Non-invasive oxidative stress detection method

Abstract

The invention relates to the technical field of biomedical optics. In particular to a non-invasive method for detecting the oxidative stress marker level in a living body and in food, and the oxidative stress state of the living body is monitored. When part of oxidative stress products accumulated in a living body are subjected to electromagnetic excitation irradiation with a specific wavelength, fluorescence with a specific wavelength can be emitted, a signal is generated, the intensity of the signal is consistent with the intensity of the measured fluorescence, and the oxidative stress level of the living body is evaluated by detecting the intensity of the signal through non-invasive irradiation on the skin tissue of the living body. The invention develops a non-invasive, rapid and convenient oxidative stress detection method, and the oxidative stress level can be monitored at any time in daily life or clinical tests under the condition that discomfort is not brought to a testee. An instrumentation specialized for such detection is also described. The method is helpful for establishing a new oxidative stress detection standard, and has important practical significance for improving the consciousness of people for preventing and treating related diseases and developing new products by enterprises.

Description

Technical Field

[0001] The present invention relates to the field of biomedical optics technology. Specifically, it relates to a non-invasive method for detecting the levels of oxidative stress markers in living organisms and foods, and monitoring the oxidative stress state of organisms. Background Art

[0002] Oxidative stress refers to the damage to cell structure and function caused by the disruption of the balance between oxidants (such as free radicals) and antioxidants (such as superoxide dismutase) in living organisms. Oxidative stress occurs when the amount of free radicals and other reactive oxygen species (ROS) generated in the body exceeds the scavenging capacity of the antioxidant system. Long-term oxidative stress can damage cell tissues, accelerate the aging process, and may lead to the occurrence of various diseases, including cardiovascular diseases, neurodegenerative diseases, cancer, and diabetes.

[0003] Currently, the detection methods of oxidative stress mainly include two strategies: direct and indirect. The direct strategy usually involves detecting free radicals and other ROS in blood, urine, or tissue samples. However, these species have a short lifespan and high reactivity, so they are difficult to detect and often require invasive sampling. The indirect strategy includes detecting biomarkers, such as lipid peroxidation products, protein oxidation products, and DNA oxidation products. The detection of these biomarkers usually requires complex laboratory procedures, such as chromatography, mass spectrometry, and enzyme-linked immunosorbent assay (ELISA). Although these methods are widely used in scientific research and clinical diagnosis, they usually require professional operators, expensive equipment, and a long processing time, which limits their application in rapid diagnosis and daily detection.

[0004] Advanced glycation end products (AGEs), advanced lipoxidation end products (ALEs), protein nitration products (such as nitrotyrosine 3-NT), and protein thioether derivatives are complex molecules formed under oxidative stress conditions. Their accumulation in the body is closely related to the development of various diseases, such as diabetes, cardiovascular diseases, and neurodegenerative diseases. They reflect the level of oxidative stress and play a key role in the formation and progression of diseases by promoting inflammation and further oxidative damage. Reducing the formation and accumulation of AGEs, ALEs, and other oxidative stress products, as well as enhancing the antioxidant defense ability in the body, is a potential strategy for preventing and treating related diseases. Therefore, developing a non-invasive oxidative stress detection method that can achieve rapid and convenient real-time monitoring of oxidative stress levels without causing discomfort to the subjects has important practical significance for improving people's health and preventing and treating related diseases. Summary of the Invention

[0005] To achieve the above objectives, the present invention has developed an innovative, non-invasive method for detecting oxidative stress, providing a new tool for individual health monitoring and also bringing an important technology to the medical and health field.

[0006] The method of the present invention is based on the autofluorescence characteristics of oxidative stress markers such as advanced glycation end products (AGEs), advanced lipoxidation end products (ALEs), protein nitration products (such as nitrotyrosine 3-NT), and protein thioether derivatives. When these substances are irradiated by electromagnetic excitation of a specific wavelength, they can emit fluorescence of a specific wavelength, and the intensity of this fluorescence is proportional to the concentration of oxidative stress products in the skin.

[0007] According to the literature, although not all oxidative stress markers have fluorescence characteristics such as CML with pathogenic effects, the oxidative stress markers with fluorescence characteristics in human skin are correlated with non-fluorescent oxidative stress markers, and the levels of skin fluorescent oxidative stress markers are related to the oxidative stress levels in human blood and tissues. The levels of skin fluorescent oxidative stress markers can be used as markers of human oxidative stress levels.

[0008] The invention relates to a detection instrument equipped with an adjustable light source as an excitation source and a multi-channel detector. The device also includes a data processing unit for analyzing the fluorescence signal and converting it into a measured value of the oxidative stress level. By non-invasively irradiating living skin tissue or the surface of an object, autofluorescence measurements of the skin tissue or surface can be obtained, and this measurement can be used as a measure of the content of oxidative stress markers such as advanced glycation end products (AGEs), advanced lipoxidation end products (ALEs), protein nitration products (such as nitrotyrosine 3-NT), etc. Among them, the living skin tissue is clinically healthy skin tissue, and local abnormal skin tissues such as moles, warts, scars, skin tissues affected by sunburn, tattoos, and very hairy skin tissues should be avoided as much as possible. To reduce the influence of photoaged skin on the measurement results, when measuring, try to select skin areas that are not often exposed to sunlight, and try to fix on the same area for multiple detections. At the same time, try to avoid the skin areas where skin care products such as moisturizing creams, lotions, and sunscreen are often used.

[0009] Such as Figure 1As shown in the figure, a measurement system for measuring the content of oxidative stress markers on the skin surface or an object includes a measurement unit 1. The light source is an LED light-emitting diode 2, which is installed in a support structure in the shape of a light-shielding housing 6. The housing 6 has a contact surface 8, which is attached to the skin or sample surface 7. An opening on the contact surface 8 forms an irradiation window 5, through which a part of the skin or object surface located behind the irradiation window and adjacent to the window opening can be irradiated. The irradiation window can be rectangular, circular or other shapes. Radiation with a longer wavelength is returned to the irradiation window 5 due to the excitation of the light sent to the skin by the components in the skin, and is received by a detector 3 located at the edge of the irradiation window. The detector 3 is connected to a spectrophotometer unit 9 through an optical fiber 4. The optical signal received by the detector 3 is transmitted to a signal processor 10, and then data processing and analysis are carried out at 11. The test result of the content of oxidative stress markers is displayed at 12.

[0010] Among them, the light source 2 can use light-emitting diodes, black lights, xenon lamps, etc., and is combined with a monochromator or filter to adjust the wavelength range of the excitation light required.

[0011] To accurately measure fluorescence signals of different intensities, the detector 3 needs to have high sensitivity and a wide dynamic range. Photomultiplier tubes (PMT), photodiodes, fiber optic detectors, multi-channel detectors, etc. can be selected. Two detectors can be used in combination to increase the detection sensitivity.

[0012] The optical fiber 4 can select quartz optical fibers, silicon-based optical fibers, etc. to ensure the effective transmission of signals and minimize signal loss.

[0013] The irradiation window 5 needs to maintain a certain distance and angle from the contact surface 8 through the light-shielding housing for convenient measurement. The area of the contact surface 8 can be 1-5 square centimeters, and the area of the contact surface 8 needs to be larger than the area of the irradiation window 5 to combine the large irradiated and measured skin or object surface with the compact measurement instrument.

[0014] For the determination of oxidative stress markers including advanced glycation end products, advanced lipid peroxidation end products, and protein nitration products, etc., if all the wavelengths used for excitation are irradiated simultaneously and the fluorescence intensity is detected simultaneously, and if all the wavelengths of the excitation irradiation are lower than all the wavelength ranges for measuring the fluorescence intensity, then the wavelength range of the excitation irradiation is preferably in the range of 300-420 nanometers, and the wavelength range for measuring fluorescence is preferably in the range of 420-680 nanometers.

[0015] Operation steps:

[0016] a. Align the excitation source of the detection instrument with the skin surface of the subject or the surface of the sample to be tested, and the excitation source emits light of a specific wavelength for excitation;

[0017] b. Receive and measure the corresponding electromagnetic fluorescence intensity emitted by the skin or the sample surface in response to the irradiation, and generate a signal representative of the fluorescence intensity;

[0018] c. The detector captures the signal emitted from the skin or the sample surface and transmits the signal to the data processing unit;

[0019] d. Analyze and determine the content of the fluorescent oxidative stress marker from the signal, and output the determined oxidative stress level.

[0020] Skin pigmentation plays an important role in the autofluorescence of the skin. Melanin can absorb ultraviolet and visible light, which affects the intensity and quality of the observed autofluorescence. Generally, darker skin (higher melanin content) absorbs more ultraviolet light, which may lead to a reduction in its autofluorescence. When measuring skin fluorescence in humans, the absorption of chromophores such as melanin and hemoglobin is often the main cause of fluorescence changes and masks the fluorescence information from the chromophores. To reduce the influence of skin color differences, skin ruddiness, and racial differences on the measurement, specific correction methods are adopted. The correction method involves adjusting the measurement algorithm of the device to compensate for the differences in light absorption and scattering caused by different skin melanin contents. Specifically, it includes using a standardized skin tone reference table or automatically focusing on the skin by the absorption and diffuse reflection data of ultraviolet light by different skin colors to adapt to the influence of skin color, or automatically adjusting the readings through a computer algorithm to consider the influence of different skin colors on the fluorescence signal. In addition, correction can be performed according to the dataset of people with different skin colors.

[0021] The oxidative stress marker detection instrument provided in the invention is compact and portable, facilitating use in various environments, including laboratories, homes, clinics, and outdoor working environments, etc. In addition to detecting oxidative stress markers, the present invention can also be extended to the detection of other biomarkers with significant fluorescence characteristics by adjusting the parameters of the instrument, further enhancing its application value in the biomedical field. The instrument design takes into account the safety of users, ensuring that the excitation light source will not cause harm to the skin, and the light intensity used throughout the detection process is within the safe range. Beneficial effects and applications

[0022] The non-invasive oxidative stress detection method of the present invention provides a safe and convenient means of health monitoring for users by detecting the autofluorescence characteristics of oxidative stress products on the skin. This method does not involve invasive operations, is simple and fast to operate, improves the comfort and detection compliance of patients, and is especially suitable for chronic disease patients who need regular monitoring.

[0023] In terms of disease prevention and health management, this method enables individuals to monitor their own oxidative stress levels in an environment without professional medical supervision, thereby contributing to the early identification of health risks and the adoption of appropriate lifestyle adjustments to achieve disease prevention and early intervention. At the same time, for the public health system, it is a cost-effective and easily deployable on a large scale health risk assessment tool, reducing the socioeconomic burden caused by chronic diseases. By precisely monitoring and analyzing the changes after using antioxidant products, enterprises can more effectively guide product R & D, ensure its innovation and effectiveness while reducing the R & D cycle and costs. In addition, the non-professional operation characteristics of this method further expand its application potential in home self-monitoring and personal health management, and are expected to play an important role in improving people's quality of life. Brief Description of the Drawings

[0024] Figure 1 Composition of a non-invasive oxidative stress biomarker detection instrument Embodiment

[0025] A method for measuring the autofluorescence of skin oxidative stress biomarkers will be described in detail below. The measurement system shown in Figure 1 is used.

[0026] First, the dark current of the detector is measured by covering the opening without light irradiation to perform a dark measurement. Then, the temperature of the measurement system is kept constant to prevent changes in the dark current.

[0027] Next, the light source 2 is turned on, and then wait for a period of time (such as several minutes) until the tube 2 produces a substantially constant light output.

[0028] After that, the probe connected to the irradiation window 8 is placed on the skin 7 of the person to be detected. For example, it can be a part of the forearm or the leg (such as the calf). The measurement is carried out, and the optical signal received by the detector 3 is transmitted to the signal processor 10, and then data processing and analysis are performed at 11.

[0029] Since the measurement results are related to factors such as the melanin content in the skin tissue and the blood volume in the skin, etc., in order to reduce the influence of skin pigmentation, blood volume and racial differences on the measurement, correction is performed during data processing and analysis.

[0030] Non-invasive autofluorescence detection of skin oxidative stress biomarkers was performed in 50 healthy subjects of different age groups using Figure 1The measurement system shown. In healthy subjects, obesity, smoking, alcoholism, use of medications, special diets, minerals, vitamins or other types of supplements, and antioxidants were excluded. Ten subjects were included in each age group, without gender distinction, and the test results were expressed in AU. The average levels of oxidative stress markers in different age groups were as follows: 20 - 29 (1.63 AU); 30 - 39 (1.79 AU); 40 - 49 (1.97 AU); 50 - 59 (2.20 AU); 60 - 69 (2.49 AU).

[0031] According to the study A Quantitative Method to Monitor Reactive Oxygen Species Production by Electron Paramagnetic Resonance in Physiological and Pathological Conditions, the production rate of reactive oxygen species (ROS) in capillary blood was detected using an electron paramagnetic resonance (EPR) instrument in the laboratory. The ROS production rate in young sedentary people (aged 18 - 44) was 1.84 μmol / min, in middle-aged sedentary people (aged 45 - 59) was 1.91 μmol / min, and in elderly sedentary people (aged 60 - 74) was 2.12 μmol / min.

[0032] There was a significant correlation (R = 0.9871) between the levels of skin oxidative stress markers detected non-invasively and the levels of reactive oxygen free radicals (ROS) in the blood.

[0033] An increase in ROS leads to the formation of more oxidative stress products. ROS is a highly reactive molecule that can cause various types of cell damage, including modifications of proteins, lipids, and nucleic acids. These modifications can lead to the formation of oxidative stress products. For example, ROS can oxidize lipids and proteins, creating a favorable environment for the formation of oxidative stress products such as AGEs and ALEs.

[0034] Conversely, an increase in oxidative stress products also leads to an increase in ROS levels. For example, once oxidative stress products such as AGEs and ALEs are formed, they interact with specific receptors such as RAGE (receptor for advanced glycation end products). This interaction activates multiple cell signaling pathways, leading to the production of ROS. The binding of oxidative stress products to receptors on the cell surface can activate NADPH oxidase, which is the main source of ROS in cells.

[0035] This interaction forms a feedback loop where an increase in ROS leads to an increase in oxidative stress products, and an increase in oxidative stress products in turn leads to an increase in ROS generation. This cycle exacerbates oxidative stress and inflammatory conditions, leading to the development of various chronic diseases. The non-invasive oxidative stress detection method of the present invention helps to quickly understand this cycle and intervene immediately, which is crucial for controlling and treating diseases related to oxidative stress.

[0036] For those skilled in the art familiar with this field, it will be clearly recognized that many other embodiments and patterns are possible within the framework of the present invention, and the present invention is not limited to the examples described above.

Claims

1. A non-invasive oxidative stress detection method, comprising the following steps: a. Align the excitation source of the detection instrument with the skin surface of the subject or the surface of the sample to be tested, and the excitation source emits light of a specific wavelength for excitation; b. Receive and measure the electromagnetic fluorescence intensity emitted in response to the irradiation on the skin or sample surface, and generate a signal representing the fluorescence intensity; c. The detector captures the signal emitted from the skin or sample surface and transmits the signal to the data processing unit; d. Analyze and determine the content of the fluorescent oxidation stress marker from the signal, and output the determined oxidation stress level.

2. The non-invasive oxidative stress detection method according to claim 1, characterized in that: The skin tissue is a clinically healthy and intact living skin tissue, and local abnormal skin tissues such as moles, warts, scars, skin tissues affected by sunburn, tattoos, and very hairy skin tissues should be avoided as much as possible. When measuring, try to select skin areas that are not often exposed to sunlight, and try to fix on the same area for multiple detections. At the same time, try to avoid the skin areas where skin care products such as moisturizing creams, lotions, and sunscreen are often used. By directly irradiating the outer surface part of the skin with the light source, it is non-invasive and irradiates as a whole at the same time.

3. The non-invasive oxidative stress detection method according to claim 1, characterized in that: The fluorescent radiation emitted in response to the irradiation is received simultaneously from an area of the irradiated skin surface of 1-5 square centimeters. The fluorescent radiation is received by the detector, and the irradiation window needs to maintain a certain distance and angle from the contact surface through the light-shielding housing for convenient measurement. The area of the contact surface needs to be larger than the area of the irradiation window.

4. An instrument device for a non-invasive oxidative stress detection method, comprising the following components: A collection unit, which includes: A light source for non-invasively irradiating the surface part of the intact skin tissue behind the irradiation window with electromagnetic excitation on the surface of the living skin; A detector for measuring only the electromagnetic fluorescence received from the irradiated skin surface part; Connected to the spectrophotometer unit through an optical fiber, used to generate a signal conforming to the signal intensity according to the measured fluorescence intensity, determine the content of the fluorescent oxidation stress marker in the skin tissue from the signal, and output the determined oxidation stress level; An irradiation window that defines a surface area and is used to transmit the fluorescent radiation received from the skin surface through this window to the detector; A spectrophotometer unit that includes: a signal processor, a data processor, and a display screen, used to receive signals and process, analyze, and output data.

5. The instrument device for a non-invasive oxidative stress detection method according to claim 4, characterized in that: The light source can use light-emitting diodes, black lights, xenon lamps, etc., and is combined with a monochromator or filter to adjust the required excitation light wavelength range.

6. The instrument device for a non-invasive oxidative stress detection method according to claim 4, characterized in that: The detector needs to have high sensitivity and a wide dynamic range. Photomultiplier tubes (PMT), photodiodes, fiber optic detectors, multi-channel detectors, etc. can be selected. Two detectors can be used in combination to increase the detection sensitivity. Optical fibers can be selected from quartz fibers, silicon-based fibers, etc. to ensure effective signal transmission and minimum signal loss.

7. The instrument device for a non-invasive oxidative stress detection method according to claim 4, characterized in that: The irradiation window needs to maintain a certain distance and angle from the contact surface through the light-shielding housing for convenient measurement. The area of the contact surface can be 1-5 square centimeters or a larger area. The area of the contact surface needs to be larger than the area of the irradiation window to combine the large irradiated and measured skin or object surface with the compact measurement instrument.

8. The instrument device for a non-invasive oxidative stress detection method according to claim 4, characterized in that: The wavelength range of the excitation irradiation can be in the range of 300-420 nanometers, and the wavelength range of the measured fluorescence can be in the range of 420-680 nanometers.

9. The instrument device for a non-invasive oxidative stress detection method according to claim 8, characterized in that: The fluorescent radiation emitted in response to the irradiation is received simultaneously from a surface area of the irradiated portion of the skin that is greater than 1 square centimeter.

Images