hours at 37°and measurements were carried out in the same reaction cell using the experimental device is shown schematically in Figure 5. The results are shown in Table 2 and figure 4.

Concentration indicators optimized independently of each indicator. Dye-based diferida dipyrromethene gives the lowest background signal, even though it was used in a higher concentration than indicators R-PE or Alexa 532. The background signal analysis is important because it is associated with sensitivity analysis. The most important source of background signal is the free fraction of labeled antibodies (indicator) in the reaction suspension. Background levels of three different indicators p is estaline in Table 2 and figure 4. The results clearly show that the best correlation of the signal:background receive indicator, labeled dye-based diferida dipyrromethene. Moreover, when comparing the analysis results with the results of measurements in solution (example 19, table 1) shows that the ratio of the two-photon fluorescence between R-D and 3 connection has changed. In measurements in solution (table 1) the ratio of the signals R-RE:compound 3 was 34 (618/18=34) in favor of R-PE, whereas the dimension of the particles (table 2) ratio amounted to only 3.2 (25,6/8=3,2) in favor of R-PE.

6,90
Table 2

The signal of two-photon fluorescence (TPF) from the surface of the microparticles in the AFP immunoassay without separation. Signal zero concentration of AFP in the coming mostly from the free indicator solution (=background signal)
The AFP concentration[ng/ml]The signal TPF (R-D indicator)The signal TPF (Alexa 532 indicator)The signal TPF (indicator 3)
01,800,720,50
12,751,350,71
23.101.401,00
54,202,401,50
103,502,00
5025,6to 12.08,00
20061,020,018,0
50064,022,038,0

Example 22

Measurement of the effect of separate pulse

Direct measurement of damping under the action of an individual pulse is very difficult to implement. However, it can be measured indirectly by determining the axial resolution in the dye solution. The deep component (z-wave) is most likely to change the resolution. Z-response is measured using the device described in figure 5. Cuvettes for sample 5 was replaced by a pair of slides. The dye solutions were placed between the two object glasses. As slow progress samples of dye solution to the focus recorded a constant increase in signal. The maximum receive signal when the focal volume completely covers the sample. For various dyes was registered interval fluorescence intensity from 20% to 80%, the data presented in table 3. The interval of the fluorescence intensity of dye-based diferida dipyrromethene less than the same values for R-phycoerythrin or Rhodamine C. Thus, the dye-based diferida dipirro tanbara offer better spatial resolution and better signal-to:background, than rodinov or R-D in the system of two-photon excitation using a laser with high efficiency two-photon excitation. This result is in good agreement with the measurements of microparticles described in Example 21.

Table 3

The interval values of the intensity of two-photon fluorescence 20%-80% of the selected two-photon fluorescent dyes
fluorescent dyeValue the fluorescence intensity of 20%-80% [a.u.]
dyes based diferida dipyrromethene
Connection 311,5
Compound 812,6
BODIPY 530/55012,2
BODIPY 558/56813,7
BODIPY 564/57614.1
Rhodamine16,5
R-REa 21.5

Example 23

Tagging oligonucleotides compound 6 and compound 8

To a solution of 5'-aminomethylpropanol of the oligonucleotide (17 bases, 28 μg, 5 nmol) in carbonate buffer (200 μl, 100 mm, pH 8.5) was added a 40-fold excess amount of tagging agent (compound 6 or 8) in anhydrous DMF (50 μl). The reaction mixture was incubated at 22°in ECENA 20 hours. Labeled oligonucleotides were purified using HPLC with reversed-phase column RP-18) and the method of gradient elution. Used solvent A: 2% acetonitrile in buffer acetate of triethylamine (50 mm, pH 7) and B: 70% acetonitrile in buffer acetate of triethylamine (50 mm, pH 7). The gradient started with 5% solvent and the amount of solvent In linearly increased up to 40% in 25 minutes. Both conjugate dye-oligonucleotide (labeled compound 6 or compound 8) was suirable between 18 and 22 minutes, while unlabeled oligonucleotide was elyuirovaniya at time 10 minutes Unbound agent tagging was removed from the column by increasing the amount of solvent to 100%.

The concentration of labeled oligonucleotides was determined spectrophotometrically. Received output tagging 25% (compound 6) and 20% (compound 8). Labeled oligonucleotides were diluted to equimolar concentrations and outputs the fluorescence was determined by methods such as single-photon and two-photon excitation. As a result, it was found that the fluorescence yield of the oligonucleotide labeled with compound 8, an order of magnitude higher in comparison with the oligonucleotide, labeled compound 6. The same result was obtained when using methods as single-photon and two-photon excitation. Higher fluorescence yield of the oligonucleotide labeled with compound 8, mo is but to explain the increased hydrophilicity used for tagging compounds and the increased distance between the label and the oligonucleotide.

Example 24

Tagging mouse IgG anti-AFP fluorescent dyes based diferida dipyrromethene (6 and 8)

To a solution of 0.2 mg (1.25 mmol) of mouse IgG anti-AFP (clone A) in 40 μl of phosphate buffered saline (10/150 nm, pH 7.4) was added 5-fold excess (of 6.25 nmol) of compound 6 or compound 8 in anhydrous N,N-dimethylformamide. Added 4 μl of NaHCO3(1M, aq.) and the mixture is incubated at room temperature for 3 hours. The product was purified on a gel filtration column (NAP-5 (Amersham Pharmacia Biotach, Uppsala, Sweden), using as eluent phosphate buffered saline (50/150, 10 mm NaN3, pH 7.4). Moving colored faction quickly gathered. The degree of labeling of these protein conjugates (compounds 6 and 8) was determined spectrophotometrically. Received the degree of labeling 2,3 (compound 6) and 2.5 (compound 8) fluorophores for protein.

Example 25

Comparison of indicators of antibodies labeled with compound 6 and compound 8 in the immunoassay using particles

AFP analysis was carried out using a 3.2 μm carboxy-modified polystyrene microparticles (Carboxy Modified Microspheres PA05N, Bangs Laboratories, Inc., Fishers, IN, USA) as a solid phase. Mouse monoclonal anti-AFP IgG (clone) covalently bound with microparticles using EDAC (1-ethyl-3-(dimethylaminopropyl)carbodiimide) method binding (TechNote 205, Bangs Laboratories, Inc., Fishers, IN, U.S.A.). The original suspension anti-AFP-coated microparticles were diluted with buffer to analyze TRIS pH 8.0 to a concentration of particles of 1.5·107particles/ml Mouse monoclonal IgG anti-AFP (clone A) was labelled with dye-based diferida dipyrromethene (compound 6 or 8) and used as an indicator. The indicators were diluted with a buffer for analysis to a final concentration of 16 nm. 5 μl of the suspension of microparticles and 5 μl of the indicator was mixed with 10 ál of the standard substances Alpha-1-Fetoprotein (X0900, Dako A/S, Denmark). The reaction mixture was incubated for 2 h at 37°and measurements were carried out in the same reaction cell using the experimental device is shown schematically in Figure 5. The results are shown in Tables 4 and 5.

22,26
Table 4

AFP immunoassay using indicator labeled compound 6. All concentrations were measured on six parallel samples.
The AFP concentration [ng/ml]2PF signal (average of 6 parallel measurements)The standard deviation (SD)The coefficient of variation (%) (CV)Signal-to-background (S-S0)
06,039,03149,660,00
0,13,020,67-3,01
16,635,1477,460,60
512,743,2825,71of 6.71
1018,183,6319,9612,15
2045,017,8317,4038,98
100141,4623,1316,35135,43
400249,4827,6611,09243,45
1000260,9831,9412,24254,95

Sensitivity analysis is often determined by calculating the standard deviation (SD) of the signal from parallel samples at zero concentration of the analyte. The lowest concentration of analyte that gives a stronger signal than 3SD (standard deviation at zero concentration of the analyte, multiplied by 3), usually used for determining the sensitivity analysis.

Sensitivity analysis is strongly influenced by the background signal (signal:background), as described in example 21, and the repeatability of the measurements.

Table 5

AFP immunoassay using indicator mechen the th connection 8. All concentrations were measured on six parallel samples.
The AFP concentration [ng/ml]2PF signal (average of 6 parallel measurements)The standard deviation (SD)The coefficient of variation (%) (CV)Signal-to-background (S-S0)
05,110,285,460,00
0,15,880,264,470,77
17,050,355,001,94
5made 13.360,795,958,25
1020,651,527,3815,54
2039,373,809,6634,26
100140,3320,2514,43135,23
400274,6916,796,11269,58
1000333,2212,453,74328,12

The results presented in tables 4 and 5 clearly show that the indicator labeled hydrophilic dye-based diferida dipyrromethene (compound 8), provides a more sensitive analysis. The difference in values 3SD between the two indicators unexpectedly large, more than 3SD connection 8=0,84 against 3SD connection 6=27). This means that the indicator labeled hydrophilic dye-based diferida dipyrromethene (compound 8) provides a sensitivity analysis on an order of magnitude higher than the indicator labeled hydrophobic dye-based diferida dipyrromethene (compound 6). On average, both indicators give an answer that depends on the concentration of the analyte, whereas the indicator labeled hydrophilic dye-based diferida dipyrromethene (compound 8) shows a slightly higher signal level. In addition, the led labeled hydrophilic dye-based diferida dipyrromethene (compound 8) provides significantly lower deviation of the signal (coefficient of variation) throughout the analysis in comparison with the indicator labeled hydrophobic dye (compound 6).

It should be clear that the methods of the present invention may include different variants of the embodiment, only some of which are disclosed in this application. Specialist in the art it should be clear that there are other ways to embodiments that do not go beyond the scope of this invention. Thus, the described variants of the embodiments of the invention are illustrative and should not be construed as ogran is sustained.

1. Bioanalytical method without separation for measurement of the analyte from a biological fluid or suspension, in which the use of microparticles of the analyte as a first biospecific reagent, the second biospecific reagent labeled with two-photon fluorescent dye-based diferida dipyrromethene, in this way focus the laser to the reaction suspension, measure two-photon-excited fluorescence from individual microparticles when they are random or directed by the pressure of the exciting laser radiation through the focal volume of the laser beam, characterized in that the aforementioned two-photon fluorescent dye-based diferida dipyrromethene has the structure (II)

where R1represents a substituted or unsubstituted phenyl, 2-thienyl, 2-pyrrolidinyl or phenylalaninol group; group R2, R3, R4, R5, R6or R7each independently is hydrogen or substituted or unsubstituted alkyl group and at least one of the groups R2, R3, R4, R5, R6or R7is substituted to obtain a reactive group selected from the group comprising carboxylic acid reactive ester of carboxylic acid, Angi the reed carboxylic acid, maleimid, Amin and isothiocyanate; and at least one of the groups R1, R2, R3, R4, R5, R6or R7is substituted for the receiving group, which imparts water-solubility selected from the group comprising ammonium salt or an alkali metal sulfonic or carboxylic acid.

2. The method according to claim 1, wherein R1represents 2-thienyl, 2-pyrrolidinyl, phenyl or phenylalaninol group, and R6represents a residue of the formula

where X+represents a cation.

3. The method according to claim 1, characterized in that the reactive group is Succinimidyl ether.

4. The method according to claim 1, characterized in that the second biospecific reagent is a biologically active molecule selected from the group comprising a hapten, oligonucleotide, antibody and antibody fragment.

5. The method according to claim 1, characterized in that the analyte is a biologically active molecule selected from the group comprising a hapten, oligonucleotide, nucleotide, nucleic acid, protein, antibody and antibody fragment.



 

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FIELD: biotechnology, medicine.

SUBSTANCE: the suggested immunoenzymatic test system for identifying the spectrum of antibodies to HIV types 1 and 2, type 1 group O and detecting antigen to HIV type 1 p24 deals with applying immunosorbent based upon HIV antigens being gp41 (env HIV-1 and HIV-2 group O), gp120 (env), p24 (gag), p31 (pol), gp36 (env HIV-2), antibodies to HIV 1 antigen p24, and detecting reagents, moreover, the above-mentioned HIV antigens and HIV antibodies should be sorbed in different cells of plotting boards for immunoenzymatic assay (IEA) and for sorbing it is necessary to apply 96-cell polystyrene dismountable or nonseparable plotting boards. The innovation provides higher sensitivity, simplification and avoiding the subjectivity in evaluating the results obtained.

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1 cl, 1 dwg, 1 ex, 8 tbl

FIELD: medicinal microbiology.

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FIELD: medicine.

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1 tbl, 4 ex

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EFFECT: improved assay method.

2 tbl, 2 ex

FIELD: pharmaceutical chemistry.

SUBSTANCE: invention relates, in particular, to a composition for interaction of ligands wherein the composition comprises a noncovalent associate of multiple separate conjugates being each of that comprises a head group and a tail group wherein tail groups of conjugates form hydrophobic aggregate. Conjugates are mobile within the associate and in the presence of ligand at least two head groups are places by a method corresponding to the epitope formation that is able to interact with ligand stronger as compared with each separate head group. Invention provides applying conjugates in combinatory approach for detecting effective combinations to induce the desirable interaction in binding in receptor-specific treatment of patients.

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14 cl, 8 tbl

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EFFECT: improved preparing method.

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EFFECT: improved preparing method.

4 cl, 4 ex

FIELD: organic synthesis.

SUBSTANCE: invention relates to organoboron compounds technology, in particular to aminoboranes and, more specifically, to trimethylaminoborane, which can be used as reducing and hydroboronizing agents as well as in color photography, in magnetic film manufacture, and as fuel additive to decrease amount of deposits in combustion chamber. Method comprises reaction of trimethylamine with gaseous diborane in organic solvent at reduced temperature. Solvent is selected from aliphatic, cycloaliphatic, and aromatic hydrocarbons with melting temperature not higher than -20°C. Reaction is conducted at temperature from -30°C to 0°C, preferably from -15 to -5°C, at trimethylamine-to-solvent volume ratio 1:(1/5-3.5).Proposed method simplifies preparation procedure owing to eliminated laborious solvent removing vacuum distillation stage and stage wherein of aqueous alkali metal hydroxide is introduced to stabilize aminoborane. Yield of desired product, characterized by high purity, achieves 95-98.6%, which is essentially higher than, for example yield (86%) of morpholinoborane regarded as prototype compound in a known process.

EFFECT: enhanced economical efficiency of process.

3 cl, 4 ex

FIELD: chemistry of complex compounds.

SUBSTANCE: invention relates to new derivatives of boranocarbonate of the formula (I): wherein X1 means -H; X3 and X2 mean similar or different substitutes that are taken among the group consisting of -H, -NHxRy at x + y = 3, or -R wherein R means substitute that is bound with nitrogen or boron atom through carbon atom, respectively, and represents methyl or ethyl group; Y means group -OH, -OH2, -OR or -NHR wherein R means substitute that is bound with nitrogen or oxygen atom through carbon atom, respectively, and represents methyl or ethyl group; or their salts. Invention provides using prepared compounds as source of carbon monoxide (CO) and as a reducing agent in preparing carbonyl metal complexes in an aqueous solution. Also, invention involves a method for preparing borane carbonate and a method for reducing with using H3BCO as a reducing agent.

EFFECT: improved method for preparing.

20 cl, 14 ex

FIELD: chemistry of organophosphorus compounds.

SUBSTANCE: invention relates to compounds with the bond C-P, namely to phosphorus-boron-containing methacrylate that can be used as inhibitor of combustion of polyvinyl alcohol-base film materials. Invention describes phosphorus-boron-containing methacrylate of the following formula: wherein n = 4-8. Polyvinyl alcohol films modified with indicated phosphorus-boron-containing methacrylate shows the enhanced refractoriness, rupture strength up to 206 kgf/cm2, water absorption up to 240% and relative elongation up to 12%.

EFFECT: valuable properties of substance.

1 tbl, 2 ex

FIELD: polymerization catalysts.

SUBSTANCE: invention relates to novel organometallic compounds and to olefin polymerization catalytic systems including such organometallic compounds, and also to a method for polymerization of olefins conduct in presence of said catalytic system. Novel organometallic compound is prepared by bringing into contact (i) compound of general formula I: (I), where Ra, Rb, Rc, and Rd, identical or different, represent hydrocarbon groups; and (ii) Lewis acid of general formula MtR

13
, where Mt represents boron atom and R1, identical or different, are selected from halogen and halogenated C6-C30-aryl groups.

EFFECT: enabled preparation of novel olefin polymerization cocatalysts, which reduce use of excess cocatalyst relative to alkylalumoxanes, do not lead to undesired by-products after activation of metallocene, and form stable catalytic compositions.

14 cl, 1 tbl, 32 ex

FIELD: chemistry of organometallic compounds.

SUBSTANCE: invention relates to a method for preparing lithium complexes salts of the general formula (I): wherein each radical R3-R6 means hydrogen atom (H) or halogen atom (F, Cl or Br). Method involves mixing a) 3-, 4-, 5-, 6-substituted phenol of the formula (III): wherein R3-R6 have above given values with chlorosulfonic acid in acceptable solvent to yield compound of the formula (IV): ; b) intermediate product of the formula (IV) from the stage a) wherein R3-R6 have values given above is subjected for interaction with chlorotrimethylsilane to yield compound of the formula (II) given in the invention description and obtained product is filtered off and subjected for differential distillation; c) intermediate product (II) from the stage b) is subjected for interaction with tetramethanolate borate lithium (1-) in acceptable solvent and the end product (I) is isolated from it. Invention provides the development of a simple method for synthesis of lithium complex salts.

EFFECT: improved preparing method.

3 cl, 4 ex

The invention relates to a method for producing salts consisting of a bulky organic cation and bulk organic anion, specifically salts triarylmethyl cations with organoboron anion

FIELD: chemistry of organometallic compounds.

SUBSTANCE: invention relates to a method for preparing lithium complexes salts of the general formula (I): wherein each radical R3-R6 means hydrogen atom (H) or halogen atom (F, Cl or Br). Method involves mixing a) 3-, 4-, 5-, 6-substituted phenol of the formula (III): wherein R3-R6 have above given values with chlorosulfonic acid in acceptable solvent to yield compound of the formula (IV): ; b) intermediate product of the formula (IV) from the stage a) wherein R3-R6 have values given above is subjected for interaction with chlorotrimethylsilane to yield compound of the formula (II) given in the invention description and obtained product is filtered off and subjected for differential distillation; c) intermediate product (II) from the stage b) is subjected for interaction with tetramethanolate borate lithium (1-) in acceptable solvent and the end product (I) is isolated from it. Invention provides the development of a simple method for synthesis of lithium complex salts.

EFFECT: improved preparing method.

3 cl, 4 ex

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