RU2633494C2 - Biosensor for non-invasive optical monitoring of biological tissues pathology - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: biosensor contains: a radiation source and receiver; an applicator, made in the form of a vessel with a biocompatible immersion agent; a radiating light guide connected to a radiation source at one end, and a receiving light guide connected to the radiation receiver at one end. The distal ends of the light guides are located inside the applicator.

EFFECT: increased depth of tissues sounding at a decreased harmful radiation effect on body tissues due to optical clarification of biological tissues, and light guides arrangement within the applicator with a biocompatible immersion agent allows to reconcile the refractive indices of the light guide end with the immersion fluid and eliminate optical reflection at the biotissue - light guide end border.

15 cl, 1 tbl, 3 dwg

Description

The invention relates to medicine, namely to endocrinology, in particular to determining the pathology of tissues of vital organs, due to the development of diabetes mellitus.

Currently existing methods for determining the degree of glycation of hemoglobin in blood and glycation of biological tissues can be divided into invasive and non-invasive ones. Invasive methods for determining the degree of glycation of hemoglobin, namely, determining the concentration of glycated hemoglobin Hb _A1c , are based on a biochemical analysis of blood taken from a patient from a vein and subsequent spectrophotometric measurement of the concentration of Hb _A1c as a product of the biochemical reaction, according to the intensity of its Soret band.

The concentration of Hb _A1c in the blood is a biochemical indicator of blood, reflecting the average blood sugar for a period of up to three months (the average life time of red blood cells), and is the percentage of hemoglobin in the blood irreversibly associated with glucose molecules (maximum in patients with severe diabetes about 15 -16% of the total hemoglobin concentration). Glycated hemoglobin results from the Mayer reaction (N. Vigneshwaran, G Bijukumar, N. Karmakar, S. Anand, and A. Misra, "Autofluorescence characterization of advanced glycation end products of hemoglobin," Spectrochimica Acta Part A 61 (1), 163 -170 (2005)). The higher the Hb _A1c level, the higher the glycemia over the past three months and, accordingly, the greater the risk of developing complications caused by diabetes mellitus, such as retinopathy, nephropathy, muscle dystrophy and, as a consequence, loss of mobility if skeletal muscles or a heart attack are affected myocardium, if myocardium is affected, as well as a number of other complications.

A device is known according to US patent US 8140147, publ. 11/15/2007, IPC А61В 6/00, consisting of a flexible probe with a light source and a radiation receiver. An optical probe may or may not be in contact with tissue. An optical probe delivers broadband light to the biological tissue and detects its diffuse fluorescence in vivo. This device is able to determine the presence of diabetes mellitus or the development of prediabetes by determining the presence of end products of glycation of collagen, measuring their concentration. The device can be applied to tissues of the skin, oral mucosa or sclera.

A device is known according to the application WO 2006009906, publ. 01/26/2006, IPC G01F 19/00, for non-invasive diagnosis of diabetes mellitus, including a light excitation source, a detector that receives a fluorescence signal, a processor for measuring attenuation of the fluorescence signal intensity. The device uses an ultra-short pulse of light excitation of UV, IR or the visible range, which is a sequence of nanosecond pulses. An excitation pulse is directed to the surface of the skin of the forearm, leg or palm of the patient. Light interacts with various layers of the skin, the absorbed light excites glycation products in the skin, generating a fluorescence signal, which is collected using a detector. The processor is connected to a detector for measuring attenuation of skin fluorescence intensity to determine the contribution of individual fluorophores to the total fluorescence signal, after which the nature and location of the fluorophores are identified. By the received fluorescence signal, the presence or absence of glycation products is determined, and accordingly, a conclusion is drawn about the risk or diagnosis of diabetes.

The principle of operation of the described devices is based on the use of fluorescence of biological tissues and protein glycation products.

The closest in technical essence is an experimental setup for non-invasive optical monitoring of the pathology of biological tissues caused by the development of diabetes mellitus, containing a radiation source and receiver, an applicator made in the form of a cuvette with a biocompatible immersion agent, an emitting fiber, connected at one end to a radiation source, and a receiving fiber connected at one end to a radiation receiver (Tuchina DK et al. Ex vivo optical measurements of glucose diffusion kinetics in native and diabetic mouse skin. 03/12/2015. J. Biophotonics 8, No. 4, p. 332-346). In this case, the ends of the LEDs are located at a certain distance from the cell.

The disadvantage of the design is the low mechanical strength, since the fibers are not protected from displacement or damage. In addition, there is a lack of sensitivity of the device caused by possible displacements during patient movement during measurements.

The technical problem to which the present invention is directed is the development of a biosensor for non-invasive optical monitoring of the pathology of biological tissues due to the development of diabetes mellitus.

The technical result is to increase the sensitivity by matching the refractive indices of the end of the fiber with the immersion liquid, which eliminates optical reflections at the interface between the biological tissue and the end of the fiber both in the irradiating and in the receiving part with an increase in mechanical strength.

This problem is solved by the fact that in the biosensor containing the source and receiver of light emitting and receiving fibers connected to the source and receiver, respectively, an applicator made in the form of a vessel containing a biocompatible immersion agent, according to the solution the distal ends of the optical fibers are located inside the applicator. The optical fibers can be parallel to each other, and the distance l between the centers of the distal ends of the emitting and receiving optical fibers is approximately double the depth of sounding of the biological tissueh, l≅2h.In another embodiment, the distal ends of the emitting and receiving fibers are opposed to each other.

As sources, receivers of radiation and immersion agent can be used in various options, described below.

The invention is illustrated by drawings, where in FIG. 1 - histograms of the dependence of the glucose diffusion coefficient in healthy and diabetic skin on the concentration of glucose solution, FIG. 2 is a diagram of a biosensor, the emitting and receiving fibers of which are parallel to each other, in FIG. 3 is a diagram of a biosensor, the emitting and receiving fibers of which are located opposite each other, where

1 - healthy skin;

2 - diabetic skin;

3 - skin;

4 - emitting (input) fiber;

5 - receiving (output) fiber;

6 - applicator with an agent;

7 - agent;

8 - water contained in biological tissue;

9 - a beam of light.

The diffusion coefficient of the immersion fluid (IL) in the biological tissue is estimated based on the kinetics of the change in the transmission or reflection of the biological tissue sample interacting with the IL. The method used in detail is described in the literature [Tuchin V.V. Optical Clearing of Tissues and Blood, PM 154, SPIE Press, Bellingham, WA, 2005; Tuchin V.V. Handbook of Optical Sensing of Glucose in Biological Fluids and Tissues, Taylor & Francis Group LLC, CRC Press, 2009].

A method for measuring the diffusion coefficient of an immersion fluid in a biological tissue is based on determining the diffusion coefficient from the kinetics of changes in collimated transmittance, light attenuation coefficient, or biological tissue scattering coefficient interacting with IL by minimizing the least squares function, which includes experimentally obtained and theoretically calculated data. The diffusion coefficient of an agent in a biological tissue is defined as the speed of the average flow of immersion agent molecules into the tissue and water from the tissue. When using the kinetics of collimated transmission, the objective function is as follows:

- теоретически рассчитанный коэффициент пропускания образца при заданном значении коэффициента диффузии D в момент времени t, сек,

- измеренное значение коэффициента пропускания в момент времени t, сек, N_t - количество экспериментальных точек временной зависимости коллимированного пропускания образца биоткани на определенной длине волны.Where

- theoretically calculated sample transmittance at a given value of the diffusion coefficient D at time t, s,

- the measured value of the transmittance at time t , sec, N _t is the number of experimental points of the time dependence of the collimated transmittance of the biological tissue sample at a specific wavelength.

The problem of estimating the diffusion rate is solved within the framework of the free diffusion model. The one-dimensional diffusion equation is the second Fick law and has the form:

,

,

four

where C (x, t) is the concentration of OPA in the sample of biological tissue, g / ml; D - diffusion coefficient ASO, cm ² / sec; t is the diffusion time, sec; x is the spatial coordinate along the thickness of the sample, see

, а решение уравнения диффузии (усредненное по объему) имеет вид:

. В случае же иммерсирования мышечной ткани граничные условия имеют вид: C(0,t)=С ₀ и C(l,t)=С ₀, тогда решение уравнения диффузии имеет вид:

. В то же время в обоих случаях решение уравнения диффузии отражает кинетику изменения концентрации ОПА в образце биоткани.Depending on the type of biological tissue and the geometry of the experiment, different boundary conditions are used to solve the diffusion equation, which leads to the fact that the diffusion equation has different solutions. So, for example, for the skin (in the case of immersion in IL), the boundary conditions are: C (0, t ) = C ₀ and

, and the solution of the diffusion equation (averaged over the volume) has the form:

. In the case of muscle tissue immersion, the boundary conditions are: C (0, t ) = C ₀ and C (l, t) = C ₀ , then the solution of the diffusion equation has the form:

. At the same time, in both cases, the solution of the diffusion equation reflects the kinetics of changes in the concentration of OPA in the biological tissue sample.

In mathematical models of biological tissues, their optical characteristics (scattering and absorption coefficients), geometric parameters (thickness and area of the sample), refractive indices and volume fractions of biological tissue components, etc. are taken into account.

The proposed biosensor does not use a fluorescence signal, but an optical signal reflected or transmitted through biological tissue 3 at one or many wavelengths and contains an emitting fiber 4 and a receiving fiber 5, as well as an applicator 6 filled with a biocompatible antireflection agent 7 (Fig. 2, 3).

The optical fiber 4 is connected at one end to the source, and the optical fiber 5 to the light receiver. In this case, the distal (free) ends pass through the walls of the applicator inward to bring them to the test biological tissue.

When registering light reflection from the skin or mucous membrane of accessible organs of the human body (for example, the oral cavity or nose), the emitting 4 and 5 receiving fibers are parallel to each other. The distance l between the centers of the distal ends of the emitting and receiving fibers is approximately double the probing depth of the biological tissue h, l ≅ 2 h. One of the opposite ends of the optical fibers is connected to a radiation source, and the other to an optical detector (Fig. 2).

When recording the transmission of a light beam 9 through a protruding area of the skin, the distal ends of the emitting and receiving optical fibers 4, 5 are simultaneously fed from one and the other side of the protruding area of the skin accessible for translucency (earlobe, skin between the fingers, skin drawn from anywhere on the human body , allowing you to start the radiation and take it "into the light"). The applicator 6 supplies the agent on both sides of the protruding area of the skin (Fig. 3). Registration of the optical signal is carried out within 10-30 minutes.

As a radiation source, a wide-band white light lamp is used, either a set of diode lasers of different wavelengths in the range of 500-2000 nm, or a set of LEDs of different wavelengths in the range of 500-1100 nm, a miniature digital spectrometer connected to a computer is used as an optical detector smartphone, or a set of miniature photodiodes tuned with filters to different wavelengths and connected to a computer or smartphone.

To ensure simplicity, reliability, low cost and maximum sensitivity of the biosensor to changes in the patient’s tissue under study, the biosensor is tuned to certain wavelengths when using one or more (two to three) diode lasers in the range 400-2300 nm, or one or more (two three) LEDs in the range 400-2300 nm, and miniature photodiodes connected to a computer or smartphone are used as optical detectors.

Implementation example

The described method was used to determine the diffusion coefficients of glucose in the skin of mice, patients with diabetes mellitus and not having this disease (control), in vitro. The collimated transmission spectra of in vitro skin samples of mice placed in aqueous glucose solutions of different concentrations in the wavelength range of 500-900 nm were measured. The diffusion coefficients D and permeability P = D / l , the characteristic diffusion time g, the thickness of the samples l before and after immersion in glucose solutions and the maximum transmission of samples Kmax obtained for glucose and calculated on the basis of the measured transmission spectra are presented in Table 1.

Table 1. Diffusion coefficients of glucose in mouse skin, corresponding permeability coefficients, characteristic diffusion time, thickness of samples before and after immersion in glucose solutions, maximum transmission of samples.

± - standard deviation

As a result, for diabetic skin, using each concentration of the solution, lower values of glucose diffusion coefficient were obtained compared to the control (Fig. 1). The characteristic time of glucose diffusion in diabetic skin is longer compared to the control.

In this case, the diffusion coefficient of glucose in healthy skin is 1.5-2.5 times higher compared with diabetic skin for glucose solutions of different concentrations, the permeability coefficient in case of diabetic skin is an order of magnitude lower, which can serve as a criterion for skin discrimination in normal and diabetes. When evaluating several parameters, for example, the coefficient of diffusion of an agent in a tissue, the coefficient of tissue permeability for an agent, the characteristic diffusion time, and the maximum light transmission through a sample, more information about the state of the tissue can be obtained. The presented experimental data allow us to reliably differentiate the state of the skin in normal conditions and with the development of diabetes. Although the use of glucose molecules as test molecules at all concentrations of an enlightening solution from 30 to 56% gives a significant difference in the values of the diffusion coefficient, permeability, or characteristic diffusion time, it is more advisable to choose a less concentrated solution. So for a 30% glucose solution, we have a ratio of glucose diffusion coefficients in healthy skin to diabetic equal to 2.7, permeability 2.1 and a characteristic diffusion time of 0.7.

Using the proposed technology, it is possible to non-invasively determine the coefficients of permeability and diffusion of the test agent in the skin, mucous membranes of the mouth and nose, sclera of the eye by measuring the kinetics of their optical enlightenment and reversible dehydration. The measured coefficients of permeability and diffusion rate of the test agent provide information on the degree of glycation of the protein components of human skin, and the presence of pathological changes in biological tissues inaccessible for direct analysis (kidney, retin, myocardium, skeletal muscle, brain tissue, etc.) can be extrapolated from these values. ) Such a test should allow monitoring the development of diabetes mellitus, determining the stage of the disease and monitoring the condition of the body during the treatment of the disease over a long period of time (months and years), which, in principle, does not allow the well-known methods for determining glycated hemoglobin based on studies of blood samples - due to red blood cell turnover every three months.

This technology should use biocompatible agents and a range of sensor wavelengths and emitter power densities that do not have a harmful effect on the human body. In this case, an invasive intervention in the body is not required, which can be inconvenient and dangerous for a diabetes patient, since even small wounds can lead to serious complications, up to gangrene. In the proposed technology, it is possible to use various biocompatible agents, thus, it is possible to take into account the individual characteristics of the patient’s skin (moisture, skin type, tendency to irritation, etc.).

As immersion agents can be used aqueous or aqueous-alcoholic solutions of glycerol with concentrations ranging from 20 to 80%, aqueous solutions of polyethylene glycol of different molecular weights from 200 to 20,000, aqueous or aqueous-alcoholic solutions of glucose, sucrose, fructose or other sugars with concentrations in the range from 20 to 60% with an alcohol content of 5 to 20%; non-ionic water-soluble radiopaque substance yogheksol.

Measurements are required every 2-3 months to be able to correlate the results with measurements of glycated hemoglobin.

To implement the method, a biosensor can be used that performs non-invasive optical monitoring of tissue pathology, including vital organs in diabetes mellitus.

The present invention allows us to judge the presence or development of pathology of the studied tissue, as well as tissues of internal vital organs (myocardium, retina, brain, cartilage of joints, kidney tissue, etc.), difficult to access for light radiation.

The proposed design significantly improves the quality of the sensor, determines its high stability, due to significantly less sensitivity to patient movements during measurements. Compactness is also important for ease of use of the biosensor. The advantage is also the mechanical strength of the biosensor, due to the lack of additional fasteners and fiber optic connectors.

Claims (15)

1. A biosensor for non-invasive optical monitoring of the pathology of biological tissues caused by the development of diabetes mellitus, containing a radiation source and receiver, an applicator made in the form of a vessel with a biocompatible immersion agent, an emitting fiber, connected at one end to a radiation source, and a receiving fiber connected at one end to the radiation receiver, characterized in that the distal ends of the optical fibers are located inside the applicator. 2. The biosensor according to claim 1, characterized in that the optical fibers are parallel to each other, and the distance l between the centers of the distal ends of the emitting and receiving optical fibers is approximately double the probing depth of the biological tissue h, l≅2h. 3. The biosensor according to claim 1, characterized in that the distal ends of the emitting and receiving optical fibers are located opposite each other. 4. The biosensor according to claim 1, characterized in that the radiation source is a broadband lamp, and the radiation receiver is a miniature digital spectrometer connected to a computer or smartphone. 5. The biosensor according to claim 1, characterized in that the radiation source is a set of diode lasers of different wavelengths in the range of 400-2300 nm. 6. The biosensor according to claim 1, characterized in that the radiation source is a set of LEDs of different wavelengths in the range of 400-2300 nm. 7. The biosensor according to claim 1, characterized in that the radiation source is a diode laser of a specific wavelength selected in the range 400-2300 nm, the radiation receiver is a miniature photodiode connected to a computer or smartphone. 8. The biosensor according to claim 1, characterized in that the radiation source is an LED of a specific wavelength selected in the range of 400-2300 nm. 9. The biosensor according to claim 1, characterized in that the radiation source is two diode lasers of certain wavelengths selected in the range 400-2300 nm, and the radiation receivers are two miniature photodiodes tuned by filters to different wavelengths and connected to a computer or smartphone. 10. The biosensor according to claim 1, characterized in that the radiation source is two LEDs of specific wavelengths selected in the range of 400-2300 nm. 11. The biosensor according to claim 1, characterized in that the radiation source is a light source of the terahertz (THz) wavelength range. 12. The biosensor according to claim 1, characterized in that as an immersion agent use aqueous or aqueous-alcoholic solutions of glycerol with concentrations in the range from 20 to 80%. 13. The biosensor according to claim 1, characterized in that as the immersion agent use aqueous solutions of polyethylene glycol of different molecular weights from 200 to 20,000. 14. The biosensor under item 1, characterized in that as an immersion agent use aqueous or aqueous-alcoholic solutions of glucose, sucrose, fructose or other sugars with concentrations in the range from 20 to 60% with an alcohol content of from 5 to 20% . 15. The biosensor according to claim 1, characterized in that the nonionic water-soluble radiopaque substance iohexol is used as the immersion agent.

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