An AML organoid model and its construction method

Abstract

The present invention discloses an AML organoid model and a construction method thereof, relating to the field of microfluidic chip technology. The present invention includes an AML organoid model, which is formed by 3D bioprinting and is constructed from top to bottom with a nutrient circulation mechanism, a tumor cell growth mechanism, a cell external stimulation mechanism, and an endothelial cell growth mechanism; a first flow channel is constructed in the nutrient circulation mechanism, and a first test electrode is installed in the first flow channel; a second flow channel is constructed in the tumor cell growth mechanism, and the second flow channel is filled with channel ink and a second test electrode is installed in the second flow channel; a cavity and an air supply channel are interconnected in the cell external stimulation mechanism, and the air supply channel is connected to the cavity; a third flow channel is constructed in the endothelial cell growth mechanism, and the inner wall of the third flow channel is filled with an outer layer of ink; so as to achieve the purpose of constructing a high-throughput drug sensitivity detection platform based on AML organoids by using microfluidics and 3D bioprinting technology for highly heterogeneous blood tumors such as AML.

Description

Technical Field

[0001] The present invention relates to the technical field of microfluidic chips, and in particular to an AML organoid model and a construction method thereof. Background Art

[0002] Acute myeloid leukemia (AML) is a clonal hematologic malignancy that arises from hematopoietic stem cells. AML is the most common form of leukemia in adults, accounting for approximately one-third of all leukemia cases worldwide. The incidence of AML is generally reported to be 3-5 per 100,000 people per year. In China, there are 85,000 new cases of AML each year, with a five-year survival rate of less than 20%. With increasing life expectancy and an aging population in China, the incidence of AML is likely to increase significantly. The survival rate for AML patients is the lowest of all leukemias. Only one-quarter of adult AML patients survive beyond five years after diagnosis. Furthermore, progress in AML treatment has been slow over the past 20 years, with no significant improvement in survival rates.

[0003] Currently, the traditional treatment for AML patients primarily utilizes the "3+7" chemotherapy regimen, which combines anthracyclines such as idarubicin (IDA), daunorubicin (DNR), and mitoxantrone (MIT) with standard doses of Ara-C. In addition, the CAG regimen (cytarabine + aclarubicin + granulocyte colony-stimulating factor) and the HAG regimen (homoharringtonine + cytarabine + granulocyte colony-stimulating factor) are also widely used as low-intensity chemotherapy regimens. However, according to multiple previous studies, the overall efficacy of traditional treatment regimens in AML patients is not ideal, showing low response rates and shorter median overall survival (OS).

[0004] This phenomenon is mainly attributed to the high heterogeneity of AML patients, their poor prognosis, the inability of some patients to tolerate intensive treatment, and the refractory nature of the disease. The fundamental reason is that AML cells themselves have extremely strong tumor heterogeneity. The presence of multiple gene mutations causes tumor cells to undergo clonal evolution and form different subclones. At the same time, the heterogeneity of the tumor microenvironment, such as differences in blood vessels and oxygen supply, and differences in immune cells, also has a significant impact on the development of drug resistance in patients. These different mutations, epigenetic variations, or downstream abnormalities may cause patients to present with similar clinical manifestations, but trigger different treatment responses, ultimately leading to poor treatment effects. Summary of the Invention

[0005] The purpose of the present invention is to provide an AML organoid model and its construction method, so as to achieve the purpose of constructing a high-throughput drug sensitivity detection platform based on AML organoids by using microfluidics and 3D bioprinting technology for AML, a highly heterogeneous blood tumor.

[0006] In order to achieve the above object, the present invention adopts the following technical means:

[0007] An AML organoid model, created using 3D bioprinting, is constructed from top to bottom with a nutrient circulation mechanism, a tumor cell growth mechanism, a cell external stimulation mechanism, and an endothelial cell growth mechanism.

[0008] A first flow channel is constructed in the nutrient circulation mechanism, and a first testing electrode is installed in the first flow channel;

[0009] A second flow channel is constructed in the tumor cell growth mechanism, the second flow channel is filled with channel ink, and a second test electrode is installed in the second flow channel;

[0010] The external cell stimulation mechanism is internally structured with a cavity and an air supply channel that are interconnected, and the air supply channel is connected to the cavity;

[0011] A third flow channel is constructed in the endothelial cell growth mechanism, and the inner wall of the third flow channel is filled with an outer layer of ink.

[0012] Preferably, the nutrient circulation mechanism, tumor cell growth mechanism, cell external stimulation mechanism and endothelial cell growth mechanism are all formed using a core / shell GelMA-TGF-β1 / gelatin ink system as a profile;

[0013] The profile is formed by coaxial nozzles, including tissue cell-carrying ink for the outer nozzle and endothelial cell-carrying sacrificial ink for the inner nozzle.

[0014] Furthermore, the channel ink is prepared in the following manner:

[0015] A1. Add porcine skin-derived gelatin to a 15% concentration in PBS and dissolve in a 50°C water bath with stirring for 30 minutes until completely dissolved.

[0016] A2. Slowly add methyl acrylate (MA) to the gelatin solution at 37°C until the MA concentration reaches 6%. After two hours of reaction, remove the MA from the gelatin solution to obtain GelMA.

[0017] A3. GelMA was dialyzed in deionized water for one week, freeze-dried, and 5 g of TGF-β1 monomer was dissolved in 15% GelMA with PBS and stirred for 5 minutes to obtain GelMA-TGF-β1.

[0018] A4. Mix GelMA-TGF-β1 and KG-1a cells to obtain the channel ink.

[0019] Furthermore, the outer layer ink is prepared in the following manner:

[0020] B1. Adipose-derived mesenchymal stem cells were extracted from 4-week-old rats;

[0021] B2. Culture the cells in complete mesenchymal stem cell medium, changing the medium every two days. Subculture the cells when they reach 80%-90% confluence. After two subcultures, use the cells for organoid culture.

[0022] B3. Every 10 5 Mesenchymal stem cells (MSCs) and 1 ml of 5% sodium alginate are mixed in equal proportions to prepare the outer layer ink.

[0023] Furthermore, the nutrient circulation mechanism, tumor cell growth mechanism, cell external stimulation mechanism and endothelial cell growth mechanism are stacked in sequence from top to bottom, and a one-way filter membrane is constructed between two adjacent layers.

[0024] Furthermore, the nutrient circulation mechanism is constructed with a vertically penetrating electrode mounting hole, which passes through the first flow channel and extends into the second flow channel. A measuring electrode is installed in the electrode mounting hole, and the part of the measuring electrode located in the first flow channel serves as the first test electrode, and the part of the measuring electrode located in the second flow channel serves as the second test electrode.

[0025] Furthermore, the measuring electrodes include a reference electrode, an enzyme sensor and a counter electrode in sequence along the flow direction of the liquid in the first flow channel.

[0026] Furthermore, the liquid inlet and liquid outlet of the first flow channel are respectively constructed on the opposite side walls of the nutrient circulation mechanism, and the ink inlet and ink outlet of the third flow channel are respectively constructed on the opposite side walls of the endothelial cell growth mechanism, and the orthographic projection of the first flow channel on the endothelial cell growth mechanism coincides with that of the third flow channel.

[0027] Furthermore, the ink inlet and ink outlet of the second flow channel are respectively constructed on opposite sides of the tumor cell growth mechanism, and the second flow channel includes a main channel and a measurement channel separately connected to each second test electrode. A dry film blocking layer is provided on the upstream side of the measurement channel relative to the second test electrode. The two ends of the main channel serve as the ink inlet and ink outlet. The main channel is also connected to a test channel, and the test channel extends from the side wall of the tumor cell growth mechanism and is connected to a nano-nozzle.

[0028] At the same time, a method for constructing the aforementioned AML organoid model comprises the following steps:

[0029] S1. Configure channel ink and outer ink;

[0030] S2. forming the endothelial cell growth mechanism by 3D printing, introducing the outer layer of ink into the third flow channel, and curing the ink using ultraviolet light;

[0031] S3. Forming the external cell stimulation mechanism on top of the endothelial cell growth mechanism using 3D printing, and curing with ultraviolet light;

[0032] S4. forming the tumor cell growth mechanism above the external cell stimulation mechanism using 3D printing, introducing the channel ink into the second flow channel, and curing it using ultraviolet light;

[0033] S5. Using 3D printing to form the nutrient circulation mechanism above the tumor cell growth mechanism;

[0034] S6. Insert electrodes to form the first test electrode and the second test electrode.

[0035] During use, the present invention has the following beneficial effects:

[0036] By combining microfluidics and 3D bioprinting technologies, AML organoid models are prepared to simulate tumor heterogeneity. By constructing an environment that can accurately reproduce the tumor microenvironment to simulate a more realistic environment, we can predict individual patients' responses to therapy and provide a research and diagnostic basis for drug screening and personalized treatment.

[0037] The model is built using specific biological ink to ensure the accuracy and reliability of the simulated environment.

[0038] It can accurately reproduce the microenvironmental conditions of tumor cells, including key factors such as oxygen concentration, nutrient supply, and pH, and effectively simulate the complex interactions between tumor cells and various cells in the surrounding microenvironment. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 Schematic diagram of the structure of the AML organoid model of the present invention.

[0040] Figure 2 for Figure 1 Schematic diagram of the explosion structure.

[0041] Figure 3 This is a schematic diagram of the cross-sectional structure of the tumor cell growth mechanism of the present invention.

[0042] Figure 4This is a schematic diagram of the first flow channel layout structure of the present invention.

[0043] Among them, 1-nutrient circulation mechanism, 2-tumor cell growth mechanism, 3-cell external stimulation mechanism, 4-endothelial cell growth mechanism, 5-first flow channel, 6-second flow channel, 7-cavity, 8-air supply channel, 9-third flow channel, 10-electrode mounting hole, 11-main flow channel, 12-measurement flow channel, 13-dry film blocking layer, 14-test channel, 15-nano nozzle. DETAILED DESCRIPTION

[0044] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Generally, the components of the embodiments of the present invention described and shown in the drawings herein can be arranged and designed in various different configurations.

[0045] Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the invention as claimed, but rather merely represents selected embodiments of the present invention. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without creative effort are also within the scope of protection of the present invention.

[0046] It should be noted that, in the absence of conflict, the embodiments and features of the embodiments of the present invention can be combined with each other.

[0047] It should be noted that similar reference numerals and letters denote similar items in the following drawings, and therefore, once an item is defined in one drawing, it does not need to be further defined or explained in subsequent drawings.

[0048] In the description of the present invention, it should be noted that the terms "center," "upper," "lower," "left," "right," "vertical," "horizontal," "inner," "outer," etc., indicating orientations or positional relationships, are based on the orientations or positional relationships shown in the accompanying drawings, or are the orientations or positional relationships in which the inventive product is typically placed when in use, or are the orientations or positional relationships commonly understood by those skilled in the art. These terms are intended only to facilitate the description of the present invention and to simplify the description, and are not intended to indicate or imply that the device or element referred to must have a specific orientation, be constructed, or operate in a specific orientation. Therefore, they should not be construed as limiting the present invention. Furthermore, the terms "first," "second," etc., are used only to distinguish descriptions and should not be construed as indicating or implying relative importance.

[0049] In the description of the present invention, it should also be noted that, unless otherwise expressly specified or limited, the terms "disposed," "installed," "connected," and "connected" should be understood in a broad sense. For example, they may refer to fixed connections, detachable connections, or integral connections; they may refer to mechanical connections or electrical connections; they may refer to direct connections or indirect connections through an intermediate medium; and they may refer to internal communication between two components. Those skilled in the art will understand the specific meanings of the above terms in the present invention based on the specific circumstances.

[0050] Please refer to Figures 1 to 4 As shown, an AML organoid model is formed by 3D bioprinting, and is constructed from top to bottom with a nutrient circulation mechanism 1, a tumor cell growth mechanism 2, a cell external stimulation mechanism 3, and an endothelial cell growth mechanism 4;

[0051] The nutrient circulation mechanism 1 is used for exchanging nutrients. Specifically, a first flow channel 5 is constructed in the nutrient circulation mechanism 1, and a first test electrode is installed in the first flow channel 5;

[0052] Nutrients circulate in the first flow channel 5 and gradually penetrate downward to the tumor cell growth mechanism 2, the cell external stimulation mechanism 3 and the endothelial cell growth mechanism 4. The aforementioned first test electrode is used to test parameters of products generated by tumor cells.

[0053] A second flow channel 6 is constructed in the tumor cell growth mechanism 2 , the second flow channel 6 is filled with channel ink, and a second test electrode is installed in the second flow channel 6 ;

[0054] The aforementioned tumor cell growth mechanism 2 is used to inoculate tumor cells for growth, thereby providing a growth environment for the tumor cells.

[0055] The external cell stimulation mechanism 3 is internally constructed with a cavity 7 and an air supply channel 8 that are interconnected, and the air supply channel 8 is connected to the cavity 7;

[0056] The air supply channel 8 of the aforementioned cell external stimulation mechanism 3 is connected to an external air pump. Under the action of the air pump, gas is regularly extracted, and negative pressure is applied toward the cavity 7, driving the stretching of the cavity 7, thereby simulating the heart beating environment and providing external stimulation to the aforementioned inoculated cells.

[0057] A third flow channel 9 is constructed in the endothelial cell growth mechanism 4 , and the inner wall of the third flow channel 9 is filled with an outer layer of ink.

[0058] The endothelial cell growth mechanism 4 simulates the in vivo endothelial cell growth environment. Furthermore, the cells in the endothelial cell growth mechanism 4 and the tumor cell growth mechanism 2 are inoculated before the entire system is activated. During system operation, only the nutrient circulation mechanism 1, located at the top layer, receives and removes culture medium for nutrient supply.

[0059] Furthermore, the nutrient circulation mechanism 1, tumor cell growth mechanism 2, cell external stimulation mechanism 3, and endothelial cell growth mechanism 4 are all formed using a core / shell type GelMA-TGF-β1 / gelatin ink system as a profile;

[0060] The profile is formed by coaxial nozzles, including tissue cell-carrying ink for the outer nozzle and endothelial cell-carrying sacrificial ink for the inner nozzle.

[0061] Specifically, a core / shell GelMA-TGF-β1 / gelatin ink system was employed, and coaxial nozzle technology was used to achieve the simultaneous release of a tissue cell-carrying ink (outer nozzle) and a sacrificial ink (inner nozzle) carrying endothelial cells. During the printing process, the sacrificial ink served as a support structure to ensure the formation of the flow channel; during the subsequent culture phase, the sacrificial ink gradually melted, forming a smooth flow channel network. Simultaneously, the endothelial cells were released from the sacrificial ink and tightly adhered to the inner wall of the flow channel, completing the vascularization process. This process effectively simulates the natural transition from a flow channel network to a vascularized network, and is closer to the real environment in the body than traditional methods.

[0062] For the aforementioned channel ink, the channel ink is prepared in the following manner:

[0063] A1. Add porcine skin-derived gelatin to a 15% concentration in PBS and dissolve in a 50°C water bath with stirring for 30 minutes until completely dissolved.

[0064] A2. Slowly add methyl acrylate (MA) to the gelatin solution at 37°C until the MA concentration reaches 6%. After two hours of reaction, remove the MA from the gelatin solution to obtain GelMA.

[0065] A3. GelMA was dialyzed in deionized water for one week, freeze-dried, and 5 g of TGF-β1 monomer was dissolved in 15% GelMA with PBS and stirred for 5 minutes to obtain GelMA-TGF-β1.

[0066] A4. Mix GelMA-TGF-β1 and KG-1a cells to obtain the channel ink.

[0067] In addition, for the outer layer ink, the outer layer ink is prepared in the following manner:

[0068] B1. Adipose-derived mesenchymal stem cells were extracted from 4-week-old rats;

[0069] B2. Culture the cells in complete mesenchymal stem cell medium, changing the medium every two days. Subculture the cells when they reach 80%-90% confluence. After two subcultures, use the cells for organoid culture.

[0070] B3. Every 10 5 Mesenchymal stem cells (MSCs) and 1 ml of 5% sodium alginate are mixed in equal proportions to prepare the outer layer ink.

[0071] Furthermore, in order to allow the nutrients in the top layer to better permeate downward in one direction, the nutrient circulation mechanism 1, tumor cell growth mechanism 2, cell external stimulation mechanism 3 and endothelial cell growth mechanism 4 are stacked in sequence from top to bottom, and a one-way filter membrane is constructed between two adjacent layers.

[0072] In order to improve the integrity of the entire system, a vertically penetrating electrode mounting hole 10 is constructed on the nutrient circulation mechanism 1. The electrode mounting hole 10 passes through the first flow channel 5 and extends into the second flow channel 6. A measuring electrode is installed in the electrode mounting hole 10. The part of the measuring electrode located in the first flow channel 5 serves as the first test electrode, and the part of the measuring electrode located in the second flow channel 6 serves as the second test electrode.

[0073] Furthermore, the measuring electrodes include a reference electrode, an enzyme sensor and a counter electrode in sequence along the liquid flow direction in the first flow channel 5 .

[0074] At the same time, the liquid inlet and liquid outlet ends of the first flow channel 5 are respectively constructed on the opposite side walls of the nutrient circulation mechanism 1, and the ink inlet and ink outlet ends of the third flow channel 9 are respectively constructed on the opposite side walls of the endothelial cell growth mechanism 4, and the orthographic projection of the first flow channel 5 on the endothelial cell growth mechanism 4 coincides with that of the third flow channel 9.

[0075] In addition, since biological samples usually contain complex matrices and salts, these components will interfere with mass spectrometry analysis. Therefore, the ink inlet and ink outlet of the second flow channel 6 are respectively constructed on opposite sides of the tumor cell growth mechanism 2. The second flow channel 6 includes a main channel 11 and a measurement channel 12 that is individually connected to each second test electrode. A dry film blocking layer 13 is provided on the upstream side of the measurement channel 12 relative to the second test electrode. The two ends of the main channel 11 serve as the ink inlet and ink outlet. The main channel 11 is also connected to a test channel 14. The test channel 14 extends from the side wall of the tumor cell growth mechanism 2 and is connected to a nano nozzle 15.

[0076] In general, the aforementioned organoid model is prepared in the following manner: S1. preparing channel ink and outer layer ink;

[0077] S2. forming the endothelial cell growth mechanism 4 by 3D printing, and introducing the outer layer of ink into the third flow channel 9, and curing it by ultraviolet light;

[0078] S3. Forming the external cell stimulation mechanism 3 above the endothelial cell growth mechanism 4 by 3D printing, and curing with ultraviolet light;

[0079] S4. Using 3D printing to form the tumor cell growth mechanism 2 above the cell external stimulation mechanism 3, and introducing the channel ink into the second flow channel 6, and curing it using ultraviolet light;

[0080] S5. Using 3D printing to form the nutrient circulation mechanism 1 above the tumor cell growth mechanism 2;

[0081] S6. Insert electrodes to form the first test electrode and the second test electrode.

[0082] Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. An AML organoid model, characterized in that Using 3D bioprinting, the structure consists of a nutrient circulation mechanism (1), a tumor cell growth mechanism (2), a cell external stimulation mechanism (3), and an endothelial cell growth mechanism (4) from top to bottom. A first flow channel (5) is constructed in the nutrient circulation mechanism (1), and a first test electrode is installed in the first flow channel (5); A second flow channel (6) is constructed in the tumor cell growth mechanism (2), the second flow channel (6) is filled with channel ink, and a second test electrode is installed in the second flow channel (6); The external cell stimulation mechanism (3) is internally structured with a cavity (7) and an air supply channel (8) that are interconnected, and the air supply channel (8) is connected to the cavity (7); A third flow channel (9) is constructed in the endothelial cell growth mechanism (4), and the inner wall of the third flow channel (9) is filled with an outer layer of ink; The channel ink is prepared in the following manner: A1. Add porcine skin-derived gelatin to a 15% concentration in PBS and dissolve in a 50°C water bath with stirring for 30 minutes until completely dissolved. A2. Slowly add methyl acrylate (MA) to the gelatin solution at 37°C until the MA concentration reaches 6%. After two hours of reaction, remove the MA from the gelatin solution to obtain GelMA. A3. GelMA was dialyzed in deionized water for one week, freeze-dried, and 5 g of TGF-β1 monomer was dissolved in 15% GelMA with PBS and stirred for 5 minutes to obtain GelMA-TGF-β1. A4. GelMA-TGF-β1 was mixed with KG-1a cells to obtain the channel ink; The outer layer ink is prepared in the following manner: B1. Adipose-derived mesenchymal stem cells were extracted from 4-week-old rats; B2. Culture the cells in complete mesenchymal stem cell medium, changing the medium every two days. Subculture the cells when they reach 80%-90% confluence. After two subcultures, use the cells for organoid culture. B3. Every 10 5 Mesenchymal stem cells (MSCs) and 1 ml of 5% sodium alginate are mixed in equal proportions to prepare the outer layer ink.

2. An AML organoid model according to claim 1, characterized in that The nutrient circulation mechanism (1), tumor cell growth mechanism (2), cell external stimulation mechanism (3), and endothelial cell growth mechanism (4) are all formed using a core / shell type GelMA-TGF-β1 / gelatin ink system as a profile; The profile is formed by coaxial nozzles, including tissue cell-carrying ink for the outer nozzle and endothelial cell-carrying sacrificial ink for the inner nozzle.

3. An AML organoid model according to claim 1, characterized in that The nutrient circulation mechanism (1), tumor cell growth mechanism (2), cell external stimulation mechanism (3) and endothelial cell growth mechanism (4) are stacked in sequence from top to bottom, and a one-way filter membrane is constructed between two adjacent layers.

4. The AML organoid model according to claim 1, characterized in that The nutrient circulation mechanism (1) is provided with an electrode mounting hole (10) extending vertically therethrough. The electrode mounting hole (10) passes through the first flow channel (5) and extends into the second flow channel (6). A measuring electrode is mounted in the electrode mounting hole (10). The portion of the measuring electrode located in the first flow channel (5) serves as the first test electrode, and the portion of the measuring electrode located in the second flow channel (6) serves as the second test electrode.

5. An AML organoid model according to claim 4, characterized in that The measuring electrodes include a reference electrode, an enzyme sensor and a counter electrode in sequence along the direction of liquid flow in the first flow channel (5).

6. The AML organoid model according to claim 1, characterized in that The liquid inlet and liquid outlet of the first flow channel (5) are respectively constructed on opposite side walls of the nutrient circulation mechanism (1), the ink inlet and ink outlet of the third flow channel (9) are respectively constructed on opposite side walls of the endothelial cell growth mechanism (4), and the orthographic projection of the first flow channel (5) on the endothelial cell growth mechanism (4) coincides with that of the third flow channel (9).

7. The AML organoid model according to claim 1, characterized in that The ink inlet and ink outlet of the second flow channel (6) are respectively constructed on opposite sides of the tumor cell growth mechanism (2). The second flow channel (6) includes a main flow channel (11) and a measurement flow channel (12) separately connected to each second test electrode. A dry film blocking layer (13) is provided on the upstream side of the measurement flow channel (12) relative to the second test electrode. The two ends of the main flow channel (11) serve as the ink inlet and ink outlet. The main flow channel (11) is also connected to a test channel (14). The test channel (14) extends from the side wall of the tumor cell growth mechanism (2) and is connected to a nano nozzle (15).

8. A method for constructing an AML organoid model according to any one of claims 1 to 7, characterized in that: The following steps are involved: S1. Configure channel ink and outer ink; S2. forming the endothelial cell growth mechanism (4) by 3D printing, introducing the outer layer ink into the third flow channel (9), and curing it by ultraviolet light; S3. forming the cell external stimulation mechanism (3) above the endothelial cell growth mechanism (4) by 3D printing, and curing the same by ultraviolet light; S4. forming the tumor cell growth mechanism (2) above the cell external stimulation mechanism (3) by 3D printing, and introducing the channel ink into the second flow channel (6), and curing it by ultraviolet light; S5. Using 3D printing to form the nutrient circulation mechanism (1) above the tumor cell growth mechanism (2); S6. Insert electrodes to form the first test electrode and the second test electrode.

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