Mitochondria screening system for research and development of anti-aging drug CRO

Abstract

The invention discloses a mitochondrial screening system oriented to anti-aging drug CRO research and development, and relates to the technical field of anti-aging drug research and development, the system comprises a light-driven proton pump module, a chemical regulation and control module, a mode switching control unit and a real-time monitoring feedback module, the light-driven proton pump module generates a proton gradient, and the chemical regulation and control module controls the mode switching control unit and the real-time monitoring feedback module; the chemical regulation and control module reduces the proton gradient, the mode switching control unit realizes reversible switching between an optical mode and a chemical mode, and the real-time monitoring feedback module detects the free radical concentration and feeds back regulation parameters; the system further integrates a cross-species verification interface, supports multi-species synchronous testing, realizes reversible regulation and control of mitochondrial free radical gradient, avoids cell adaptability deviation, improves screening accuracy and repeatability, meets the requirements of CRO platform for high throughput and multi-model verification, and provides a reliable screening platform for research and development of anti-aging drugs.

Description

Technical Field

[0001] This invention relates to the field of anti-aging drug development technology, specifically a mitochondrial screening system for CRO development of anti-aging drugs. Background Technology

[0002] Mitochondria, as core organelles in cellular energy metabolism and aging regulation, are closely associated with various age-related diseases due to their dysfunction. Currently, there is a growing demand for mitochondrial phenotype-based screening in anti-aging drug development. However, existing technologies still have limitations in the precise regulation and dynamic monitoring of mitochondrial free radicals, making it difficult to meet the requirements of CRO platforms for high throughput, reproducibility, and physiological mimicry.

[0003] Currently, mitochondrial function regulation mainly relies on genetic or chemical interventions. For example, the patent (CN102952796A) from Beijing University of Science and Technology, entitled "A Method for Screening Active Substances and Anti-aging Products that Quench Free Radicals by Expressing Deformed Purple Red Protein in the Mitochondrial Inner Membrane," uses photosensitive proteins to induce proton concentration gradients to generate free radicals. However, this technology can only achieve unidirectional and irreversible free radical activation, and long-term light exposure can easily lead to cell adaptation bias, affecting the reliability of the screening results. Furthermore, another representative technology, such as the patent "A New Anti-aging Reagent and Its Identification Method" (CN102612564B), uses a cell quiescence model to screen anti-aging compounds. However, it relies on a static cell model and lacks the ability to monitor and regulate the dynamic function of mitochondria in real time. Existing technologies have not resolved the core contradiction between the irreversibility of free radical generation and cell adaptation, making it difficult to achieve gradient simulation and precise evaluation of mitochondrial function in drug screening.

[0004] Given the shortcomings of existing technologies, the core problem this invention aims to solve is that existing free radical induction technologies, due to their unidirectional and irreversible proton gradient generation method, cannot dynamically regulate free radical concentration. Long-term use of these technologies leads to cellular adaptation bias, thereby interfering with the accuracy and reproducibility of anti-aging drug screening. This problem directly restricts the standardized application of mitochondrial screening systems in CRO platforms.

[0005] To address the aforementioned issues, this invention aims to provide a mitochondrial screening system that achieves reversible gradient regulation of mitochondrial free radicals through a photochemical dual-mode synergistic regulation mechanism. This overcomes the defects of cell adaptation and irreversible damage caused by unidirectional induction, providing a more reliable and efficient screening platform for CRO research and development of anti-aging drugs. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a mitochondrial screening system for CRO research and development of anti-aging drugs. This system generates a proton gradient through a light-driven proton pump module, reduces the proton gradient through a chemical regulation module, and achieves reversible switching between light and chemical modes by combining a mode switching control unit. Furthermore, it dynamically adjusts parameters based on a real-time monitoring and feedback module, enabling reversible regulation of the mitochondrial free radical gradient. This avoids cell adaptation bias and meets the requirements of anti-aging drug CRO research and development for screening accuracy, reproducibility, and high throughput, thus overcoming the deficiencies of existing technologies.

[0007] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a mitochondrial screening system for CRO research and development of anti-aging drugs, comprising a light-driven proton pump module, a chemical regulation module, a mode switching control unit and a real-time monitoring and feedback module; The light-driven proton pump module includes a Janus metal-organic monolayer structure embedded in a mitochondrial membrane biomimetic liposome, which drives proton transmembrane transport under light to generate a proton gradient. The chemical regulation module injects rotenone through a microfluidic channel to inhibit the mitochondrial electron transport chain and reduce the proton gradient. The mode switching control unit integrates a photosensitive element and a chemical reagent injection module. It dynamically adjusts the light intensity and rotenone concentration through a programmable controller to achieve reversible switching between light mode and chemical mode. The real-time monitoring and feedback module uses a fluorescent probe to detect free radical levels and collects data through a confocal microscope. The feedback signal automatically adjusts the microfluidic injection rate and illumination parameters to maintain the free radical concentration within the target range.

[0008] Furthermore, the operation method of the mode switching control unit is based on the following free radical gradient reversible control algorithm: in, Indicates time The proton gradient intensity, measured in millivolts. This represents the light intensity, with values ​​ranging from 0 to 100 milliwatts per square centimeter. The proton transport efficiency of the Janus metal-organic monolayer structure is defined as the number of protons transported per square nanometer per second. This indicates the concentration of rotenone, ranging from 0 to 10 micromoles per liter. This represents the inhibition coefficient of rotenone on the electron transport chain; This represents the natural decay constant of the proton gradient. and These are the weighting coefficients for light-driven and chemically regulated reactions, which are dynamically adjusted by the programmed controller based on the real-time monitored free radical concentration.

[0009] Furthermore, the proton transport flux of the Janus metal-organic monolayer structure in the light-driven proton pump module is determined by the following proton transmembrane kinetic model: in, This represents the proton flux through the mitochondrial membrane per unit time. This represents the proton mobility in the Janus metal-organic monolayer structure. This represents the effective membrane area of ​​the biomimetic liposome. This indicates the pH value on the mitochondrial matrix side. This indicates the pH value of the intermembrane space of the mitochondria; The proton transmembrane dynamics model dynamically optimizes illumination parameters using real-time monitored pH differences to linearly generate the proton gradient.

[0010] Furthermore, the inhibition efficiency of rotenone in the chemical regulation module is quantified by the following electron flow interruption equation: in, This indicates the inhibition efficiency of rotenone on the electron transport chain. This represents the inhibition rate constant of rotenone. This indicates that rotenone is at time [time missing]. concentration, Indicates the duration of the chemical regulation mode; The microfluidic channel adjusts the rotenone injection rate in real time according to the inhibition efficiency, so that the proton gradient is reset to the target baseline.

[0011] Furthermore, the dynamic equilibrium of free radical concentration in the real-time monitoring feedback module is achieved through the following feedback control equation: in, It represents the change in free radical concentration per unit time. This indicates the concentration of free radicals detected in real time. Indicates the proton gradient intensity. This indicates the amount of antioxidant compensation provided by the anti-aging drug being tested. This represents the contribution coefficient of the proton gradient to the generation of free radicals. This represents the attenuation coefficient of antioxidant compensation; The output of the feedback control equation is used to dynamically adjust the light intensity and rotenone concentration, so that the free radical concentration is stabilized within a preset range.

[0012] Furthermore, the mode switching control unit supports timing configuration of optical mode, chemical mode and hybrid mode; In light mode, the programmed controller activates the light intensity to the set value and keeps the rotenone concentration at zero; In chemical mode, the programmed controller reduces the light intensity to zero and increases the rotenone concentration to the set value; In hybrid mode, the programmed controller alternately switches the timing of light and chemical stimulation, with a switching cycle of 1 to 10 minutes, and the duration ratio of light mode to chemical mode within each cycle is adjustable between 0.5 and 2.

[0013] Furthermore, the real-time monitoring feedback module integrates the fluorescent probe MitoSOXRed with a confocal microscope, and the monitoring data is subjected to noise filtering and signal enhancement through a convolutional neural network; The input layer of the convolutional neural network receives the original fluorescence intensity sequence, the hidden layer performs temporal feature extraction, and the output layer generates the purified free radical concentration value. The network weights are updated online using the gradient descent method to maintain detection accuracy in different cell models.

[0014] Furthermore, the system also includes a cross-species verification interface for simultaneously testing zebrafish embryos, nematodes, and mammalian cell lines; The cross-species validation interface connects to the microfluidic channel via an adapter, adjusting the flow rate and culture medium composition to suit the osmotic pressure and metabolic needs of different species. The real-time monitoring and feedback module performs standardized processing on free radical data from different species, making cross-species mitochondrial response curves comparable.

[0015] Compared with existing technologies, this mitochondrial screening system for CRO-developed anti-aging drugs has the following beneficial effects: I. This invention utilizes the coordinated operation of a light-driven proton pump module, a chemical regulation module, and a mode switching control unit to achieve proton transmembrane transport and generate a proton gradient through light-driven means. Chemical regulation inhibits the mitochondrial electron transport chain to reduce the proton gradient. Furthermore, a programmed controller dynamically adjusts light intensity and rotenone concentration to achieve reversible switching between light and chemical modes. Simultaneously, a real-time monitoring and feedback module dynamically detects free radical concentration and performs closed-loop parameter adjustment, constructing a reversible free radical gradient regulation mechanism. This mechanism dynamically adjusts free radical concentration, avoiding cell adaptation bias caused by long-term unidirectional induction, thereby ensuring the accuracy and reproducibility of mitochondrial function assessment during anti-aging drug screening.

[0016] Second, this invention integrates a cross-species validation interface and uses an adapter to adjust the flow rate of the microfluidic channel and the composition of the culture medium to adapt to the osmotic pressure and metabolic requirements of different species models, enabling simultaneous testing of cells or embryos from multiple species. At the same time, the real-time monitoring and feedback module uses fluorescent probes combined with convolutional neural networks to filter data noise and enhance signals, achieving accurate detection of free radical concentrations. It also performs standardized processing on monitoring data from different species, which can improve the high-throughput level of drug screening and the comparability of cross-species results, thereby meeting the technical requirements of CRO platforms for multi-model validation and high detection accuracy.

[0017] Other advantages, objectives and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination or study, or may be learned from the practice of the invention. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.

[0019] Figure 1 This is a schematic diagram of the system module structure and collaborative operation of the present invention; Figure 2 This is a free radical dynamic regulation cycle diagram of the present invention; Figure 3 This is a flowchart illustrating the operation of the present invention. Detailed Implementation

[0020] To further illustrate the technical means and effects of the present invention in achieving its intended purpose, the following detailed description of the specific implementation methods, structures, features, and effects of the present invention, in conjunction with the accompanying drawings and preferred embodiments, is provided below.

[0021] Example 1 like Figure 1 and Figure 2As shown in this embodiment, a specific implementation of a mitochondrial screening system for CRO research and development of anti-aging drugs is disclosed. The system aims to achieve precise reversible regulation and dynamic monitoring of mitochondrial free radicals through the coordinated operation of a light-driven proton pump module, a chemical regulation module, a mode switching control unit, and a real-time monitoring feedback module. It combines a free radical gradient reversible regulation algorithm, a proton transmembrane dynamics model, an electron flow interruption equation, and a feedback control equation. The system also supports cross-species validation using zebrafish embryos, nematodes, and mammalian cell lines, meeting the CRO platform's requirements for high-throughput, reproducibility, and physiological simulation in drug screening.

[0022] The system hardware setup and module configuration are implemented as follows: Construction of a light-driven proton pump module: A Janus metal-organic monolayer structure was prepared using Au-TiO2 composite organic framework material. This structure possesses unilateral photoresponsiveness and proton transport characteristics and can be embedded into biomimetic liposomes via self-assembly. The biomimetic liposomes were prepared using phosphatidylcholine lipids via thin-film dispersion to ensure biocompatibility and structural similarity to the natural mitochondrial membrane, forming a carrier that can simulate the mitochondrial membrane environment. The prepared Janus metal-organic monolayer structure-biomimetic liposome complex was used as a mitochondrial functional simulation model and placed in a constant-temperature incubation environment to maintain a stable temperature and avoid interference with proton transport efficiency.

[0023] Chemical regulation module construction: A microfluidic chip is fabricated using PDMS material. The chip is equipped with independent reagent storage chambers, transport channels, and reaction chambers. The inner diameter and length of the transport channels are adapted to the precise transport requirements of rotenone reagent. Rotenone stock solution is stored in the reagent storage chamber and connected to the transport channel via a micro-injection pump to achieve quantitative injection of rotenone. The reaction chamber is connected to the mitochondrial simulation model placement area of ​​the light-driven proton pump module to ensure that the injected rotenone can quickly contact the mitochondrial simulation model and act on the electron transport chain.

[0024] Mode switching control unit configuration: An embedded MCU is selected as the programmable controller. This controller has multi-channel data acquisition and output control functions and can be connected to both the photosensitive element and the chemical reagent injection module simultaneously. The photosensitive element is a silicon-based photoelectric sensor, installed on the side of the mitochondrial simulation model, which collects ambient light intensity data in real time and transmits it to the programmable controller. The chemical reagent injection module is linked with the micro-injection pump of the microfluidic chip and receives instructions from the programmable controller to adjust the injection rate. At the same time, the controller has a built-in timing control program that supports parameter configuration and switching between light mode, chemical mode and mixed mode.

[0025] The real-time monitoring and feedback module was constructed as follows: The fluorescent probe MitoSOXRed was loaded into the mitochondrial simulation model at the conventional experimental concentration. This probe can specifically bind to free radicals generated by mitochondria and emit fluorescent signals. The excitation and emission wavelengths of the confocal microscope were set to the characteristic wavelengths of MitoSOXRed. The lens was aimed at the mitochondrial simulation model area to acquire fluorescence intensity images in real time and convert them into digital signals. A convolutional neural network was deployed in the data processing unit. The input layer of the network received the raw fluorescence intensity sequence transmitted by the confocal microscope. The hidden layer extracted the temporal features in the sequence through 3×3 convolution kernels. The output layer generated the purified free radical concentration value. The network weights were updated online using the gradient descent method to adapt to the signal characteristics of different cell models and maintain detection accuracy.

[0026] Cross-species validation interface configuration: A replaceable adapter is connected to the rear end of the reaction chamber of the microfluidic chip. This adapter can change the liquid flow rate by adjusting the flow resistance of the internal channel to adapt to the different metabolic rates and osmotic pressure requirements of zebrafish embryos, nematodes, and mammalian cell lines. At the same time, multiple culture medium storage units are configured to store zebrafish embryo-specific embryonic culture medium, nematodegrowth medium (NGM) for nematodes, and DMEM culture medium for mammalian cells, respectively. Through the linkage between the adapter and the microfluidic channel, the precise delivery of different culture media can be achieved. The data processing unit is equipped with a standardized algorithm to convert the free radical concentration data of different species into relative concentration values ​​based on the blank control group, ensuring the comparability of cross-species mitochondrial response curves.

[0027] The system operation process and the implementation of the mathematical model are as follows: Proton gradient generation of the light-driven proton pump module: When the light-driven proton pump module is started, the programmable controller sends a command to the photosensitive element to activate the light source and adjust the light intensity to the initial set value; the Janus metal-organic monolayer structure generates photogenerated carriers under the action of light, driving protons to be transported across the membrane from the membrane gap of the biomimetic liposome to the matrix side, thus generating a proton gradient.

[0028] In this process, the proton transport flux of the Janus metal-organic monolayer structure is regulated by a proton transmembrane dynamics model, the model expression of which is as follows: In the formula, It represents the proton flux through the mitochondrial membrane per unit time. Its value directly reflects the efficiency of proton transmembrane transport. Too high or too low flux will affect the stability of the proton gradient. This parameter represents the proton mobility within the Janus metal-organic monolayer structure. It is determined by the material composition and microstructure of the Janus structure. In this embodiment, the Au-TiO2 composite organic framework... The values ​​were calibrated through previous material characterization experiments to ensure they matched the proton transport properties of the mitochondrial membrane. The effective membrane area of ​​the biomimetic liposome is controlled by the liposome preparation process. In this embodiment, the liposome particle size is measured by a dynamic light scattering instrument, and the effective membrane area is calculated to ensure the uniformity of the membrane area and maintain the consistency of proton transport. This indicates the pH value on the mitochondrial matrix side. The pH value represents the intermembrane pH of the mitochondrial membrane, and both are monitored in real time using a pH electrode.

[0029] When detected and When the difference increases, the programmed controller appropriately reduces the light intensity based on the calculation results of the proton transmembrane dynamics model to slow down the proton transmembrane rate and ensure linear generation of the proton gradient; when the difference decreases, the light intensity is increased to maintain the linear trend of gradient generation and avoid sudden increases or decreases in the proton gradient that could lead to abnormal function of the mitochondrial simulation model.

[0030] Proton gradient suppression by the chemical regulation module: When it is necessary to reduce the proton gradient, the programmable controller switches to chemical regulation mode and sends a command to the microinjection pump of the microfluidic chip to inject rotenone reagent; rotenone binds to the electron transport chain complex in the mitochondrial simulation model, inhibiting the electron transport process and thus reducing the proton gradient.

[0031] The inhibition efficiency of rotenone was quantified by an electron flow interruption equation, as shown below: In the formula, The value represents the inhibition efficiency of rotenone on the electron transport chain, ranging from 0 to 1, where 0 indicates no inhibition and 1 indicates complete inhibition. In this embodiment, the value is taken when the proton gradient decreases to the target baseline. As a control threshold; The inhibition rate constant of rotenone is represented by the parameter, which is determined by the batch purity of rotenone and the active state of the electron transport chain in the mitochondrial simulation model. The effect of different concentrations of rotenone on the electron transport rate was determined through preliminary experiments, and the result was obtained by fitting the data. value; Indicates time The concentration of rotenone was calculated from the injection rate and reaction chamber volume of the microfluidic chip. This indicates the duration of the chemical regulation mode, which is dynamically adjusted by the programmable controller based on the monitoring results of the inhibition efficiency.

[0032] When detected When the flow rate is below a threshold, the programmable controller increases the injection rate of the microinjection pump, thereby increasing... To improve inhibition efficiency; when When the threshold is reached, the injection rate is reduced to maintain the concentration, so that the proton gradient is stably reset to the target baseline.

[0033] Multi-mode operation of the mode switching control unit: The mode switching control unit realizes the timing configuration and switching of optical mode, chemical mode and hybrid mode through a programmable controller. The operating logic of the three modes is as follows: Light mode: The programmable controller sends a command to the light source to activate the light intensity to the initial set value, and at the same time controls the micro-injection pump to stop the injection, so that the rotenone concentration is kept at zero. At this time, the system generates a proton gradient only through the light-driven proton pump module. Chemical mode: The programmed controller shuts off the light source, reduces the light intensity to zero, and simultaneously starts the micro-injection pump to inject rotenone, increasing the concentration to the initial set value. At this time, the system only suppresses the proton gradient through the chemical regulation module. Hybrid mode: The programmed controller loads a timing control program to alternately switch between light and chemical stimulation. The switching cycle is set to 1 to 10 minutes, and the duration ratio of light mode to chemical mode in each cycle is set to 0.5 to 2, which can be adjusted according to screening requirements.

[0034] The switching between the three modes is based on the free radical gradient reversible modulation algorithm to achieve precise control of the proton gradient. The algorithm expression is as follows: In the formula, Indicates time The proton gradient intensity, measured in millivolts, is a core parameter reflecting the mitochondrial membrane potential state, and its value is calibrated in real time using a membrane potential detection kit. This represents the light intensity, ranging from 0 to 100 milliwatts per square centimeter, and is collected in real time by a photosensitive element and fed back to the controller. The proton transport efficiency of the Janus metal-organic monolayer structure is defined as the number of protons transported per square nanometer per second. This parameter is determined by the material properties and fabrication process of the Janus structure and is obtained through proton conductivity measurement experiments. The concentration of rotenone is indicated, ranging from 0 to 10 micromoles per liter, and is monitored in real time by the concentration sensor of the microfluidic chip. The inhibition coefficient of rotenone on the electron transport chain is determined by the active state of the electron transport chain in the mitochondrial simulation model and is obtained by fitting the relationship between the electron transport rate and the concentration of rotenone. The natural decay constant of the proton gradient is determined by factors such as experimental temperature and culture medium composition. It is calculated by measuring the decay rate of the proton gradient through a blank control experiment. value; and These are the weighting coefficients for light-driven and chemically regulated reactions, respectively. Their values ​​are dynamically adjusted based on real-time monitoring of the free radical concentration—when the free radical concentration exceeds a preset range, the programmed controller increases the value. and reduce Reduce by enhancing chemical regulation This reduces the generation of free radicals; when the free radical concentration is below a preset range, it increases... and reduce Enhance the light-driven effect to improve Promotes the generation of free radicals and ensures Co-stabilization with free radical concentration.

[0035] The free radical dynamic balance of the real-time monitoring feedback module: The real-time monitoring feedback module collects free radical signals through a fluorescent probe and confocal microscope. After processing by a convolutional neural network, it achieves the dynamic balance of free radical concentration based on a feedback control equation. The specific process is as follows: After the fluorescent probe MitoSOXRed is loaded onto a mitochondrial simulation model, it specifically binds to free radicals and emits fluorescence. A confocal microscope acquires fluorescence images at set time intervals, converts the images into fluorescence intensity sequences, and transmits them to a data processing unit. A convolutional neural network performs noise filtering and signal enhancement on the original fluorescence intensity sequences. The input layer receives the sequence data, the hidden layer extracts temporal features from the sequence through convolution operations, and the output layer generates the purified free radical concentration values. The network weights are updated online using the gradient descent method, with each update based on the detection error adjustment of the previous cycle, ensuring that detection accuracy is maintained in different models such as zebrafish embryos, nematodes, and mammalian cell lines.

[0036] The dynamic equilibrium of free radical concentration is achieved through a feedback control equation, expressed as follows: In the formula, It represents the change in free radical concentration per unit time, and its positive or negative sign reflects the rising or falling trend of free radical concentration; The concentration of free radicals detected in real time is output by a convolutional neural network; This represents the proton gradient intensity, as described in the previous algorithm. Maintain consistency; This indicates the amount of antioxidant compensation provided by the anti-aging drug under test, which is determined by the concentration and activity of the drug under test and is calibrated through a drug concentration gradient experiment. The contribution coefficient of the proton gradient to free radical generation is determined by the activity of the mitochondrial respiratory chain, and is obtained by measuring different... The free radical generation rate was obtained by fitting the data. The attenuation coefficient, representing antioxidant compensation, is determined by the metabolic rate of antioxidants and is calculated by monitoring the attenuation process of free radical concentration after drug action.

[0037] When the output of the feedback control equation When the value is positive, it indicates that the free radical concentration is increasing, and the programmed controller will reduce the light intensity. Or increase rotenone concentration By reducing Reduce free radical generation; when When it is negative, increase or reduce By improving Promotes the generation of free radicals, ultimately leading to It remains stable within the preset range.

[0038] Collaborative operation of the cross-species validation interface: The cross-species validation interface, through an adapter, links with microfluidic channels and culture medium storage units to achieve synchronous testing of different species models. For zebrafish embryos, the adapter adjusts the flow rate of the microfluidic channel to match the rate of embryonic development, and the culture medium storage unit provides zebrafish embryo-specific culture medium to maintain the normal developmental environment of the embryos; for nematodes, the flow rate is adjusted to match the rate of nematode movement, and NGM culture medium is used; for mammalian cell lines, the flow rate is adjusted to match the rate of cell adhesion and growth, and DMEM culture medium is used.

[0039] After collecting free radical data from different species, the real-time monitoring and feedback module converts the data into uniform relative values ​​through a standardized algorithm, eliminating the influence of metabolic differences between different species on the data. This allows for direct comparison of mitochondrial response curves from zebrafish embryos, nematodes, and mammalian cell lines, providing data support for cross-species activity assessment of drugs.

[0040] In summary, this embodiment achieves precise and reversible regulation and dynamic monitoring of mitochondrial free radicals through the coordinated operation of the light-driven proton pump module, chemical regulation module, mode switching control unit, and real-time monitoring feedback module, combined with the proton transmembrane dynamics model, electron flow interruption equation, free radical gradient reversible regulation algorithm, and feedback control equation. Simultaneously, the configuration of a cross-species verification interface supports simultaneous testing of multiple species models, solving the cell adaptation bias problem caused by unidirectional induction in existing technologies.

[0041] Example 2 like Figure 3 As shown in Example 1, this example details the specific steps of a mitochondrial screening system for anti-aging drug CRO development during operation. The specific steps are as follows: 1. System initialization and parameter settings: Start the system's various modules, including the light-driven proton pump module, the chemical regulation module, the mode switching control unit, and the real-time monitoring and feedback module.

[0042] Preset parameters are loaded through a programmable controller, including initial light intensity, rotenone concentration, target free radical concentration range, and mode switching sequence.

[0043] Check whether the microfluidic channels, light sources, sensors, and data acquisition devices are in normal working order.

[0044] 2. Select and activate the operation mode: Depending on the screening requirements, select the light mode, chemical mode, or mixed mode in the programmable controller: Light mode: Activate the light source and set the light intensity; rotenone injection is paused and the concentration remains at zero.

[0045] Chemical mode: Turn off the light source; start the micro-injection pump and inject rotenone to the set concentration.

[0046] Mixed Mode: Sets the alternation cycle and duration ratio of light and chemical stimulation.

[0047] 3. Perform light-driven proton gradient generation: During the illumination phase in either the light mode or the hybrid mode, the Janus metal-organic monolayer structure drives proton transmembrane transport under illumination, forming a proton gradient.

[0048] The pH values ​​on both sides of the membrane are monitored in real time, and the light intensity is dynamically adjusted by a programmed controller to maintain the stable generation of the proton gradient.

[0049] 4. Perform chemical regulation to suppress the proton gradient: In the chemical phase of either chemical or mixed-mode operation, rotenone is injected into the microfluidic channel to inhibit the mitochondrial electron transport chain.

[0050] Based on the real-time detected proton gradient intensity, the rotenone injection rate is adjusted to gradually reset the gradient to the target baseline.

[0051] 5. Real-time monitoring of free radical concentration: A mitochondrial simulation model was labeled with the fluorescent probe MitoSOXRed, and fluorescence signals were continuously acquired using a confocal microscope.

[0052] The collected fluorescence intensity sequence is processed by a convolutional neural network to filter out noise and output the purified free radical concentration value.

[0053] 6. Dynamic feedback and parameter adjustment: Compare real-time free radical concentration with the target range: If the concentration is too high, the programmed controller will reduce the light intensity or increase the rotenone injection rate.

[0054] If the concentration is too low, increase the light intensity or reduce the rotenone injection rate.

[0055] The system continuously monitors and adjusts to keep the free radical concentration within a preset range.

[0056] 7. Cross-species testing and data standardization: The microfluidic channel flow rate and culture medium type can be adjusted by replacing the adapter, making it suitable for zebrafish embryos, nematodes or mammalian cell lines.

[0057] Free radical response data from different species were collected, processed by a standardized algorithm, and converted into relative concentration values ​​to facilitate cross-species comparisons.

[0058] 8. Data Output and System Shutdown: Record the free radical concentration curves, proton gradient intensity, and parameter adjustment logs for each mode.

[0059] After completing the test, gradually turn off the light source, stop the reagent injection, save the data, and reset the system.

[0060] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A mitochondrial screening system for anti-aging drug CRO R&D, comprising a light-driven proton pump module, a chemical regulation module, a mode switching control unit and a real-time monitoring feedback module, characterized in that ； The light-driven proton pump module comprises a Janus metal organic monolayer structure embedded in a mitochondrial membrane mimetic liposome, which drives proton transport across the membrane under light to generate a proton gradient; The chemical regulation module injects rotenone through a microfluidic channel to inhibit the mitochondrial electron transport chain and reduce the proton gradient; The mode switching control unit integrates a photosensitive element and a chemical reagent injection module, dynamically adjusts the light intensity and rotenone concentration through a programmed controller, and realizes reversible switching between the light mode and the chemical mode; The real-time monitoring feedback module uses a fluorescent probe to detect the level of free radicals and collects data through a confocal microscope, and the feedback signal automatically adjusts the microfluidic injection rate and light parameters to maintain the free radical concentration within the target interval.

2. The mitochondrial screening system for anti-aging drug CRO R&D according to claim 1, wherein, The operation method of the mode switching control unit is based on the following reversible free radical gradient control algorithm: wherein, represents the proton gradient intensity at time , in millivolt, represents the light intensity, ranging from 0 to 100 milliwatts per square centimeter, represents the proton transport efficiency of the Janus metal organic monolayer structure, defined as the number of protons transported per square nanometer per second, represents the concentration of rotenone, ranging from 0 to 10 micromoles per liter, represents the inhibition coefficient of rotenone on the electron transport chain; represents the natural decay constant of the proton gradient, and are the weight coefficients of light-driven and chemical-regulated, respectively, dynamically adjusted by the programmed controller according to the real-time monitored free radical concentration.

3. The mitochondrial screening system for anti-aging drug CRO R&D according to claim 1, characterized in that, The proton transport flux of the Janus metal organic monolayer structure in the light-driven proton pump module is determined by the following proton transport kinetics model: wherein, represents the proton flux through the mitochondrial membrane per unit time, represents the mobility of protons in the Janus metal organic monolayer structure, represents the effective membrane area of the biomimetic liposome, represents the pH value on the mitochondrial matrix side, represents the pH value of the mitochondrial membrane gap; The proton transport kinetics model dynamically optimizes the light parameters based on the real-time monitored pH difference, which is used for linear generation of the proton gradient.

4. The mitochondrial screening system for anti-aging drug CRO R&D according to claim 1, characterized in that, The inhibition efficiency of rotenone in the chemical regulation module is quantified by the following electron flow interruption equation: wherein, represents the inhibition efficiency of rotenone on the electron transport chain, represents the inhibition rate constant of rotenone, represents the concentration of rotenone at time t, represents the duration of the chemical modulation pattern; The microfluidic channel adjusts the rotenone injection rate in real time according to the inhibition efficiency to reset the proton gradient to the target baseline.

5. The mitochondrial screening system for anti-aging drug CRO R&D according to claim 1, characterized in that, The dynamic balance of free radical concentration in the real-time monitoring feedback module is achieved through the following feedback control equation: wherein, represents the amount of change in the concentration of free radicals per unit time, represents the concentration of free radicals detected in real time, represents the intensity of the proton gradient, represents the amount of antioxidant compensation provided by the anti-aging drug to be tested, represents the contribution coefficient of the proton gradient to the generation of free radicals, represents the decay coefficient of antioxidant compensation; The output of the feedback control equation is used to dynamically adjust the light intensity and rotenone concentration to stabilize the free radical concentration within the preset interval.

6. The mitochondrial screening system for anti-aging drug CRO R&D according to claim 1, wherein, The mode switching control unit supports the timing configuration of the light mode, the chemical mode, and the mixed mode; In the light mode, the programmed controller activates the light intensity to the set value and keeps the rotenone concentration at zero; In the chemical mode, the programmed controller reduces the light intensity to zero and increases the rotenone concentration to the set value; In the mixed mode, the programmed controller alternately switches the timing of light and chemical stimulation, with a switching period of 1 to 10 minutes, and the duration ratio of light mode to chemical mode within each period is adjustable between 0.5 and 2.

7. The mitochondrial screening system for anti-aging drug CRO R&D according to claim 1, characterized in that, The real-time monitoring feedback module integrates the fluorescent probe MitoSOX Red and the confocal microscope, and the monitoring data is processed through a convolutional neural network for noise filtering and signal enhancement; The input layer of the convolutional neural network receives the original fluorescence intensity sequence, the hidden layer performs time series feature extraction, and the output layer generates the purified free radical concentration value. The network weights are updated online through the gradient descent method to maintain detection accuracy in different cell models.

8. The mitochondrial screening system for anti-aging drug CRO R&D according to claim 1, characterized in that, The system also includes a cross-species verification interface for simultaneous testing of zebrafish embryos, nematodes, and mammalian cell lines; The cross-species verification interface connects the microfluidic channel through an adapter, adjusts the flow rate and medium composition to adapt to the osmotic pressure and metabolic needs of different species; The real-time monitoring feedback module performs standardization processing on free radical data of different species to make the cross-species mitochondrial response curves comparable.

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