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

Abstract

Provided in the present application are a radiotherapy system, an auxiliary system and method for assessing free radical damage, and the uses thereof. The auxiliary system comprises: a cuvette, wherein a tube body of the cuvette comprises side surfaces, a top surface and a bottom surface, the top surface is provided with an opening matching a light-shielding cover, and the side surfaces comprise a first side surface, a second side surface, a third side surface and a fourth side surface; a first optical fiber, one end of the first optical fiber being connected to a laser assembly, and the other end of the first optical fiber being connected to the first side surface; an irradiation assembly, provided on one side of the top surface; a fluorescence filter, provided on the third side surface or the fourth side surface; a second optical fiber, one end of the second optical fiber being connected to the fluorescence filter, and the other end of the second optical fiber being connected to a photometric assembly; and a chamber, used for accommodating the cuvette and the fluorescence filter. For FLASH radiotherapy, the present application can implement real-time and accurate measurement of fluorescence intensity by optimizing the measurement environment, thereby providing a reliable platform for assessing free radical damage.

Description

Radiotherapy systems, auxiliary systems for assessing free radical damage, methods and their applications

[0001] This application claims priority to Chinese Patent Application No. 202411564444.8, filed on November 5, 2024, which is incorporated herein by reference in its entirety. Technical Field

[0002] This application relates to the technical field of free radical damage assessment, such as to radiotherapy systems, auxiliary systems, methods, and applications for assessing free radical damage. Background Technology

[0003] 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.

[0004] 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.

[0005] 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.

[0006] Based on this, this application provides a radiotherapy system, an auxiliary system for assessing free radical damage, a method thereof, and their applications. Summary of the Invention

[0007] The purpose of this application is to provide a radiotherapy system, an auxiliary system for assessing free radical damage, a method thereof, and its application, which can achieve accurate detection of fluorescence emphasis by optimizing the detection environment, and provide a reliable platform for assessing free radical damage.

[0008] The objective of this application is achieved through the following technical solution:

[0009] Firstly, this application provides an auxiliary system for assessing free radical damage, including...

[0010] A laser assembly for generating a laser to excite fluorescent molecules;

[0011] 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.

[0012] 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;

[0013] An irradiation assembly for generating rays and disposed on one side of the top surface;

[0014] A fluorescent filter, wherein the fluorescent filter is disposed on the third side or the fourth side;

[0015] A photometric component, the photometric component being used to measure fluorescence intensity;

[0016] 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;

[0017] A chamber for housing the cuvette and the fluorescent filter.

[0018] Furthermore, the auxiliary system also includes:

[0019] An excitation light filter is disposed on the first side and connected to the first optical fiber;

[0020] A reflector, which is disposed on the third or fourth side surface and is positioned opposite to the fluorescent filter;

[0021] An optical trap is disposed on the second side.

[0022] 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°.

[0023] The wavelength range allowed by the excitation filter is the center wavelength of the fluorescent molecule excitation light ± the second threshold;

[0024] The wavelength range allowed by the fluorescent filter is the center wavelength of fluorescence emitted by the fluorescent molecule ± the second threshold.

[0025] Furthermore, the auxiliary system also includes a base, the base comprising:

[0026] A lifting frame, wherein the lifting frame is disposed in the shielded room whose height can be adjusted by the lifting frame;

[0027] An upright plate is mounted on the lifting frame so that the height of the upright plate can be adjusted by the lifting frame;

[0028] A support is provided on the upright plate for placing the cuvette, the filter, and fixing the first and second optical fibers.

[0029] Furthermore, the photometric component includes:

[0030] A photometer, wherein the photometer is connected to the second optical fiber;

[0031] Terminal server, which is communicatively connected to the photometer;

[0032] The laser component includes:

[0033] A laser power supply, wherein the laser power supply is located outside the shielded room;

[0034] A first wire, which is connected to the laser power supply and passes through the wall of the shielded room;

[0035] 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;

[0036] The irradiation assembly includes:

[0037] A radiation power supply, wherein the radiation power supply is located outside the shielding room;

[0038] The second wire is connected to the radiation power supply and passes through the wall of the shielded room;

[0039] A radiation source, wherein the radiation source is connected to the second wire;

[0040] 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.

[0041] Both the first optical fiber and the second optical fiber are single-mode optical fibers with a diameter of 10-1500μm.

[0042] 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.

[0043] Secondly, this application provides a method for assessing free radical damage, the method being implemented based on the aforementioned auxiliary system;

[0044] The tube contains a test solution, which includes an indicator, and the volume of the test solution is 80%-100% of the tube volume.

[0045] The method includes the following steps:

[0046] Activate the laser assembly to allow the laser to pass through the first side and irradiate the solution to be tested;

[0047] 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.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] Furthermore, the test solution also includes test cells, which can be normal cells or cancer cells.

[0052] Furthermore, the volume of the cuvette is 0.1-3 mL;

[0053] The angle between the incident light and the outgoing light of the laser is 60-90°;

[0054] The power of the laser component is 1μW-1W;

[0055] The wavelength of the laser is the maximum excitation wavelength of the fluorescent molecules ± the first threshold.

[0056] The angle between the ray and the normal direction of the top surface is 0°;

[0057] 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.

[0058] The concentration of the indicator is 1 nmol / L-50 mmol / L;

[0059] 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.

[0060] Furthermore, the power of the laser component is 1-50mW;

[0061] The first threshold is 50nm;

[0062] The concentration of the indicator is 1-50 mmol / L;

[0063] The indicator is hydroxyphenylfluorescein or aminophenylfluorescein;

[0064] The radiation dose rate generated by the irradiation component is 10-100 Gy / s, and the total radiation dose is 1-80 Gy;

[0065] Furthermore, the power of the laser component is 5mW;

[0066] The wavelength of the laser is 488nm;

[0067] The concentration of the indicator is 10 mmol / L;

[0068] The first threshold is 5nm;

[0069] The indicator is hydroxyphenylfluorescein;

[0070] The radiation dose rate generated by the irradiation component is 40 Gy / s, and the total radiation dose is 80 Gy.

[0071] Thirdly, this application provides the application of the above-mentioned method in the detection of cell death rate, cell viability, and cell damage during cancer cell treatment.

[0072] Fourthly, this application provides a radiotherapy system, which includes the aforementioned auxiliary system for assessing free radical damage.

[0073] The radiotherapy system, auxiliary system for assessing free radical damage, method, and application of this application have at least the following advantages:

[0074] The auxiliary system chamber of this application 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.

[0075] The method of this application 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 the 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

[0076] The present application will be further described below with reference to the accompanying drawings and embodiments.

[0077] Figure 1 shows a partial structural schematic diagram of an auxiliary system for assessing free radical damage according to an embodiment of this application.

[0078] Figure 2 shows a schematic diagram of an auxiliary system for assessing free radical damage according to an embodiment of this application.

[0079] Figure 3 shows a schematic diagram of the structure of the base according to an embodiment of this application.

[0080] Figure 4 shows a partial structural schematic diagram of the cuvette according to an embodiment of this application.

[0081] Figure 5 shows a flowchart illustrating the method for evaluating free radical damage according to an embodiment of this application.

[0082] Figure 6 shows the curves relating the indicator concentration to the fluorescence intensity of this application.

[0083] Figure 7 shows a graph of fluorescence intensity monitored in real time in Example 1 of this application.

[0084] Figure 8 shows a graph of fluorescence intensity monitored in real time in Embodiment 2 of this application.

[0085] Figure 9 shows a graph of fluorescence intensity monitored in real time in Embodiment 3 of this application.

[0086] Figure 10 shows a graph of fluorescence intensity monitored in real time in Embodiment 4 of this application.

[0087] Figure 11 shows a graph of fluorescence intensity monitored in real time in Embodiment 5 of this application.

[0088] Figure 12 shows a graph of fluorescence intensity monitored in real time in Comparative Example 1 of this application.

[0089] 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

[0090] The technical solutions in this application will be described below with reference to the accompanying drawings and specific embodiments. It should be noted that, without conflict, the various embodiments or technical features described below can be arbitrarily combined to form new embodiments.

[0091] In the embodiments of this application, "at least one" refers to one or more, and "more than one" refers to two or more. "And / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A alone, A and B simultaneously, or B alone, where A and B can be singular or plural. The character " / " generally indicates that the preceding and following related objects are in an "or" relationship. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single or plural items. For example, at least one of a, b, or c can represent: a, b, c, a and b, a and c, b and c, a and b and c, where a, b, and c can be single or multiple. It is worth noting that "at least one" can also be interpreted as "one or more".

[0092] It should also be noted that, in the embodiments of this application, the words "exemplary" or "for example" are used to indicate that they are examples, illustrations, or descriptions. Any implementation or design scheme described as "exemplary" or "for example" in the embodiments of this application should not be construed as being more preferred or advantageous than other implementations or design schemes. Specifically, the use of words such as "exemplary" or "for example" is intended to present the relevant concepts in a specific manner.

[0093] Under the same particle type, energy, and total dose conditions, this application shows no significant difference in the direct cell-killing effect (direct effect) between FLASH particle radiotherapy (also known as flash therapy) and conventional particle radiotherapy. However, the indirect cell-killing effects (indirect effects) differ considerably between the two. 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.

[0094] The auxiliary system for assessing free radical damage in this embodiment 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 fluorescence filter 32, a reflector 5, and an optical trap 6. Even further, the auxiliary system may also include a base.

[0095] Referring to Figure 4, the cuvette 7 of this application includes a tube body and a light-shielding cap. The tube body includes side surfaces and opposing top and bottom surfaces 71 and 72. Furthermore, 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. Additionally, the side surfaces include opposing first and second side surfaces 731 and 732, and opposing third and fourth side surfaces 734.

[0096] In one embodiment, the cuvette 7 can be various shapes such as cuboid, cube, or cylinder. In another possible implementation, 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.

[0097] Referring to Figure 3, the base of this application includes: a lifting frame 81, a vertical plate 82, and a bracket 83. In application, the base is placed inside the chamber 100.

[0098] In one embodiment, the upright plate 82 has an L-shaped structure. One side of the L-shaped upright plate 82 is mounted on the lifting frame 81, and the other side of the L-shaped upright plate 82 is freely mounted on the lifting frame 81. A bracket 83 is 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 X-rays to better irradiate the liquid inside the cuvette 7, thereby improving the accuracy and precision of the detection results.

[0099] Referring to Figure 2, the laser assembly 1 of this application includes: a laser power supply 11, a first wire 12, and a laser diode 13. In application, the laser diode 13 is disposed inside the 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.

[0100] In one embodiment, a first through-hole 201 matching the first wire 12 is provided on the shielded chamber 200. The first wire 12 passes through the first through-hole 201 and penetrates the wall of the shielded chamber 200. One end of the laser diode 13 (e.g., the third input end, i.e., 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 other end of the laser diode 13 (e.g., the third output end, i.e., the output end of the laser diode 13) is connected to one end of the first optical fiber 21 (e.g., the first input end, i.e., the input end of the first optical fiber 21), and the other end of the first optical fiber 21 (e.g., the first output end, i.e., the output end of the first optical fiber 21) is connected to the first side 731 to transmit the incident laser light into the cuvette 7 through the first optical fiber 21. In application, to increase the optical signal intensity, the diameter of the optical fiber is increased. Specifically, the diameter of the first optical fiber 21 is a single-mode optical fiber with a diameter of 10-1500 μm. In one possible implementation, the diameter of the first optical fiber 21 is 600 μm.

[0101] In one embodiment, to prevent non-ideal wavelength laser light from entering the cuvette 7, an excitation 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 by the excitation filter 31 is the center wavelength of the fluorescent molecule excitation light ± a second threshold, where the second threshold is 0-15 nm. In one possible implementation, the second threshold is 2 nm. In practical applications, the angle between the incident and emitted laser light is 60-90°.

[0102] In one embodiment, the fluorescence filter 32 is disposed on the fourth side 734. Further, one end of the second optical fiber 22 (e.g., the second input end, i.e., the input end of the second optical fiber 22) is connected to the fluorescence filter 32, and the other end of the second optical fiber 22 (e.g., the second output end, i.e., the output end of the second optical fiber 22) is connected to the photometric component 9.

[0103] Referring to Figure 2, the photometric component 9 of this application 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.

[0104] In one embodiment, the photometer 91 is a communication-enabled photometer 91, 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. In one possible implementation, the diameter of the second optical fiber 22 is 200 μm, and its length is 30 m.

[0105] In one embodiment, 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 fluorescence filter 31 allows a wavelength range of ± the center wavelength of fluorescence emitted by the fluorescent molecule, where the second threshold is 0-15 nm. In one possible implementation, the second threshold is 2 nm.

[0106] Referring to Figure 1 in one embodiment, the fluorescence generated by the fluorescent molecules 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. In one possible implementation, 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.

[0107] In one embodiment, the output of the photometer 91 is 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.

[0108] The irradiation assembly 4 of this application 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.

[0109] In one embodiment, a second through-hole 202 matching a second wire is provided on the shielded chamber 200. The second wire passes through the second through-hole 202 and penetrates the wall of the shielded 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°. In one possible implementation, 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.

[0110] The method for assessing free radical damage described in this application 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's volume.

[0111] In one embodiment, 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. In one possible implementation, the indicator is hydroxyphenyl fluorescein, and the concentration of the indicator is 10 mmol / L.

[0112] 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. Referring to Figure 6, within the indicator concentration range of 0-10 μmol / L, the fluorescence intensity shows a linear positive correlation with the indicator concentration, indicating that the free radical reaction is not saturated at this point. Specifically, the fitted curve in Figure 6 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 this application.

[0113] Referring to Figure 5, the method of this application includes steps S1-S5.

[0114] Step S1: Activate the laser assembly 1 so that the laser passes through the first side 731 and irradiates the solution to be tested.

[0115] 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.

[0116] 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.

[0117] In practical applications, the power of laser component 1 is 1μW-1W, and more specifically, the power of laser component 1 is 1-50mW. In one possible implementation, 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, where the first threshold is 50nm, and more specifically, the first threshold is 5nm. In one possible implementation, the wavelength of the laser is 488nm.

[0118] 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.

[0119] 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.

[0120] 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. In one possible implementation, the radiation dose rate generated by the irradiation component 4 is 40 Gy / s, and the total radiation dose is 80 Gy.

[0121] 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.

[0122] When applying the application, the preset duration is calculated as the total radiation dose divided by the radiation dose rate.

[0123] 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.

[0124] When applying the application, the preset intensity change rate is the noise amplitude before irradiation per unit time.

[0125] 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.

[0126] The method described in this application can be applied to the detection of cell death rate, cell viability, and cell damage during cancer cell treatment.

[0127] Furthermore, the auxiliary system of this application 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.

[0128] Furthermore, the auxiliary system of this application can be used in research on radiosensitization or reduction of radiation damage. By adjusting radiotherapy parameters, it can regulate the intensity of oxidative damage in different cells, which helps to optimize radiotherapy strategies and improve patient benefits.

[0129] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0130] Based on the auxiliary system of this application, steps 1-5 of the above method are used to complete Examples 1-5 and Comparative Example 1.

[0131] Example 1

[0132] 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.

[0133] The auxiliary system does not have a laser filter; the fluorescent filter 31 allows a wavelength of 514nm ± 2nm to pass through.

[0134] 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.

[0135] The laser component 1 has a power of 5mW, a laser wavelength of 488nm, and a laser half-width of 1.5nm.

[0136] The radiation dose rate was 6.0046 Gy / s, the total radiation dose was 260 Gy, and the irradiation time was 43.3 s.

[0137] The fluorescence intensity monitored in real time in Example 1 is shown in Figure 7.

[0138] In Figure 7, time point 041 indicates the start of radiation irradiation; time point 043 indicates the shutdown of irradiation component 4; during the 042 phase between time points 041 and 043, the indicator is continuously oxidized, generating cumulative fluorescence intensity.

[0139] As shown in Figure 7, after the irradiation component 4 is turned off, the reaction between the indicator and free radicals basically stops, and no more fluorescent molecules are produced.

[0140] Example 2

[0141] The difference between Example 2 and Example 1 is as follows:

[0142] The test solution also contains ferrous ions, and the concentration of ferrous ions is 1 mmol / L.

[0143] The radiation dose rate is 10 Gy / s, and the total radiation dose is 40 Gy.

[0144] The fluorescence intensity monitored in real time in Example 2 is shown in Figure 8.

[0145] In Figure 8, time point 801 indicates the start of irradiation; time point 802 indicates the end of irradiation by shutting off irradiation component 4. During the period from the start of irradiation to the end of irradiation, the indicator is continuously oxidized, generating accumulated fluorescence intensity.

[0146] As shown in Figure 8, the presence of ferrous ions significantly increases the rate of the free radical reaction. In application, the ferrous ion-catalyzed Fenton reaction of hydrogen peroxide enables its detection, resulting in a significantly improved signal-to-noise ratio.

[0147] As shown in Figure 8, after the irradiation component 4 is turned off, the reaction between the indicator and free radicals basically stops, and no more fluorescent molecules are produced.

[0148] Example 3

[0149] The difference between Example 3 and Example 1 is as follows:

[0150] The indicator is aminophenylfluorescein.

[0151] The test solution also contains ferrous ions, and the concentration of ferrous ions is 1 mmol / L.

[0152] The radiation dose rate is 10 Gy / s, and the total radiation dose is 40 Gy.

[0153] The fluorescence intensity monitored in real time in Example 3 is shown in Figure 9.

[0154] In Figure 9, time point 901 indicates the start of irradiation; time point 902 indicates the end of irradiation by shutting off irradiation component 4. During the period from the start of irradiation to the end of irradiation, the indicator is continuously oxidized, generating accumulated fluorescence intensity.

[0155] As shown in Figure 9, the presence of ferrous ions significantly increases the rate of 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.

[0156] As shown in Figure 9, after the irradiation component 4 is turned off, the reaction between the indicator and free radicals basically stops, and no more fluorescent molecules are produced.

[0157] Example 4

[0158] The difference between Example 4 and Example 1 is as follows:

[0159] The diameter of the first optical fiber 21 is 1500μm.

[0160] The second optical fiber 22 has a diameter of 600 μm.

[0161] The indicator is aminophenylfluorescein.

[0162] The test solution also contains ferrous ions, and the concentration of ferrous ions is 1 mmol / L.

[0163] The radiation dose rate was 40 Gy / s, and the total radiation dose was 80 Gy.

[0164] The fluorescence intensity monitored in real time in Example 4 is shown in Figure 10.

[0165] In Figure 10, time point 1001 indicates the start of irradiation; time point 1002 indicates the end of irradiation by shutting off irradiation component 4. During the period from the start of irradiation to the end of irradiation, the indicator is continuously oxidized, resulting in accumulated fluorescence intensity.

[0166] As shown in Figure 10, the presence of ferrous ions significantly increases the rate of 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.

[0167] Referring to Figure 10, it can be further seen 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, and therefore it is 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).

[0168] As shown in Figure 10, after the irradiation component 4 is turned off, the reaction between the indicator and free radicals basically stops, and no more fluorescent molecules are produced.

[0169] Example 5

[0170] The difference between Example 5 and Example 1 is as follows:

[0171] The indicator is 2',7'-dichlorodihydrofluorescein diacetate.

[0172] The test solution also contains ferrous ions, and the concentration of ferrous ions is 1 mmol / L.

[0173] The radiation dose rate was 40 Gy / s, and the total radiation dose was 80 Gy.

[0174] The fluorescence intensity monitored in real time in Example 5 is shown in Figure 11.

[0175] In Figure 11, time point 1101 indicates the start of irradiation; time point 1102 indicates the end of irradiation by shutting off irradiation component 4. During the period from the start of irradiation to the end of irradiation, the indicator is continuously oxidized, generating accumulated fluorescence intensity.

[0176] As shown in Figure 11, the presence of ferrous ions significantly increases the rate of the free radical reaction. In practical applications, the ferrous ion-catalyzed Fenton reaction of hydrogen peroxide enables its detection, significantly improving the signal-to-noise ratio. However, in real-world applications, 2',7'-dichlorodihydrofluorescein diacetate is unstable and prone to auto-oxidation in air during storage, generating free radicals. This results in a higher background fluorescence intensity of the test solution in cuvette 7, reducing detection accuracy.

[0177] As shown in Figure 11, after the irradiation component 4 is turned off, the reaction between the indicator and free radicals basically stops, and no more fluorescent molecules are produced.

[0178] Comparative Example 1

[0179] The difference between Comparative Example 1 and Example 1 is as follows:

[0180] The auxiliary system in Comparative Example 1 did not have a shielded room of 200.

[0181] The fluorescence intensity monitored in real time for Comparative Example 1 is shown in Figure 12.

[0182] In Figure 12, time point 1201 indicates the start of irradiation; time point 1202 indicates the shutdown of irradiation component 4 and the end of irradiation.

[0183] As shown in Figure 12, due to the absence of a shielding chamber 200, the components of the photometric component 9 are affected by radiation, resulting in inaccurate fluorescence intensity detection results.

[0184] In summary, the embodiments of this application 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 this application possesses radiation interference resistance capabilities to ensure that relevant information on free radical damage, such as assessment results, can be provided stably and accurately during radiotherapy.

[0185] This application 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 application 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 radiotherapy system's particle accelerator in real time based on the assessment results of free radical damage. Therefore, the radiotherapy method can achieve more precise treatment.

[0186] Although embodiments of this application have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting this application. 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 this application, and all such changes should fall within the protection scope of the claims of this application.

Claims

1. An auxiliary system for assessing free radical damage, comprising: A laser assembly (1) is used to generate a laser that excites 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 to 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 to each other, and a third side surface (733) and a fourth side surface (734) that are opposite to 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 surface (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), said photometric component (9) is used to measure fluorescence intensity; A second optical fiber (22) is connected at one end to the fluorescent filter (32) and at the other end to the photometric component (9). A chamber (100) for accommodating the cuvette (7) and the fluorescence filter (32).

2. The auxiliary system for assessing free radical damage according to claim 1, wherein, 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, wherein, 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, wherein, The auxiliary system also includes a base, the base comprising: A lifting frame (81) is installed inside the shielding 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 disposed 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, wherein, The photometric component (9) includes: A photometer (91) is connected to the second optical fiber (22); A terminal server (93) is communicatively connected to the photometer (91); The laser component (1) includes: A laser power supply (11) is disposed 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), 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 10-1500 μm.

6. The auxiliary system for assessing free radical damage according to claim 5, wherein, 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, said method being 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 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; The photometric component (9) is activated, and combined with the fluorescence filter (32), the background fluorescence intensity of the test solution in the cuvette (7) is detected; 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 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 (4) is turned off. 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, wherein, 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, wherein, 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, wherein, 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, wherein, 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. The use of the method according to any one of claims 7-11 in the detection of cell death rate, the detection of cell viability, and the detection of cell damage during cancer cell treatment.

13. A radiotherapy system comprising an auxiliary system for assessing free radical damage according to any one of claims 1-6.

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