Microchip detection device based on three-dimensional blood-brain barrier organoid and preparation method and use method of microchip detection device

Abstract

The invention discloses a microchip detection device based on a three-dimensional blood brain barrier organ and a preparation method and a use method thereof, the microchip detection device comprises an interdigital electrode and a three-dimensional star-shaped nano structure electrically deposited on the interdigital electrode, and further comprises a PDMS microporous film arranged on the surface of the interdigital electrode, and in combination with the electrochemical impedance spectroscopy, the evaluation of the physiological state of the three-dimensional blood-brain barrier organ is realized. On the basis of the device, non-destructive and label-free rapid detection is carried out on neuroinflammation blood brain barrier organs, the detection efficiency and accuracy are improved, and a powerful tool is provided for central nervous system disease research.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a three-dimensional blood-brain barrier organoid microchip detection device, a preparation method thereof, and a usage method thereof. Background Art

[0002] Central nervous system diseases, including cerebrovascular diseases, neurodegenerative diseases, and neuroinflammatory diseases, etc., in their pathophysiological mechanisms of onset, blood-brain barrier (BBB) dysfunction plays a key role. The BBB is composed of endothelial cells, astrocytes, pericytes, and other cellular components, jointly forming a precisely regulated neurovascular unit (NVU) to maintain brain homeostasis and resist pathogen invasion. During the progression of neurodegenerative diseases, neuroinflammation is significant, specifically manifested as the degradation of tight junction proteins in the BBB, which further exacerbates the permeability of the BBB. Therefore, constructing a BBB inflammatory injury model to screen therapeutic drugs is crucial for preventing neurodegenerative diseases.

[0003] Traditionally, genetically engineered animal models have been used in BBB research. Although they can simulate the physiological environment of the human BBB, genetic heterogeneity may lead to deviations in experimental results in human applications. The classic Transwell model, although having high throughput, repeatability, and simple construction operation, is difficult to fully reproduce the physiological characteristics of the BBB due to the lack of cell-cell interactions.

[0004] To overcome this limitation, in vitro 3D cell models, especially organoid models, have become a research hotspot. Organoids are formed by one or more types of cells through adhesion, aggregation, and self-assembly into 3D cell clusters, breaking through the limitations of traditional monolayer cell culture, being closer to real tissues, and being easy to produce in high throughput. However, evaluating the integrity of the BBB model has become a new challenge.

[0005] Currently, the transendothelial electrical resistance (TEER) technology combined with the Transwell model is used to evaluate BBB permeability by measuring electrical resistance. Although the operation is simple, it is not applicable to three-dimensional culture models. Although optical microscopy imaging can accurately characterize the physiological characteristics of the model, it takes a long time and requires labeling of samples, which is not conducive to real-time detection. Although the electrical sphere detection method can achieve real-time evaluation, it is difficult to be applied in high throughput. Electrochemical impedance spectroscopy (EIS) technology, as a non-destructive and label-free detection method, although having potential, is limited by the insufficient sensitivity of ordinary electrodes, and the detection performance is limited.

[0006] In view of the limitations of the above electrochemical impedance spectroscopy technology, it is urgent to develop an efficient and accurate detection scheme to solve the current problem of difficult evaluation of the BBB model. Summary of the Invention

[0007] The object of the present invention is to provide a microchip detection device with high conductivity and high sensitivity, which is specially designed for non-destructive, label-free and rapid detection of pathological blood-brain barrier (BBB) organoids.

[0008] To achieve this object, the present invention provides the following solutions:

[0009] A microchip detection device based on three-dimensional blood-brain barrier organoids, comprising interdigital electrodes and three-dimensional star-shaped nanostructures electro-deposited on the interdigital electrodes, and further comprising a PDMS microporous film disposed on the surface of the interdigital electrodes and used for impedance detection of three-dimensional blood-brain barrier organoids.

[0010] The present invention also provides a preparation method of a microchip detection device based on three-dimensional blood-brain barrier organoids, comprising the following steps:

[0011] Step 1: Fabricate interdigital electrodes by MEMS process, ultrasonically process the interdigital electrodes in acetone, absolute ethanol and ultrapure water for 4 - 6 min respectively, and clean them in a plasma cleaner for 1 - 3 min; use the interdigital electrodes as the working electrode, a platinum wire as the counter electrode, and silver / silver chloride as the reference electrode, and in a gold solution containing ethylenediaminetetraacetic acid, K 2 HPO 4 、Na 2 SO 3 and HAuCl 4 ·3H 2 O, perform constant potential deposition at -0.8 V for 500 s, and dry to obtain a chip;

[0012] Step 2: Fabricate a PDMS microporous film with a thickness of 5 mm and a micropore diameter of 5 mm; perform plasma treatment on the PDMS microporous film for 2 min and bond it to the chip by plasma to obtain the microchip detection device.

[0013] Preferably, in the step 1, the pH of the gold solution is 6.5.

[0014] Preferably, in the step 1, the specific steps of the MEMS process are: design the shape of the interdigital electrodes by using a mask plate structure, grow a 500 Å titanium-tungsten adhesion layer and a 2500 Å gold conductive layer on the glass surface through photolithography, wet etching and magnetron sputtering, and perform cleaning and dicing to obtain the interdigital electrodes.

[0015] The present invention also provides a usage method of a microchip detection device based on three-dimensional blood-brain barrier organoids, comprising the following steps:

[0016] Step 1: Obtain a three-dimensional blood-brain barrier organoid model by culturing cells on a 1% agarose substrate;

[0017] Step 2: Treat the three-dimensional blood-brain barrier organoid model with tumor necrosis factor to obtain a blood-brain barrier neuroinflammatory pathology model;

[0018] Step 3: Use the above-mentioned microchip detection device based on three-dimensional blood-brain barrier organoids to perform impedance detection on the three-dimensional blood-brain barrier organoid model and the blood-brain barrier neuroinflammatory pathology model and conduct data analysis.

[0019] Preferably, in Step 2, the tumor necrosis factor is TNF-α.

[0020] Preferably, in Step 3, one end of the microchip detection device based on three-dimensional blood-brain barrier organoids is used as the working electrode, and the other end is used as the counter electrode and reference electrode, which are connected to a VersaSTAT4 electrochemical workstation. The impedance detection and data analysis of the three-dimensional blood-brain barrier organoid model and the blood-brain barrier neuroinflammatory pathology model are carried out by using the EIS method.

[0021] Preferably, the cell culture includes the following steps:

[0022] Step 1: Weigh 400 - 500 mg of agarose powder and mix it with 40 - 50 mL of ultrapure water, and heat it in a microwave oven to make a 0.1% agarose substrate. Then place it in an autoclave at 121 °C and autoclave for 30 min. After that, add 50 μL into a 96-well plate and solidify it to obtain a low-adhesion substrate;

[0023] Step 2: Add 1500 human brain microvascular endothelial cells and astrocytes each in a 1:1 ratio into the 96-well plate containing the low-adhesion substrate, then add organoid medium, and place it in an incubator to culture for 45 - 50 h to self-assemble into a three-dimensional blood-brain barrier organoid model.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows: By modifying three-dimensional star nanostructures on the interdigitated electrodes, the microchip detection device for three-dimensional blood-brain barrier organoids is obtained, realizing the detection of blood-brain barrier (BBB) organoids. Combining with the EIS method, it realizes the non-destructive and label-free rapid detection of impedance differences in the BBB model, improves the detection efficiency and accuracy, and provides a powerful tool for the research of central nervous system diseases. Description of the Drawings

[0025] Figure 1 Scanning electron microscope images before electrodeposition, during the 50 s nucleation stage of electrodeposition, and during the 500 s growth stage of electrodeposition of the interdigitated electrodes;

[0026] Figure 2 Optical microscope image of the self-assembled three-dimensional blood-brain barrier organoid model;

[0027] Figure 3 It is the equivalent circuit diagram of the microchip detection device for detecting the three-dimensional blood-brain barrier organoid model;

[0028] Figure 4 It is the equivalent circuit diagram of the microchip detection device during the blank test sample;

[0029] Figure 5 It is the impedance data diagram of 10 2 -10 5 Hz of the microchip detection device for detecting the organoid models of Examples 1-5;

[0030] Figure 6 It is the impedance data diagram of the microchip detection device for detecting the organoid models of Examples 1-5 at 100 Hz;

[0031] Figure 7 It is the impedance information diagram of the microchip detection device for detecting the pathologically treated organoid models of Examples 6-8;

[0032] Figure 8 It is the fluorescence quantification diagram of the tight junction protein expression of the pathologically treated organoid models of Examples 6-8;

[0033] Figure 9 It is the impedance detection data diagram of the microchip detection device for detecting the pathologically treated organoid models of Examples 9-12;

[0034] Figure 10 It is the fluorescence quantification diagram of the tight junction protein expression of the pathologically treated organoid models of Examples 9-12. Detailed implementation manners

[0035] To make the technical solutions and advantages of the present invention clearer, the following will, in conjunction with specific embodiments, clearly and completely describe the technical solutions of the present invention. Obviously, the described embodiments are some but not all of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts fall within the scope of protection of the present invention.

[0036] In the first aspect according to the present invention, a microchip detection device is provided, which includes interdigital electrodes and three-dimensional star-shaped nanostructures electro-deposited on the interdigital electrodes, and further includes a PDMS microporous film disposed on the surface of the interdigital electrodes. By modifying the interdigital electrodes with three-dimensional star-shaped nanostructures in the present invention, a microchip detection device is obtained, realizing the detection of blood-brain barrier (BBB) organoids. Combining the EIS method and the modified electrode, the non-destructive and label-free rapid detection of impedance differences in the BBB model is achieved, providing a powerful tool for the research of central nervous system diseases.

[0037] In a second aspect of the present invention, there is also provided a method for preparing a detection device based on a three-dimensional blood-brain barrier organoid microchip, comprising the following steps:

[0038] Step 1: Fabricate interdigitated electrodes using MEMS technology. Ultrasonically treat the interdigitated electrodes in acetone, absolute ethanol, and ultrapure water for 4 - 6 minutes respectively, and clean them in a plasma cleaner for 1 - 3 minutes. Use the interdigitated electrodes as the working electrode, a platinum wire as the counter electrode, and silver / silver chloride as the reference electrode. In a gold solution containing ethylenediaminetetraacetic acid, K 2 HPO 4 、Na 2 SO 3 and HAuCl 4 ·3H 2 O, perform potentiostatic deposition at -0.8V for 500s, and dry to obtain a chip;

[0039] Step 2: Fabricate a PDMS microporous film with a thickness of 5 mm and a micropore diameter of 5 mm. Plasma-treat the PDMS microporous film for 2 minutes and bond it to the chip by pressing the plasma key to obtain a microchip detection device.

[0040] In an embodiment according to the present invention, in Step 1, the pH of the gold solution is 6.5.

[0041] In an embodiment according to the present invention, in Step 1, the specific steps of the MEMS technology are as follows: Design the shape of the interdigitated electrodes using a mask plate structure, grow a 500 Å titanium-tungsten adhesion layer and a 2500 Å gold conductive layer on the glass surface through photolithography, wet etching, and magnetron sputtering, and perform cleaning and dicing to obtain the interdigitated electrodes.

[0042] In a third aspect of the present invention, there is provided a method for using a microchip detection device based on a three-dimensional blood-brain barrier organoid, comprising the following steps:

[0043] Step 1: Obtain a three-dimensional blood-brain barrier organoid model by culturing cells on a 1% agarose substrate;

[0044] Step 2: Treat the three-dimensional blood-brain barrier organoid model with tumor necrosis factor to obtain a blood-brain barrier neuroinflammation pathological model;

[0045] Step 3: Use the above-mentioned microchip detection device based on a three-dimensional blood-brain barrier organoid to perform impedance detection on the three-dimensional blood-brain barrier organoid model and the blood-brain barrier neuroinflammation pathological model and perform data analysis.

[0046] In an embodiment according to the present invention, in Step 2, the tumor necrosis factor is TNF-α.

[0047] In one embodiment of the present invention, in step three, one end of the microchip detection device based on the three-dimensional blood-brain barrier organoid is used as the working electrode, and the other end is used as the counter electrode and the reference electrode, which are connected to the VersaSTAT4 electrochemical workstation. The impedance detection of the three-dimensional blood-brain barrier organoid model and the blood-brain barrier neuroinflammatory pathological model is carried out by the EIS method and data analysis is performed.

[0048] In one embodiment of the present invention, cell culture includes the following steps:

[0049] Step one: Weigh 400 - 500 mg of agarose powder and mix it with 40 - 50 mL of ultrapure water, and heat it in a microwave oven to make a 0.1% agarose substrate. Then place it in an autoclave at 121 °C and autoclave for 30 min. After that, add 50 μL to a 96-well plate and solidify it to obtain a low-adhesion substrate.

[0050] Step two: Add 1500 human brain microvascular endothelial cells and astrocytes each in a 1:1 ratio to a 96-well plate containing the low-adhesion substrate, then add the organoid culture medium, and place it in an incubator to culture for 45 - 50 h to self-assemble into a three-dimensional blood-brain barrier organoid model.

[0051] The present invention will be further described below through specific embodiments.

[0052] Preparation of the microchip detection device:

[0053] Step one: Use the MEMS process to fabricate interdigitated electrodes with a pitch and width of 10 μm. Ultrasonically treat the interdigitated electrodes in acetone, absolute ethanol, and ultrapure water for 5 min each, and clean them in a plasma cleaner for 2 min. Use the interdigitated electrodes as the working electrode, a platinum wire as the counter electrode, and silver / silver chloride as the reference electrode. In a gold solution containing ethylenediaminetetraacetic acid, K 2 HPO 4 、Na 2 SO 3 and HAuCl 4 ·3H 2 O, perform potentiostatic deposition at -0.8 V for 500 s, and dry it to obtain the chip.

[0054] Step two: Put the PDMS and the curing agent solution into a freeze dryer to evacuate and remove bubbles, then put it in an oven at 90 °C and bake and cure for 90 min to obtain a PDMS film. Punch micropores in the interior of the PDMS film to obtain a PDMS microporous film. Plasma-treat the PDMS microporous film for 2 min and bond it to the chip by pressing the plasma key to obtain the microchip detection device.

[0055] Establishment of the BBB organoid model:

[0056] Step 1: Weigh 500 mg of agarose powder and mix it with 50 mL of ultrapure water, and heat it in a microwave oven to make a 0.1% agarose substrate. Then place it in an autoclave at 121 °C and autoclave for 20 min. After that, add 50 μL to a 96-well plate and wait for it to solidify. Add 1500 human brain microvascular endothelial cells and astrocytes each in a ratio of 1:1 to the 96-well plate, and add an appropriate amount of organoid medium. Place it in an incubator and culture for 48 h to self-assemble into a three-dimensional blood-brain barrier organoid model.

[0057] Step 2: Obtain an inflammatory pathological model by treating the three-dimensional blood-brain barrier organoid model with tumor necrosis factor.

[0058] Step 3: Use a microchip detection device to detect impedance information of the inflammatory pathological model and analyze and process it.

[0059] The following are the data parameters of the examples. The rest are the same as those in Example 1 and will not be elaborated here.

[0060] Table 1

[0061]

[0062]

[0063] Performance test:

[0064] For the BBB organoid models of Examples 6 - 12, two duplicates are prepared. One uses a microchip detection device to detect impedance information of the inflammatory pathological model and analyze and process it, and the other performs fluorescence characterization on the protein and analyze and process it.

[0065] The specific steps of fluorescence characterization are as follows:

[0066] (1) After the self-assembled and plump and round organoids in bright-field imaging are treated with the same-parameter drug, use a 200 μL pipette to take them out of the 96-well plate and place them in a 0.5 mL centrifuge tube. First, add 0.4 mL of 4% paraformaldehyde solution to the centrifuge tube and incubate at room temperature for 10 min. Then remove the paraformaldehyde solution and add 0.4 mL of PBS solution to wash three times, 2 min each time.

[0067] (2) Fixation: Add 0.4 ml of 0.25% Triton X-100 solution to the centrifuge tube with the fixed organoids to completely cover them. After standing at room temperature for 10 min, wash three times with PBS solution, 2 min each time.

[0068] (3) Blocking: Add 0.4 mL of 5% BSA solution to a culture dish to completely immerse the cells and let it stand at room temperature for 1 h. No washing is required after completion.

[0069] (4) Staining: The Alexa Fluor 594-labeled ZO-1 monoclonal antibody was uniformly mixed with 1% BSA solution at a ratio of 1:200 and added to the centrifuge tube in the absence of light. The staining solution was co-incubated with the organoids for 3 h, and then rinsed 3 times with PBS for 3 min each time.

[0070] (5) Photographing: The stained organoid samples were transferred to a confocal dish and placed on a scanning laser confocal microscope for observation and photographing.

[0071] (6) Analysis: The fluorescence staining results were quantitatively analyzed using Image J software.

[0072] Note: For each rinse, centrifuge with a microcentrifuge to precipitate and then carefully aspirate the supernatant.

[0073] It can be seen from Figure 1 that the present invention can electrochemically deposit a three-dimensional star-shaped nanostructure on the interdigitated electrodes, and characterized the trigonal pyramid shape in the nucleation stage and the star shape in the growth stage of the formation process.

[0074] It can be seen from Figure 2 that the present invention obtained a three-dimensional blood-brain barrier organoid model formed by self-assembly, and its formed morphology is a round and three-dimensional spherical shape.

[0075] It can be seen from Figure 3 and Figure 4 that the present invention analyzed the impedance equivalent circuit diagram for detecting organoids based on the interdigitated electrode EIS method, which includes R ct : charge transfer resistance; C dl : double-layer capacitance; R int : cell resistance; C m : cell membrane capacitance; R ext : cell junction resistance; R s : solution resistance. And it is divided into the equivalent circuit diagrams for sample detection and blank control without sample.

[0076] It can be seen from Figure 5 that the device of the present invention detected the impedance data diagrams of 3 - 25 organoids. The impedance results showed that as the number of detected organoids increased, the impedance data also showed an upward trend.

[0077] It can be seen from Figure 6 that the impedance data of different numbers of organoids were analyzed at a frequency of 100 Hz, which further illustrated the positive correlation between the impedance data and the detection number, and demonstrated the feasibility of the device of the present invention for impedance sensing of different numbers of organoids.

[0078] Figure 7 and Figure 8 as well asFigure 9 and Figure 10 As can be seen from the correspondence between each group, the data of the fluorescence quantification graph corroborate the impedance data graph, and it is analyzed that the change of the impedance data is associated with the damage of ZO-1. The decrease of the impedance result represents the aggravation of ZO-1 dissociation, which not only shows that the microchip detection device of the present invention is accurate in detecting impedance information for the inflammatory pathological model, but also verifies that this device can convert the signal of the physiological state damage of the organoid into an intuitively quantifiable impedance signal.

[0079] According to the disclosure and teachings of the above specification, those skilled in the art to which the present invention pertains are also able to make changes and modifications to the above embodiments. Therefore, the present invention is not limited to the above specific embodiments, and any obvious improvements, substitutions or variations made by those skilled in the art on the basis of the present invention all fall within the protection scope of the present invention. In addition, although some specific terms are used in this specification, these terms are only for convenience of description and do not constitute any limitation to the present invention.

Claims

1. A microchip detection device based on three-dimensional blood-brain barrier organoids, characterized in that: The invention comprises interdigital electrodes and three-dimensional star-shaped nanostructures electro-deposited on the interdigital electrodes, and also comprises a PDMS microporous film arranged on the surface of the interdigital electrodes, and is used for impedance detection of three-dimensional blood-brain barrier organoids.

2. A method for preparing a microchip detection device based on a three-dimensional blood-brain barrier organoid, characterized in that: The following steps are involved: Step 1: using MEMS technology to make interdigital electrodes, ultrasonically treating the interdigital electrodes in acetone, anhydrous ethanol and ultrapure water for 4 to 6 minutes respectively, and cleaning them in a plasma cleaner for 1 to 3 minutes; using the interdigital electrodes as working electrodes, platinum wire as counter electrodes, and silver / silver chloride as reference electrodes, in a gold solution containing ethylenediaminetetraacetic acid, K2HPO4, Na2SO3 and HAuCl4·3H2O, depositing at a constant potential of -0.8V for 500 seconds, and drying to obtain a chip; Step 2: Prepare a PDMS microporous film with a thickness of 5 mm and a micropore diameter of 5 mm; plasma treat the PDMS microporous film for 2 minutes and plasma bond it to the chip to obtain a microchip detection device.

3. The method for preparing a microchip detection device based on a three-dimensional blood-brain barrier organoid according to claim 2, characterized in that: In step one, the specific steps of the MEMS process are: using a mask structure to design the shape of the interdigitated electrode, growing a 500A titanium tungsten adhesion layer and a 2500A gold conductive layer on the glass surface by photolithography, wet etching, and magnetron sputtering, and then cleaning and slicing to obtain the interdigitated electrode.

4. A method for using a microchip detection device based on a three-dimensional blood-brain barrier organoid, characterized in that: The microchip detection device based on three-dimensional blood-brain barrier organoids according to claim 1 comprises the following steps: Step 1: Obtain a three-dimensional blood-brain barrier organoid model by culturing cells on a 1% agarose substrate; Step 2: treating the three-dimensional blood-brain barrier organoid model with tumor necrosis factor to obtain a blood-brain barrier neuroinflammation pathology model; Step 3: Use a microchip detection device based on a three-dimensional blood-brain barrier organoid to perform impedance detection on the three-dimensional blood-brain barrier organoid model and the blood-brain barrier neuroinflammatory pathology model and perform data analysis.

5. The method for using the microchip detection device based on three-dimensional blood-brain barrier organoids according to claim 4, characterized in that: In the step three, one end of the microchip detection device based on the three-dimensional blood-brain barrier organoid is used as the working electrode, and the other end is used as the counter electrode and reference electrode, connected to the VersaSTAT4 electrochemical workstation, and the EIS method is used to perform impedance detection and data analysis on the three-dimensional blood-brain barrier organoid model and the blood-brain barrier neuroinflammatory pathology model.

6. The method for using the microchip detection device according to claim 4, characterized in that: The cell culture comprises the following steps: Step 1: Weigh 400-500 mg of agarose powder and mix it with 40-50 mL of ultrapure water, and heat it in a microwave oven to make a 0.1% agarose base, and put it in an autoclave at 121°C for 30 minutes, then add 50 μL to a 96-well plate and solidify to obtain a low-adhesion base; Step 2: Add 1500 human brain microvascular endothelial cells and 1500 astrocytes at a ratio of 1:1 to a 96-well plate containing the low-adhesion substrate, add organoid culture medium, and culture in an incubator for 45 to 50 hours to allow it to self-assemble into a three-dimensional blood-brain barrier organoid model.

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