A device for detecting in real time the penetration of a drug through the blood-brain barrier and a method for operating the same

Abstract

The application belongs to the technical field of real-time detection of the penetration amount of central nervous system disease treatment drugs, and particularly relates to a device for real-time detection of drug penetration of the blood-brain barrier, which comprises an electrochemical sensor, a blood-brain organ chip and a clamp, the electrochemical sensor is fixedly connected with the blood-brain organ chip through the clamp, the blood-brain organ chip is located above the electrochemical sensor, and the electrochemical sensor is modified with a specific sensitive film. The application can measure the current response of the drug in the penetration process in real time under the change of different flow rates, so as to determine the penetration amount of the drug penetration of the blood-brain barrier, and has important significance for revealing and analyzing the process of the drug penetration of the blood-brain barrier for central nervous system disease treatment. In addition, a working method of the device is also disclosed.

Description

Technical Field

[0001] This invention belongs to the technical field of real-time detection of drug penetration for the treatment of central nervous system diseases, and specifically relates to a device for real-time detection of drug penetration across the blood-brain barrier and its working method. Background Technology

[0002] Currently, many drugs used in the medical field to treat central nervous system diseases, such as levodopa (L-dopa, C9H), are... 11 Levodopa (NO4) crosses the blood-brain barrier and enters brain tissue. It is then decarboxylated by dopa decarboxylase and converted into dopamine, exerting its effects. It is suitable for primary Parkinson's disease and non-drug-induced Parkinson's syndrome. However, excessive use of levodopa can lead to involuntary movements, hallucinations, delusions, anxiety, and depression, as well as potential cardiovascular and gastrointestinal complications, severely impacting patients' daily lives. Therefore, real-time monitoring of levodopa's penetration across the blood-brain barrier is crucial for timely prevention of related side effects.

[0003] Current methods for detecting drugs used in the treatment of central nervous system diseases, such as chromatography and capillary zone electrophoresis, require drug extraction and pretreatment at the endpoint. Furthermore, these methods are relatively complex and cannot meet the demands of real-time detection. Therefore, a novel technical solution is urgently needed to address these issues. Summary of the Invention

[0004] One of the objectives of this invention is to provide a device for real-time detection of drug penetration into the blood-brain barrier, addressing the shortcomings of existing technologies. This device utilizes an electrochemical sensor modified with a specific sensitive film to culture endothelial cells on a blood-brain organochip to form a complete and dense blood-brain barrier layer. This allows for efficient, sensitive, and real-time detection of the process by which drugs for treating central nervous system diseases penetrate the blood-brain barrier.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A device for real-time detection of drug penetration across the blood-brain barrier includes an electrochemical sensor, a blood-brain organ-on-a-chip, and a clamp. The electrochemical sensor is fixedly connected to the blood-brain organ-on-a-chip via the clamp, and the blood-brain organ-on-a-chip is located above the electrochemical sensor. The electrochemical sensor is modified with a specific sensitive film.

[0007] As an improvement to the device for real-time detection of drug penetration across the blood-brain barrier described in this invention, the specific sensitive membrane is used to specifically identify the drug.

[0008] As an improvement to the device for real-time detection of drug penetration into the blood-brain barrier described in this invention, the blood-brain organochip is composed of an upper structure, a lower structure, and a culture layer bonded together. The culture layer is located between the upper structure and the lower structure. The upper flow channels in the upper structure and the lower flow channels in the lower structure are arranged in a cross shape. The culture layer serves as the interface for the formation of the blood-brain barrier.

[0009] As an improvement to the device for real-time detection of drug penetration across the blood-brain barrier described in this invention, the upper structure is provided with an upper flow channel, the length, width and height of which are 10000 μm, 2000 μm and 150 μm, respectively.

[0010] As an improvement to the device for real-time detection of drug penetration across the blood-brain barrier described in this invention, the lower structure is provided with a lower flow channel, the length, width and height of which are 10000 μm, 2000 μm and 2000 μm respectively.

[0011] As an improvement to the device for real-time detection of drug penetration across the blood-brain barrier described in this invention, the culture layer is a polycarbonate membrane with multiple pores. The diameter of the pores is set to 400 nm. In practical applications, the pores are used to observe drug penetration within the upper and lower flow channels.

[0012] As an improvement to the device for real-time detection of drug penetration across the blood-brain barrier described in this invention, the electrochemical sensor has a three-electrode system structure, which includes a counter electrode, a reference electrode, and a working electrode. The working electrode is indium tin oxide, the counter electrode is a platinum wire, the reference electrode is silver / silver chloride, and the working electrode is modified with the specific sensitive film.

[0013] As an improvement to the device for real-time detection of drug penetration across the blood-brain barrier described in this invention, the working electrode is located below the blood-brain organochip. The counter electrode and the reference electrode should extend through the inlet and outlet of the lower channel in the lower structure and directly contact the liquid in the lower channel. The counter electrode and the reference electrode are not in contact with the upper structure and the culture layer. The counter electrode, the reference electrode, and the working electrode are used to connect to an electrochemical workstation and then to a PC host computer.

[0014] The second objective of this invention is to provide a method for operating the aforementioned device for real-time detection of drug penetration across the blood-brain barrier, comprising the following steps:

[0015] Step 1: Form a complete and dense blood-brain barrier layer on the blood-brain organ-on-a-chip, connect the electrochemical sensor to the electrochemical workstation, and then connect it to the PC host computer.

[0016] Step 2: Inject a matrix solution at the connection point between the electrochemical sensor and the blood-brain organ-on-a-chip. Introduce an experimental solution of a set concentration into the input terminal of the blood-brain organ-on-a-chip. The drug in the experimental solution that has permeated into the connection point and the matrix solution reforms into a recombinant experimental solution. The recombinant experimental solution is oxidized and loses electrons on the surface of the specific sensitive film.

[0017] Step 3: The electrons lost in Step 2 are transmitted to the PC host computer through the electrochemical sensor to form a response current, thereby realizing the real-time detection of drug penetration.

[0018] As an improvement to the working method described in this invention, step three further includes: scanning at the phase connection point, wherein the scanning method is to scan once every 5 minutes at a constant potential of 0.4V.

[0019] As an improvement to the working method described in this invention, the matrix solution in step two is used to support the drug.

[0020] As an improvement to the working method described in this invention, the experimental solution in step two is a mixture of the drug and the matrix solution.

[0021] As an improvement to the working method described in this invention, the method for forming the blood-brain barrier layer in step one is as follows: combining endothelial cells with glial cells or pericytes, introducing fluid shear stress into the endothelial cell layer, and completing the growth and formation of the blood-brain barrier layer.

[0022] The beneficial effects of this invention are that the device for real-time detection of drug penetration across the blood-brain barrier includes an electrochemical sensor, a blood-brain organochip, and a clamp. The electrochemical sensor is fixedly connected to the blood-brain organochip via the clamp, and the blood-brain organochip is positioned above the electrochemical sensor. The electrochemical sensor is modified with a specific sensitive film. By fixing the electrochemical sensor and the blood-brain organochip to form a miniature electrolytic cell, this invention can measure the current response during the penetration process of drugs for treating central nervous system diseases in real time under different flow rate changes, thereby determining the amount of drugs penetrating the blood-brain barrier. This invention is beneficial for revealing and analyzing the process of drugs for treating central nervous system diseases penetrating the blood-brain barrier. Attached Figure Description

[0023] The features, advantages, and technical effects of exemplary embodiments of the present invention will now be described with reference to the accompanying drawings.

[0024] Figure 1 This is a schematic diagram of the structure of the present invention.

[0025] Figure 2 This is a schematic diagram of the blood-brain organ-on-a-chip in this invention.

[0026] Figure 3 This is a schematic diagram of the electrochemical sensor in this invention.

[0027] Figure 4 A schematic diagram of the system in the permeation process of levodopa solution according to the present invention.

[0028] Figure 5 This is an optical imaging image of the blood-brain barrier in Embodiment 1 of the present invention.

[0029] Figure 6 This is a current response curve at different concentrations and times in Example 2 of the present invention.

[0030] Figure 7 This is a current response curve of uric acid, glucose, ascorbic acid and levodopa in Example 3 of the present invention.

[0031] Figure 8 This is an optical imaging image of the blood-brain barrier in Embodiment 4 of the present invention.

[0032] Figure 9 The figures shown are the current response curve and permeability comparison graph in Embodiment 5 of the present invention.

[0033] The reference numerals in the attached figures are explained as follows:

[0034] 1-Electrochemical sensor; 11-Counter electrode; 12-Reference electrode; 13-Working electrode; 14-Specific sensitive film;

[0035] 2-Blood-brain organ-on-a-chip; 21-Upper structure; 211-Upper flow channel; 22-Lower structure; 221-Lower flow channel; 23-Culture layer;

[0036] 3-Clamp; 31-Fixing plate; 32-Screw;

[0037] 4-Levodopa. Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this application, not all of them. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0039] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains; the terminology used in the description of this application is for the purpose of describing particular embodiments only and is not intended to limit the application; the terms "comprising" and "having," and any variations thereof, in the description, claims, and foregoing drawings of this application are intended to cover non-exclusive inclusion. The reference to "implementation" in this application means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The appearance of this phrase in various places in the description does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments.

[0040] In the description of this application, it should be noted that, unless otherwise expressly specified and limited, the terms "installation," "connection," "linking," and "attachment" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal communication between two components. Those skilled in the art can understand the specific meaning of the above terms in this application according to the specific circumstances.

[0041] The inventors discovered that currently used methods for detecting drugs for the treatment of central nervous system diseases, such as chromatography and capillary zone electrophoresis, require drug extraction and pretreatment at the endpoint, and the detection systems are relatively complex and cannot meet the needs of real-time detection.

[0042] In view of this, the present application provides a technical solution that integrates an electrochemical sensor into a blood-brain organ-on-a-chip on which the blood-brain barrier is grown. A specific sensitive film is modified on the electrochemical sensor, and endothelial cells are cultured on the integrated blood-brain organ-on-a-chip to form a complete and dense blood-brain barrier layer, thereby constructing a device that can efficiently, sensitively and in real time detect the process of drug penetration into the blood-brain barrier.

[0043] The following is in conjunction with the appendix Figures 1-9 The present invention will be further described in detail with specific embodiments, but this is not intended to limit the present invention. In this embodiment, the central nervous system disease treatment drug detected in real time is levodopa. The device structure and working method in this embodiment are set according to levodopa. It can be understood that when the central nervous system disease treatment drug changes, the present invention only needs to make adaptive changes and is equally applicable.

[0044] like Figure 1As shown, a device for real-time detection of drug penetration across the blood-brain barrier includes an electrochemical sensor 1, a blood-brain organochip 2, and a clamp 3. The electrochemical sensor 1 is fixedly connected to the blood-brain organochip 2 via the clamp 3. The blood-brain organochip 2 is located above the electrochemical sensor 1. The clamp 3 includes two fixing plates 31 fixedly connected by screws 32. The electrochemical sensor 1 and the blood-brain organochip 2 are located between the two fixing plates 31.

[0045] like Figure 2 As shown, the blood-brain organochip 2 is composed of an upper structure 21, a lower structure 22, and a culture layer 23 bonded together. The culture layer 23 is located between the upper structure 21 and the lower structure 22. The upper flow channel 211 in the upper structure 21 and the lower flow channel 221 in the lower structure 22 are arranged in a cross shape. The culture layer 23 is used as an interface for the formation of the blood-brain barrier. Endothelial cells combine with glial cells or pericytes to establish a blood-brain barrier that is more relevant to the human body. Fluid shear stress is introduced into the endothelial cell layer to simulate the effect of blood flow in blood vessels. Based on this, the growth and formation of the blood-brain barrier can be completed. This setting makes the blood-brain organochip 2 have good biocompatibility and operability.

[0046] Preferably, the upper structure 21 has an upper flow channel 211 with a length, width, and height of 10000 μm, 2000 μm, and 150 μm, respectively; the lower structure 22 has a lower flow channel 221 with a length, width, and height of 10000 μm, 2000 μm, and 2000 μm, respectively; the culture layer 23 is a polycarbonate film with a thickness of 10 μm and an area of ​​36 mm². 2 The culture layer 23 has multiple pores with a diameter of 400 nm. The pores are designed to facilitate observation of the levodopa permeation in the upper and lower channels during use.

[0047] Preferred, such as Figure 3 As shown, the electrochemical sensor 1 has a three-electrode system structure, which includes a counter electrode 11, a reference electrode 12 and a working electrode 13. The working electrode 13 is indium tin oxide, the counter electrode 11 is a platinum wire, the reference electrode 12 is silver / silver chloride, and a specific sensitive film 14 is modified on the working electrode 13.

[0048] In this embodiment, in order to detect the permeation of levodopa 4 in real time, the specific sensitive membrane 14 is a single-walled carbon nanotube-gold nanoparticle-polypyrrole / tyrosinase sensitive membrane, which is used to specifically identify levodopa 4. It is understood that in some other embodiments, the specific sensitive membrane 14 can be replaced according to the change of the drug.

[0049] In this embodiment, the working electrode 13 is located below the blood-brain organochip 2. The counter electrode 11 and the reference electrode 12 should extend through the inlet and outlet of the lower flow channel 221 in the lower structure 22 and directly contact the liquid in the lower flow channel 221. The counter electrode 11, the reference electrode 12, and the working electrode 13 are used to connect to the electrochemical workstation and then to the PC host computer. In actual application, levodopa 4 contacts the electrochemical sensor 1. By applying a certain potential or current, levodopa 4 loses electrons and is oxidized into levodopaquinone. At the same time, the electrode surface will sense the change in electrons and transmit it to the PC host computer to measure the magnitude of the response current during the reaction process. According to Faraday's law, a quantitative relationship between the concentration of levodopa and the magnitude of the response current is established, thereby realizing the real-time detection of the permeation of levodopa 4.

[0050] working methods

[0051] like Figure 4 As shown, a method for operating a device for real-time detection of drug penetration across the blood-brain barrier as described above includes the following steps:

[0052] Step 1: Form a complete and dense blood-brain barrier layer on the blood-brain organ-on-chip 2, and connect the electrochemical sensor 1 to the electrochemical workstation and then to the PC host computer.

[0053] Step 2: Inject the matrix solution into the lower flow channel 221. Introduce the experimental solution with the set concentration into the upper flow channel 211 through the inlet of the upper flow channel 211. The levodopa 4 in the experimental solution that has penetrated into the lower flow channel 221 reforms with the matrix solution to form a recombinant experimental solution. The counter electrode 11, reference electrode 12 and working electrode 13 simultaneously come into contact with the recombinant experimental solution. The recombinant experimental solution is oxidized and loses electrons on the surface of the special sensitive film 14.

[0054] Step 3: The electrons lost in Step 2 are transmitted to the PC host computer through electrochemical sensor 1, forming a response current to realize the real-time detection of the permeation of levodopa 4.

[0055] It should be noted that, Figure 4 This is a cross-sectional view of a device for detecting the permeation of levodopa 4. In the actual structure, the counter electrode 11 and the reference electrode 12 extend through the inlet and outlet of the lower flow channel 221 and do not contact the upper flow channel 211 and the blood-brain barrier layer.

[0056] In this embodiment, the matrix solution is a phosphate buffer solution used to dissolve levodopa 4. Both the experimental solution and the recombinant experimental solution are levodopa solutions. In other embodiments, the solutions can be replaced according to the different drugs.

[0057] In this embodiment, the solution concentration is set to 5 μM. This set solution concentration is obtained by calculating the concentration of levodopa 4 in the human body based on the actual situation. It can be adjusted according to the specific application and is not specifically set.

[0058] Step 3 also includes scanning the solution at the lower flow channel 221. The scanning method is to scan once every 5 minutes at a constant potential of 0.4V. The above steps are actually the detection process using chronoamperometry to detect levodopa that has penetrated below the blood-brain barrier layer. Since the time from voltage application to peak current generation is less than 1 second, the use of chronoamperometry for measurement is beneficial for rapid real-time detection.

[0059] The method for forming the blood-brain barrier layer in step one is as follows: endothelial cells are combined with glial cells or pericytes, and fluid shear stress is introduced into the endothelial cell layer to complete the growth and formation of the blood-brain barrier layer.

[0060] To better illustrate the beneficial effects of the above-mentioned device and working method, the blood-brain organochip 2 was placed in an incubator at 37°C and 5% CO2 to ensure cell growth. The electrochemical sensor 1 was connected to a PC via an electrochemical workstation to complete the construction of a real-time permeation detection device suitable for levodopa 4. The levodopa solution 4 was introduced into the upper flow channel 211, and the liquid in the lower flow channel 221 was scanned every 5 minutes at a constant potential of 0.4V. The levodopa 4 that permeated through the blood-brain barrier lost electrons on the surface of the specific sensitive membrane and was oxidized to levodopaquinone. The lost electrons were quickly transferred to the PC via the electrode surface, generating a response current, thereby realizing the real-time detection of levodopa.

[0061] Based on the above-described device construction and operating method, the following embodiments are provided:

[0062] Example 1

[0063] Endothelial cells were cultured on culture layer 23 of the blood-brain organochip 2. After calcein staining, optical imaging analysis of the blood-brain barrier inside the blood-brain organochip 2 was performed on days 1, 2, 3, and 4 of culture. The results are as follows: Figure 5 As shown in the image, it can be seen that the blood-brain barrier layer gradually becomes denser with increasing culture time, confirming the biocompatibility and operability of the blood-brain organ-on-a-chip 2.

[0064] Example 2

[0065] Different concentrations of levodopa solutions were introduced into the upper flow channel 211, and the response current was detected. The results are as follows: Figure 6 As shown, calculations show that the device in this embodiment can linearly detect levodopa from 10 μM to 500 μM, with a sensitivity of 7.29 nA / μM.

[0066] Example 3

[0067] A solution containing uric acid, glucose, ascorbic acid, and levodopa was introduced into the upper flow channel 211, and the response current was detected. The results are as follows: Figure 7 As shown, the peak values ​​of the oxidative response to interfering substances such as uric acid, glucose, and ascorbic acid are significantly different from those of levodopa, indicating that the device of this embodiment can specifically detect levodopa in the presence of interfering substances.

[0068] In the diagram, UA represents uric acid; AA represents ascorbic acid; Glu represents glucose; and L-Dopa represents levodopa.

[0069] Example 4

[0070] After calcein staining of endothelial cells, optical imaging analysis was performed on the blood-brain barrier within the blood-brain organ-on-a-chip 2 before and after levodopa-4 infiltration. The results are as follows: Figure 8 As shown, it can be demonstrated that the device of the present invention does not damage the blood-brain barrier layer during the real-time detection of levodopa 4 penetration.

[0071] Example 5

[0072] The devices of bloodless brain organ-on-a-chip 2 and blood-containing brain organ-on-a-chip 2 were continuously permeated with levodopa solution at flow rates of 0.05 μL / min, 0.20 μL / min, and 1.00 μL / min for 30 minutes, respectively, to obtain the following results: Figure 9 The current response shown demonstrates that the device in this invention can efficiently, sensitively, and in real time detect changes in levodopa 4 concentration, and also reveals that the permeability of levodopa 4 increases with increasing flow rate.

[0073] Production method

[0074] A method for manufacturing a device for real-time detection of drug penetration across the blood-brain barrier as described above includes the following steps:

[0075] Step 1: Using indium tin oxide as the working electrode 13, platinum wire as the counter electrode 11, and silver / silver chloride as the reference electrode 12, the electrode is ultrasonically cleaned for 5 minutes with acetone, anhydrous ethanol, and ultrapure water, and then dried at room temperature.

[0076] Step 2: Place the working electrode 13 in the mixed solution, deposit it at a constant potential of 0.85V for 230s, and dry it in air. Then, drop 5.0μL of 1mg / mL tyrosinase solution onto the surface of the semi-finished product and let it stand in a refrigerator at 4℃ to obtain the electrochemical sensor 1.

[0077] Step 3: Using polydimethylsiloxane, an upper structure 21 with an upper flow channel 211 having a length, width and height of 10000 μm, 2000 μm and 150 μm respectively, and a lower structure 22 with a lower flow channel 221 having a length, width and height of 10000 μm, 2000 μm and 2000 μm respectively are fabricated. The upper structure 21 and the lower structure 22 are bonded to a polycarbonate film to form a blood-brain organ-on-a-chip 2.

[0078] Step 4: Fix the blood-brain organ-on-chip 2 and the electrochemical sensor 1 using the clamp 3 to obtain the device.

[0079] The mixed solution is a solution of 0.05% (effective addition amount) single-walled carbon nanotubes, 4 μg / mL gold nanoparticles and 0.1 mol / L pyrrole prepared from ultrapure water.

[0080] Based on the disclosure and teachings of the foregoing specification, those skilled in the art can make changes and modifications to the above embodiments. Therefore, the present invention is not limited to the specific embodiments described above, and any obvious improvements, substitutions, or modifications made by those skilled in the art based on the present invention are within the scope of protection of the present invention. Furthermore, although some specific terms are used in this specification, these terms are only for convenience of explanation and do not constitute any limitation on the present invention.

Claims

1. A device for real-time detection of drug penetration across the blood-brain barrier, characterized in that, The device includes an electrochemical sensor (1), a blood-brain organ-on-a-chip (2), and a clamp (3). The electrochemical sensor (1) is fixedly connected to the blood-brain organ-on-a-chip (2) via the clamp (3). The blood-brain organ-on-a-chip (2) is located above the electrochemical sensor (1). The electrochemical sensor (1) is modified with a specific sensitive film (14). The blood-brain organochip (2) is composed of an upper structure (21), a lower structure (22) and a culture layer (23) bonded together. The culture layer (23) is located between the upper structure (21) and the lower structure (22). The upper flow channel (211) in the upper structure (21) and the lower flow channel (221) in the lower structure (22) are arranged in a cross shape. The culture layer (23) is used as the interface for the growth of the blood-brain barrier. A matrix solution is injected into the lower flow channel (221), and an experimental solution with a set concentration is introduced into the upper flow channel (211) through the inlet of the upper flow channel (211). The drug in the experimental solution that has penetrated into the lower flow channel (221) and the matrix solution reforms into a recombinant experimental solution. The counter electrode (11), the reference electrode (12), and the working electrode (13) simultaneously come into contact with the recombinant experimental solution. The recombinant experimental solution is oxidized and loses electrons on the surface of the special sensitive film (14). The specific sensitive film (14) is used to specifically identify drugs.

2. The device for real-time detection of drug penetration across the blood-brain barrier as described in claim 1, characterized in that, The electrochemical sensor (1) has a three-electrode system structure, which is provided with a counter electrode (11), a reference electrode (12) and a working electrode (13). The counter electrode (11) is a platinum wire, the reference electrode (12) is silver / silver chloride, the working electrode (13) is indium tin oxide, and the working electrode (13) is modified with the specific sensitive film (14).

3. The device for real-time detection of drug penetration across the blood-brain barrier as described in claim 2, characterized in that, The working electrode (13) is located below the blood-brain organ-on-chip (2). The counter electrode (11) and the reference electrode (12) should extend through the inlet and outlet of the lower channel (221) in the lower structure (22) and directly contact the liquid in the lower channel (221). The counter electrode (11), the reference electrode (12) and the working electrode (13) are used to connect to the electrochemical workstation and then to the PC host computer.

4. A method for operating the device for real-time detection of drug penetration across the blood-brain barrier as described in any one of claims 1-3. Its features are, Includes the following steps: Step 1: Form a complete and dense blood-brain barrier layer on the blood-brain organ-on-chip (2), and connect the electrochemical sensor (1) to the electrochemical workstation and then to the PC host computer. Step 2: Inject a matrix solution at the connection point between the electrochemical sensor (1) and the blood-brain organochip (2), and introduce an experimental solution with a set concentration into the input terminal of the blood-brain organochip (2). The drug in the experimental solution permeates into the connection point, and the drug in the experimental solution and the matrix solution reform a recombinant experimental solution. The recombinant experimental solution reacts on the surface of the specific sensitive film (14) to cause electronic changes. Step 3: The electronic changes in Step 2 are transmitted to the PC host computer through the electrochemical sensor (1) to form a response current and realize the real-time detection of drug penetration.

5. The working method as described in claim 4, characterized in that, Step three also includes scanning at the phase connection point by scanning the current every 5 minutes at a constant potential of 0.4V using an electrochemical workstation.

6. The working method as described in claim 4, characterized in that, The matrix solution in step two is used to support the drug.

7. The working method as described in claim 4, characterized in that, In step two, the experimental solution is a mixture of the drug and the matrix solution.

8. The working method as described in claim 4, characterized in that, The method for forming the blood-brain barrier layer in step one is as follows: endothelial cells are combined with glial cells or pericytes, and fluid shear stress is introduced into the endothelial cell layer to complete the growth and formation of the blood-brain barrier layer.

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