RU2429465C1 - Optical diffuser for analysis of biologically active substance transfer, analytical system for determining biologically active substance in fluid and method of determining biologically active substance concentration in fluid - Google Patents

Abstract

FIELD: physics. ^ SUBSTANCE: optical diffuser comprises analytical system to determine biologically active substance in analysed fluid by registered optical biosensor pickup dichroism signal variation proceeding from DNA molecular structures. Said biosensor comprises sensitive element displaying circular dichroism in visible spectrum. Proposed system comprises narrow-band light source emitting on wavelength corresponding to maximum optical activity of sensitive element, wavelength selector made up of narrow-band interference filter, polariser to generate linearly polarised light flux, polarisation modulator, photo detector, registration and processing system and computer to calculate biologically active substance concentration proceeding from results of measuring circular dichroism signal differences obtained before contact of biosensor with analysed fluid and in preset interval of biosensor contact with analysed fluid. ^ EFFECT: higher accuracy of determination of concentration. ^ 5 cl, 5 dwg

Description

Technical field

The present invention relates to medical equipment and biotechnologies, and more specifically to devices for determining substances or compounds exhibiting circular dichroism in the analyzed samples (hereinafter referred to as CD), including those that implement biosensor technologies for determining biologically active substances (hereinafter BAS), and can be used in medical and clinical biochemistry, as well as in molecular pharmacology, in the food, pharmaceutical and biotechnological industries and ecology, and most effectively in clinical biochemistry missions.

State of the art

Various devices are known that implement biosensor technologies based on the registration of optical signals of biologically active substances that exhibit abnormal circular dichroism in the analyzed liquids.

A known spectropolarimeter company Jasco Corporation, Japan (Jasco J-710/720 Spectropolarimeter, Instruction Manual) containing a light source, selector, polarizer, polarization modulator, cell with the sample under study, photodetector, synchronous amplifier, DC amplifier, computer, in which register the value of circular dichroism (hereinafter referred to as CD), proportional to the concentration of biologically active substances in the sample. However, the lack of signal accumulation mode, which means insufficient sensitivity for determining biologically active substances (10-7 moles), high weight, dimensions and power consumption, high cost of the device, and lack of mobility limit the scope of this spectropolarimeter.

A known device for determining the biologically active substance in the analyzed fluid (RU, 2107280, C1) and its two technical implementations in the form of a utility model (DE, 10035709, C2) and a portable dichrometer SKD-2 (Uspekhi Fizicheskikh Nauk, 2004, v.174, No. 6, p. 686-690), containing a sequentially installed light source, a selector, a polarizer, a polarization modulator, a cell for placing a test sample containing a biosensor based on cholesteric liquid crystal DNA dispersion (hereinafter referred to as CLCD DNA) in contact with the analyzed liquid w, photodetector, lock-in amplifier, the signal processing means, the control unit.

When passing through the test sample, in which the biosensor exhibits the properties of anomalous circular dichroism, the light flux becomes modulated in intensity, due to which an electric signal arises at the photodetector output, the variable component of which at the radiation polarization modulation frequency is proportional to the value of the CD signal. Then the signal is fed to the input of a digital registration system and, after amplification, filtering and conversion to a digital code, is fed to a computer. The interface board based on the microcontroller provides the necessary interaction of all the nodes of the device, the collection and preliminary processing of the CD signal, data transfer to the computer, as well as testing the parameters of all dichrometer systems.

The operation of the SKD-2 dichrometer is carried out using a software package that implements various modes of the dichrometer and supports a library of methods for determining various biologically active substances, so the user can select from the menu the substance whose concentration is currently required, after which the program is in mode the dialogue “leads” the researcher through all the actions prescribed by the method and gives the result in the form of the concentration value of the determined compound in the studied image tse.

However, the disadvantages of the above devices include:

- non-optimal design of the lamp light source, leading to the formation of ozone inside the spectral block in the UV range near 200 nm;

- insufficiently reliable and difficult to manufacture and configure the electrodynamic drive (positional type) rotation of the diffraction grating of the selector;

- insufficient stability of the characteristics and insufficient resistance to external influences of the photoelastic polarization modulator, as well as

- the influence of the electronic modulator excitation circuit on other systems of the device, which reduces the sensitivity of registration of CD, and, accordingly, the measurement of the concentration of biologically active substances.

The presence of a spurious CD signal due to the stresses in the windows of the optical cell with the sample under study and imperfections in the cell fixation unit, as well as excessive noise in the CD recording channel, lead to insufficient reliability and stability of the results of measurements of the CD signal.

The amplification of the resulting DC signal by direct current in the path of the synchronous amplifier leads to a temperature drift of the signal, while the presence of an analog low-pass filter in the synchronous amplifier significantly reduces the possibility of further processing of the useful signal due to the limited set of filter time constants.

The presence of a wavelength selector in the form of a monochromator and a separate thermostatic control unit for the cell with the breakdown leads to excessive bulkiness of the dichrometer.

The above-mentioned disadvantages of devices for determining biologically active substances were eliminated in a dichrometer for determining a biologically active substance in liquids, gels, and films (RU, 92959, U1) and in a dichrometer for determining a biologically active substance in a analyzed liquid (RU, 92960, U1). In these dichrometers, in order to achieve higher accuracy in determining the concentrations of the components to be determined in any analyzed samples, including liquids, gels, films, an improved design of a lamp light source is proposed, which sharply reduces the level of ozone generated in the UV range near and below 200 nm , a technologically advanced stepper motor for turning the diffraction grating, the design of the device for placing the analyzed sample is improved, which allows sliding the movement of the cell with the sample relative to the body of the device without the appearance of stresses in the material of the cell walls, the constructive implementation of the photoelastic polarization modulator in combination with stabilization of its operating current, which ensured high stability of the modulator characteristics and its resistance to external mechanical influences.

In addition, the proposed devices for determining biologically active substances implement the principle of modularity, in which the functional modules of the device for determining biologically active substances contain microcontrollers that provide control of functional modules in coordinated modes and their interaction with each other using a standard interface like I2C. The operation of the analytical device is controlled using an external or built-in computer, while the microcontroller of the digital registration system module provides the necessary operations by microcontrollers of other functional modules, the primary signal processing, its transmission via USB to the specified computer, the reception and transmission of computer commands to other functional modules.

The indicated technical solutions allowed to slightly reduce the dimensions of the proposed analytical devices by including the thermostatting unit of the cell with the sample in the optical (spectral) unit.

In addition, it has become possible to use biosensors based on molecular DNA constructs that retain their abnormal optical activity for a long time.

Using the proposed analytical devices allows more accurately and with better reproducibility to determine the value of CD, and hence the concentration of various biologically active substances in the liquid, while achieving a higher sensitivity of their determination (up to 10-9 M / l) and it becomes possible to significantly expand the class of defined biologically active substances.

At the same time, there are many analytical problems for the solution of which multifunctional wide-band dichrometers are not required and which could be solved with the help of even simpler and cheaper specialized instruments that use a specific specific biosensor to determine only these biologically active substances or biologically active ones, aimed at determining only these BAS. In this case, it would be possible to abandon such cumbersome structural elements of multifunctional dichrometers as a powerful tube radiation source, which is required to ensure a wide range of wavelengths of its operation, a radiation wavelength selector in the form of a monochromator, and a highly sensitive radiation detector based on a photoelectron multiplier.

Such an opportunity appeared with the creation of biosensors containing particles of molecular DNA constructs (MK DNA) (Liquid crystal dispersions and DNA nanoconstructions (edited by Yu.M. Evdokimov) - M., Radio engineering, 2008). MK DNA is characterized not only by anomalous optical activity, manifested in the form of an intense band in the CD spectrum in the region of absorption of nitrogenous DNA bases at a wavelength of ~ 270 nm, but also by additional anomalous optical activity in the region of absorption of sensitive elements of the biosensor, for example, “crosslinking” nanobridges MK DNA, for example, nanobridges containing antibiotic chromophores as structural elements at a wavelength of ~ 520 nm. The magnitude of the anomalous optical activity of MK DNA biosensors in this band remains unchanged for a long time and can decrease (up to complete disappearance) under the influence of biologically active substances, the “target” of which are the structural elements of nanobridges, which are, in fact, nanosensors. The presence and concentration of biologically active substances in the analyzed liquid is determined by the magnitude of the recorded changes under the influence of biologically active substances (as a result of its interaction with MK DNA) of the CD sensor signal in only one of the above bands in the visible range of the spectrum using a pre-recorded calibration graph.

Disclosure of invention

The basis of the present invention was the task of creating a compact specialized biosensor analytical system for determining biologically active substances in the analyzed liquid samples, the “target” of which are reactive, structurally sensitive MK DNA structural elements that are sensitive to a defined biological substance, having anomalous optical activity in the visible spectrum, without which or loss of sensitivity of registration of the CD signal, with high accuracy in determining the concentration of determined biologically active substances, including ultra-low concentration (d about ~ 10 ^-9 moles), in any analyzed samples, including liquids, gels, films, including biological fluids, such as blood plasma, whole blood, and others.

The problem is solved by creating an analytical system for determining the biologically active substance in the analyzed fluid by the magnitude of the recorded change in the optical signal of the circular dichroism of the biosensor under the action of the substance to be determined, containing the following in sequence:

- source of light radiation;

- a selector adapted to form a luminous flux with a wavelength corresponding to the region of optical activity of the biosensor, manifested in the spectrum of its circular dichroism;

- a polarizer adapted to form a linearly polarized light flux;

- a spectral gap adapted to isolate a linearly polarized light flux with a certain direction of the polarization vector;

a polarization modulator adapted to convert said linearly polarized light flux into a circularly polarized light flux with a periodically changing direction of rotation of the polarization vector;

- a device for placing a sample containing a biosensor or an analyzed liquid in contact with the biosensor in an optically permeable cuvette containing a thermostatic unit for said cuvette;

- a photodetector adapted for recording optical signals of circular dichroism of the biosensor and converting them into a proportional electrical signal;

- a digital recording system adapted to isolate and amplify said electrical signal and convert it to digital form;

- a means of processing the received electrical signal and calculating the concentration of biologically active substances in a liquid, characterized in that the system:

- adapted to determine the biologically active substance in the analyzed fluid by the magnitude of the recorded change in the optical signal of the circular dichroism of the biosensor based on molecular DNA constructs containing a sensitive element that exhibits circular dichroism in the visible spectrum;

- as a source of light radiation contains a narrow-band light source of the visible range, emitting at a wavelength corresponding to the maximum optical activity of the sensitive element of the biosensor, manifested by him in the visible region of the spectrum of circular dichroism, and equipped with a collimating lens placed in the light beam of the radiation source,

- as a wavelength selector contains a narrow-band interference filter located in the light beam of the radiation source after the collimating lens;

- the processing means is adapted to calculate the concentration of the biologically active substance based on the measurement results of the difference in the values of the circular dichroism signals of the sensitive element of the biosensor, made before the biosensor contacts the analyte and after a predetermined period of time the biosensor contacts the analyte.

The problem was also solved by the development of a method for determining the concentration of a biologically active substance in an analyte using a biosensor in contact with it containing a sensitive element that exhibits circular dichroism, in which the analyte with a biosensor is irradiated with a circularly polarized light flux in the visible region of the spectrum at a wavelength corresponding to the maximum optical activity of the sensitive element of the biosensor, manifested by him in the visible region of the spectrum of circular dichroism oism, in which the concentration of the substance is determined by the magnitude of the recorded change in the circular dichroism signal of the sensitive element of the biosensor under the action of the substance to be determined by comparing the results of measurements recorded before contact and when the biosensor is in contact with the analyzed liquid, with calibration data, characterized in that the signal is measured circular dichroism is carried out repeatedly after a predetermined period of time of contact of the biosensor with the analyzed liquid, less than mja complete diffusion of the liquid being analyzed in the biosensor, and thus is used to characterize the proper analytical system above according to the invention for determining the active substance in an analyzed liquid.

Moreover, according to the invention, it is advisable that in the specified analytical system the light source contains two additional narrow-band radiation sources adapted to work alternately with the first source and emitting at wavelengths at which the circular dichroism signal of the sensitive element of the biosensor of the test sample is minimal with respect to the optical signal circular dichroism of the sensitive element of the biosensor at the wavelength of the first radiation source, while one of the indicated additional It was adapted to emit light at a shorter one, and the other of these additional sources was adapted to emit light at a longer wavelength with respect to the wavelength of the first radiation source, each of these additional radiation sources being equipped with a collimating lens placed in the light beam of the corresponding additional radiation source, and the wavelength selector contained two additional interference filters located in the light beam of each of the additional radiation sources after the corresponding collimating lens, and that the system contains means for directing collimated radiation flows of additional sources along the radiation path of the first source.

Moreover, in the method for determining the concentration of a biologically active substance according to the invention, it is advisable to additionally irradiate the analyzed liquid with a biosensor with a circularly polarized light flux at wavelengths shorter and longer than the wavelength at which the circular dichroism signal of the sensitive element of the biosensor is minimal with respect to the optical signal of circular dichroism of the sensitive element biosensor at a wavelength corresponding to the specified maximum optical activity senses an element of the biosensor, and at the same time use the corresponding analytical system described above according to the invention for determining the biologically active substance in the analyzed liquid

The problem was also solved by creating an optical diffusometer for analyzing the mechanisms of transport of biologically active substances and determining their diffusion coefficients in a biosensor based on molecular DNA constructs containing a sensitive element that exhibits the properties of circular dichroism when exposed to a light flux in the visible region of the spectrum containing the analytical system described above according to the invention for determining the biologically active substance in the analyzed fluid.

Brief Description of the Drawings

In the future, the analytical system for determining the biologically active substance in the analyzed fluid according to the invention is illustrated by examples of its structural implementation and application and the accompanying drawings.

Figure 1 is an optical diagram of an analytical system according to the invention, an embodiment.

Figure 2 is an optical diagram of an analytical system according to the invention, an embodiment.

Figure 3 - CD spectrum of a biosensor based on MK DNA "crosslinked" by DAU-Cu nanobridges.

Figure 4 - Dependence of the magnitude of the relative change in the signal of circular dichroism ΔA / ΔA _{max of the} biosensor based on MK DNA generated by it at a wavelength of ~ 515 nm, on the time of its treatment with hyporamine, characterizing the dynamics of the destruction of the MK DNA biosensor under the action of hyporamine.

Figure 5 - Calibration dependence of ΔA / ΔA _max on hyporamine concentration, corresponding to a 10-minute treatment with hyporamine biosensor based on MK DNA.

However, the presented examples of the implementation and application of the analytical system according to the invention do not limit other possibilities of its implementation and application, not beyond the scope of the claims.

Best Mode for Carrying Out the Invention

Figure 1 and Figure 2 shows the structural diagrams of two embodiments of the analytical system according to the invention for determining a biologically active substance (BAS) in the analyzed fluid using a biosensor containing a sensitive element exhibiting circular dichroism in the visible spectral region of a circularly polarized light flux, the magnitude of the recorded change in the optical signal of the biosensor under the action of the substance to be determined.

The analytical system according to the invention, made according to the first embodiment (Figure 1), contains one narrow-band radiation source 1 of the visible wavelength range, a collimating lens 2 placed in the light beam of the radiation source 1, and a selector made in the form of a narrow-band interference filter 3, placed in a beam of light coming from a collimating lens 2.

In this case, the radiation source 1 must be adapted for radiation at a wavelength in the region of optical activity of the sensitive element of the biosensor exhibiting circular dichroism in a narrow visible region of the spectrum. As a narrow-band radiation source 1, it is advisable to use a miniature and cheap light emitting diode with a high brightness of radiation, for example, the diode S12LG2C-B.

The ability to use only the visible wavelength range for measurements expands the selection of sources that are numerous in this range, including laser ones, and also eliminates the problems associated with the use of UV radiation (absorption in air, ozone formation).

Narrow-band diode radiation source 1 replaces the broad-band (usually tube) radiation source and wavelength selector based on a monochromator with their complex and dimensional design and elements, which are traditionally used in multifunctional dichrometers, and thereby dramatically simplify, miniaturize, and cheapen the analytical system for determining biologically active substances in the analyzed fluid.

To conduct more accurate measurements of the magnitude of the change under the influence of the biologically active substance of the optical signal of the sensitive element of the biosensor at the wavelength of the maximum of the intense (anomalous) band in the visible region of the CD spectrum, it is advisable to use an analytical system containing, in addition to the narrow-band radiation source 1 emitting two narrow-band sources a source operating alternately with the first radiation source 1 and emitting at wavelengths, where the CD signal of the biosensor in absolute value is mini small: one source at a shorter wavelength, another source at a longer wavelength relative to the position of the maximum of this band in the CD spectrum of the sensitive element of the biosensor.

The introduction of two additional miniature radiation sources slightly complicates the design of the analytical system, but allows one to take into account the contribution of background signals to the measured CD signal and, accordingly, significantly increase the accuracy of measuring changes in the optical properties of the biosensor during its interaction with biologically active substances, as well as the presence and concentration of biologically active substances in the analyzed liquid.

In this case, collimating lenses and interference filters can be installed in the light beam of each additional narrow-band light source, and for subsequent polarization, polarization conversion, and transmission of the test sample, it is advisable to combine and direct the streams of collimated narrow-band radiation of two additional light sources along the radiation path of the first ( working) source 1.

The analytical system according to the invention, made according to the second embodiment (FIG. 2), contains three narrow-band radiation sources 1, 1a, 1b of the visible wavelength range, their corresponding collimating lenses 2, 2a, 2b, placed in the light beam of the corresponding sources 1, 1a and 1b radiation, and, accordingly, three narrow-band interference filters 3, 3a and 3b of the selector, placed in beams of light coming from collimating lenses 2, 2a and 2b, respectively. In addition, the analytical system (Figure 2) contains two translucent plane-parallel mirrors A and B, made, for example, of quartz, for directing the streams of collimated narrow-band radiation of sources 1a and 1b along the path of the radiation flux of source 1.

Moreover, the radiation sources 1, 1a and 1b are adapted for radiation at different wavelengths:

- radiation source 1 - at a working wavelength corresponding to the maximum of the optical signal of the CD of the sensitive element of the biosensor in the visible region of the CD spectrum;

- radiation source 1a - at a shorter relative to the working wavelength of the source 1, at which the optical signal CD of the sensitive element of the biosensor has a minimum;

- radiation source 1b - at a wavelength longer with respect to the working wavelength of source 1, at which the optical signal CD of the sensitive element of the biosensor also has a minimum.

As sources of radiation 1, 1a and 1b, for example, LEDs of the type S12LB2C-B, S12LG2C-B, NCSU033A and NCSU034A can be used.

It is desirable that one of the plane-parallel surfaces of mirrors A and B to prevent interference effects from being collimated onto the collimated radiation has an antireflection coating, while the other reflects the radiation of the radiation source corresponding to these mirrors and is transparent to the radiation of other narrow-band light sources.

Instead of translucent mirrors, completely reflecting mirrors can be used, which, in accordance with the algorithm for measuring changes in the CD signal and the following sequence of sequentially switching each of the radiation sources, would be introduced into the light flux alternately, respectively, alternately switched on by additional radiation sources.

To solve problems associated only with the determination of the presence of biologically active substances or a rough estimate of the concentration of biologically active substances in the test fluid (for example, testing biocides or determining the suitability of the fluid for use), and when using known quality biosensors, the introduction of an additional analytical source to the source emitting at the working length in the analytical system waves, radiation sources and other elements are not required, and the system remains the simplest and most compact.

When using a narrow-band radiation source (1, 1a, 1b), it is most effective as a wavelength selector of its radiation to narrow the emission band even more if it is not narrow enough, use a narrow-band interference filter (3, 3a, 3b) with a multilayer dielectric coating, but other types of spectral filters are also possible.

The collimating lenses 2, 2a, 2b are used to compensate for the divergence of the radiation of a narrow-band light source and can be performed in any way known to a person skilled in the art.

Moreover, the analytical systems according to the invention (FIG. 1, FIG. 2) also contain:

- a polarizer 4, forming from the specified radiation a linearly polarized light flux;

- spectral gap 5, emitting an "ordinary" beam;

- polarization modulator 6, which converts a linearly polarized light flux into a circularly polarized one with a periodically changing direction of rotation of the polarization vector;

- a device 7 for placing an analyzed sample containing a biosensor in the analyzed liquid or a biosensor;

- photodetector 8, converting changes in the anomalous optical signal of the CD of the sensitive element of the biosensor under the influence of biologically active substances into an electrical signal proportional to it;

- digital registration system 9;

- means 10 for processing the received electrical signal and calculating the concentration of biologically active substances in the analyzed liquid or determining the optical properties of the biosensor;

- power supply 11 of the photodetector 8.

The polarizer 4 (FIG. 1, FIG. 2) is adapted to form a linearly polarized light flux and can be prismatic (for example, from a nonlinear potassium dihydrogen phosphate crystal), film (for example, NPF-S type), liquid crystal (for example, based on ferroelectric liquid crystals SZhK-582) or made by any other method known to the person skilled in the art.

When using a prism or film polarizer 4, it is advisable to convert a linearly polarized light flux into a circularly polarized one with a periodically changing direction of rotation of the polarization vector in an electro-optical modulator 6 of circular polarization of photoelastic type.

Instead of the photoelastic type modulator 6, other types of electro-optical modulators can be used, for example, a combination of a liquid crystal-based modulator that provides alternating (periodic) output of two mutually orthogonal linear polarizations, and a quarter-wave plate (λ / 4) from crystalline quartz that converts linearly polarized light flux into a circularly polarized with a periodically changing direction of rotation of the polarization vector.

For better separation of orthogonal polarizations in the case of using a prism polarizer made of non-linear crystalline material, a spectral gap 5 can be used, which is located immediately after the prism polarizer 4 (Figure 1) or after the electro-optical circular polarization modulator 6 (Figure 2).

As a device 7 for placing a sample, it is advisable to use an optically permeable thermostatic, for example, using a temperature controller 7a based on Peltier elements and temperature sensors of a cuvette or a well of a thermostatic standard microplate with a minimum residual dichroism of a transparent material, respectively, of the walls of the cuvette or the bottom of the holes, adapted to accommodate in them a biosensor or sample containing the analyzed fluid in contact with the biosensor.

Due to the high brightness of the light emitting diodes used as radiation sources 1, 1a, 1b, as a photodetector 8, adapted for recording optical signals of circular dichroism of the biosensor and converting them into a proportional electric signal, instead of expensive photoelectronic multipliers (for example, HAMAMATSU R1464 or R7400U- 04) can be used miniature standard photodiodes, for example, FD-24.

It is advisable that in the photodetector circuit 8, which converts changes in the CD signal under the influence of ALS into an electric signal, there is an electrical feedback in order to regulate its sensitivity, arranged in such a way that for any change in the intensity of circularly polarized light radiation irradiating the test sample, the constant component the voltage at the photodetector output remained unchanged, while the variable component of the voltage will be proportional to the value of cr ug dichroism of the test. Thanks to this approach, it is possible to reduce the level of interference associated with changes in radiation intensity and increase the sensitivity of the registration system and the analytical system as a whole.

It is advisable to obtain a stable mode of operation of the photodetector 8 to use a stable adjustable power source 11 and the design of the photodetector 8, which provides reliable shielding of the photodetector 8 from external electromagnetic fields and stray light.

Functional devices of the analytical system according to the invention are controlled, for example, using a compact personal computer (laptop), an integrated microcomputer, or other similar means with appropriate software.

It is advisable that the principle of modularity be implemented in the proposed analytical system, the necessary operations by the above-described functional modules of the system are carried out using microcontrollers, and the circuitry solutions provide the possibility of using both an external and an integrated computer to control the device. The computer of the processing means 10 turns on and controls the operation of radiation sources 1, 1a, 1b, circular polarization modulator 6, cuvette 7 temperature regulator 7a, photodetector 8 power supply 11, digital registration system 9.

The software manages the operating modes of the analytical system according to the invention, visually displays the measurement results on a personal computer screen, saves and documents the measurement results, performs various actions to process the experimental results (smoothing, accumulation, comparison with other results), calibrates the hardware of the device, calculates unknown concentration of biologically active substances in the fluid using pre-built calibration graphs. The reception and transmission of commands from the processing means 10 to the microcontrollers of the functional devices is carried out through the microcontroller of the digital registration system 9. Communication between the modules is carried out via a standard digital interface type I2C. The circuitry solution provides the possibility of using both an external and an embedded computer via the USB interface to control the system.

Thus, the present invention through the use of biosensors based on molecular DNA constructs and new technical solutions allows us to make an analytical device for determining biologically active substances in a liquid, without compromising its sensitivity, simpler, more compact and more mobile with the possibility of express analysis of biologically active substances containing liquids.

The implementation of the analytical system according to the invention allows it to be carried out in a compact design in a single package, to provide ease of maintenance and mobility in various operating conditions.

The use of the analytical system according to the invention can be illustrated by the example of determining the presence and concentration of biologically active substances in the analyzed fluid and measuring the diffusion rate of biologically active substances in the biomaterial.

The operation of the analytical system according to the invention, performed according to the second embodiment (Figure 2), is shown by the example of determining the presence and concentration of hyporamine in the analyzed liquid (human blood) using a MK DNA biosensor made in the form of an integrated microchip representing a CLCD DNA particle , in which neighboring nucleic acid molecules are “crosslinked” by nanobridges, consisting of alternating Cu ^{2+ ions} and daunomycin antibiotic molecules, which form polymer chelate crosslinks (RU, 2139933, C1).

The use of these particles of CLCD DNA as an integral biosensor is due to the fact that the molecules of many chemical and biologically active compounds form complex compounds with Cu ^{2+ ions} , "pulling" these ions out of the structure of the chelate complex with 4 oxygen atoms in the two subsequent molecules of the anthracycline compound. This process is accompanied not only by the violation of the integrity of the entire polymer "crosslinking", but also by a corresponding decrease in the anomalous optical activity of the biosensor (Figure 3). The magnitude of the decrease in anomalous optical activity is related to the concentration of a chemical or biologically active compound present in the analyzed fluid and destroying the polymer chelate crosslinking. Therefore, an abnormal optical signal can be used as an analytical criterion for determining analytes whose “target” is the “building blocks” of the microchip, in particular, the structural elements of nanobridges, in particular Cu ^{2+ ions} and antibiotic molecules. In this case, the antibiotic molecule is a sensitive element of the biosensor, exhibiting a circular dichroism of different magnitude before and after the integrity of the indicated “crosslinking” - nanobridge when irradiated in the visible spectral region in the absorption band of daunomycin at a wavelength of ~ 515 nm.

For these studies used an analytical system (Figure 2) containing three radiation sources:

- source 1, emitting at a wavelength of ~ 515 nm, corresponding to the maximum of the intense negative band in the CD spectrum of the sensitive element of the biosensor - daunomycin,

- source 1A, emitting at a wavelength of ~ 450 nm, less than the wavelength of the specified maximum;

- a source 16 emitting at a wavelength of ~ 700 nm, a longer wavelength of the indicated maximum.

As sources of radiation 1, 1a, and 1b, containing collimating lenses 2, 2a, and 2b, LEDs of the types S12LG2C-B, S12LB2C-B, and NCSU034A were used.

At the same time, the analytical system also contained:

- three narrow-band interference filters 3, 3a, 3b, narrowing the emission band from sources 1, 1a, 1b; semitransparent plane-parallel plates A and B, while one of the plane-parallel surfaces of mirrors A and B had a multilayer antireflection coating, and the other reflected radiation as sources 1a or 1b as possible and was transparent for radiation from other narrow-band sources, for example, in mirror B it was transparent for radiation sources 1 and 1a;

- polarizer 4, made in the form of a prism from a nonlinear crystalline material;

- spectral gap 5;

- modulator 6 of circular polarization of photoelastic type, mounted behind the spectral gap 5;

- a device 7 for the placement of the test sample, made in the form of a thermostatic microplate with the help of the unit 7a with a minimum residual dichroism of the transparent material of the bottom of the wells, adapted to accommodate a biosensor or sample containing the analyzed liquid in contact with the biosensor;

- photo detector 8;

- a digital system 9 for recording an electrical signal;

- means 10 for processing an electrical signal, calculating the concentration of the determined biologically active substances or determining the optical properties of the biosensor and for controlling the functional modules of the proposed analytical system, made in the form of an integrated microcomputer;

- power supply 11 of the photodetector 8.

Photo detector 8 has two outputs 12 and 13; the output 13 is connected to the input of the power source 11 of the photodetector 8, and the output of the power source 11 is connected, in turn, to the input of the photodetector 8 to organize electrical feedback in order to regulate its sensitivity. The output 12 of the photodetector 17 is connected to the first input 14 of the digital registration system 9, the output 15 of which is connected to the computer input of the processing means 10. To control the system, the computer outputs of the processing means 10 are connected to radiation sources 1, 1a, 1b, to control the sequential operation of three sources with the temperature regulator 7a of the cell temperature 7, with the input of a circular polarization modulator 6 to control the modulation parameters and stabilization current, and the output of the modulator 6 is connected in turn, with the input of a digital registration system 9 for synchronizing the frequency and phase of the modulation and amplification.

Moreover, the analytical system according to the invention (Figure 2) works as follows.

Sources 1, 1A and 1B of radiation work alternately:

- the light source 1 generates a narrow-band light stream for a predetermined time in the region of the maximum of the negative band in the CD spectrum of the sensitive element of the biosensor — daunomycin at a wavelength of about 515 nm, this stream was collimated by lens 2 and after cutting with an interference filter 3 an even narrower band in the spectrum radiation at a wavelength of 515 nm was sent through translucent plates A and B to polarizer 4;

- the light radiation source 1a generates a narrow-band light stream at a shorter wavelength for a predetermined time, at which the CD signal of the sensitive element of the biosensor daunomycin is minimal, for example, at a wavelength of 450 nm, its luminous flux is collimated by lens 2a, then after cutting with an interference filter 3a an even narrower band in the radiation spectrum is reflected from the translucent plate A and enters, passing the translucent plate B, into the polarizer 4 along the radiation path from source 1;

- the light emission source 1b generates for a given time a narrow-band light flux at a wavelength at which the CD signal of the sensitive element of the biosensor daunomycin is minimal from the side of longer waves, for example 700 nm, its luminous flux is collimated by lens 2b, after the interference filter 3b cuts out a narrower band in the radiation spectrum is reflected from the translucent plate B and enters the polarizer 4 along the radiation path of sources 1 and 1a.

Alternate inclusion of all three radiation sources 1, 1a, 1b for predetermined time intervals is carried out by the computer of the processing means 10. The total time of irradiation (exposure) of the investigated liquid is selected so that changes in the magnitude of the anomalous CD signal of the biosensor under the influence of the biologically active substance contained in the liquid can occur.

After passing through the polarizer 4, the light flux becomes linearly polarized. The spectral gap 5 extracts from the linearly polarized radiation flux only the component corresponding to the “ordinary” ray emerging from the polarizer 4. Passing the spectral gap 5, the radiation enters the polarization modulator 6, passing through which it becomes circularly polarized with a periodically changing direction of rotation of the polarization vector. After passing the device 7 with the sample located in it, containing a biosensor having an abnormal circular dichroism, the light flux becomes modulated in intensity.

Under the action of light, an electrical signal appears at the outputs of the photodetector 8, and at the first output 12, a variable component is recorded proportional to the value ΔA of the signal due to the anomalous optical activity of the biosensor (ΔA = A _R -A _L , where A _R and A _{L are} respectively the absorption in the radiation substance with right and left circular polarization), and at the second output 13 is a constant component proportional to the value of the signal characterizing the absorption of the sample biomaterial (A≈A _R ≈A _L ), while the frequency of the variable component equal to the modulation frequency of the circular polarization of the radiation.

In the specified analytical system, the constant component is maintained at a constant level by adjusting the supply voltage of the photodetector 8, for which the signal of the constant component from the output 13 of the photodetector 8 is input to the input of the photodetector power supply 11, i.e., the constant component is stabilized using negative feedback on the constant component with the simultaneous measurement of the variable component, which is equivalent to measuring their ratio (ΔA / A), and therefore, measuring the signal circularly of the original sample dichroism alternately on each of the three wavelengths (515, 450 and 700 nm).

From the output 12 of the photo detector 8, an electrical signal corresponding to the CD value is fed to the first input 14 of the digital registration system 9, to the second input 15 of which a reference signal with a modulation frequency of circular polarization is applied. The registration system 9 amplifies the signals, converts them into direct current, and supplies processing means 10 to the computer for processing according to a certain algorithm. The resulting signal is the original sample KD KD _MC at a wavelength of 515 nm, calculated on the basis of background signal CD at wavelengths 450 nm and 750 is stored in the memory means 10 processing.

The determination of the concentration of biologically active substances of hyporamine was carried out as follows.

Initially, the residual circular dichroism of the CD _{OST of the} transparent bottom material of the wells was measured to place the studied samples of the microplate of device 7 at a wavelength corresponding to the maximum of the intense negative band in the CD spectrum of the sensitive element of the biosensor - daunomycin at a wavelength of ~ 515 nm, taking into account the background signals of the CD of _OST on wavelengths of ~ 450 nm and ~ 700 nm.

Then, the initial characteristics of the biosensor were determined. For this, from a certain amount of biomaterial containing the sensitive elements of the biosensor, the initial samples of the biosensor were prepared by placing identical portions of the biomaterial into the wells and the anomalous circular dichroism signal inherent in each source sample with the biosensor was measured at the same wavelength ~ 515 nm, adjusting its value by the measured value residual circular dichroism of the bottom material of the wells. For all samples, the resulting CD signals (ΔA _max ), calculated taking into account the background CD signals at wavelengths of ~ 450 nm and ~ 700 nm, were high and relatively the same, indicating a high quality of the biosensors used.

Figure 3 shows the CD spectrum in the visible region of the used biosensor based on molecular DNA constructs in which the DNA molecules in the particles of the liquid crystal lyotropic dispersion are “crosslinked” with nanobridges containing daunomycin and copper (DAU-Cu) (C _DNA ~ 5.6 μg / ml; With _PEG = 170 mg / ml, mol.weight PEG = 4000 Yes; With _DAU ~ 27.18 · 10 ^-6 M; With _Cu ~ 1 · 10 ^-5 M; 0.3 M NaCl + 10 ^-2 M Na ⁺ phosphate buffer). The resulting CD signals of all initial samples of the biosensor (ΔA _max ) were stored in the computer memory of processing means 10: CD spectra of molecular DNA constructs before (curve 1, C _Hyporamine = 0) and after (curve 2, C _Hyporamine = 4.0 μg / ml) hyporamine treatment; the CD spectrum of CLCD DNA – DAE complexes (Curve 3. ΔА = (A _L -A _R ) · 10 ^-6 optical units; L = 1 cm; T = 22 ° C).

Then, a series of calibration samples was prepared by treating the wells of the aforementioned initial samples of the biosensor with a liquid containing a biologically active substance in various predetermined concentrations, and measured at predetermined time intervals Δt less than the time of complete diffusion of the biologically active substance into the biosensor, the circular dichroism signals ΔA _{t of} each test sample on wavelength ~ 515 nm, taking into account the background signals at wavelengths of ~ 450 nm and ~ 750 nm. The values of the resulting signals ΔA _t at a wavelength of ~ 515 nm for given identical periods of time and for each of the studied samples with different hyporamine concentrations, calculated taking into account the background signals at wavelengths of ~ 450 nm and ~ 700 nm, stored in the computer memory processing means 10 , where they then correlated with the values of the resulting CD signals of all initial biosensor samples ΔA _max . As a result, we obtained the dependences of the relative change in the circular dichroism signal (ΔА _t / ΔА _max ) generated by biosensors based on MK DNA (DNA from cattle thymus (Sigma, USA); molecular weight ~ (0.3- 0.7) · 10 ⁶ Yes; With _DNA = 19.92 μg / ml; With _bis-MM = 170 mg / ml; mol. Mass of bis-macromonomer PEG = 6000 Yes; DAU (Sigma, USA); C _DAU = 25.764 × 10 ^-6 M; CuCl ₂ x · 2H ₂ O (Aldrich, USA); With _Cu = 9.901х · 10 ^-6 M; 0.3 M NaCl + 10 ^-2 M Na ⁺ phosphate buffer ; pH ~ 7.0. hydrogel sizes ~ 6 × 1 × 20 mm; temperature 22 ° C) at a wavelength of ~ 515 nm, from the time they were treated with hyporamine at different concentrations (Figure 4), characterizing the dynamics of the destruction of MK DNA biosensor under the action of homocysteine: curve 1 - C _Hyporamine = 0.5 μg / ml; curve 2 - C _hyporamine = 1.0 μg / ml; curve 3 - C _hyporamine = 2.0 μg / ml; curve 4 - C _hyporamine = 3.0 μg / ml; curve 5 - C _hyporamine = 0.4 μg / ml; ΔА × 10 ^-6 - circular dichroism (optical units).

Using the values of the relative changes in the circular dichroism signal (ΔА / ΔА _max ) generated by MK DNA biosensors at a wavelength of ~ 515 nm (similar to those described above), processed with a liquid containing a determined biologically active substance in various given concentrations, obtained over a specified time interval ΔТ , constructed a calibration curve changes depending on the time interval? T signal (ΔA / ΔA _max) on the concentration hiporamin (5) corresponding to the 10-minute treatment biosensor based on DNA MK hypo amine, which is stored in the memory means 10, the processing computer.

For determination in an analyzed liquid sample of unknown concentration of the biologically active substances initially in accordance with the procedure described above was prepared biomaterial sample with a biosensor, which in well with the known value KD _ost material its bottom placed is the same as for the above initial sample with a biosensor, the quantity of biomaterial containing a biosensor, and using the analytical system (Figure 2), we measured its circular dichroism signal (ΔA _max ) when samples were irradiated with circularly polarized light at a working wavelength ~ 520 nm taking into account background signals at wavelengths of ~ 450 and ~ 700 nm in accordance with the above algorithm.

Then, the prepared biomaterial sample with the biosensor was subjected to the treatment with the analyzed liquid, presumably containing hyporamine in an unknown concentration, and after the exposure time ΔT, the change in the signal of the circular dichroism of the biosensor (ΔА) was measured over the time interval ΔT upon irradiation with circularly polarized light at an operating wavelength of ~ 515 nm and two additional wavelengths (~ 450 and ~ 700 nm).

The measured value of the relative change under the action of hyporamine during ΔT of the circular dichroism signal (ΔA / ΔA _max ) generated by gel biosensors based on MK DNA at a wavelength of ~ 515 nm, the computer was compared with a calibration curve (Figure 5) and displayed on the monitor corresponding to it the concentration of hyporamine in the analyzed fluid containing it.

The result of measuring the presence and concentration of biologically active substances in the liquid can be presented in the form of a form containing information about the time and place of the analysis, as well as the type of substance to be determined and its content in the analyzed liquid. In addition, the calibration curve is shown on the form on which the experimentally measured and used to determine the concentration of biologically active substances in the analyzed liquid indicates the value of the resulting CD signal equal to the ratio (ΔA / ΔA _max ) during the exposure time ΔT.

As can be seen from Figure 4, the time for determining the concentration of biologically active substances in the analyzed liquid can be significantly reduced if measurements of the CD values of solid-state biosensors treated with the biologically active substances containing the analyzed liquid are made without waiting for the complete diffusion of the biologically active substances in the biomaterial, at small equal time intervals ΔТ, sufficient for the manifestation of a characteristic dependence of the change in the anomalous CD signal of the biosensor over time during the interaction of a biologically active substance containing biologically active substances with sensitive elements of such a biosensor ka. In this case, the calibration curves used to determine the unknown concentration of biologically active substances in the analyzed liquid should be recorded exactly in accordance with the above procedure for measuring CD signals, i.e., at the same defined equal time intervals ΔT.

The diffusion rate of the biologically active substance contained in the analyzed liquid at a certain low concentration into the biosensor is made by the same computer of the processing means 10 also on the basis of the results obtained in accordance with the procedure described above for measuring the magnitude of changes in the CD signal recorded before and after processing the solid-state biosensor with the indicated liquid , i.e. measuring ΔA original sample after complete penetration of biologically active substances into the biosensor (? T = T _diff), or at certain intervals of time ΔT (the exposure time) n after the start of the liquid treatment. Such capabilities of the analytical system according to the invention make it possible to use it in an optical diffusometer to determine the dynamics of biologically active substances in molecular DNA constructs.

The computer of the processing means 10 also performs the necessary interaction of all elements of the device, implements the required control and processing algorithm, generates control signals to turn on the radiation sources 1, 1a, 1b, generates a voltage with a modulation frequency for operation of the polarization modulator 6, generates a reference signal at the modulation frequency for the registration system 9, it controls the temperature controller 7a of the temperature of the device 7 for placing the sample.

Thus, the analytical system according to the invention through the use of biosensors based on molecular DNA constructs and new technical solutions allows you to quickly, accurately and with high sensitivity to determine in various fluids, including biological fluids, for example in the blood of patients, the presence and concentration of various biologically active substances (antitumor drugs, antibiotics, proteins, etc.) and will help save the health and life of patients in cases where other methods are not applicable or do not give adlezhaschego effect.

In addition, the present invention, designed to determine the presence and concentration of practically important classes of biologically active substances, which in one way or another violate the structural integrity of the molecular structures of the biosensor DNA, makes it possible to make an analytical system for determining biologically active substances in liquids simpler, more compact, more mobile and simple in service without compromising its sensitivity.

It is possible to use the analytical system according to the invention as a compact analytical system for determining the presence and concentration of biologically active substances in a liquid and as an optical diffusometer for analyzing the biological transport mechanisms of biologically active substances that destroy the biosensor and determining their diffusion coefficients.

Industrial applicability

The present invention can be used in medical and clinical biochemistry, as well as in molecular pharmacology, in the study of pharmacokinetics of significant biologically active compounds, in the pharmaceutical industry and ecology. Most effective is its use in clinical biochemistry.

The present invention allows in many cases to replace complex and expensive equipment, eliminating the use of highly qualified personnel, and can be used in medical and clinical biochemistry, in the practice of oncology, surgery, gynecology, in biomedical screening, as well as in molecular pharmacology in the study pharmacokinetics of significant biologically active compounds in the pharmaceutical industry and ecology. Its most effective use in clinical medicine and biochemistry.

Claims (5)

1. The analytical system for determining the biologically active substance in the analyzed fluid by the magnitude of the recorded changes in the optical signal of the circular dichroism of the biosensor under the action of the substance to be determined, containing placed in series:
light source;
a selector adapted to generate a luminous flux with a wavelength corresponding to the region of optical activity of the biosensor, manifested in the spectrum of its circular dichroism;
a polarizer adapted to form a linearly polarized light flux;
a spectral gap adapted to isolate a linearly polarized light flux with a specific direction of the polarization vector;
a polarization modulator adapted to convert said linearly polarized light flux into a circularly polarized light flux with a periodically changing direction of rotation of the polarization vector;
a device for placing a sample containing a biosensor or an analyzed liquid in contact with the biosensor in an optically permeable cuvette containing a thermostatic unit for said cuvette;
a photo detector adapted to register optical signals of circular dichroism of the biosensor and convert them into a proportional electrical signal;
a digital recording system adapted to isolate and amplify said electrical signal and convert it to digital form;
means for processing the obtained electrical signal and calculating the concentration of the biologically active substance in the liquid,
characterized in that:
adapted to determine the biologically active substance in the analyzed fluid by the magnitude of the recorded change in the optical signal of the circular dichroism of the biosensor based on molecular DNA constructs containing a sensitive element exhibiting circular dichroism in the visible region of the spectrum;
as a source of light radiation contains the first narrow-band light source of the visible range, emitting at a wavelength corresponding to the maximum optical activity of the sensitive element of the biosensor, manifested by him in the visible region of the spectrum of circular dichroism, and equipped with a collimating lens placed in the light beam of the radiation source,
as a wavelength selector, comprises a narrow-band interference filter placed in the light beam of the radiation source after the collimating lens;
the processing means is adapted to calculate the concentration of the biologically active substance based on the measurement results of the difference in the values of the circular dichroism signals of the sensitive element of the biosensor, made before the biosensor contacts the analyte and after a specified period of time the biosensor contacts the analyte. 2. The analytical system according to claim 1, characterized in that the light radiation source contains two additional narrow-band radiation sources adapted to operate alternately with the first source and emitting at wavelengths at which the circular dichroism signal of the sensitive element of the biosensor of the test sample is minimal with respect to optical signal of circular dichroism of the sensitive element of the biosensor at the wavelength of the first radiation source, while one of these additional sources is adapted for emitting light at a shorter, and the other of these additional sources is adapted to emit light at a longer wavelength with respect to the wavelength of the first radiation source, each of these additional radiation sources equipped with a collimating lens placed in the light beam of the corresponding additional radiation source, and the wavelength selector contains two additional interference filters located in the light beam of each of these additional radiation sources after e corresponding collimating lens and the system comprises means for directing collimated radiation fluxes additional sources of the first source of radiation flux path. 3. A method for determining the concentration of a biologically active substance in an analyte using a biosensor in contact with it containing a sensitive element exhibiting circular dichroism, in which the analyte with a biosensor is irradiated with a circularly polarized light flux in the visible spectrum at a wavelength corresponding to the maximum optical activity of the sensitive element of the biosensor, manifested by him in the visible region of the spectrum of circular dichroism, in which the concentration of the substance is determined the magnitude of the recorded change in the circular dichroism signal of the sensitive element of the biosensor under the action of the substance to be determined by comparing the measurement results recorded before contact and when the biosensor comes in contact with the analyzed liquid, with calibration data, characterized in that the circular dichroism signal is measured repeatedly after a specified interval contact time of the biosensor with the analyzed liquid, less than the time of complete diffusion of the analyzed liquid into the biodate chick, and using an analytical system to determine the biologically active substance in the analyzed fluid, as described in claim 1. 4. The method according to claim 3, characterized in that they additionally irradiate the analyzed liquid with a biosensor with a circularly polarized light flux at wavelengths shorter and longer than the wavelength at which the circular dichroism signal of the sensitive element of the biosensor is minimal with respect to the optical signal of circular dichroism of the sensitive element a biosensor at a wavelength corresponding to the indicated maximum optical activity of the sensitive element of the biosensor, and an analytical system is used to distribution of the biologically active substance in the analyzed fluid, characterized in paragraph 2. 5. An optical diffusometer for analyzing the mechanisms of transport of biologically active substances and determining their diffusion coefficients in a biosensor based on molecular DNA constructs containing a sensitive element that exhibits the properties of circular dichroism when exposed to light in the visible region of the spectrum, containing an analytical system for determining the biologically active substance in the analyzed fluid, characterized in claim 1 or 2.

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