In-situ biological tissue cell free radical detection device and method based on EPR

Abstract

The invention relates to the technical field of biological tissue cell detection, in particular to an in-situ biological tissue cell free radical detection device and method based on EPR. The technical problems that in the prior art, a fluorescent probe is prone to being oxidized, the detection accuracy is affected, a traditional EPR method needs to freeze or fix a sample, a cell sample is prone to being inactivated, and free radicals in the sample are inactivated or attenuated are solved. According to the technical scheme, the in-situ biological tissue cell free radical detection device based on the EPR comprises a culture cabin, a central controller, a culture dish, a cleaning fluid storage rack, a control assembly, a detection assembly and a temperature control assembly; through the design of the movable sterile liquid suction head and the liquid conveying channel, automatic sampling and conveying are achieved, the problem that free radicals are inactivated or attenuated in traditional EPR detection is solved, the authenticity and reliability of a detection result are ensured, and real-time, dynamic and in-situ detection of biological free radicals is achieved through automatic operation of a central control system.

Description

Technical Field

[0001] This invention relates to the field of biological tissue and cell detection technology, and in particular to an in situ biological tissue and cell free radical detection device and method based on EPR. Background Technology

[0002] Biological free radicals (such as reactive oxygen species) play a key role in oxidative stress and disease mechanisms, but their short lifespan (milliseconds) and low concentration make detection difficult. Current technologies mainly rely on chemical probes (such as fluorescence methods) or EPR non-in situ detection. However, fluorescent probes are easily oxidized, affecting detection accuracy. Traditional EPR methods require freezing or fixing samples, and the materials used are incompatible with biological samples, which can easily cause cell sample inactivation, leading to problems such as inactivation or attenuation of free radicals in the sample. Therefore, to address the aforementioned issues, we propose an in-situ biological tissue cell free radical detection device and method based on EPR. Through the design of a movable sterile aspiration head and infusion channel, automated sample collection and delivery are achieved, improving experimental efficiency. Since there is no need to freeze or fix the samples, the problem of free radical inactivation or attenuation in traditional EPR detection is avoided, ensuring the authenticity and reliability of the detection results. Through the automated operation of the central control system, researchers can adjust experimental conditions in real time during the culture process and simultaneously record free radical change data, realizing real-time, dynamic, and in-situ detection of biological free radicals. Summary of the Invention

[0003] To overcome the problems of existing biological free radical detection devices and methods, such as fluorescent probes being easily oxidized, affecting detection accuracy, traditional EPR methods requiring frozen or fixed samples, and the materials used being incompatible with biological samples, which can easily cause cell sample inactivation, leading to the inactivation or attenuation of free radicals in the sample.

[0004] The technical solution of this invention is as follows: an in-situ biological tissue cell free radical detection device based on EPR, comprising a culture chamber, a central controller, culture dishes, a cleaning solution storage rack, a control component, a detection component, and a temperature control component. The central controller is located on the outside of the culture chamber and is used to synchronously control culture parameters and detection timing. The control component is located on the inside of the culture chamber, along with culture dishes and a cleaning solution storage rack. The detection component is located on the inside of the culture chamber, and the temperature control component is located on the outside of the detection component. The components include a UV sterilizing lamp, a culture plate, a carbon dioxide inlet, a humidity regulator, a temperature and humidity sensor, and a concentration detector. The UV sterilizing lamp, which has a ring-shaped structure, is installed on the inner top surface of the culture chamber. A culture plate is located on the inner side of the culture chamber, and a carbon dioxide inlet is located on the side wall. A humidity regulator, a temperature and humidity sensor, and a concentration detector are all located on the inner side of the culture chamber. These components are integrated on the top surface of the culture chamber. The temperature and humidity sensor is used for… The system collects temperature and humidity data within the chamber, and a concentration detector collects carbon dioxide concentration data. The detection components include a first linear guide rail, a first linear motor, an electric telescopic rod, a connecting plate, a suction head, a delivery tube, a second linear guide rail, a second linear motor, a sample syringe, a trapping agent storage tank, a trapping agent injection tube, and a capillary tube. The first linear guide rail is located inside the culture chamber, and the first linear motor is located outside the first linear guide rail. An electric telescopic rod is located below the first linear motor, and a connecting plate is located at the lower end of the electric telescopic rod. The connecting plate contains... A suction head is provided on the side, and a delivery tube is provided at the upper end of the suction head. A second linear guide rail is provided on the inner side of the culture chamber, and a second linear motor is provided on the outer side of the second linear guide rail. A sample injector is provided on one side of the second linear motor. The sample injector and the delivery tube are connected to each other. A capture agent storage tank is provided on the inner bottom surface of the culture chamber. A capture agent injection tube is provided on the inner side of the capture agent storage tank. A capillary is provided below the capture agent injection tube. The inner wall of the capillary is coated with PEG. An EPR resonant cavity is provided on the outer side of the capillary. The temperature control component is located on the outer side of the capillary.

[0005] Preferably, a circular sterilization UV lamp at 15mW / cm² is used. 2Radiation intensity cyclic sterilization is employed. A first linear motor drives the suction head along a first linear guide rail for positioning, with an electric telescopic rod enabling Z-axis fine-tuning. During sampling, the suction head descends at 2 mm / s, contacts the liquid surface, and then rises 0.2 mm, utilizing surface tension to create bubble-free sampling. A sterile filter membrane provides dual protection against contamination. A heparin coating on the inner wall of the delivery tube ensures no residue risk during the transport of high-viscosity biological samples. The humidity regulator and sterile distilled water storage tank are linked, employing ultrasonic atomization technology to inject sterile distilled water into the chamber. This is further enhanced by temperature and humidity control. The sensor forms a closed-loop control. When the temperature drift is greater than 0.2℃, the Peltier element, driven by the PID temperature controller, stabilizes the capillary temperature to 37±0.1℃, ensuring the working stability of the EPR resonant cavity. The carbon dioxide cylinder injects gas into the chamber through the first conduit, and the concentration is dynamically balanced with the PID algorithm. When the free radical concentration changes abruptly, the central controller starts the gas compensation program to compensate for the gas. After the detection is completed, the first linear motor drives the suction head to the cleaning liquid storage rack, and the waste liquid is transported to the waste liquid tank by the second linear motor, reducing the risk of cross-contamination.

[0006] Preferably, the moving speed range of the first linear motor and the second linear motor is 5~10mm / s, the suction end of the suction head is equipped with a sterile filter membrane with a thickness of 0.2μm, and the inner wall of the delivery tube is coated with heparin.

[0007] Preferably, a waste liquid tank is installed on the bottom of the culture chamber, a carbon dioxide cylinder is installed on the outside of the culture chamber, a first conduit is installed on the inside of the carbon dioxide cylinder, the first conduit is connected to the carbon dioxide inlet, a sterile distilled water storage tank is installed on the outside of the culture chamber, a second conduit is installed on the inside of the sterile distilled water storage tank, and the second conduit is connected to the culture chamber.

[0008] Preferably, the temperature control assembly includes a Peltier element, a temperature sensor, a mounting bracket, and a PID temperature controller. The Peltier element and the temperature sensor are located on the outer side of the capillary tube, the mounting bracket is located on the outer side of the EPR resonant cavity, and the PID temperature sensor is located on the outer side of the mounting bracket.

[0009] Preferably, the central controller performs the following linkage control: A1: When the rate of change in free radical concentration is detected to be ≥5% / min, adjust the O2 concentration in the culture chamber at a rate of 0.5% / s. A2: Synchronize the EPR signal with the timestamp and the culture environment parameters to establish a dynamic correlation database.

[0010] The method for detecting free radicals in situ biological tissue cells based on the above-mentioned EPR-based in-situ biological tissue cell free radical detection device includes the following steps: S1: Turn on the circular ultraviolet sterilization lamp with a radiation intensity of 15mW / cm². 2 The process is repeated three times, introducing a 5% concentration of carbon dioxide mixed gas into the culture chamber. The temperature range is set to 37±0.1℃, the humidity range to 95±1%RH, and the oxygen concentration gradient is adjustable from 5% to 20%. S2: The first linear motor moves linearly to control the aspiration head to move above the liquid surface of the culture dish, aspirate the sample, input the sample into the sample syringe through the delivery tube and inject it into the capillary, and at the same time inject the capture agent into the capillary through the capture agent injection tube. The capture agent is phosphate buffer. The sample and the capture agent mix to form a free radical and capture agent complex. S3: Initiate EPR scan and calculate free radical concentration in real time. The calculation formula is as follows: C(t) = k × ∫ g'(B)dB, where k is the calibration coefficient and g'(B) is the integral value of the first derivative spectrum; S4: Establish a time series matrix: M(t)=[T(t),H(t),CO2(t),O2(t),C(t)], and trigger environmental parameter adjustment when the rate of change of C(t) ΔC / Δt ≥ 5% / min, specifically: If the O2 concentration fluctuates by more than 0.3%, gas compensation is performed; if the temperature drift is greater than 0.2°C, the Peltier element is activated. S5: After each test, the first linear motor moves linearly to the top of the cleaning fluid storage rack, and the second linear motor moves linearly to the top of the waste liquid tank. The cleaning fluid is drawn up by the suction head to clean the suction head, delivery tube and sample syringe respectively, and the waste liquid is discharged into the waste liquid tank.

[0011] Preferably, the sampling control step in step S2 is as follows: the suction head descends at a speed of 2 mm / s, and after contacting the liquid surface, it is lifted by 0.2 mm to form surface tension sampling; wherein the actual sampling volume is calculated using the formula: V actual = Vnom ×(1+0.02×(T-37)), where V nom T represents the nominal volume, and T represents the real-time temperature.

[0012] As a preferred option, the dynamic adjustment strategy for the EPR parameter in step S3 specifically includes: When the signal strength S(t) > 2000 a.u., the microwave power automatically drops to 5mW; When the signal-to-noise ratio (SNR) is less than 20 dB, the number of scans increases from 1 to 3. Modulation amplitude is calculated according to formula A mod =0.5+0.3×exp(-S(t) / 1000) Dynamic adjustment.

[0013] Preferably, the gas compensation step in step S4 specifically involves: setting the target oxygen concentration to O. 2 target =O 2 current ×(1-0.05×ΔC / Δt); The adjustment rate is limited to ±0.5% / s; When ΔC / Δt is greater than 10% / min for 3 consecutive times, switch to hypoxia mode, where O2 ≤ 10%.

[0014] Preferably, in step S3, the calibration coefficient k is determined through online calibration, specifically as follows: S301: During the first run of each test, inject a standard of known concentration; S302: Record the peak-to-peak value of the EPR signal. std Establish a linear regression equation k=Σ(C std ×S std ) / Σ(S std 2 ); S303: Requirement R 2 ≥0.995, an alarm is triggered when the deviation of k value is >2%.

[0015] The beneficial effects of this invention are: 1. This invention utilizes a ring-shaped ultraviolet sterilization lamp to cover the entire culture chamber, killing microorganisms. It injects a 5% CO2 mixed gas, and dynamically adjusts the concentration using a PID algorithm to simulate standard cell culture conditions. A humidity regulator and ultrasonic atomization technology work together to atomize and inject sterile distilled water into the chamber, maintaining a temperature and humidity level of 95±1%. RH humidity; the Peltier element, driven by a PID controller, stabilizes the capillary temperature at 37±0.1℃, ensuring the stability of the EPR resonant cavity. The aspiration head is equipped with a 0.2μm sterile filter membrane at the aspiration end. Combined with an electric telescopic rod, it descends at a speed of 2mm / s, contacts the liquid surface, and then rises 0.2mm, utilizing surface tension to achieve bubble-free sampling. The inner wall of the delivery tube is coated with heparin to avoid residues of high-viscosity biological samples. After detection, the aspiration head automatically moves to the cleaning solution storage rack, and the waste liquid is transported to the waste liquid bucket via a second linear motor, reducing the risk of cross-contamination and ensuring the sterility of the experimental environment and the reliability of the experimental results. It is suitable for multiple fields such as biomedical research, drug screening, and environmental toxicology, realizing automated sample sampling and delivery, avoiding the contamination risks of traditional manual operation, and improving experimental efficiency. Since there is no need to freeze or fix the sample, it also avoids the problem of free radical inactivation or attenuation in traditional EPR detection, ensuring the authenticity and reliability of the detection results. 2. Through the automated operation of the central control system, the present invention allows researchers to adjust experimental conditions in real time during the culture process and record free radical change data simultaneously, realizing in-situ real-time detection of free radicals during the culture of biological tissues and cells, providing strong technical support for the study of oxidative stress, disease mechanisms, etc. Attached Figure Description

[0016] Figure 1 The diagram shown is a first three-dimensional structural schematic of the EPR-based in-situ biological tissue cell free radical detection device of the present invention. Figure 2 The diagram shown is a second three-dimensional structural schematic of the EPR-based in-situ biological tissue cell free radical detection device of the present invention. Figure 3 The diagram shown is a first cross-sectional view of the in-situ biological tissue cell free radical detection device based on EPR of the present invention. Figure 4 The diagram shown is a second cross-sectional view of the EPR-based in-situ biological tissue cell free radical detection device of the present invention. Figure 5 The diagram shown is a planar cross-sectional view of the EPR-based in-situ biological tissue cell free radical detection device of the present invention. Figure 6 The diagram shows the steps of the in-situ biological tissue cell free radical detection method based on EPR of the present invention. Figure 7 The image shown is a free radical map of an embodiment of the EPR-based in situ biological tissue cell free radical detection method of the present invention; Figure labeling: 1. Culture chamber; 2. Central controller; 4. Petri dish; 5. Cleaning solution storage rack; 101. Ultraviolet sterilization lamp; 102. Culture plate; 103. Carbon dioxide inlet; 104. Humidity regulator; 105. Temperature and humidity sensor; 106. Concentration detector; 201. First linear guide rail; 202. First linear motor; 203. Electric telescopic rod; 204. Connecting plate; 205. Suction head; 206. Delivery tube; 20 7. Second linear guide rail; 208. Second linear motor; 209. Sample syringe; 210. Capturing agent storage tank; 211. Capturing agent injection tube; 212. Capillary tube; 213. EPR resonant cavity; 301. Peltier element; 302. Temperature sensor; 303. Fixture; 304. PID temperature controller; 6. Waste liquid tank; 7. Carbon dioxide cylinder; 701. First conduit; 8. Sterile distilled water storage tank; 802. Second conduit. Detailed Implementation

[0017] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0018] Please see Figure 1 , Figure 2 , Figure 3 , Figure 4 and Figure 5This invention provides an embodiment of an in-situ biological tissue cell free radical detection device based on EPR, comprising a culture chamber 1, a central controller 2, culture dishes 4, a cleaning solution storage rack 5, a control component, a detection component, and a temperature control component. The central controller 2 is located on the outer side of the culture chamber 1 and is used to synchronously control culture parameters and detection timing. The control component, culture dishes 4, and cleaning solution storage rack 5 are located on the inner side of the culture chamber 1. The detection component is located on the inner side of the culture chamber 1, and the temperature control component is located on the outer side of the detection component. The control component includes an ultraviolet sterilization lamp 101, a culture plate 102, a carbon dioxide inlet 103, and a humidity control unit. The culture chamber 1 includes a humidity regulator 104, a temperature and humidity sensor 105, and a concentration detector 106. An ultraviolet sterilization lamp 101 with a ring structure is installed on the top surface of the chamber. A culture plate 102 is installed on the inner side of the culture chamber 1. A carbon dioxide inlet 103 is provided on the side wall of the culture chamber 1. The humidity regulator 104, temperature and humidity sensor 105, and concentration detector 106 are integrated on the top surface of the culture chamber 1. The temperature and humidity sensor 105 is used to collect temperature and humidity data inside the chamber, and the concentration detector 106 is used to collect data from the chamber. The internal carbon dioxide concentration data detection component includes a first linear guide rail 201, a first linear motor 202, an electric telescopic rod 203, a connecting plate 204, a suction head 205, a delivery tube 206, a second linear guide rail 207, a second linear motor 208, a sample syringe 209, a trapping agent storage tank 210, a trapping agent injection tube 211, and a capillary tube 212. The first linear guide rail 201 is located inside the culture chamber 1, and the first linear motor 202 is located outside the first linear guide rail 201. The electric telescopic rod 203 is located below the first linear motor 202, and the connecting plate 204 is located at the lower end of the electric telescopic rod 203. The suction head 205 is located inside the connecting plate 204. The upper end of the aspiration head 205 is provided with a delivery tube 206. The inner side of the culture chamber 1 is provided with a second linear guide rail 207. The outer side of the second linear guide rail 207 is provided with a second linear motor 208. A sample injector 209 is provided on one side of the second linear motor 208. The sample injector 209 and the delivery tube 206 are connected to each other. The bottom surface of the culture chamber 1 is provided with a capture agent storage tank 210. The inner side of the capture agent storage tank 210 is provided with a capture agent injection tube 211. The lower part of the capture agent injection tube 211 is provided with a capillary tube 212. The inner wall of the capillary tube 212 is coated with PEG. The outer side of the capillary tube 212 is provided with an EPR resonant cavity 213. The temperature control component is located on the outer side of the capillary tube 212.

[0019] Preferably, a circular sterilization UV lamp at 15mW / cm² is used. 2Radiation intensity cyclic sterilization is performed. The first linear motor 202 drives the suction head 205 to be positioned along the first linear guide rail 201. The electric telescopic rod 203 enables Z-axis fine-tuning. During sampling, the suction head 205 descends at a speed of 2 mm / s, contacts the liquid surface, and then rises 0.2 mm, utilizing surface tension to create bubble-free sampling. A sterile filter membrane provides double contamination protection. The heparin coating on the inner wall of the delivery tube 206 ensures no residue risk during the delivery of high-viscosity biological samples. The humidity regulator 104 and the sterile distilled water storage tank 8 are linked and use ultrasonic atomization technology to inject sterile distilled water into the chamber. This is further enhanced by the temperature and humidity sensor 105. A closed-loop control is formed. When the temperature drift is greater than 0.2℃, the Peltier element 301, driven by the PID temperature controller 304, stabilizes the temperature of the capillary tube 212 to 37±0.1℃, ensuring the working stability of the EPR resonant cavity 213. The carbon dioxide cylinder 7 injects gas into the chamber through the first conduit 701. The concentration is dynamically balanced with the PID algorithm. When the free radical concentration changes abruptly, the central controller 2 starts the gas compensation program to perform gas compensation. After the detection is completed, the first linear motor 202 drives the suction head 205 to the cleaning liquid storage rack 5, and the waste liquid is transported to the waste liquid tank 6 through the second linear motor 208, reducing the risk of cross-contamination.

[0020] Preferably, the moving speed range of the first linear motor 202 and the second linear motor 208 is 5~10mm / s, the suction end of the suction head 205 is equipped with a sterile filter membrane with a thickness of 0.2μm, and the inner wall of the delivery tube 206 is coated with heparin.

[0021] Preferably, a waste liquid tank 6 is provided on the inner bottom surface of the culture chamber 1, a carbon dioxide cylinder 7 is provided on the outer side of the culture chamber 1, a first conduit 701 is provided on the inner side of the carbon dioxide cylinder 7, the first conduit 701 is connected to the carbon dioxide inlet 103, a sterile distilled water storage tank 8 is provided on the outer side of the culture chamber 1, a second conduit 8 is provided on the inner side of the sterile distilled water storage tank 8, and the second conduit 8 is connected to the culture chamber 1.

[0022] Preferably, the temperature control assembly includes a Peltier element 301, a temperature sensor 302, a mounting bracket 303, and a PID temperature controller 304. The Peltier element 301 and the temperature sensor 302 are disposed on the outer side of the capillary tube 212, the mounting bracket 303 is disposed on the outer side of the EPR resonant cavity 213, and the PID temperature sensor 302 is disposed on the outer side of the mounting bracket 303.

[0023] Preferably, the central controller 2 performs the following linkage control: A1: When the rate of change in free radical concentration is detected to be ≥5% / min, adjust the O2 concentration in the culture chamber at a rate of 0.5% / s. A2: Synchronize the EPR signal with the timestamp and the culture environment parameters to establish a dynamic correlation database.

[0024] Please see Figure 6 and Figure 7 The method for detecting free radicals in situ biological tissue cells based on the above-mentioned EPR-based in-situ biological tissue cell free radical detection device includes the following steps: S1: Turn on the circular ultraviolet sterilization lamp 101 with a radiation intensity of 15mW / cm². 2 The process is repeated three times. A 5% concentration of carbon dioxide mixed gas is introduced into the culture chamber 1. The temperature range is set to 37±0.1℃ and the humidity range is 95±1%RH. The oxygen concentration gradient is adjustable from 5% to 20%. S2: The first linear motor 202 moves linearly to control the aspiration head 205 to move above the liquid surface of the culture dish 4, aspirate the sample, and input the sample into the sample syringe 209 through the delivery tube 206 and inject it into the capillary tube 212. At the same time, the capture agent is injected into the capillary tube 212 through the capture agent injection tube 211. The capture agent is phosphate buffer. The sample and the capture agent mix to form a free radical and capture agent complex. S3: Initiate EPR scan and calculate free radical concentration in real time. The calculation formula is as follows: C(t) = k × ∫ g'(B)dB, where k is the calibration coefficient and g'(B) is the integral value of the first derivative spectrum; S4: Establish a time series matrix: M(t)=[T(t),H(t),CO2(t),O2(t),C(t)], and trigger environmental parameter adjustment when the rate of change of C(t) ΔC / Δt ≥ 5% / min, specifically: If the O2 concentration fluctuates by more than 0.3%, gas compensation is performed; if the temperature drift is greater than 0.2°C, the Peltier element 301 is activated. S5: After each test, the first linear motor 202 moves linearly to the top of the cleaning fluid storage rack 5, and the second linear motor 208 moves linearly to the top of the waste liquid tank 6. The cleaning fluid is drawn up by the suction head 205 to clean the suction head 205, the delivery tube 206 and the sample syringe 209 respectively, and the waste liquid is discharged into the waste liquid tank 6.

[0025] Preferably, the sampling control step in step S2 is as follows: the suction head 205 descends at a speed of 2 mm / s, and after contacting the liquid surface, it is lifted by 0.2 mm to form surface tension sampling; wherein the actual sampling volume is calculated using the formula: V actual = Vnom ×(1+0.02×(T-37)), where V nom T represents the nominal volume, and T represents the real-time temperature.

[0026] As a preferred option, the dynamic adjustment strategy for the EPR parameter in step S3 specifically includes: When the signal strength S(t) > 2000 a.u., the microwave power automatically drops to 5mW; When the signal-to-noise ratio (SNR) is less than 20 dB, the number of scans increases from 1 to 3. Modulation amplitude is calculated according to formula A mod =0.5+0.3×exp(-S(t) / 1000) Dynamic adjustment.

[0027] Preferably, the gas compensation step in step S4 specifically involves: setting the target oxygen concentration to O. 2 target =O 2 current ×(1-0.05×ΔC / Δt); The adjustment rate is limited to ±0.5% / s; When ΔC / Δt is greater than 10% / min for 3 consecutive times, switch to hypoxia mode, where O2 ≤ 10%.

[0028] Preferably, in step S3, the calibration coefficient k is determined through online calibration, specifically as follows: S301: During the first run of each test, inject a standard of known concentration; S302: Record the peak-to-peak value of the EPR signal. std Establish a linear regression equation k=Σ(C std ×S std ) / Σ(S std 2 ); S303: Requirement R 2 ≥0.995, an alarm is triggered when the deviation of k value is >2%.

[0029] Example Optionally, in tumor pharmacology research, it is necessary to assess the impact of novel anticancer drugs on the oxidative stress level of cancer cells. Traditional methods, due to sample fixation leading to the decay of free radicals, struggle to capture real-time dynamic changes. This device, through in-situ detection, can monitor the dynamic fluctuations of intracellular free radicals in real time after drug action, providing precise data for drug action mechanism research. The specific implementation steps are as follows: Q1: Seed the liver cancer cell line into culture dish 4, add DMEM medium containing 10% fetal bovine serum, and place it in culture chamber 1; Q2: Set the culture environment to 37℃, 5% CO2, and 95% humidity using the central controller 2, and sterilize 3 times using UV sterilization lamp 101 in a cycle; Q3: Inject different concentrations (0.1, 1, 10 μM) of anticancer drugs into culture dish 4, with 3 replicates for each group; Q4: Set to automatically sample once every 10 minutes. The aspiration head 205 descends at a speed of 2 mm / s to contact the liquid surface and uses surface tension to aspirate 20 μL of supernatant. The supernatant is then injected into the capillary 212 through the heparin-coated delivery tube 206, and a capture agent (phosphate buffer containing DMPO, final concentration 50 mM) is injected at the same time. Q5: Initial scan microwave power is 10mW. If the signal strength S(t) > 2000 a.u., it will automatically decrease to 5mW. When the signal-to-noise ratio is < 20dB, the number of scans will increase to 3. Q6: During the initial test, inject TEMPOL standard at a known concentration, record the peak value Sstd, and calculate the k value; if R 2 If the value is <0.995 or the k-value deviation is >2%, an alarm will be triggered and recalibration will be performed. Q7: Calculate the free radical concentration using the formula C(t)=k×∫g'(B)dB, and establish a time series matrix M(t)=[T, H,CO2, O2, C(t)]; Q8: After 15 minutes of drug action, the rate of change of C(t) ΔC / Δt was detected to be 8% / min (exceeding the threshold of 5% / min). The central controller 2 automatically executes the following: Gas compensation: Target oxygen concentration adjusted to O 2 target =O 2 current ×(1-0.05×8%)=19.4%, adjustment rate 0.5% / s; Temperature control: When the temperature drift is greater than 0.2℃, the Peltier element 301 is activated to maintain the temperature of the capillary tube 212 at 37±0.1℃; Q9: Synchronously record EPR signals and environmental parameters; Q10: After the test is completed, the aspiration head 205 is moved to the cleaning solution storage rack 5. The aspiration head 205, the delivery tube 206 and the syringe are cleaned in sequence with PBS and 70% ethanol. The waste liquid is discharged into the bucket to avoid cross-contamination. The following results can be obtained by following the steps above: Experimental Results and Effects 1. In three independent experiments, the deviation of the k value was <1.5%, R 2 ≥0.997, meeting the alarm threshold requirements, with a minimum detectable free radical concentration of 0.5μM, which is 3 times more sensitive than the traditional fluorescence method; In the 2.1 μM drug group, the peak concentration of ·OH reached 8.2 μM after 30 min of treatment, and then decreased due to the activation of the cellular antioxidant system. In the 10 μM group, cell apoptosis was induced by excessive oxidative stress, and the free radical concentration dropped sharply after 45 min. After dynamic adjustment of O2 concentration, the drug-induced oxidative stress intensity decreased by 18±3%, which verified the alleviating effect of hypoxia on drug toxicity. 3. A single experiment can complete fully automated detection. The sterile filter membrane and heparin coating design ensure a contamination rate of <0.1% and a cell survival rate of >95%.

[0030] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention.

Claims

1. An in-situ biological tissue cell free radical detection device based on EPR, comprising a culture chamber (1), characterized in that: It also includes a central controller (2), petri dishes (4), a cleaning solution storage rack (5), a control component, a detection component, and a temperature control component. The central controller (2) is located on the outside of the culture chamber (1). The central controller (2) is used to synchronously control the culture parameters and detection sequence. The control component is located on the inside of the culture chamber (1). The petri dishes (4) are located on the inside of the culture chamber (1). The cleaning solution storage rack (5) is located on the inside of the culture chamber (1). The detection component is located on the inside of the culture chamber (1). The temperature control component is located on the outside of the detection component. The control component includes an ultraviolet sterilization lamp (101), a culture plate (102), a carbon dioxide inlet (103), a humidity regulator (104), a temperature and humidity sensor (105), and a concentration detector (106). 106), an ultraviolet sterilization lamp (101) is installed on the top surface of the culture chamber (1). The ultraviolet sterilization lamp (101) has a ring structure. A culture plate (102) is installed on the inner side of the culture chamber (1). A carbon dioxide inlet (103) is opened on the side wall of the culture chamber (1). A humidity regulator (104) is installed on the inner side of the culture chamber (1). A temperature and humidity sensor (105) is installed on the inner side of the culture chamber (1). A concentration detector (106) is installed on the inner side of the culture chamber (1). The humidity regulator (104), temperature and humidity sensor (105) and concentration detector (106) are integrated on the top surface of the culture chamber (1). The temperature and humidity sensor (105) is used to collect temperature and humidity data inside the chamber. The concentration detector (106) is used to collect temperature and humidity data inside the chamber. The carbon dioxide concentration detection component includes a first linear guide rail (201), a first linear motor (202), an electric telescopic rod (203), a connecting plate (204), a suction head (205), a delivery tube (206), a second linear guide rail (207), a second linear motor (208), a sample syringe (209), a trapping agent storage tank (210), a trapping agent injection tube (211), and a capillary tube (212). The first linear guide rail (201) is located inside the culture chamber (1), and the first linear motor (202) is located outside the first linear guide rail (201). The electric telescopic rod (203) is located below the first linear motor (202), and the connecting plate (204) is located at the lower end of the electric telescopic rod (203). A suction head (205) is provided on the inner side of the connecting plate (204), and a delivery tube (206) is provided on the upper end of the suction head (205). A second linear guide rail (207) is provided on the inner side of the culture chamber (1), and a second linear motor (208) is provided on the outer side of the second linear guide rail (207). A sample syringe (209) is provided on one side of the second linear motor (208). The sample syringe (209) and the delivery tube (206) are connected to each other. A capture agent storage tank (210) is provided on the inner bottom surface of the culture chamber (1). A capture agent injection tube (211) is provided on the inner side of the capture agent storage tank (210). A capillary tube (212) is provided below the capture agent injection tube (211). The inner wall of the capillary tube (212) is coated with PEG.An EPR resonant cavity (213) is disposed on the outside of the capillary tube (212), and the temperature control component is located on the outside of the capillary tube (212).

2. The in-situ biological tissue cell free radical detection device based on EPR according to claim 1, characterized in that: The first linear motor (202) and the second linear motor (208) have a moving speed range of 5~10 mm / s. The suction end of the suction head (205) is equipped with a sterile filter membrane with a thickness of 0.2 μm. The inner wall of the delivery tube (206) is coated with heparin.

3. The in-situ biological tissue cell free radical detection device based on EPR according to claim 1, characterized in that: The bottom of the culture chamber (1) is provided with a waste liquid tank (6), the outside of the culture chamber (1) is provided with a carbon dioxide cylinder (7), the inside of the carbon dioxide cylinder (7) is provided with a first conduit (701), the first conduit (701) and the carbon dioxide inlet (103) are connected to each other, the outside of the culture chamber (1) is provided with a sterile distilled water storage tank (8), the inside of the sterile distilled water storage tank (8) is provided with a second conduit (8), the second conduit (8) and the culture chamber (1) are connected to each other.

4. The in-situ biological tissue cell free radical detection device based on EPR according to claim 1, characterized in that: The temperature control assembly includes a Peltier element (301), a temperature sensor (302), a mounting bracket (303), and a PID temperature controller (304). The Peltier element (301) is disposed on the outside of the capillary tube (212), the temperature sensor (302) is disposed on the outside of the capillary tube (212), the mounting bracket (303) is disposed on the outside of the EPR resonant cavity (213), and the PID temperature sensor (302) is disposed on the outside of the mounting bracket (303).

5. The in-situ biological tissue cell free radical detection device based on EPR according to claim 1, characterized in that: The central controller (2) performs the following linkage control: A1: When the free radical concentration change rate is detected to be ≥5% / min, adjust the O2 concentration in the culture chamber (1) at a rate of 0.5% / s; A2: Synchronize the EPR signal with the timestamp and the culture environment parameters to establish a dynamic correlation database.

6. The method for detecting free radicals in situ biological tissue cells based on EPR using the EPR-based in situ biological tissue cell free radical detection device according to claims 1-5, characterized in that: It includes the following steps: S1: Turn on the ring-shaped ultraviolet sterilization lamp (101) with a radiation intensity of 15mW / cm². 2 The process was repeated three times, and a 5% concentration of carbon dioxide mixed gas was introduced into the culture chamber (1). The temperature range was set to 37±0.1℃ and the humidity range was 95±1%RH. S2: The first linear motor (202) moves linearly to control the aspiration head (205) to move above the liquid surface of the culture dish (4), and the sample is aspirated. The sample is input into the sample syringe (209) through the delivery tube (206) and injected into the capillary (212). At the same time, the capture agent is injected into the capillary (212) through the capture agent injection tube (211). The capture agent is phosphate buffer. The sample and the capture agent are mixed to form a free radical and capture agent complex. S3: Initiate EPR scan and calculate free radical concentration in real time. The calculation formula is as follows: C(t) = k × ∫ g'(B)dB, where k is the calibration coefficient and g'(B) is the integral value of the first derivative spectrum; S4: Establish a time series matrix: M(t)=[T(t),H(t),CO2(t),O2(t),C(t)], and trigger environmental parameter adjustment when the rate of change of C(t) ΔC / Δt ≥ 5% / min, specifically: If the O2 concentration fluctuates by more than 0.3%, gas compensation is performed; if the temperature drift is greater than 0.2°C, the Peltier element (301) is activated. S5: After each test is completed, the first linear motor (202) moves linearly to the top of the cleaning fluid storage rack (5), and the second linear motor (208) moves linearly to the top of the waste liquid tank (6). The cleaning fluid is drawn up by the suction head (205) to clean the suction head (205), the delivery tube (206) and the sample syringe (209) respectively, and the waste liquid is discharged into the waste liquid tank (6).

7. The method for in-situ detection of free radicals in biological tissues and cells based on EPR according to claim 6, characterized in that: The sampling control step in step S2 is as follows: the suction head (205) descends at a speed of 2 mm / s, and after contacting the liquid surface, it rises 0.2 mm to form surface tension sampling; the actual sampling volume is calculated using the formula: V actual = Vnom ×(1+0.02×(T-37)), where V nom T represents the nominal volume, and T represents the real-time temperature.

8. The method for detecting free radicals in in situ biological tissue cells based on EPR according to claim 6, characterized in that: The dynamic adjustment strategy for EPR parameters in step S3 specifically includes: When the signal strength S(t) > 2000 a.u., the microwave power automatically drops to 5mW; When the signal-to-noise ratio (SNR) is less than 20 dB, the number of scans increases from 1 to 3. Modulation amplitude is calculated according to formula A mod =0.5+0.3×exp(-S(t) / 1000) Dynamic adjustment.

9. The method for detecting free radicals in in situ biological tissue cells based on EPR according to claim 6, characterized in that: The gas compensation step in step S4 specifically involves: the target oxygen concentration being O 2 target =O 2 current ×(1-0.05×ΔC / Δt); The adjustment rate is limited to ±0.5% / s; When ΔC / Δt is greater than 10% / min for 3 consecutive times, switch to hypoxia mode, where O2 ≤ 10%.

10. The method for detecting free radicals in in situ biological tissue cells based on EPR according to claim 6, characterized in that: In step S3, the calibration coefficient k is determined through online calibration, specifically as follows: S301: During the first run of each test, inject a standard of known concentration; S302: Record the peak-to-peak value of the EPR signal. std Establish a linear regression equation k=Σ(C std ×S std ) / Σ(S std 2 ); S303: Requirement R 2 ≥0.995, an alarm is triggered when the deviation of k value is >2%.

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