Radiotherapy system, auxiliary system and method for evaluating free radical injury and application of auxiliary system and method

Abstract

The invention discloses a radiotherapy system, an auxiliary system and method for evaluating free radical damage and application thereof, a tube body of a cuvette of the auxiliary system comprises side surfaces, a top surface and a bottom surface, the top surface is provided with an opening matched with a shading cover, and the side surfaces comprise a first side surface, a second side surface, a third side surface and a fourth side surface; one end of the first optical fiber is connected with the laser assembly, and the other end of the first optical fiber is connected with the first side surface; the irradiation assembly is arranged on one side of the top surface; the fluorescent light filter is arranged on the third side surface or the fourth side surface; one end of the second fiber is connected with the fluorescent filter, and the other end of the second fiber is connected with the luminosity assembly; the chamber is used for accommodating the cuvette and the fluorescent light filter; for FLASH radiotherapy, the real-time accurate detection of the fluorescence intensity can be realized by optimizing the detection environment, and a reliable platform is provided for free radical damage assessment.

Description

Technical Field

[0001] The present invention relates to the technical field of free radical damage assessment, and in particular to a radiotherapy system, an auxiliary system and method for assessing free radical damage, and applications thereof. Background Art

[0002] In the medical field, radiotherapy is an important means of treating tumors, and improving its efficacy has always been a hot topic of research. Although traditional radiotherapy methods can effectively inhibit tumor growth to a certain extent, they also cause a certain degree of damage to normal tissues. With the advancement of science and technology, the development of new and efficient radiotherapy methods, such as FLASH proton therapy, aims to improve the treatment effect while reducing damage to normal tissues, which has become the focus of current research. The development of such new methods is not only expected to benefit more patients, but also to improve the overall level of tumor treatment.

[0003] The cell-killing effect of radiotherapy is mainly divided into two mechanisms: direct killing and indirect killing. Direct killing refers to the direct effect of high-energy rays on biological macromolecules in cells, such as DNA, causing structural damage and loss of function. Indirect killing, on the other hand, is the attack of biological macromolecules in cells by free radicals generated by ionizing radiation, causing damage. Therefore, accurately assessing the degree of free radical damage during radiotherapy is of great significance for optimizing radiotherapy plans and improving treatment effects.

[0004] Currently, the evaluation of free radical damage mainly relies on time-delayed monitoring methods, but this method has many limitations. Since free radical reactions are usually fast, time-delayed monitoring often cannot accurately capture the generation and change process of free radicals, thus affecting the accuracy of the evaluation. Developing a method that can monitor free radical production in real time is crucial for evaluating the killing effect of radiotherapy on tumors.

[0005] Therefore, the present application proposes a radiotherapy system, an auxiliary system for evaluating free radical damage, a method and applications thereof. Summary of the invention

[0006] The purpose of the present invention is to provide a radiotherapy system, an auxiliary system for evaluating free radical damage, a method and its application, which can achieve accurate detection of fluorescence emphasis by optimizing the detection environment, and provide a reliable platform for free radical damage evaluation.

[0007] The purpose of the present invention is achieved by the following technical solutions:

[0008] In a first aspect, the present invention provides an auxiliary system for assessing free radical damage, comprising:

[0009] A laser assembly, the laser assembly being used to generate laser light for exciting fluorescent molecules;

[0010] A cuvette, the cuvette comprising a tube body and a light shielding cover, the tube body comprising a side surface and a top surface and a bottom surface opposite to each other, the top surface is provided with an opening matching the light shielding cover, the side surfaces comprising a first side surface and a second side surface opposite to each other, and a third side surface and a fourth side surface opposite to each other;

[0011] A first optical fiber, one end of which is connected to the laser assembly, and the other end of which is connected to the first side surface;

[0012] An irradiation component, which is used to generate radiation and is arranged on one side of the top surface;

[0013] a fluorescent filter, wherein the fluorescent filter is disposed on the third side surface or the fourth side surface;

[0014] A photometric component, wherein the photometric component is used to measure fluorescence intensity;

[0015] A second optical fiber, one end of the second optical fiber is connected to the fluorescent filter, and the other end of the second optical fiber is connected to the photometric component;

[0016] A chamber is used to accommodate the cuvette and the fluorescent filter.

[0017] Furthermore, the auxiliary system also includes:

[0018] an excitation light filter, the excitation light filter being disposed on the first side surface and connected to the first optical fiber;

[0019] a reflector, wherein the reflector is disposed on the third side surface or the fourth side surface and is disposed opposite to the fluorescent filter;

[0020] A light trap is disposed on the second side surface.

[0021] Furthermore, the tube body contains a solution to be tested, the solution to be tested includes an indicator, the ray is used to irradiate the solution to be tested to generate free radicals, the free radicals are used to react with the indicator to generate the fluorescent molecules, and the angle between the ray and the normal direction of the top surface is 0-30°;

[0022] The wavelength range allowed by the excitation light filter is the central wavelength of the excitation light of the fluorescent molecule ± the second threshold;

[0023] The wavelength range allowed by the fluorescence filter is the central wavelength of the fluorescence emitted by the fluorescent molecule ± the second threshold.

[0024] Furthermore, the auxiliary system also includes a base, and the base includes:

[0025] A lifting frame, the lifting frame is arranged in the shielding room whose height can be adjusted by the lifting frame;

[0026] A vertical plate, wherein the vertical plate is arranged on the lifting frame so that the height of the vertical plate can be adjusted by the lifting frame;

[0027] A bracket is arranged on the vertical plate and is used for placing the cuvette and the filter, and fixing the first optical fiber and the second optical fiber.

[0028] Further, the photometric component includes:

[0029] a photometer connected to the second optical fiber;

[0030] A terminal server, the terminal server being communicatively connected to the photometer;

[0031] The laser assembly comprises:

[0032] A laser power supply, wherein the laser power supply is arranged outside the shielding room;

[0033] A first electric wire, which is connected to the laser power supply and passes through the wall of the shielding room;

[0034] a laser diode, one end of which is connected to the first electric wire, and the other end of which is connected to the first optical fiber;

[0035] The irradiation assembly comprises:

[0036] A ray power supply, wherein the ray power supply is arranged outside the shielding room;

[0037] A second electric wire, the second electric wire is connected to the radiation power supply and passes through the wall of the shielding room;

[0038] a radiation source connected to the second electric wire;

[0039] The shielding room is provided with a first through hole matching the first electric wire, a second through hole matching the second electric wire, and a third through hole matching the second optical fiber, and the second optical fiber passes through the wall of the shielding room through the third through hole;

[0040] The first optical fiber and the second optical fiber are both single-mode optical fibers with a diameter of 10-1500 μm.

[0041] Furthermore, the second optical fiber is a light-transmitting optical fiber with a diameter of 160-1250 μm, and the light-transmitting optical fiber is a glass optical fiber or a plastic optical fiber.

[0042] In a second aspect, the present invention provides a method for evaluating free radical damage, wherein the method is implemented based on the auxiliary system described above;

[0043] The tube body is filled with a solution to be tested, the solution to be tested includes an indicator, and the volume of the solution to be tested is 80%-100% of the volume of the tube body;

[0044] The method comprises the following steps:

[0045] Starting the laser assembly to allow the laser to pass through the first side surface and irradiate the solution to be tested;

[0046] Starting the photometric component and combining it with the fluorescent filter to detect the background fluorescence intensity of the solution to be tested in the cuvette;

[0047] In response to the change speed of the background fluorescence intensity being less than the preset intensity change speed, the irradiation component is started, so that the radiation generated by the irradiation component irradiates the solution to be tested to generate free radicals, and the free radicals react with the indicator to generate fluorescent molecules;

[0048] Real-time detection of the fluorescence intensity of the fluorescent molecules, when the irradiation time of the ray irradiating the solution to be tested is greater than or equal to a preset time, turning off the irradiation component;

[0049] In response to the fact that the change rate of the fluorescence intensity monitored in real time is less than the preset intensity change rate, the free radical concentration is determined according to the detected fluorescence intensity and the background fluorescence intensity.

[0050] Furthermore, the solution to be tested also includes cells to be tested, and the cells to be tested are normal cells or cancer cells.

[0051] Furthermore, the volume of the cuvette is 0.1-3 mL;

[0052] The angle between the incident light of the laser and the outgoing light thereof is 60-90°;

[0053] The power of the laser component is 1μW-1W;

[0054] The wavelength of the laser is the maximum excitation wavelength of the fluorescent molecule ± the first threshold;

[0055] The angle between the ray and the normal direction of the top surface is 0°;

[0056] The indicator comprises one or a combination of calcein-AM, ethidium homodimer-1, aminophenyl fluorescein, hydroxyphenyl fluorescein, 2',7'-dichlorodihydrofluorescein diacetate, dihydroethidium and derivatives thereof;

[0057] The concentration of the indicator is: 1nmol / L-50mmol / L;

[0058] The radiation dose rate generated by the irradiation component is 1Gy / s-100Gy / s, and the total radiation dose is 0.1-260Gy.

[0059] Furthermore, the power of the laser assembly is 1-50 mW;

[0060] The first threshold is 50 nm;

[0061] The concentration of the indicator is 1-50mmol / L;

[0062] The indicator is hydroxyphenylfluorescein or aminophenylfluorescein;

[0063] The radiation dose rate generated by the irradiation assembly is 10-100 Gy / s and the total radiation dose is 1-80 Gy;

[0064] Furthermore, the power of the laser assembly is 5 mW;

[0065] The wavelength of the laser is 488nm;

[0066] The concentration of the indicator is 10mmol / L;

[0067] The first threshold is 5 nm;

[0068] The indicator is hydroxyphenylfluorescein;

[0069] The radiation dose rate generated by the irradiation assembly is 40 Gy / s and the total radiation dose is 80 Gy.

[0070] In a third aspect, the present invention provides the application of the above method in the detection of cell mortality, the detection of cell viability and the detection of cell damage during the treatment of cancer cells.

[0071] In a fourth aspect, the present invention provides a radiotherapy system, comprising the auxiliary system for evaluating free radical damage described above.

[0072] Compared with the prior art, the beneficial effects of the present invention include at least:

[0073] The chamber of the auxiliary system of the present invention can reduce the interference of external light on the detection results; the shielding room can not only shield the interference of radiation on the circuit and the photometric component, but also further reduce the interference of external light on the detection results; the light shielding cover of the cuvette (or the light shielding cover of the cavity where the cuvette is installed) can prevent light leakage and interference from external light sources, such as light used for lighting, and is easy to open and close, convenient for adding the solution to be tested and cleaning; the rays of the irradiation component are designed to be parallel to the normal direction of the top surface or at a certain angle, such as 0-30°, which is conducive to the rays penetrating from the top surface and exiting from the bottom surface, and contacting with the solution to be tested. The solution achieves maximum contact, so that the radiation can evenly irradiate the solution to be tested in the cuvette, further improving the uniformity and efficiency of free radical generation; the laser component guides the laser to the first side of the cuvette through the first optical fiber, ensuring the generation of fluorescent molecules; a fluorescent filter is set on the third side, which can effectively filter out background light and stray light, and only allow fluorescence of a specific wavelength to pass, thereby improving the sensitivity and accuracy of fluorescence detection; the second optical fiber transmits the fluorescent signal to the photometric component, which can measure the fluorescence intensity in real time and accurately, providing reliable data support for free radical damage assessment. Real-time monitoring can not only provide key information such as the generation rate and concentration of free radicals, but also help doctors more accurately understand the dynamic changes of free radicals during radiotherapy, providing strong support for the formulation of personalized treatment plans.

[0074] The method of the present invention utilizes the radiation of the irradiation component to irradiate the solution to be tested, generating free radicals, which react with the indicator to generate fluorescent molecules, and utilizes the photometric component in combination with the fluorescent filter to detect the fluorescence intensity of the fluorescent molecules in real time and accurately, thereby realizing an accurate assessment of the concentration of free radicals. Furthermore, the volume of the solution to be tested is accurately controlled at 80%-100% of the volume of the tube body, which can reduce the interference caused by the movement of bubbles generated by the insufficient volume of the solution in the cuvette during the experiment, and the detection error caused by it. In addition, by detecting the background fluorescence intensity, the influence of background fluorescence on the detection results can be eliminated, and the reliability of the data can be improved. Furthermore, by real-time monitoring of the change rate of the fluorescence intensity, and determining the free radical concentration when the change rate is less than the preset value, the actual concentration of the free radical can be more accurately reflected, and the deviation caused by time delay or measurement error can be reduced. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] Figure 1 It is a partial structural schematic diagram of an auxiliary system for evaluating free radical damage according to an embodiment of the present invention.

[0076] Figure 2 It is a structural schematic diagram of an auxiliary system for evaluating free radical damage according to an embodiment of the present invention.

[0077] Figure 3 It is a schematic structural diagram of a base according to an embodiment of the present invention.

[0078] Figure 4 It is a partial structural schematic diagram of a cuvette according to an embodiment of the present invention.

[0079] Figure 5 4 is a flow chart of a method for evaluating free radical damage according to an embodiment of the present invention.

[0080] Figure 6 It is a curve showing the relationship between the indicator concentration and the fluorescence intensity of the present invention.

[0081] Figure 7 It is a curve chart of the fluorescence intensity monitored in real time in Example 1 of the present invention.

[0082] Figure 8 It is a curve chart of the fluorescence intensity monitored in real time in Example 2 of the present invention.

[0083] Fig. 9 It is a curve chart of the fluorescence intensity monitored in real time in Example 3 of the present invention.

[0084] Fig.10 It is a curve chart of the fluorescence intensity monitored in real time in Example 4 of the present invention.

[0085] Fig.11 It is a curve chart of the fluorescence intensity monitored in real time in Example 5 of the present invention.

[0086] Fig.12 It is a curve chart of the fluorescence intensity monitored in real time in Comparative Example 1 of the present invention.

[0087] In the figure: 1. laser assembly; 10. sunshade; 11. laser power supply; 12. first wire; 13. laser diode; 21. first optical fiber; 22. second optical fiber; 31. excitation light filter; 32. fluorescence filter; 4. irradiation assembly; 5. reflector; 6. light trap; 7. cuvette; 71. top surface; 72. bottom surface; 731. first side surface; 732. second side surface; 733. third side surface; 734. fourth side surface; 81. lifting frame; 82. vertical plate; 83. bracket; 9. photometric assembly; 91. photometer; 92. third wire; 93. terminal server; 100. chamber; 200 shielding room; 201. first through hole; 202. second through hole. DETAILED DESCRIPTION

[0088] Example embodiments will now be described more fully with reference to the accompanying drawings. However, example embodiments can be implemented in a variety of forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided to make the present invention more comprehensive and complete and to fully convey the concepts of example embodiments to those skilled in the art. The same reference numerals in the figures represent the same or similar structures, and thus their repeated description will be omitted.

[0089] The words expressing positions and directions described in the present invention are all explained with reference to the accompanying drawings as examples, but they can be changed as needed, and all such changes are included in the protection scope of the present invention.

[0090] Under the same particle type, energy and total dose conditions, there is no significant difference in the direct effect of killing cells between FLASH particle radiotherapy (also known as flash therapy) and conventional particle radiotherapy. However, the indirect effect of killing cells is very different between the two. This is mainly because different dose rates affect the generation of free radicals in the solution and the rate of complex reactions, thereby determining the strength of the indirect effect. The particles may include but are not limited to protons.

[0091] The auxiliary system for evaluating free radical damage in the embodiment of the present invention includes a laser component 1, a cuvette 7, a first optical fiber 21, an irradiation component 4, a photometric component 9, a second optical fiber 22, and a chamber 100. Further, the auxiliary system of the invention may also include a shielding chamber 200. Further, the auxiliary system of the present invention may also include an excitation light filter 31, a reflector 5, and a light trap 6. Further, the auxiliary system of the present invention may also include a base.

[0092] refer to Figure 4 The cuvette 7 of the present invention includes a tube body and a light shielding cover. The tube body includes a side surface and a top surface 71 and a bottom surface 72 that are opposite to each other. Further, the top surface 71 is provided with an opening that matches the light shielding cover, and the light shielding cover is buckled on the opening to form a sealed cavity with the tube body. In addition, the side surface includes a first side surface 731 and a second side surface 732 that are opposite to each other, and a third side surface 733 and a fourth side surface 734 that are opposite to each other.

[0093] In some preferred embodiments, the cuvette 7 can be in various shapes such as a cuboid, a cube or a cylinder. More preferably, the cuvette 7 is a centrifuge tube, and the volume of the centrifuge tube is 0.1-3 mL. When used, the tube body of the cuvette 7 is filled with a solution to be tested, and the solution to be tested includes an indicator. The radiation of the irradiation component 4 irradiates the solution to be tested to generate free radicals, and the free radicals react with the indicator to generate fluorescent molecules.

[0094] refer to Figure 3 The base of the present invention comprises: a lifting frame 81, a vertical plate 82 and a bracket 83. When used, the base is arranged in the chamber 100.

[0095] In some preferred embodiments, the vertical plate 82 is an L-shaped structure, one side of the L-shaped vertical plate 82 is arranged on the lifting frame 81, the other side of the L-shaped vertical plate 82 is freely arranged on the lifting frame 81, and the bracket 83 is installed on the free edge of the L-shaped vertical plate 82. When used, the bracket 83 matches the size of the cuvette 7 and is used to place the cuvette 7. In actual application, the height between the cuvette 7 and the ground can be adjusted by adjusting the lifting frame 81 according to actual needs, so that the laser and the ray can better irradiate the liquid in the cuvette 7, thereby improving the accuracy and precision of the detection result.

[0096] refer to Figure 2 The laser assembly 1 of the present invention comprises: a laser power supply 11, a first electric wire 12 and a laser diode 13. When used, the laser diode 13 is arranged in the shielding room 200, and the laser power supply 11 is arranged outside the shielding room 200, so as to avoid interference of the irradiation assembly 4 on the laser power supply 11 and related circuits, so as to improve the accuracy of the parameters of the laser emission.

[0097] In some preferred embodiments, a first through hole 201 matching the first wire 12 is provided on the shielding room 200, and the first wire 12 passes through the wall of the shielding room 200 through the first through hole 201. The input end of the laser diode 13 is connected to the laser power supply 11 through the first wire 12 to convert the electrical signal into a laser optical signal. Further, the output end of the laser diode 13 is connected to one end of the first optical fiber 21, and the other end of the first optical fiber 21 is connected to the first side surface 731 to transmit the incident light of the laser to the cuvette 7 through the first optical fiber 21. When applied, in order to increase the amount of optical signal, the diameter of the optical fiber is increased. Specifically, the diameter of the first optical fiber 21 is a single-mode optical fiber of 10-1500μm, and preferably, the diameter of the first optical fiber 21 is 600μm.

[0098] In some preferred embodiments, in order to prevent laser light of non-ideal wavelength from entering the cuvette 7, an excitation light filter 31 is provided on the first side 731, and the excitation light filter 31 filters the light signal in the first optical fiber 21. When used, the wavelength range allowed to pass by the excitation light filter 31 is the central wavelength of the excitation light of the fluorescent molecule ± the second threshold, the second threshold is 0-15nm, and preferably, the second threshold is 2nm. In actual application, the angle between the incident light of the laser and its outgoing light is 60-90°.

[0099] refer to Figure 2 The photometric component 9 of the present invention includes: a photometer 91, a third wire 92 and a terminal server 93. When used, the photometric component 9 is arranged outside the shielding room 200 to avoid interference of the irradiation component 4 on the components and related circuits of the photometric component 9 and influence on the detection results.

[0100] In some preferred embodiments, the photometer 91 is a photometer 91 capable of communication transmission, and the input end of the photometer 91 is connected to the second optical fiber 22. When used, in order to increase the amount of light signal, the diameter of the optical fiber is increased. Specifically, the second optical fiber 22 is a single-mode optical fiber with a diameter of 10-1500 μm. Further, the second optical fiber 22 is a light-transmitting optical fiber with a diameter of 160-1250 μm, and the light-transmitting optical fiber is a glass optical fiber or a plastic optical fiber. Preferably, the diameter of the second optical fiber 22 is 200 μm and the length is 30 m.

[0101] In some preferred embodiments, in order to reduce the interference signal in the optical signal of the second optical fiber 22, a fluorescence filter 31 is provided on the third side 733 or the fourth side 734, and the optical signal filtered by the fluorescence filter is transmitted to the photometer 91 through the second optical fiber 22. Further, the wavelength range allowed to pass by the fluorescence filter 31 is the central wavelength of the fluorescence emitted by the fluorescent molecule ± a second threshold value, the second threshold value is 0-15 nm, and preferably, the second threshold value is 2 nm.

[0102] In some preferred embodiments, reference Figure 1 The fluorescence generated by the fluorescent molecules generated by the laser on the liquid in the cuvette 7 is non-directional. In order to increase the amount of the fluorescent light signal entering the second optical fiber 22, a reflector 5 is set on the fourth side 734 or the third side 733. When used, the reflector 5 is arranged opposite to the fluorescent filter 31: when the fluorescent filter 31 is arranged on the third side 733, the reflector 5 is arranged on the fourth side 734; when the fluorescent filter 31 is arranged on the fourth side 734, the reflector 5 is arranged on the third side 733. Preferably, when the fluorescent filter 31 of the present invention is arranged on the third side 733, the reflector 5 is arranged on the fourth side 734. In addition, in order to further reduce the noise signal, a light trap 6 is arranged on the second side 732 to absorb the laser passing through the solution.

[0103] In some preferred embodiments, the output end of the photometer 91 is connected to the terminal server 93 through a third wire 92. When used, the terminal server 93 is a user end such as a computer, which can store real-time fluorescence intensity data for subsequent analysis of the detection data.

[0104] The irradiation assembly 4 of the present invention includes: a ray power supply, a second wire and a radiation source. When used, the ray power supply is arranged outside the shielding room 200, and the radiation source is arranged inside the shielding room 200 to avoid interference of the radiation source on the ray power supply and related circuits, so as to improve the accuracy of irradiation parameters.

[0105] In some preferred embodiments, a second through hole 202 matching the second electric wire is provided on the shielding room 200, and the second electric wire passes through the wall of the shielding room 200 through the second through hole 202, and the radiation source is connected to the ray power supply through the second electric wire to generate rays. When applied, the radiation source is arranged on one side of the top surface 71, and the angle between the generated ray and the normal direction of the top surface 71 is 0-30°. Preferably, the angle between the ray and the normal direction of the top surface 71 is 0°. In actual application, a shading plate 10 is also provided between the ray and the shading cover to further reduce the influence of external light on the detection results.

[0106] The method for evaluating free radical damage of the present invention can be implemented by the auxiliary system mentioned above. In addition, before using the auxiliary system, the tube body is filled with a test solution, the test solution includes an indicator, and the volume of the test solution is 80%-100% of the volume of the tube body.

[0107] In some preferred embodiments, the indicator is one or more combinations of calcein-AM, ethidium homodimer-1, aminophenyl fluorescein, hydroxyphenyl fluorescein, 2',7'-dichlorodihydrofluorescein diacetate, dihydroethidium and its derivatives, and the concentration of the indicator is: 1nmol / L-50mmol / L. Further, the indicator is hydroxyphenyl fluorescein or aminophenyl fluorescein, and the concentration of the indicator is 1-50mmol / L. Preferably, the indicator is hydroxyphenyl fluorescein, and the concentration of the indicator is 10mmol / L.

[0108] As the concentration of the indicator increases, the possibility of the indicator reacting with free radicals increases, thereby obtaining more fluorescent molecules. Therefore, within a certain concentration range, the fluorescence intensity is positively correlated with the concentration of the indicator. Figure 6 , when the indicator concentration is in the range of 0-10μmol / L, the fluorescence intensity is positively correlated with the indicator concentration, which shows that the free radical reaction is not saturated. Figure 6 The fitting curve in is: y=a+bx, where y represents the fluorescence intensity, x represents the indicator concentration, a is the intercept, and is 25.46097±5.83655, b is the slope, and is -38.34387±32.75755, and the Pearson correlation coefficient is 0.95126, which is close to 1, indicating that there is a strong positive correlation between the fluorescence intensity and the indicator concentration, and also illustrating the feasibility of the auxiliary system in the embodiment of the present invention.

[0109] refer to Figure 5 The method of the present invention includes steps S1 to S5.

[0110] Step S1: Start the laser assembly 1 to allow the laser to pass through the first side surface 731 and irradiate the solution to be tested.

[0111] When used, the test solution may also include cells to be tested or ferrous ions. The cells to be tested are normal cells or cancer cells. When used, the mortality rate or vitality of normal cells and cancer cells is evaluated by calcein-AM staining and red fluorescent ethidium homodimer-1 staining, which can characterize cell damage more quickly and provide guidance for subsequent clinical experiments.

[0112] In practical application, if the solution to be tested also includes ferrous ions, the concentration of ferrous ions is 0.1 μmol / L-10 mmol / L, and the Fenton reaction is used to convert hydrogen peroxide into detectable hydroxyl radicals. Those skilled in the art can adjust the concentration of cells to be tested according to actual conditions.

[0113] In practical application, the power of the laser component 1 is 1μW-1W, further, the power of the laser component 1 is 1-50mW, preferably, the power of the laser component 1 is 5mW. The wavelength of the laser is the maximum excitation wavelength of the fluorescent molecule ± the first threshold, the first threshold is 50nm, further, the first threshold is 5nm, preferably, the wavelength of the laser is 488nm.

[0114] Step S2: Start the photometric component 9 and detect the background fluorescence intensity of the solution to be tested in the cuvette 7 in combination with the fluorescence filter 31 .

[0115] Step S3: In response to the background fluorescence intensity changing speed being less than the preset intensity changing speed (at this time, the reaction system reaches a stable state, generally 0-150s after starting the photometric component 9), start the irradiation component 4, so that the radiation generated by the irradiation component 4 irradiates the solution to be tested to generate free radicals, which react with the indicator to generate fluorescent molecules.

[0116] When used, the radiation dose rate generated by the irradiation component 4 is 1Gy / s-100Gy / s, and the total radiation dose is 0.1-260Gy. Further, the radiation dose rate generated by the irradiation component 4 is 10-100Gy / s, and the total radiation dose is 1-80Gy. Preferably, the radiation dose rate generated by the irradiation component 4 is 40Gy / s, and the total radiation dose is 80Gy.

[0117] Step S4: Real-time detection of the fluorescence intensity of the fluorescent molecules, when the irradiation time of the radiation irradiating the solution to be tested is greater than or equal to a preset time, the irradiation component 4 is turned off.

[0118] When applied, the preset duration is calculated as the total radiation dose divided by the radiation dose rate.

[0119] Step S5: In response to the change rate of the real-time monitored fluorescence intensity being less than the preset intensity change rate (at this time, the free radical reaction can be considered complete, generally 0-500s after turning off the irradiation component 4), the free radical concentration is determined based on the detected fluorescence intensity and the background fluorescence intensity.

[0120] When applied, the preset intensity change speed is the noise amplitude before irradiation per unit time.

[0121] In practical applications, based on the positive correlation between fluorescence intensity and the total amount of free radical damage, the total amount of free radical damage is determined by measuring the fluorescence intensity; the free radical generation rate is further calculated based on the measurement of the free radical concentration; the oxidative damage rate generated by free radicals can be reflected by the rate of increase in fluorescence intensity.

[0122] The method of the present invention can be applied to the detection of cell mortality rate, the detection of cell viability and the detection of cell damage during the treatment of cancer cells.

[0123] Furthermore, the auxiliary system of the present invention can be used to develop new radiotherapy methods, and optimize the treatment plan by testing free radical parameters under different conditions. Specifically, the treatment plan with the greatest efficacy or the least cell killing can be found through experiments to improve the effect and safety of radiotherapy.

[0124] In addition, the auxiliary system of the present invention can be used for research on radiosensitization or reduction of radiation damage. By adjusting radiotherapy parameters to regulate the intensity of oxidative damage to different cells, it helps to optimize radiotherapy strategies and enhance patient benefits.

[0125] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0126] Based on the auxiliary system of the present invention, the above-mentioned steps 1 to 5 are used to complete Examples 1-5 and Comparative Example 1.

[0127] Example 1

[0128] The first optical fiber 21 has a diameter of 600 μm, and the second optical fiber 22 has a diameter of 200 μm and a length of 30 m.

[0129] The auxiliary system is not provided with a laser filter; the fluorescence filter 31 allows the wavelength to pass through to be 514nm±2nm.

[0130] The volume of the cuvette 7 is 1 mL, the volume of the solution to be tested is 0.9 mL, the indicator is hydroxyphenyl fluorescein, and the indicator concentration is 10 mmol / L.

[0131] The power of the laser component 1 is 5 mW, the laser wavelength is 488 nm, and the laser half-peak width is 1.5 nm.

[0132] The radiation dose rate was 6.0046 Gy / s, the total radiation dose was 260 Gy, and the irradiation time was 43.3 s.

[0133] Example 1 The fluorescence intensity monitored in real time is as follows Figure 7 shown.

[0134] Figure 7 In the figure, time point 041 indicates the start of radiation irradiation; time point 043 indicates the irradiation component 4 is turned off; in stage 042 between time point 041 and time point 043, the indicator is continuously oxidized to generate cumulative fluorescence intensity.

[0135] Combination Figure 7 It can be seen that after the irradiation component 4 is turned off, the reaction between the indicator and the free radicals basically stops, and no more fluorescent molecules are generated.

[0136] Example 2

[0137] The difference between Example 2 and Example 1 is that:

[0138] The solution to be tested also includes ferrous ions, and the concentration of ferrous ions is 1 mmol / L.

[0139] The radiation dose rate was 10 Gy / s and the total radiation dose was 40 Gy.

[0140] Example 2 The fluorescence intensity monitored in real time is as follows Figure 8 shown.

[0141] Figure 8 In the figure, time point 801 indicates the start of irradiation; time point 802 indicates the end of irradiation when the irradiation component 4 is turned off. During the period from the start of irradiation to the end of irradiation, the indicator is continuously oxidized to generate cumulative fluorescence intensity.

[0142] Combination Figure 8 It can be seen that the free radical reaction rate is significantly increased due to the presence of ferrous ions. When applied, ferrous ions catalyze the Fenton reaction of hydrogen peroxide, allowing hydrogen peroxide to be detected, and the signal-to-noise ratio is significantly improved.

[0143] Combination Figure 8 It can be seen that after the irradiation component 4 is turned off, the reaction between the indicator and the free radicals basically stops, and no more fluorescent molecules are generated.

[0144] Example 3

[0145] The difference between Example 3 and Example 1 is that:

[0146] The indicator is aminophenylfluorescein.

[0147] The solution to be tested also includes ferrous ions, and the concentration of ferrous ions is 1 mmol / L.

[0148] The radiation dose rate was 10 Gy / s and the total radiation dose was 40 Gy.

[0149] Example 3 Real-time monitoring of fluorescence intensity Fig. 9 shown.

[0150] Fig. 9 In the figure, time point 901 indicates the start of irradiation; time point 902 indicates the end of irradiation when the irradiation component 4 is turned off. During the period from the start of irradiation to the end of irradiation, the indicator is continuously oxidized to generate cumulative fluorescence intensity.

[0151] Combination Fig. 9 It can be seen that due to the presence of ferrous ions, the free radical reaction rate is significantly increased. When applied, ferrous ions catalyze the Fenton reaction of hydrogen peroxide, allowing hydrogen peroxide to be detected, and the signal-to-noise ratio is significantly improved. In actual application, the fluorescence intensity generated by aminophenyl fluorescein is stronger than that of hydroxyphenyl fluorescein, further improving the signal-to-noise ratio.

[0152] Combination Fig. 9 It can be seen that after the irradiation component 4 is turned off, the reaction between the indicator and the free radicals basically stops, and no more fluorescent molecules are generated.

[0153] Example 4

[0154] The difference between Example 4 and Example 1 is that:

[0155] The diameter of the first optical fiber 21 is 1500 μm.

[0156] The diameter of the second optical fiber 22 is 600 μm.

[0157] The indicator is aminophenylfluorescein.

[0158] The solution to be tested also includes ferrous ions, and the concentration of ferrous ions is 1 mmol / L.

[0159] The radiation dose rate was 40 Gy / s and the total radiation dose was 80 Gy.

[0160] Example 4 Real-time monitoring of fluorescence intensity Fig.10 shown.

[0161] Fig.10 In the figure, time point 1001 indicates the start of irradiation; time point 1002 indicates the end of irradiation when the irradiation component 4 is turned off. During the period from the start of irradiation to the end of irradiation, the indicator is continuously oxidized to generate cumulative fluorescence intensity.

[0162] Combination Fig.10It can be seen that due to the presence of ferrous ions, the free radical reaction rate is significantly increased. When applied, ferrous ions catalyze the Fenton reaction of hydrogen peroxide, allowing hydrogen peroxide to be detected, and the signal-to-noise ratio is significantly improved. In actual application, the fluorescence intensity generated by aminophenyl fluorescein is stronger than that of hydroxyphenyl fluorescein, further improving the signal-to-noise ratio.

[0163] Combination Fig.10 , it can be further known that: a larger diameter optical fiber can improve the signal strength of the output signal, thereby achieving the purpose of noise reduction. Based on the relationship between the diameter of the optical fiber and its bending resistance: the larger the diameter of the optical fiber, the worse its flexibility, so it is more susceptible to damage or performance degradation when bent. Through comparative experiments, it can be known that since the second optical fiber 22 is relatively long, the use of a second optical fiber 22 with a diameter of 600μm can effectively improve the output signal strength while taking into account the durability of the cost (not easy to break).

[0164] Combination Fig.10 It can be seen that after the irradiation component 4 is turned off, the reaction between the indicator and the free radicals basically stops, and no more fluorescent molecules are generated.

[0165] Example 5

[0166] The difference between Example 5 and Example 1 is that:

[0167] The indicator is 2',7'-dichlorodihydrofluorescein diacetate.

[0168] The solution to be tested also includes ferrous ions, and the concentration of ferrous ions is 1 mmol / L.

[0169] The radiation dose rate was 40 Gy / s and the total radiation dose was 80 Gy.

[0170] Example 5 Real-time monitoring of fluorescence intensity Fig.11 shown.

[0171] Fig.11 In the figure, time point 1101 indicates the start of irradiation; time point 1102 indicates the end of irradiation when the irradiation assembly 4 is turned off. During the period from the start of irradiation to the end of irradiation, the indicator is continuously oxidized to generate a cumulative fluorescence intensity.

[0172] Combination Fig.11 It can be seen that due to the presence of ferrous ions, the free radical reaction rate is significantly increased. When used, ferrous ions catalyze the hydrogen peroxide Fenton reaction so that hydrogen peroxide can be detected, and the signal-to-noise ratio is significantly improved. In actual application, since 2',7'-dichlorodihydrofluorescein diacetate is unstable, it is easy to self-oxidize in the air during storage, generating free radicals, making the background fluorescence intensity of the solution to be tested in the cuvette 7 containing the solution to be tested higher, reducing the accuracy of the detection.

[0173] Combination Fig.11 It can be seen that after the irradiation component 4 is turned off, the reaction between the indicator and the free radicals basically stops, and no more fluorescent molecules are generated.

[0174] Comparative Example 1

[0175] The difference between Comparative Example 1 and Example 1 is that:

[0176] The auxiliary system of Comparative Example 1 is not provided with the shielding room 200 .

[0177] Comparative Example 1 The fluorescence intensity monitored in real time is as follows Fig.12 shown.

[0178] Fig.12 In the figure, time point 1201 indicates the start of irradiation; at time point 1202, the irradiation component 4 is turned off and the irradiation ends.

[0179] Combination Fig.12 It can be seen that, since the shielding room 200 is not provided, the components of the photometric assembly 9 are disturbed by the radiation, resulting in inaccurate fluorescence intensity detection results.

[0180] In summary, the embodiments of the present invention are capable of real-time monitoring of free radical generation and accurate evaluation of the auxiliary system, method and application of free radical damage during radiotherapy, which are of great significance for improving the efficacy of radiotherapy and reducing normal tissue damage. At the same time, the auxiliary system in the embodiments of the present invention has the ability to resist radiation interference to ensure that relevant information on free radical damage, such as evaluation results, can be provided stably and accurately during radiotherapy.

[0181] An embodiment of the present invention further provides a radiotherapy system, which includes the auxiliary system for evaluating free radical damage and a particle accelerator for generating particles. The aforementioned irradiation component 4 for generating rays may be a part of a particle accelerator, and the particles are used to treat a patient's tumor, and may include but are not limited to protons. An embodiment of the present invention further provides a radiotherapy method, which includes implementing the aforementioned method for evaluating free radical damage to obtain an evaluation result of free radical damage; based on the evaluation result of free radical damage, adjusting the particle dose of the particle accelerator of the radiotherapy system in real time. Therefore, the radiotherapy method can achieve more precise treatment.

[0182] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, substitute and modify the above embodiments within the scope of the invention without departing from the principles and purpose of the present invention. All such changes should fall within the scope of protection of the claims of the present invention.

Claims

1. An auxiliary system for evaluating free radical damage, characterized in that: include A laser component (1), wherein the laser component (1) is used to generate laser light for exciting fluorescent molecules; A cuvette (7), the cuvette (7) comprising a tube body and a light shielding cover, the tube body comprising a side surface and a top surface (71) and a bottom surface (72) opposite to each other, the top surface (71) being provided with an opening matching the light shielding cover, the side surface comprising a first side surface (731) and a second side surface (732) opposite to each other, and a third side surface (733) and a fourth side surface (734) opposite to each other; A first optical fiber (21), one end of the first optical fiber (21) being connected to the laser assembly (1), and the other end of the first optical fiber (21) being connected to the first side surface (731); an irradiation component (4), the irradiation component (4) being used to generate radiation and being arranged on one side of the top surface (71); a fluorescent filter (32), the fluorescent filter (32) being arranged on the third side surface (733) or the fourth side surface (734); A photometric component (9), wherein the photometric component (9) is used to measure fluorescence intensity; A second optical fiber (22), one end of the second optical fiber (22) being connected to the fluorescent filter (32), and the other end of the second optical fiber (22) being connected to the photometric component (9); A chamber (100), wherein the chamber (100) is used to accommodate the cuvette (7) and the fluorescent filter (32).

2. The auxiliary system for evaluating free radical damage according to claim 1, characterized in that: The auxiliary system also includes: an excitation light filter (31), the excitation light filter (31) being arranged on the first side surface (731) and connected to the first optical fiber (21); a reflector (5), the reflector (5) being arranged on the third side surface (733) or the fourth side surface (734) and being arranged opposite to the fluorescent filter (32); a light trap (6), the light trap (6) being arranged on the second side surface (732); A shielding room (200), the shielding room (200) is used to accommodate part of the laser component (1), part of the irradiation component (4) and the chamber (100).

3. The auxiliary system for evaluating free radical damage according to claim 2, characterized in that: The tube body contains a solution to be tested, the solution to be tested includes an indicator, the radiation is used to irradiate the solution to be tested to generate free radicals, the free radicals are used to react with the indicator to generate the fluorescent molecules, and the angle between the radiation and the normal direction of the top surface (71) is 0-30°; The wavelength range allowed to pass by the excitation light filter (31) is the central wavelength of the excitation light of the fluorescent molecule ± a second threshold value; The wavelength range allowed to pass by the fluorescence filter (32) is the central wavelength of fluorescence emitted by the fluorescent molecule ± a second threshold.

4. The auxiliary system for evaluating free radical damage according to claim 2, characterized in that: The auxiliary system further comprises a base, wherein the base comprises: A lifting frame (81), wherein the lifting frame (81) is arranged in the shielding room (200); A vertical plate (82), wherein the vertical plate (82) is arranged on the lifting frame (81), so that the height of the vertical plate (82) can be adjusted by the lifting frame (81); A bracket (83), the bracket (83) is arranged on the vertical plate (82), and is used for placing the cuvette (7), the filter, and fixing the first optical fiber (21) and the second optical fiber (22).

5. The auxiliary system for assessing free radical damage according to claim 2, characterized in that: The photometric component (9) comprises: a photometer (91), the photometer (91) being connected to the second optical fiber (22); A terminal server (93), the terminal server (93) being communicatively connected to the photometer (91); The laser assembly (1) comprises: A laser power supply (11), wherein the laser power supply (11) is arranged outside the shielding room (200); a first electric wire (12), the first electric wire (12) being connected to the laser power supply (11) and passing through a wall of the shielding room (200); a laser diode (13), one end of the laser diode (13) being connected to the first electric wire (12), and the other end of the laser diode (13) being connected to the first optical fiber (21); The irradiation assembly (4) comprises: A radiation power supply, the radiation power supply being arranged outside the shielding room (200); a second electric wire, the second electric wire being connected to the radiation power supply and passing through the wall of the shielding room (200); a radiation source connected to the second electric wire; The shielding room (200) is provided with a first through hole (201) matching the first electric wire (12), a second through hole (202) matching the second electric wire, and a third through hole matching the second optical fiber (22); the second optical fiber (22) passes through the wall of the shielding room (200) through the third through hole; The first optical fiber (21) and the second optical fiber (22) are both single-mode optical fibers with a diameter of 10-1500 μm.

6. The auxiliary system for assessing free radical damage according to claim 5, characterized in that: The second optical fiber (22) is a light-transmitting optical fiber with a diameter of 160-1250 μm, and the light-transmitting optical fiber is a glass optical fiber or a plastic optical fiber.

7. A method for evaluating free radical damage, characterized in that The method is implemented based on the auxiliary system according to any one of claims 1 to 6; The tube body is filled with a solution to be tested, the solution to be tested includes an indicator, and the volume of the solution to be tested is 80%-100% of the volume of the tube body; The method comprises the following steps: Starting the laser assembly (1) to allow the laser to pass through the first side surface (731) and irradiate the solution to be tested; Starting the photometric component (9) and combining it with the fluorescent filter (32) to detect the background fluorescence intensity of the solution to be tested in the cuvette (7); In response to the change speed of the background fluorescence intensity being less than a preset intensity change speed, the irradiation component (4) is started, so that the radiation generated by the irradiation component (4) irradiates the solution to be tested to generate free radicals, which react with the indicator to generate fluorescent molecules; Real-time detection of the fluorescence intensity of the fluorescent molecules, when the irradiation time of the radiation irradiating the solution to be tested is greater than or equal to a preset time, the irradiation component (4) is turned off; In response to the fact that the change rate of the fluorescence intensity monitored in real time is less than the preset intensity change rate, the free radical concentration is determined according to the detected fluorescence intensity and the background fluorescence intensity.

8. The method for assessing free radical damage according to claim 7, characterized in that: The solution to be tested also includes cells to be tested, and the cells to be tested are normal cells or cancer cells.

9. The method for assessing free radical damage according to claim 7, characterized in that: The volume of the cuvette is 0.1-3 mL; The angle between the incident light of the laser and the outgoing light thereof is 60-90°; The power of the laser component (1) is 1 μW-1 W; The wavelength of the laser is the maximum excitation wavelength of the fluorescent molecule ± the first threshold; The angle between the ray and the normal direction of the top surface (71) is 0°; The indicator comprises one or a combination of calcein-AM, ethidium homodimer-1, aminophenyl fluorescein, hydroxyphenyl fluorescein, 2',7'-dichlorodihydrofluorescein diacetate, dihydroethidium and derivatives thereof; The concentration of the indicator is: 1nmol / L-1mmol / L; The irradiation component (4) generates a radiation dose rate of 1 Gy / s-100 Gy / s and a total radiation dose of 0.1-260 Gy.

10. The method for evaluating free radical damage according to claim 9, characterized in that: The power of the laser component (1) is 1-50 mW; The first threshold is 50 nm; The concentration of the indicator is 1-50mmol / L; The indicator is hydroxyphenylfluorescein or aminophenylfluorescein; The radiation dose rate generated by the irradiation component (4) is 10-100 Gy / s and the total radiation dose is 1-80 Gy.

11. The method for assessing free radical damage according to claim 10, characterized in that: The power of the laser component (1) is 5 mW; The wavelength of the laser is 488nm; The concentration of the indicator is 10mmol / L; The first threshold is 5 nm; The indicator is hydroxyphenylfluorescein; The irradiation component (4) generates a radiation dose rate of 40 Gy / s and a total radiation dose of 80 Gy.

12. Use of the method according to any one of claims 7 to 11 in detecting cell mortality, cell viability and cell damage during the treatment of cancer cells.

13. A radiotherapy system, characterized in that: The radiotherapy system comprises the auxiliary system for assessing free radical damage according to any one of claims 1-6.

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