Radiotherapy system, auxiliary system for assessing free radical damage, method and use thereof

Abstract

The application discloses a radiotherapy system, an auxiliary system for evaluating free radical damage, a method and application thereof, wherein a tube body of a cuvette of the auxiliary system comprises a side surface, a top surface and a bottom surface, the top surface is provided with an opening matched with a light shielding cover, and the side surface comprises a first side surface, a second side surface, a third side surface and a fourth side surface; a first optical fiber is connected at one end with a laser assembly and at the other end with the first side surface; an irradiation assembly is arranged at one side of the top surface; a fluorescent filter is arranged on the third side surface or the fourth side surface; one end of a second optical fiber is connected with the fluorescent filter, and the other end of the second optical fiber is connected with a photometric assembly; and a cavity is used for accommodating the cuvette and the fluorescent filter. For FLASH radiotherapy, the application can realize real-time and accurate detection of fluorescence intensity by optimizing a detection environment, and provides a reliable platform for free radical damage evaluation.

Description

Technical Field

[0001] This invention relates to the technical field of free radical damage assessment, and more particularly to radiotherapy systems, auxiliary systems and methods for assessing free radical damage, and their applications. Background Technology

[0002] In the medical field, radiotherapy, as a crucial method for treating tumors, has always been a hot research topic in terms of improving its efficacy. While traditional radiotherapy methods can effectively inhibit tumor growth to some extent, they also cause some damage to normal tissues. With advancements in technology, developing novel and highly effective radiotherapy methods, such as FLASH proton therapy, aimed at improving treatment outcomes while minimizing damage to normal tissues, has become a key research focus. The development of these new methods not only holds the promise of benefiting more patients but also enhancing the overall level of tumor treatment.

[0003] The cell-killing effects of radiotherapy are mainly divided into two mechanisms: direct killing and indirect killing. Direct killing refers to the direct action of high-energy rays on intracellular biomolecules, such as DNA, leading to structural damage and loss of function. Indirect killing, on the other hand, involves free radicals generated by ionizing radiation attacking intracellular biomolecules, causing damage. Therefore, accurately assessing the degree of free radical damage during radiotherapy is of great significance for optimizing radiotherapy protocols and improving treatment outcomes.

[0004] Currently, the assessment of free radical damage mainly relies on time-delayed monitoring methods, but this approach has many limitations. Because free radical reactions are typically rapid, time-delayed monitoring often fails to accurately capture the generation and changes of free radicals, thus affecting the accuracy of the assessment. Developing a method capable of real-time monitoring of free radical generation is crucial for evaluating the tumor-killing effects of radiotherapy regimens.

[0005] Therefore, this application proposes a radiotherapy system, an auxiliary system for assessing free radical damage, a method thereof, and its application. Summary of the Invention

[0006] The purpose of this invention is to provide a radiotherapy system, an auxiliary system for assessing free radical damage, a method thereof, and their applications, which can achieve accurate detection of fluorescence emphasis by optimizing the detection environment, thus providing a reliable platform for assessing free radical damage.

[0007] The objective of this invention is achieved through the following technical solution:

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

[0009] A laser assembly for generating a laser to excite fluorescent molecules;

[0010] A cuvette, comprising a tube and a light-shielding cap, wherein the tube comprises a side surface and a top surface and a bottom surface opposite to each other, the top surface having an opening that matches the light-shielding cap, and the side surface 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 assembly for generating rays and disposed on one side of the top surface;

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

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

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

[0016] A chamber for housing the cuvette and the fluorescent filter.

[0017] Furthermore, the auxiliary system also includes:

[0018] An excitation light filter is disposed on the first side and connected to the first optical fiber;

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

[0020] An optical trap is disposed on the second side.

[0021] Furthermore, the tube contains a test solution, which includes an indicator. The radiation is used to irradiate the test solution to generate free radicals. The free radicals react with the indicator to generate the fluorescent molecules. The angle between the radiation and the normal direction of the top surface is 0-30°.

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

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

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

[0025] A lifting frame, wherein the lifting frame is disposed in the shielded room whose height can be adjusted by the lifting frame;

[0026] An upright plate is mounted on the lifting frame so that the height of the upright plate can be adjusted by the lifting frame;

[0027] A support is provided on the upright plate for placing the cuvette, the filter, and fixing the first and second optical fibers.

[0028] Furthermore, the photometric component includes:

[0029] A photometer, wherein the photometer is connected to the second optical fiber;

[0030] Terminal server, which is communicatively connected to the photometer;

[0031] The laser component includes:

[0032] A laser power supply, wherein the laser power supply is located outside the shielded room;

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

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

[0035] The irradiation assembly includes:

[0036] A radiation power supply, wherein the radiation power supply is located outside the shielding room;

[0037] The second wire is connected to the radiation power supply and passes through the wall of the shielded room;

[0038] A radiation source, wherein the radiation source is connected to the second wire;

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

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

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

[0042] Secondly, the present invention provides a method for assessing free radical damage, the method being implemented based on the aforementioned auxiliary system;

[0043] The tube contains a test solution, which includes an indicator, and the volume of the test solution is 80%-100% of the tube volume.

[0044] The method includes the following steps:

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

[0046] The photometric component is activated, and in conjunction with the fluorescence filter, the background fluorescence intensity of the solution to be tested in the cuvette is detected.

[0047] When the rate of change of the background fluorescence intensity is less than the preset rate of change of intensity, the irradiation component is activated, and the rays generated by the irradiation component irradiate the test solution, generating free radicals. The free radicals react with the indicator to generate fluorescent molecules.

[0048] The fluorescence intensity of fluorescent molecules is detected in real time, and the irradiation component is turned off when the irradiation time of the test solution is greater than or equal to a preset duration.

[0049] When the rate of change of fluorescence intensity monitored in real time is less than the preset rate of change of intensity, the free radical concentration is determined based on the detected fluorescence intensity and the background fluorescence intensity.

[0050] Furthermore, the test solution also includes test cells, which can be normal cells or cancer cells.

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

[0052] The angle between the incident light and the outgoing light of the laser 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 molecules ± the first threshold.

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

[0056] The indicator includes one or more of the following: calcein-AM, ethidium homodimer-1, aminophenyl fluorescein, hydroxyphenyl fluorescein, 2',7'-dichlorodihydrofluorescein diacetate, ethidium dihydrofluorescein and its derivatives.

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

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

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

[0060] The first threshold is 50nm;

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

[0062] The indicator is hydroxyphenylfluorescein or aminophenylfluorescein;

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

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

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

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

[0067] The first threshold is 5nm;

[0068] The indicator is hydroxyphenylfluorescein;

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

[0070] Thirdly, the present invention provides the application of the above method in the detection of cell death rate, the detection of cell viability, and the detection of cell damage during the treatment of cancer cells.

[0071] Fourthly, the present invention provides a radiotherapy system comprising the aforementioned auxiliary system for assessing free radical damage.

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

[0073] The chamber of the auxiliary system of this invention can reduce the interference of external light on the detection results; the shielding chamber not only shields the circuit and photometric components from radiation interference, but also further reduces 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 lighting light, while being easy to open and close, facilitating the addition of the test solution and cleaning; the irradiation component's rays are designed to be parallel to or at a certain angle to the normal direction of the top surface, such as 0-30°, which facilitates the rays to enter from the top surface and exit from the bottom surface, interacting with the test solution. The solution achieves maximum contact, allowing the radiation to uniformly irradiate the test solution within the cuvette, further improving the uniformity and efficiency of free radical generation. The laser assembly guides the laser to the first side of the cuvette via a first optical fiber, ensuring the generation of fluorescent molecules. A fluorescence filter on the third side effectively filters out background and stray light, allowing only specific wavelengths of fluorescence to pass through, thereby improving the sensitivity and accuracy of fluorescence detection. A second optical fiber transmits the fluorescence signal to a photometric assembly, which can measure fluorescence intensity in real time and accurately, providing reliable data support for free radical damage assessment. Real-time monitoring not only provides crucial information such as the rate and concentration of free radical generation but also helps doctors more accurately understand the dynamic changes of free radicals during radiotherapy, providing strong support for developing personalized treatment plans.

[0074] The method of this invention utilizes the radiation from an irradiation component to irradiate the test solution, generating free radicals. These free radicals react with an indicator to produce fluorescent molecules. By using a photometric component combined with a fluorescence filter, the fluorescence intensity of the fluorescent molecules can be detected in real time and accurately, thereby achieving an accurate assessment of the free radical concentration. Furthermore, the volume of the test solution is precisely controlled to 80%-100% of the tube volume, reducing interference caused by bubble movement due to insufficient solution volume in the cuvette during the experiment, thus minimizing detection errors. In addition, by detecting background fluorescence intensity, the influence of background fluorescence on the detection results can be eliminated, improving data reliability. Furthermore, by monitoring the rate of change of fluorescence intensity in real time and determining the free radical concentration when the rate of change is less than a preset value, the actual free radical concentration can be reflected more accurately, reducing deviations caused by time delays or measurement errors. Attached Figure Description

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

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

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

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

[0079] Figure 5 This is a flowchart illustrating the method for evaluating free radical damage according to an embodiment of the present invention.

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

[0081] Figure 7 This is a graph showing the fluorescence intensity monitored in real time according to Embodiment 1 of the present invention.

[0082] Figure 8 This is a graph showing the fluorescence intensity monitored in real time in Embodiment 2 of the present invention.

[0083] Figure 9 This is a graph showing the fluorescence intensity monitored in real time in Embodiment 3 of the present invention.

[0084] Figure 10 This is a graph showing the fluorescence intensity monitored in real time in Embodiment 4 of the present invention.

[0085] Figure 11 This is a graph showing the fluorescence intensity monitored in real time in Embodiment 5 of the present invention.

[0086] Figure 12 This is a graph showing the fluorescence intensity monitored in real time in Comparative Example 1 of this invention.

[0087] In the diagram: 1. Laser component; 10. Light shield; 11. Laser power supply; 12. First wire; 13. Laser diode; 21. First optical fiber; 22. Second optical fiber; 31. Excitation light filter; 32. Fluorescent filter; 4. Irradiation component; 5. Reflector; 6. Optical 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. Support; 9. Photometric component; 91. Photometer; 92. Third wire; 93. Terminal server; 100. Chamber; 200. Shielded room; 201. First through hole; 202. Second through hole. Detailed Implementation

[0088] Exemplary embodiments will now be described more fully with reference to the accompanying drawings. However, these exemplary embodiments can be implemented in many forms and should not be construed as limited to the embodiments set forth herein; rather, they are provided to make the invention more comprehensive and complete, and to fully convey the concept of the exemplary embodiments to those skilled in the art. The same reference numerals in the drawings denote the same or similar structures, and therefore repeated descriptions of them will be omitted.

[0089] The terms used to express position and direction in this invention are illustrated with reference to the accompanying drawings, but changes can be made as needed, and all such changes are included within the scope of protection of this invention.

[0090] Under the same particle type, energy, and total dose conditions, the direct cell-killing effect (direct effect) is not significantly different between FLASH particle radiotherapy (also known as flash therapy) and conventional particle radiotherapy. However, their indirect cell-killing effects (indirect effects) differ considerably. This is mainly because different dose rates affect the generation and recombination rate of free radicals in solution, thus determining the strength of the indirect effect. The particles may include, but are not limited to, protons.

[0091] The auxiliary system for assessing free radical damage in this embodiment of the invention includes a laser assembly 1, a cuvette 7, a first optical fiber 21, an irradiation assembly 4, a photometric assembly 9, a second optical fiber 22, and a chamber 100. Further, the auxiliary system may also include a shielding chamber 200. Further, the auxiliary system may also include an excitation light filter 31, a reflector 5, and an optical trap 6. Even further, the auxiliary system 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 cap. The tube body includes side surfaces and a top surface 71 and a bottom surface 72 that are opposite each other. Further, the top surface 71 has an opening that matches the light-shielding cap, which snaps onto the opening to form a sealed cavity with the tube body. In addition, the side surfaces include a first side surface 731 and a second side surface 732 that are opposite each other, and a third side surface 733 and a fourth side surface 734 that are opposite each other.

[0093] In some preferred embodiments, the cuvette 7 can be various shapes such as cuboid, cube, or cylinder. More preferably, the cuvette 7 is a centrifuge tube with a volume of 0.1-3 mL. In application, the cuvette 7 contains a test solution, which includes an indicator. The irradiation component 4 irradiates the test solution to generate free radicals, which react with the indicator to produce fluorescent molecules.

[0094] refer to Figure 3 The base of the present invention includes: a lifting frame 81, a vertical plate 82, and a support 83. In application, the base is disposed within the chamber 100.

[0095] In some preferred embodiments, the upright plate 82 has an L-shaped structure, with one side of the L-shaped upright plate 82 mounted on the lifting frame 81 and the other side of the L-shaped upright plate 82 freely mounted on the lifting frame 81, and a bracket 83 installed on the free side of the L-shaped upright plate 82. In application, the bracket 83 matches the size of the cuvette 7 and is used to hold the cuvette 7. In practical applications, the height between the cuvette 7 and the ground can be adjusted by adjusting the lifting frame 81 according to actual needs, allowing the laser and radiation to better irradiate the liquid inside the cuvette 7, thereby improving the accuracy and precision of the detection results.

[0096] refer to Figure 2 The laser assembly 1 of the present invention includes a laser power supply 11, a first wire 12, and a laser diode 13. In application, the laser diode 13 is disposed inside a shielded room 200, and the laser power supply 11 is disposed outside the shielded room 200, so as to avoid interference from the irradiation assembly 4 to the laser power supply 11 and related circuits, thereby improving the accuracy of the laser emission parameters.

[0097] In some preferred embodiments, a first through-hole 201 matching the first wire 12 is provided on the shielding chamber 200. The first wire 12 passes through the first through-hole 201 and penetrates the wall of the shielding chamber 200. 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 731 to transmit the incident laser light to the cuvette 7 through the first optical fiber 21. In application, the diameter of the optical fiber is increased to improve the optical signal intensity. Specifically, the diameter of the first optical fiber 21 is a single-mode optical fiber with a diameter of 10-1500 μm, preferably 600 μm.

[0098] In some preferred embodiments, to prevent non-ideal wavelength laser light from entering the cuvette 7, an excitation light filter 31 is provided on the first side 731, which filters the optical signal in the first optical fiber 21. In application, the wavelength range allowed to pass through the excitation light filter 31 is the center wavelength of the fluorescent molecule excitation light ± a second threshold, where the second threshold is 0-15 nm, preferably 2 nm. In practical applications, the angle between the incident and emitted laser 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. In application, the photometric component 9 is placed outside the shielded room 200 to avoid interference from the irradiation component 4 on the components and related circuits of the photometric component 9, as well as its impact on the detection results.

[0100] In some preferred embodiments, the photometer 91 is a photometer capable of communication transmission, and its input end is connected to the second optical fiber 22. In application, to increase the optical signal intensity, 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 transparent optical fiber with a diameter of 160-1250 μm, which can be glass or plastic optical fiber. Preferably, the diameter of the second optical fiber 22 is 200 μm and its length is 30 m.

[0101] In some preferred embodiments, to reduce interference signals 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. 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 by the fluorescence filter 31 is the center wavelength of fluorescence emitted by the fluorescent molecule ± a second threshold, where the second threshold is 0-15 nm, preferably 2 nm.

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

[0103] In some preferred embodiments, the output of the photometer 91 is communicatively connected to the terminal server 93 via a third wire 92. In application, the terminal server 93 is a user terminal such as a computer, capable of storing real-time detected fluorescence intensity data for subsequent analysis.

[0104] The irradiation assembly 4 of the present invention includes: a radiation power supply, a second wire, and a radiation source. In application, the radiation power supply is located outside the shielded room 200, and the radiation source is located inside the shielded room 200 to avoid interference from the radiation source to the radiation power supply and related circuits, thereby improving the accuracy of the irradiation parameters.

[0105] In some preferred embodiments, the shielding chamber 200 has a second through-hole 202 that matches the second wire. The second wire passes through the second through-hole 202 and penetrates the wall of the shielding chamber 200. The radiation source is connected to a radiation power supply through the second wire to generate radiation. In application, the radiation source is located on one side of the top surface 71, and the angle between the generated radiation and the normal direction of the top surface 71 is 0-30°. Preferably, the angle between the radiation and the normal direction of the top surface 71 is 0°. In practical applications, a light-shielding plate 10 is also provided between the radiation and the light-shielding cover to further reduce the influence of external light on the detection results.

[0106] The method for assessing free radical damage according to the present invention can be implemented using the aforementioned auxiliary system. Furthermore, before using the auxiliary system, the tube is filled with a test solution containing an indicator, and the volume of the test solution is 80%-100% of the tube volume.

[0107] In some preferred embodiments, the indicator is one or a combination of several selected from calcein-AM, ethidium homodimer-1, aminophenyl fluorescein, hydroxyphenyl fluorescein, 2',7'-dichlorodihydrofluorescein diacetate, ethidium dihydrofluorescein, and their derivatives, and the concentration of the indicator is 1 nmol / L-50 mmol / L. Further, the indicator is hydroxyphenyl fluorescein or aminophenyl fluorescein, and the concentration of the indicator is 1-50 mmol / L. Preferably, the indicator is hydroxyphenyl fluorescein, and the concentration of the indicator is 10 mmol / L.

[0108] As the indicator concentration increases, the likelihood of reaction between the indicator and free radicals increases, resulting in more fluorescent molecules. Consequently, within a certain concentration range, the fluorescence intensity is positively correlated with the indicator concentration. (Reference) Figure 6 Within the concentration range of 0-10 μmol / L, the fluorescence intensity of the indicator showed a positive linear correlation with the indicator concentration, indicating that the free radical reaction was not saturated at this point. Specifically, Figure 6 The fitted curve is: y = a + bx, where y represents fluorescence intensity, x represents indicator concentration, a is the intercept (25.46097 ± 5.83655), b is the slope (-38.34387 ± 32.75755), and the Pearson correlation coefficient is 0.95126, close to 1, indicating a strong positive correlation between fluorescence intensity and indicator concentration, and also demonstrating the feasibility of the auxiliary system in the embodiments of the present invention.

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

[0110] Step S1: Activate the laser assembly 1 so that the laser passes through the first side 731 and irradiates the solution to be tested.

[0111] In application, the test solution may also include test cells or ferrous ions. The test cells can be normal cells or cancer cells. When applied, calcein-AM staining and etidium homodimer-1 staining (which emits red fluorescence) are used to assess the mortality or viability of normal and cancer cells, enabling faster characterization of cell damage and providing guidance for subsequent clinical trials.

[0112] In practical applications, if the test solution also contains ferrous ions (0.1 μmol / L–10 mmol / L), the Fenton reaction can be used to convert hydrogen peroxide into detectable hydroxyl radicals. Those skilled in the art can adjust the concentration of the test cells according to the specific circumstances.

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

[0114] Step S2: Activate the photometric component 9, and in conjunction with the fluorescence filter 31, detect the background fluorescence intensity of the test solution in the cuvette 7.

[0115] Step S3: When the rate of change of background fluorescence intensity is less than the preset rate of change of intensity (at this time, the reaction system reaches a stable state, generally 0-150s after the photometric component 9 is started), the irradiation component 4 is started, so that the rays generated by the irradiation component 4 irradiate the test solution, generating free radicals. The free radicals react with the indicator to generate fluorescent molecules.

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

[0117] Step S4: Real-time detection of fluorescence intensity of fluorescent molecules. When the irradiation time of the test solution is greater than or equal to the preset time, turn off the irradiation component 4.

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

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

[0120] When applying the application, the preset intensity change rate 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 fluorescence intensity; based on the measurement of free radical concentration, the free radical generation rate is further calculated; the rate of increase in fluorescence intensity can reflect the rate of oxidative damage caused by free radicals.

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

[0123] Furthermore, the auxiliary system of this invention can be used to develop new radiotherapy methods by optimizing treatment protocols through testing free radical parameters under different conditions. Specifically, it can be used to experimentally identify treatment protocols with the greatest efficacy or the least cell-killing effect, thereby improving the effectiveness and safety of radiotherapy.

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

[0125] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0126] Based on the auxiliary system of the present invention, steps 1-5 of the above method 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 does not have a laser filter; the fluorescent filter 31 allows a wavelength of 514nm ± 2nm to pass through.

[0130] The volume of cuvette 7 is 1 mL, the volume of the test solution is 0.9 mL, the indicator is hydroxyphenylfluorescein, and the indicator concentration is 10 mmol / L.

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

[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: Real-time monitoring of fluorescence intensity as follows Figure 7 As shown.

[0134] Figure 7 In the diagram, time point 041 indicates the start of radiation irradiation; time point 043 indicates the shutdown of irradiation component 4; and in the 042 phase between time points 041 and 043, the indicator is continuously oxidized, producing cumulative fluorescence intensity.

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

[0136] Example 2

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

[0138] The test solution also contains ferrous ions, and the concentration of ferrous ions is 1 mmol / L.

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

[0140] Example 2: Real-time monitoring of fluorescence intensity as follows Figure 8 As shown.

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

[0142] Combination Figure 8 It is evident that the presence of ferrous ions significantly accelerates the free radical reaction. In practical applications, the ferrous ion-catalyzed Fenton reaction of hydrogen peroxide enables its detection, resulting in a significantly improved signal-to-noise ratio.

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

[0144] Example 3

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

[0146] The indicator is aminophenylfluorescein.

[0147] The test solution also contains ferrous ions, and the concentration of ferrous ions is 1 mmol / L.

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

[0149] Example 3: Real-time monitoring of fluorescence intensity as follows Figure 9 As shown.

[0150] Figure 9 In the diagram, time point 901 indicates the start of radiation irradiation; time point 902 indicates the irradiation component 4 is turned off, ending the irradiation. During the period from the start of irradiation to the end of irradiation, the indicator is continuously oxidized, producing a cumulative fluorescence intensity.

[0151] Combination Figure 9 It is evident that the presence of ferrous ions significantly accelerates the free radical reaction. In practical applications, the ferrous ion-catalyzed Fenton reaction of hydrogen peroxide enables its detection, resulting in a significantly improved signal-to-noise ratio. Furthermore, in real-world applications, aminophenyl fluorescein exhibits stronger fluorescence intensity than hydroxyphenyl fluorescein, further enhancing the signal-to-noise ratio.

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

[0153] Example 4

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

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

[0156] The second optical fiber 22 has a diameter of 600 μm.

[0157] The indicator is aminophenylfluorescein.

[0158] The test solution also contains 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 as follows Figure 10 As shown.

[0161] Figure 10 In the diagram, time point 1001 indicates the start of radiation irradiation; time point 1002 indicates the irradiation component 4 is turned off, ending the irradiation. During the period from the start of irradiation to the end of irradiation, the indicator is continuously oxidized, producing a cumulative fluorescence intensity.

[0162] Combination Figure 10It is evident that the presence of ferrous ions significantly accelerates the free radical reaction. In practical applications, the ferrous ion-catalyzed Fenton reaction of hydrogen peroxide enables its detection, resulting in a significantly improved signal-to-noise ratio. Furthermore, in real-world applications, aminophenyl fluorescein exhibits stronger fluorescence intensity than hydroxyphenyl fluorescein, further enhancing the signal-to-noise ratio.

[0163] Combination Figure 10 Furthermore, it can be seen that larger diameter optical fibers can improve the signal strength of the output signal, thereby achieving noise reduction. Based on the relationship between optical fiber diameter and its bending resistance: the larger the diameter of the optical fiber, the worse its flexibility, and therefore the more susceptible to damage or performance degradation when bent. Through comparative experiments, it can be seen that, since the second optical fiber 22 is relatively long, using a second optical fiber 22 with a diameter of 600μm can effectively improve the output signal strength while taking into account durability (not easily broken).

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

[0165] Example 5

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

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

[0168] The test solution also contains 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 as follows Figure 11 As shown.

[0171] Figure 11 In the diagram, time point 1101 indicates the start of radiation irradiation; time point 1102 indicates the irradiation component 4 is turned off, ending the irradiation. During the period from the start of irradiation to the end of irradiation, the indicator is continuously oxidized, producing a cumulative fluorescence intensity.

[0172] Combination Figure 11 It is evident that the presence of ferrous ions significantly accelerates the free radical reaction. In practical applications, the ferrous ion-catalyzed Fenton reaction of hydrogen peroxide enables its detection, resulting in a significantly improved signal-to-noise ratio. However, in real-world applications, the instability of 2',7'-dichlorodihydrofluorescein diacetate, which readily undergoes auto-oxidation in air during storage, generates free radicals. This leads to a higher background fluorescence intensity of the test solution in cuvette 7, reducing detection accuracy.

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

[0174] Comparative Example 1

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

[0176] The auxiliary system in Comparative Example 1 did not have a shielded room of 200.

[0177] Comparative Example 1: Real-time monitoring of fluorescence intensity as follows Figure 12 As shown.

[0178] Figure 12 In the diagram, time point 1201 indicates the start of irradiation; time point 1202 indicates the shutdown of irradiation component 4, marking the end of irradiation.

[0179] Combination Figure 12 It can be seen that, due to the lack of a shielding chamber 200, the components of the photometric component 9 are affected by radiation, resulting in inaccurate fluorescence intensity detection results.

[0180] In summary, the embodiments of the present invention provide an auxiliary system, method, and application for real-time monitoring of free radical generation and accurate assessment of free radical damage during radiotherapy, which is of great significance for improving the efficacy of radiotherapy and reducing damage to normal tissues. Furthermore, the auxiliary system in the embodiments of the present invention has the ability to resist radiation interference, ensuring that relevant information on free radical damage, such as assessment results, can be provided stably and accurately during radiotherapy.

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

[0182] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the invention without departing from the principles and spirit of the invention, and all such changes should fall within the protection scope of the claims of the present invention.

Claims

1. An auxiliary system for assessing free radical damage, characterized in that, include Laser component (1), said laser component (1) is used to generate a laser to excite fluorescent molecules; A cuvette (7) includes a tube and a light-shielding cap. The tube includes a side surface and a top surface (71) and a bottom surface (72) that are opposite each other. The top surface (71) has an opening that matches the light-shielding cap. The side surface includes a first side surface (731) and a second side surface (732) that are opposite each other, and a third side surface (733) and a fourth side surface (734) that are opposite each other. A first optical fiber (21) is connected at one end to the laser assembly (1) and at the other end to the first side (731). Irradiation assembly (4), which is used to generate rays and is disposed on one side of the top surface (71); A fluorescent filter (32) is disposed on the third side (733) or the fourth side (734); Photometric component (9), the photometric component (9) being used to measure fluorescence intensity; The second optical fiber (22) has one end connected to the fluorescent filter (32) and the other end connected to the photometric component (9). A chamber (100) for accommodating the cuvette (7) and the fluorescent filter (32).

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

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

4. The auxiliary system for assessing free radical damage according to claim 2, characterized in that, The auxiliary system also includes a base, the base comprising: A lifting frame (81) is installed inside the shielded room (200); A vertical plate (82) is mounted on the lifting frame (81) so that the height of the vertical plate (82) can be adjusted by the lifting frame (81); A support (83) is provided on the upright plate (82) 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) includes: A photometer (91) is connected to the second optical fiber (22); Terminal server (93), which is communicatively connected to the photometer (91); The laser component (1) includes: A laser power supply (11) is located outside the shielded room (200); The first wire (12) is connected to the laser power supply (11) and passes through the wall of the shielding room (200); A laser diode (13) is provided, one end of which is connected to the first wire (12), and the other end of which is connected to the first optical fiber (21). The irradiation assembly (4) includes: A radiation power supply is provided outside the shielding room (200); The second wire is connected to the radiation power source and passes through the wall of the shielding room (200); A radiation source, wherein the radiation source is connected to the second wire; The shielding chamber (200) is provided with a first through hole (201) matching the first wire (12), a second through hole (202) matching the second wire, and a third through hole matching the second optical fiber (22). The second optical fiber (22) passes through the wall of the shielding chamber (200) through the third through hole. Both the first optical fiber (21) and the second optical fiber (22) are single-mode optical fibers with a diameter of 10-1500μm.

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 160-1250μm, which is a glass optical fiber or a plastic optical fiber.

7. A method for assessing free radical damage, characterized in that, The method is implemented based on the auxiliary system described in any one of claims 1-6; The tube contains a test solution, which includes an indicator, and the volume of the test solution is 80%-100% of the tube's volume. The method includes the following steps: Activate the laser assembly (1) to allow the laser to pass through the first side (731) and irradiate the solution to be tested; Activate the photometric component (9), and in conjunction with the fluorescence filter (32), detect the background fluorescence intensity of the test solution in the cuvette (7); When the rate of change of the background fluorescence intensity is less than the preset rate of change of intensity, the irradiation component (4) is activated, and the rays generated by the irradiation component (4) irradiate the test solution, generating free radicals. The free radicals react with the indicator to generate fluorescent molecules. The fluorescence intensity of the fluorescent molecules is detected in real time. When the irradiation time of the test solution by the ray is greater than or equal to the preset time, the irradiation component is turned off (4). When the rate of change of fluorescence intensity monitored in real time is less than the preset rate of change of intensity, the free radical concentration is determined based on 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 test solution also includes test cells, which can be 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 and the outgoing light of the laser is 60-90°; The power of the laser component (1) is 1μW-1W; The wavelength of the laser is the maximum excitation wavelength of the fluorescent molecules ± the first threshold. The angle between the ray and the normal direction of the top surface (71) is 0°; The indicator includes one or more of the following: calcein-AM, ethidium homodimer-1, aminophenyl fluorescein, hydroxyphenyl fluorescein, 2',7'-dichlorodihydrofluorescein diacetate, ethidium dihydrofluorescein and its derivatives. The concentration of the indicator is 1 nmol / L - 1 mmol / L; The radiation dose rate generated by the irradiation component (4) is 1 Gy / s-100 Gy / s, and the total radiation dose is 0.1-260 Gy.

10. The method for assessing free radical damage according to claim 9, characterized in that, The power of the laser component (1) is 1-50mW; The first threshold is 50nm; The concentration of the indicator is 1-50 mmol / 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 5mW; The wavelength of the laser is 488nm; The concentration of the indicator is 10 mmol / L; The first threshold is 5nm; The indicator is hydroxyphenylfluorescein; The radiation dose rate generated by the irradiation component (4) is 40 Gy / s, and the total radiation dose is 80 Gy.

12. A radiotherapy system, characterized in that, The radiotherapy system includes an auxiliary system for assessing free radical damage as described in any one of claims 1-6.

Images