Liver fibrosis organ chip sensing device for pathological assessment and drug screening

Abstract

The invention discloses a hepatic fibrosis organ chip sensing device for pathological evaluation and drug screening, which belongs to the technical field of microfluidics and comprises a top cover, an upper culture layer, a porous membrane, a lower culture layer and a glass substrate electrochemical electrode. The sensing device is combined with a hepatic fibrosis organ chip and a three-electrode detection system, gel permeability change is detected on the basis of electrochemical cyclic voltammetry, the hepatic fibrosis process is indirectly evaluated, and then in-vitro rapid screening of hepatic fibrosis potential drugs is carried out. The liver fibrosis organ chip sensing device provided by the invention provides a novel and efficient research means for real-time monitoring and quantitative evaluation of a liver fibrosis in-vitro model. The problems that in the prior art, due to the fact that dynamic and cross-scale substance transmission characteristics in a precise quantification three-dimensional tissue model are still limited, microenvironment changes dominated by extracellular matrix reconstruction are difficult to reflect in a non-invasive and in-situ mode, and the hepatic fibrosis process is difficult to detect are solved.

Description

Technical Field

[0001] This invention relates to the field of microfluidics, and more specifically to an organ-on-a-chip sensing device for pathological assessment and drug screening of liver fibrosis. Background Technology

[0002] Liver fibrosis is a pathological process of the extracellular matrix (ECM) in the liver caused by factors such as chronic liver injury. It is mainly characterized by excessive production and scarring of ECM, including collagen and fibrous tissue, following activation of hepatic stellate cells, ultimately leading to irreversible end-stage diseases such as cirrhosis. Currently, the specific pathogenesis of liver fibrosis is unclear, the process is difficult to monitor, and effective direct-acting anti-fibrotic drugs are lacking.

[0003] Organ-on-a-chip (OoC) technology combines microfluidics with tissue engineering, simulating the local spatial structure of microscale organ microunits through structural design and providing physiologically simulated shear flow to promote improved cell population distribution and functional expression, offering highly biomimetic in vitro models for drug and disease research. However, traditional methods for analyzing OoC models, such as immunofluorescence staining, Western blotting, and enzyme-linked immunosorbent assay (ELISA), often require the fixation, pretreatment, and dye incubation of biological samples. These procedures are cumbersome, time-consuming, and make it difficult to continuously monitor in-situ and dynamic changes during the experimental process.

[0004] Electrochemical analysis techniques, with their rapid response and real-time monitoring capabilities, are widely used in cell biology research for the detection of lactic acid, glucose, and various enzyme metabolites. Previous studies have combined methods such as cell electrical impedance sensing and transepithelial / endothelial resistance measurement in organ-on-a-chip systems to achieve the determination of cell barrier permeability.

[0005] However, the aforementioned traditional electrochemical methods still have limitations in accurately quantifying the dynamic, cross-scale material transport characteristics within three-dimensional tissue models. They are difficult to reflect microenvironmental changes dominated by extracellular matrix remodeling in a non-invasive and in situ manner, and the process of liver fibrosis is not easy to detect. Summary of the Invention

[0006] To address this, the present invention provides an organ-on-a-chip sensing device for pathological assessment and drug screening of liver fibrosis, thereby solving the problem that existing technologies are limited in accurately quantifying the dynamic and cross-scale material transport characteristics within three-dimensional tissue models, making it difficult to non-invasively and in situ reflect changes in the microenvironment dominated by extracellular matrix remodeling, and thus difficult to detect the process of liver fibrosis.

[0007] To achieve the above objectives, the present invention provides the following technical solution: According to a first aspect of the present invention, an organ-on-a-chip sensing device for pathological assessment and drug screening of liver fibrosis is provided. The organ-on-a-chip sensing device includes: an organ-on-a-chip and an electrochemical detection module; the organ-on-a-chip includes, from top to bottom: a top cover 1, an upper culture layer 2, a porous membrane 3, a lower culture layer 4, and a glass-substrate electrochemical electrode 5; the electrochemical detection module includes a conventional single-channel electrochemical workstation; the top cover and the upper culture layer are provided with liquid passage holes 6, and the upper culture layer and the lower culture layer are provided with liquid flow channels 7; the glass-substrate electrochemical electrode is connected to a corresponding electrode clamp of the electrochemical workstation.

[0008] According to a second aspect of the present invention, an application of an organ-on-a-chip sensing device for pathological assessment and drug screening of liver fibrosis is provided. The sensing device combines a liver fibrosis organ-on-a-chip with a three-electrode detection system to indirectly assess the progress of liver fibrosis based on changes in gel permeability detected by electrochemical cyclic voltammetry, thereby enabling rapid in vitro screening of potential drugs for liver fibrosis.

[0009] Furthermore, the electrolyte detected by the electrochemical cyclic voltammetry is a gel obtained by gelling a prepolymer of cells and collagen I solution.

[0010] Furthermore, the assessment includes the following steps: S1. Establish the concentration-current response relationship through cyclic voltammetry curves to calibrate the detection system; S2. Assess the progression of liver fibrosis by measuring the concentration of electroactive substances and based on the concentration-current response relationship; S3. Measure the cyclic voltammetric curves at different osmosis durations, and evaluate the osmosis rate by fitting the curves to assess the progression of liver fibrosis.

[0011] Further, step S1 includes the following steps: Different concentrations of electroactive substances were added to the culture layer under the liver organ microarray, and the electrochemical response was measured by cyclic voltammetry. Cyclic voltammetry curves corresponding to different concentrations were obtained, the oxidation peak current was extracted, the concentration-current response relationship was established, and a linear fitting equation was obtained.

[0012] Further, step S2 includes the following steps: Electrochemically measured concentrations of electroactive substances reflect changes in permeability. Electroactive substances are added to the culture layers on organ-on-a-chip devices with and without liver fibrosis. The concentration of electroactive substances permeating from the upper culture layer to the lower culture layer is measured. Based on the concentration-current response relationship, changes in permeability are assessed using the oxidation peak current, thereby evaluating the progress of liver fibrosis.

[0013] Further, step S3 includes the following steps: Furthermore, the permeation rate was introduced, and cyclic voltammetric curves were measured under different permeation times. The fitting relationship between the oxidation peak current and the permeation time was established. The permeation rate was evaluated by using the slope of the fitted straight line, and the liver fibrosis process was evaluated by combining the changes in permeability.

[0014] Furthermore, the drug screening includes the following steps: adding different drug stimuli to the upper culture layer of the liver fibrosis organ-on-a-chip, culturing for a certain period of time, comparing the groups with and without drug stimulation, evaluating the effect of the applied drugs on the liver fibrosis pathological model, and conducting in vitro rapid screening of potential drugs for liver fibrosis by evaluating changes in permeability after drug stimulation.

[0015] This invention provides a method for preparing an organ-on-a-chip sensor for pathological assessment and drug screening of liver fibrosis. The method comprises the following steps: Preparation of S1, top cover, upper culture layer, lower culture layer and flow channel: The mold was cleaned sequentially with ethanol and ultrapure water. The polydimethylsiloxane stock solution and its curing agent were mixed at a mass ratio of 10:1 and poured onto the plastic mold. Vacuum was drawn in a freeze dryer to remove air bubbles from the mixture of polydimethylsiloxane stock solution and curing agent. After baking and curing, the polydimethylsiloxane chip was peeled off and cut. Liquid passage holes were punched in the upper culture layer and the top cover. Then the chip was treated with oxygen plasma. S2. Preparation of glass-substrate electrochemical electrode: A 200 nm thick gold electrode was formed on a glass substrate by magnetron sputtering using a standard lift-off process. The diced electrodes were then sequentially immersed in acetone solution, ethanol, and ultrapure water for ultrasonic cleaning. S3, integration of organ-on-a-chip; The components are bonded and fixed according to the structure from top to bottom: top cover, upper culture layer, porous membrane, lower culture layer, and glass substrate electrochemical electrode. S4. Sterilization: The integrated chip is sterilized with ultraviolet light, and ethanol and phosphate buffer solution are added sequentially to the flow channel to clean each flow channel. S5. Fabrication of liver organ-on-a-chip: The lower culture layer was vented with 2×10⁻⁶ ions under low temperature conditions. 6 mL -1 A cell suspension with a density of 1 × 10⁻⁶ was mixed with a 6 mg / mL collagen I solution at a volume ratio of 1:1 to obtain a cell density of 1 × 10⁻⁶. 6 mL -1 A low-temperature collagen-cell mixture with a collagen concentration of 3 mg / mL was used to simulate the periepithelial sinusoidal space side; culture medium was introduced into the upper culture layer and maintained for a certain period of time to form a liver organ microarray, which was used to simulate the endothelial vascular side of the liver sinusoidal structure. S6. Preparation of organ-on-a-chip for liver fibrosis: Adding liver fibrosis-inducing drugs to the culture layer on a liver organ-on-a-chip simulates the secretion process of pathogenic factors. After continuous treatment for an appropriate period of time, the liver cells in the culture layer are activated induction, forming a liver fibrosis organ-on-a-chip. S7. Fabrication of the sensing device: Connect the three electrodes to the corresponding electrode clamps of the electrochemical workstation, and connect the liver fibrosis organ-on-a-chip to the electrochemical detection module to obtain the sensing device.

[0016] Furthermore, the liver fibrosis-inducing drug mentioned in step S6 is TGF-β1.

[0017] The present invention has the following advantages: The liver fibrosis organ-on-a-chip sensing device prepared in this invention can simulate the disease process of liver fibrosis. It is formed by introducing a collagen-cell mixture into the lower culture layer and supplementing the upper culture layer with culture medium, maintaining this process for a certain period. Then, by introducing a culture medium containing an appropriate concentration of inducing drugs into the upper culture layer and continuously inducing for an appropriate period, a liver fibrosis organ-on-a-chip is formed. The organ-on-a-chip integrates electrochemical electrodes, enabling electrochemical detection and providing a novel and efficient research method for real-time monitoring and quantitative assessment of in vitro models of liver fibrosis. This study utilizes electrochemical cyclic voltammetry to dynamically assess the permeability of the extracellular matrix (ECM) during the liver fibrosis process, enabling indirect detection of liver fibrosis progression based on this method. Simulating microscale fibrotic liver tissue, the study also achieves real-time detection of the sclerosis process of the perisinusoidal space ECM on slides, with results reflecting changes in ECM permeability after fibrosis occurs. The permeability assessment method based on voltammetric properties can sensitively quantify the structural evolution of the fibrotic ECM, allowing for real-time indirect detection of liver fibrosis progression. This provides a novel and effective analytical strategy for liver fibrosis and its extracellular matrix research, facilitating rapid in vitro screening of potential drugs for liver fibrosis. Attached Figure Description

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

[0019] The structures, proportions, sizes, etc. illustrated in this specification are only for the purpose of assisting those skilled in the art in understanding and reading the content disclosed herein, and are not intended to limit the conditions under which the present invention can be implemented. Therefore, they have no substantial technical significance. Any modifications to the structure, changes in the proportions, or adjustments to the size, without affecting the effects and objectives that the present invention can produce, should still fall within the scope of the technical content disclosed in the present invention.

[0020] Figure 1 This is a schematic diagram of the organ-on-a-chip structure provided in an embodiment of the present invention; Figure 2 This is a top view of the organ-on-a-chip provided in an embodiment of the present invention; Figure 3 This is a schematic diagram of the structural layers of an organ-on-a-chip provided in an embodiment of the present invention; Figure 4 This is a physical image of the glass-substrate electrochemical electrode in the organ-on-a-chip provided in this embodiment of the invention; Figure 5 This is a physical image of the organ-on-a-chip sensing device for pathological assessment and drug screening in liver fibrosis provided in an embodiment of the present invention. Figure 6 These are fluorescence microscopy images of LX-2 cells under fibrotic culture conditions provided in this embodiment of the invention, wherein... Figure 6 A shows the nuclear staining pattern of LX-2 cells. Figure 6 B is a staining diagram of the actin cytoskeleton. Figure 6 C represents the color merge diagram; Figure 7 These are the cyclic voltammetric response curves and the linear fitting relationship between the oxidation peak current and the concentration of hexaammineruthenium trichloride obtained by the organ-on-a-chip sensing device for liver fibrosis provided in this embodiment of the invention; wherein, Figure 7 A represents the cyclic voltammetric response curve of 0.1–2 mM hexaammineruthenium trichloride. Figure 7 B is a graph showing the linear fit between the oxidation peak current and the concentration of hexaammineruthenium trichloride; Figure 8 This is a comparison of the voltammetric characteristics of the control group and the fibrosis model after 120 min of 2mM hexaammineruthenium trichloride permeation into the organ-on-a-chip sensing device provided in this embodiment of the invention. Figure 9 This is a cyclic voltammetry curve measured at 30, 60, 90 and 120 min during the RuHex permeation liver fibrosis organ-on-a-chip sensing device provided in this embodiment of the invention. Figure 10 This is a linear fitting graph of the oxidation value current of an organ-on-a-chip sensor under conditions of liver fibrosis and non-liver fibrosis.

[0021] In the picture: 1. Top cover; 2. Upper culture layer; 3. Porous membrane; 4. Lower culture layer; 5. Glass substrate electrochemical electrode; 6. Liquid passage hole; 7. Liquid flow channel. Detailed Implementation

[0022] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] According to a first aspect of the present invention, an organ-on-a-chip sensing device for pathological assessment and drug screening of liver fibrosis is provided. The organ-on-a-chip sensing device includes: an organ-on-a-chip and an electrochemical detection module; the organ-on-a-chip includes, from top to bottom: a top cover 1, an upper culture layer 2, a porous membrane 3, a lower culture layer 4, and a glass-substrate electrochemical electrode 5; the electrochemical detection module includes a conventional single-channel electrochemical workstation; the top cover and the upper culture layer are provided with liquid passage holes 6, and the upper culture layer and the lower culture layer are provided with liquid flow channels 7; the glass-substrate electrochemical electrode is connected to a corresponding electrode clamp of the electrochemical workstation.

[0024] According to a second aspect of the present invention, an application of an organ-on-a-chip sensing device for pathological assessment and drug screening of liver fibrosis is provided. The sensing device combines a liver fibrosis organ-on-a-chip with a three-electrode detection system, and indirectly assesses the progress of liver fibrosis based on the detection of changes in gel permeability using electrochemical cyclic voltammetry, thereby enabling rapid in vitro screening of potential drugs for liver fibrosis.

[0025] The electrolyte detected by the electrochemical cyclic voltammetry was a gel obtained by gelling a prepolymer of cells and collagen I solution.

[0026] The evaluation includes the following steps: S1. Establish the concentration-current response relationship through cyclic voltammetry curves to calibrate the detection system; S2. Assess the progression of liver fibrosis by measuring the concentration of electroactive substances and based on the concentration-current response relationship; S3. Measure the cyclic voltammetric curves at different osmosis durations, and evaluate the osmosis rate by fitting the curves to assess the progression of liver fibrosis.

[0027] Step S1 includes the following steps: Different concentrations of electroactive substances were added to the culture layer under the liver organ microarray, and the electrochemical response was measured by cyclic voltammetry. Cyclic voltammetry curves corresponding to different concentrations were obtained, the oxidation peak current was extracted, the concentration-current response relationship was established, and a linear fitting equation was obtained.

[0028] Step S2 includes the following steps: Electrochemically measured concentrations of electroactive substances reflect changes in permeability. Electroactive substances are added to the culture layers on organ-on-a-chip devices with and without liver fibrosis. The concentration of electroactive substances permeating from the upper culture layer to the lower culture layer is measured. Based on the concentration-current response relationship, changes in permeability are assessed using the oxidation peak current, thereby evaluating the progress of liver fibrosis.

[0029] Step S3 includes the following steps: Furthermore, the permeation rate was introduced, and cyclic voltammetric curves were measured under different permeation times. The fitting relationship between the oxidation peak current and the permeation time was established. The permeation rate was evaluated by using the slope of the fitted straight line, and the liver fibrosis process was evaluated by combining the changes in permeability.

[0030] The drug screening process includes the following steps: adding different drug stimuli to the upper culture layer of the liver fibrosis organ microarray, culturing for a certain period of time, comparing the groups with and without drug stimulation, evaluating the effect of the applied drugs on the liver fibrosis pathological model, and conducting rapid in vitro screening of potential drugs for liver fibrosis by evaluating changes in permeability after drug stimulation.

[0031] This invention provides a method for preparing an organ-on-a-chip sensor for pathological assessment and drug screening of liver fibrosis. The method comprises the following steps: Preparation of S1, top cover, upper culture layer, lower culture layer and flow channel: The mold was cleaned sequentially with ethanol and ultrapure water. A mixture of polydimethylsiloxane stock solution and its curing agent was prepared at a mass ratio of 10:1 and poured onto the plastic mold. A vacuum was then applied in a freeze dryer to remove air bubbles from the mixture. After baking and curing, the polydimethylsiloxane chips were peeled off and cut. Fluid passages were made in the upper culture layer and top cover. Finally, the chips were subjected to oxygen plasma treatment. S2. Preparation of glass-substrate electrochemical electrode: A 200 nm thick gold electrode was formed on a glass substrate by magnetron sputtering using a standard lift-off process. The diced electrodes were then sequentially immersed in acetone solution, ethanol, and ultrapure water for ultrasonic cleaning. S3, integration of organ-on-a-chip; The components are bonded and fixed according to the structure from top to bottom: top cover, upper culture layer, porous membrane, lower culture layer, and glass substrate electrochemical electrode. S4. Sterilization: The integrated chip is sterilized with ultraviolet light, and ethanol and phosphate buffer solution are added sequentially to the flow channel to clean each flow channel. S5. Fabrication of liver organ-on-a-chip: The lower culture layer was vented with 2×10⁻⁶ ions under low temperature conditions. 6 mL -1 A cell suspension with a density of 1 × 10⁻⁶ was mixed with a 6 mg / mL collagen I solution at a volume ratio of 1:1 to obtain a cell density of 1 × 10⁻⁶. 6 mL -1 A low-temperature collagen-cell mixture with a collagen concentration of 3 mg / mL was used to simulate the periepithelial sinusoidal space side; culture medium was introduced into the upper culture layer and maintained for a certain period of time to form a liver organ microarray, which was used to simulate the endothelial vascular side of the liver sinusoidal structure. S6. Preparation of organ-on-a-chip for liver fibrosis: Adding liver fibrosis-inducing drugs to the culture layer on a liver organ-on-a-chip simulates the secretion process of pathogenic factors. After continuous treatment for an appropriate period of time, the liver cells in the culture layer are activated induction, forming a liver fibrosis organ-on-a-chip. S7. Fabrication of the sensing device: Connect the three electrodes to the corresponding electrode clamps of the electrochemical workstation, and connect the liver fibrosis organ-on-a-chip to the electrochemical detection module to obtain the sensing device.

[0032] In step S6, the drug used to induce liver fibrosis is TGF-β1.

[0033] To better illustrate the inventiveness of this invention, the following embodiments are provided.

[0034] Example This embodiment includes the following steps: S1, Preparation of the top cover and upper and lower flow channels: The mold was cleaned sequentially with 75% ethanol and ultrapure water. Polydimethylsiloxane (PDMS) stock solution and its SYLGARD 184 were mixed at a mass ratio of 10:1 and poured onto the plastic mold. After vacuuming to remove air bubbles, the mixture was baked at 85°C for 2 hours to cure. Further, the peeled and slit cured PDMS chips were subjected to oxygen plasma treatment. S2. Preparation of glass-substrate electrochemical electrode: A 200 nm thick gold electrode was formed on a glass substrate by magnetron sputtering using a standard lift-off process. The diced electrodes were then sequentially immersed in acetone solution, 75% ethanol and ultrapure water for ultrasonic cleaning for 5 min each. The electrode side was then bonded to the lower flow channel side of the organ-on-a-chip. S3, integration of organ-on-a-chip; The components are bonded and fixed according to the structure from top to bottom: top cover, upper culture layer, porous membrane, lower culture layer, and glass substrate electrochemical electrode. S4. Sterilization: The integrated chip was sterilized with ultraviolet light for 30 minutes, and the flow channel was cleaned by adding 75% ethanol and phosphate buffer solution for 15 minutes each. S5. Fabrication of liver organ-on-a-chip: Passaged hepatic stellate cells (LX-2 cells) were digested with trypsin and resuspended, centrifuged, and then resuspended in DMEM medium, adjusted to 2×10⁶. 6 mL -1 Prepare a solution with the specified density; mix rat tail collagen I (maintained at low temperature), 10x concentrated PBS, 1M NaOH, and ultrapure water to prepare a 6 mg / mL collagen I solution, and then mix with 2×10... 6 mL -1 Mix equal volumes of cell suspensions of 1×10⁻⁶ density to obtain a low-temperature collagen-cell mixture; [The text abruptly ends here, likely due to an incomplete sentence or missing information.] 6 mL -1 A low-temperature collagen-cell mixture with a collagen concentration of 3 mg / mL was introduced into the lower culture layer, and the upper culture layer was supplemented with an equal amount of DMEM medium and placed in a 37°C incubator. After maintaining the culture for 24 hours, a perisinusoidal space ECM structure of hepatic stellate cells was formed, resulting in a liver organ microarray.

[0035] S6. Preparation of organ-on-a-chip for liver fibrosis: After culturing the collagen-cell mixture for 24 hours, the culture medium of the upper culture layer was replaced with a medium containing 10 ng / mL TGF-β1 to induce the activation of LX-2 cells in the chip, causing fibrosis in the perisinusoidal space model. The control group was cultured under physiological conditions in DMEM medium. The corresponding fresh medium was replaced daily, and the induction was continued for 72 hours to obtain liver fibrosis organ microarrays. S7. Fabrication of the sensing device: Connect the three electrodes to the corresponding electrode clamps of the electrochemical workstation, and connect the liver fibrosis organ-on-a-chip to the electrochemical detection module to obtain the sensing device.

[0036] The schematic diagram, top view, and structural layer diagram of the organ-on-a-chip prepared by this invention are as follows: Figure 1-3 As shown. Figure 4 and Figure 5 The images show physical images of the glass-substrate electrochemical electrode in the organ-on-a-chip provided by this invention, and the physical image of the organ-on-a-chip sensing device for pathological evaluation and drug screening in liver fibrosis. In the images: 1. Top cover; 2. Upper culture layer; 3. Porous membrane; 4. Lower culture layer; 5. Glass-substrate electrochemical electrode; 6. Fluid passage; 7. Fluid flow channel. It can be seen that the glass-substrate electrochemical electrode of the organ-on-a-chip prepared by this invention adopts a three-electrode structure.

[0037] Test Example 1 The activation status of hepatic stellate cells in the liver fibrosis organ-on-a-chip prepared in the examples was characterized by staining and imaging. The specific steps included: after 96 h of cell seeding, 4% paraformaldehyde was introduced into the upper flow channel for 30 min to fix the cells, and the cells were washed three times with PBS; DAPI dye and phalloidin dye were diluted with PBS at volume ratios of 1:1000 and 1:200, respectively, and the cell nuclei and cytoskeleton (fibrous actin, F-actin) were stained for 2 h; after washing three times with PBS, the cells were observed under a confocal microscope and layered scanning fluorescence images were captured, and morphological statistical analysis was performed using ImageJ software.

[0038] Figure 6 These are fluorescence microscopy images of LX-2 cells under fibrotic culture conditions provided in this embodiment of the invention, wherein... Figure 6 A shows the nuclear staining pattern of LX-2 cells. Figure 6 B is a staining diagram of the actin cytoskeleton. Figure 6 C is the combined staining image; after 72 hours of TGF-β1 induction, the fluorescence staining results of LX-2 cell nuclei and actin cytoskeleton are as follows. Figure 6 As shown, the cell body and cytoskeleton are extended, and actin is reorganized into a highly contractile bundle of microfilaments, indicating that hepatic stellate cells are activated and transform into a myofibroblast phenotype, marking the occurrence of liver fibrosis. Further analysis of the area, aspect ratio, and nucleocytoplasmic ratio of activated LX-2 cells revealed significant morphological remodeling. Compared to the control group, the activated hepatic stellate cells showed an average area increase of 292.1%, with a corresponding decrease in aspect ratio and nucleocytoplasmic ratio. This indicates that activated LX-2 cells are more densely distributed in three-dimensional space, further validating the remodeling of the extracellular matrix under liver fibrosis conditions.

[0039] Test Example 2 This invention achieves dynamic monitoring of perisinusoidal space (ECM) permeability by introducing an electroactive substance solution into the upper endothelial culture layer and measuring the concentration change of the electroactive substance permeating to the electrochemical electrode surface in the lower culture layer using electrochemical cyclic voltammetry. This allows for rapid assessment of the impact of liver fibrosis on the ECM. In this test example, hexaammineruthenium trichloride (RuHex) was selected as the electroactive tracer for measuring electrochemical permeability, verifying the concentration-current response relationship for calibration of the detection system. Compared to commonly used electrochemical probes such as methylene blue or potassium ferricyanide, RuHex exhibits good electrochemical stability and biocompatibility, making it suitable for long-term detection.

[0040] This invention establishes the relationship between the concentration of an electroactive substance (RuHex) and the current response of an electrochemical voltammetry method. The specific steps are as follows: using DMEM medium, RuHex is prepared into solutions with different concentrations; 0.1, 0.5, 1.0, 1.5 and 2.0 mM RuHex solutions are introduced into the lower culture layer; the electrochemical response of RuHex is measured using cyclic voltammetry, with a scan range of -0.4 V to 0.2 V and a scan rate of 50 mV / s; and a linear equation is obtained by fitting the oxidation peak current with the RuHex concentration.

[0041] The measurement results are as follows Figure 7 As shown, where, Figure 7 A represents the cyclic voltammetric response curve of 0.1–2 mM hexaammineruthenium trichloride. Figure 7 B shows the linear relationship between the oxidation peak current and the concentration of hexaammineruthenium trichloride. It can be seen that as the RuHex concentration increases, the peak values ​​of both the oxidation and reduction currents significantly increase; and a good linear relationship is observed within the concentration range of 0.1 mM–2 mM. The oxidation peak current I… o The linear equation obtained by fitting (µA) to the RuHex concentration c (mM) is I. o =0.025+0.442c, correlation coefficient R 2 =0.983, proving that the electrochemical gold electrode designed in this invention can stably detect the concentration of electroactive ions and provide a standard for subsequent detection of concentration changes to reflect permeability.

[0042] Test Example 3 RuHex was used as an electroactive tracer to detect the permeability of fibrotic organ-on-a-chip (ECM) after 72 hours of TGF-β1 induction. This monitored the electrochemical behavior of electroactive substances permeating into the perisinusoidal space (ECM) during the liver fibrosis process, thereby monitoring ECM sclerosis and density changes. The specific steps included: constructing a liver fibrosis model and a control model without liver fibrosis; introducing a culture medium solution containing 2 mM RuHex into the upper culture layer; measuring the voltammetric characteristics of RuHex slowly permeating into the fibrotic perisinusoidal space ECM; and evaluating the permeability characteristics of the fibrotic perisinusoidal space ECM.

[0043] The cyclic voltammetric curves after 120 min of 2 mM RuHex permeation are as follows: Figure 8As shown, after induction with 10 ng / mL TGF-β1 for 72 h, both the CV oxidation peak current and reduction peak current values ​​of the perisinusoidal space ECM in the fibrotic group decreased. The oxidation peak current in the fibrotic group decreased by 28.3% compared with the control group, reflecting the decrease in the permeability of RuHex in the fibrotic perisinusoidal space ECM. This indicates that the activation of hepatic stellate cells induced by TGF-β1 stimulation promoted the dense distribution of cells in three-dimensional space, hindering the permeation of RuHex from the perisinusoidal space ECM to the electrode surface.

[0044] Test Example 4 This invention further introduces permeation rate evaluation, using a sensing device to detect changes in the peak oxidation current at different permeation times to assess the permeation rate, and then combining permeability assessment to analyze the properties of the local spatial structure of the perisinusoidal space (ECM) in fibrosis. The specific process includes: constructing a liver fibrosis model by treating a liver organ-on-a-chip sensing device with 10 ng / mL TGF-β1, and using a liver organ-on-a-chip sensing device without liver fibrosis as a control model; passing a culture medium solution containing 2 mM RuHex through the upper culture layer; and measuring CV curves at 30, 60, 90, and 120 min during a 120-minute detection period.

[0045] CV measurement curve (cyclic voltammetry curve) as follows Figure 9 As shown, the peak redox current in the perisinusoidal space ECM during fibrosis was significantly lower than that in the control group. Further analysis revealed that the peak CV oxidation current I measured under both constructed and non-constructed liver fibrosis model conditions within a 120-minute infiltration time... oc The value (µA) was fitted to the permeation time t (min) of RuHex, and the results are as follows: Figure 10 As shown. The infiltration rate of RuHex into the perisinusoidal space ECM per unit time is evaluated by the slope (μA / min) of the fitted straight line. The linear fitting results reflect the uniform changes in the three-dimensional space of the perisinusoidal space ECM. The infiltration rate of RuHex in the fibrotic perisinusoidal space ECM per unit time is k. 10ng =0.00495μA / min, significantly lower than the control group (k control = 0.00768 μA / min). Statistical analysis of the change rate of peak CV oxidation current with permeation time using an electrochemical sensor revealed that the permeation rate of RuHex in the activated perisinusoidal space ECM decreased by 36.3%, reflecting the increased density of the three-dimensionally cultured fibrotic perisinusoidal space ECM after TGF-β1-induced activation of hepatic stellate cells. This indicates that as liver fibrosis progresses, activated LX-2 cells morphologically extend and may secrete more collagen, leading to extracellular matrix hardening, thereby increasing ECM density and thus reducing the permeation rate of electroactive substances.

[0046] In summary, the organ-on-a-chip (ECM) sensing device for pathological assessment and drug screening provided in this application can sensitively analyze the permeability and structural changes of the perisinusoidal space (ECM) in fibrosis, providing a new analytical method for assessing ECM sclerosis in the process of liver fibrosis, and further providing a method for rapid in vitro screening of liver fibrosis drugs.

[0047] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. An organ-on-a-chip sensor for liver fibrosis used in pathological assessment and drug screening, characterized in that, The organ-on-a-chip sensing device includes an organ-on-a-chip and an electrochemical detection module. The organ-on-a-chip, from top to bottom, includes a top cover (1), an upper culture layer (2), a porous membrane (3), a lower culture layer (4), and a glass-based electrochemical electrode (5). The electrochemical detection module includes a conventional single-channel electrochemical workstation. The top cover and the upper culture layer are provided with liquid passage holes (6), and the upper culture layer and the lower culture layer are provided with liquid flow channels (7). The glass-based electrochemical electrode is connected to the corresponding electrode clamp of the electrochemical workstation.

2. The application of an organ-on-a-chip sensor for pathological evaluation and drug screening of liver fibrosis, using the organ-on-a-chip sensor for pathological evaluation and drug screening of liver fibrosis as described in claim 1, characterized in that... The sensing device combines an organ-on-a-chip for liver fibrosis with a three-electrode detection system. It detects changes in gel permeability using electrochemical cyclic voltammetry, indirectly assessing the progress of liver fibrosis and enabling rapid in vitro screening of potential drugs for liver fibrosis.

3. The application of the organ-on-a-chip sensing device for pathological evaluation and drug screening in liver fibrosis as described in claim 2, characterized in that, The electrolyte detected by the electrochemical cyclic voltammetry is a gel obtained by gelling a prepolymer of cells and collagen I solution.

4. The application of the organ-on-a-chip sensing device for pathological evaluation and drug screening in liver fibrosis as described in claim 2, characterized in that, The assessment includes the following steps: S1. Establish the concentration-current response relationship through cyclic voltammetry curves to calibrate the detection system; S2. Assess the progress of liver fibrosis by measuring the concentration of electroactive substances and based on the concentration-current response relationship. S3. Measure the cyclic voltammetric curves at different osmosis durations, and evaluate the osmosis rate by fitting the curves to assess the progression of liver fibrosis.

5. The application of the organ-on-a-chip sensing device for pathological evaluation and drug screening in liver fibrosis as described in claim 4, characterized in that, Step S1 includes the following steps: Different concentrations of electroactive substances were added to the culture layer under the liver organ microarray, and the electrochemical response was measured by cyclic voltammetry. Cyclic voltammetry curves corresponding to different concentrations were obtained, the oxidation peak current was extracted, the concentration-current response relationship was established, and a linear fitting equation was obtained.

6. The application of the organ-on-a-chip sensing device for pathological evaluation and drug screening in liver fibrosis as described in claim 4, characterized in that, Step S2 includes the following steps: Electrochemically measured concentrations of electroactive substances reflect changes in permeability. Electroactive substances are added to the culture layers on organ-on-a-chip devices with and without liver fibrosis. The concentration of electroactive substances permeating from the upper culture layer to the lower culture layer is measured. Based on the concentration-current response relationship, changes in permeability are assessed using the oxidation peak current, thereby evaluating the progress of liver fibrosis.

7. The application of the organ-on-a-chip sensing device for pathological evaluation and drug screening in liver fibrosis as described in claim 4, characterized in that, Step S3 includes the following steps: Furthermore, the permeation rate was introduced, and cyclic voltammetric curves were measured under different permeation times. The fitting relationship between the oxidation peak current and the permeation time was established. The permeation rate was evaluated by using the slope of the fitted straight line, and the liver fibrosis process was evaluated by combining the changes in permeability.

8. The application of the organ-on-a-chip sensing device for pathological evaluation and drug screening in liver fibrosis as described in claim 2, characterized in that, The drug screening includes the following steps: adding different drug stimuli to the upper culture layer of the liver fibrosis organ microarray, continuously culturing, comparing the groups with and without drug stimulation, evaluating the effect of the applied drugs on the liver fibrosis pathological model, and conducting rapid in vitro screening of potential drugs for liver fibrosis by evaluating changes in permeability after drug stimulation.

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