Micro-fluidic chip, preparation method thereof and detection method based on micro-fluidic chip

Abstract

The embodiment of the invention discloses a micro-fluidic chip, a preparation method of the micro-fluidic chip and a test method based on the micro-fluidic chip. The chip comprises an injection layer, a porous membrane layer and an organoid culture layer which are sequentially stacked, the injection layer is provided with a first flow channel, a first inlet and a first outlet which are communicated with the first flow channel, and an introduction port; the porous membrane layer is provided with an injection hole and a connecting port, and the connecting port is communicated with the introduction port; the second inlet, the second outlet and the organoid culture chamber are respectively communicated with the second flow channel, the organoid culture chamber is communicated with the injection hole, and the second inlet is communicated with the connecting port; the first inlet is used for providing a tumor drug and an ATP nano sensor for the organoid culture chamber through the first flow channel and the injection hole; the introduction port is used for providing tumor cells for in-vitro culture of the organoids for the organoid culture chamber through the connection port, the second inlet and the second flow channel. According to the micro-fluidic chip disclosed by the embodiment of the invention, the whole PDO-based drug reaction test process can be executed in a single micro-fluidic chip, and the ATP in cells can be identified under the condition that the cells are not damaged through the injected ATP nano sensor.

Description

Technical Field

[0001] The embodiments of the present disclosure belong to the field of biotechnology, and specifically relate to a microfluidic chip and a preparation method thereof, and a detection method based on the microfluidic chip. Background Art

[0002] Patient-derived cancer organoids (PDOs) have great potential to become a drug screening platform with broad applications because they are highly consistent with the genetic material of the primary tumor, including gene expression profiles, proliferation rates, and drug responses. PDO drug responses have been shown to have a strong correlation with clinical drug efficacy, and accurate analysis of PDO-based drug responses can help select the best drugs for personalized treatment, improve overall survival, and reduce overtreatment. Specifically, the accuracy of analyzing the growth status of PDOs after exposure to various drug treatments will affect the effectiveness of evaluating PDO-based drug responses.

[0003] Image-based observations, including fluorescent staining for apoptosis or cell death, immunostaining, and histology, can visualize the growth status of PDOs in terms of organoid size, shape, characteristics, and number. However, image-based observations reflect morphological changes rather than metabolic fluctuations, and changes in metabolic fluctuations are critical for quantifying the drug effects of PDOs.

[0004] Compared with direct observation of morphology, molecular assays to detect cellular energy metabolism levels can improve the accuracy of characterizing the growth status of PDOs. Adenosine triphosphate (ATP), as a molecule that directly reflects the fluctuation of organoid energy metabolism, plays a vital role in regulating cell viability and metabolic activity, and exhibits sensitivity to drug-induced apoptosis. Therefore, ATP detection has been widely used in existing PDO-based drug response tests. However, due to the necessary cell lysis experimental steps, all commercial ATP detection kits can only be applied to a single time point for ATP detection, thus leading to the neglect of the dynamic changes of PDOs. Many factors, including drug induction and changes in the intracellular / extracellular environment, may trigger mitochondrial changes or intracellular pH, affecting intracellular ATP readings at different time intervals. Therefore, the lack of full-time ATP monitoring may affect the accuracy of PDO-based drug response tests. At the same time, the number of PDOs that can be constructed from a single patient is limited. Therefore, it is impractical to test all feasible detection time points to find the optimal detection time point.

[0005] Compared with detecting the growth status of PDO at a single time point, the whole process monitoring of PDO growth at the molecular level may improve the accuracy of PDO-based drug response testing. Recently, there have been studies to monitor the growth status of organoids in real time, including measuring external microenvironmental parameters (pH, oxygen, temperature), current signals, and acidic products of organoids. Although indirect detection of indicators located in the external environment of organoids can reflect the growth status of organoids, direct monitoring of internal metabolic fluctuations of organoids has not yet been achieved. Summary of the invention

[0006] The embodiments of the present disclosure aim to solve at least one of the technical problems existing in the prior art, and disclose a microfluidic chip and a preparation method thereof, and a detection method based on the microfluidic chip.

[0007] In a first aspect, an embodiment of the present disclosure provides a microfluidic chip, the microfluidic chip comprising: an injection layer, a porous membrane layer, and an organoid culture layer stacked in sequence; The injection layer is provided with a first flow channel and a first inlet and a first outlet respectively connected with the first flow channel, and the injection layer is also provided with an introduction port; The porous membrane layer is provided with an injection hole and a connection port, and the connection port corresponds to the position of the introduction port and is connected; The organoid culture layer is provided with a second flow channel and a second inlet, a second outlet and an organoid culture chamber respectively connected to the second flow channel, the organoid culture chamber corresponds to and is connected to the injection hole, and the second inlet corresponds to and is connected to the connection port; wherein, The first inlet is used to provide tumor drugs and ATP nanosensors to the organoid culture chamber through the first flow channel and the injection hole; The introduction port is used to provide tumor cells for in vitro culturing of organoids to the organoid culture chamber through the connection port, the second inlet and the second flow channel.

[0008] In some embodiments, the first flow channel is disposed on a side of the injection layer facing the organoid culture layer; and / or the second flow channel and the organoid culture chamber are disposed on a side of the organoid culture layer facing the injection layer.

[0009] In some embodiments, the first flow channel extends outward at a position corresponding to the organoid culture chamber to match the organoid culture chamber.

[0010] In some embodiments, the first inlet and the first outlet are symmetrically distributed on both sides of the injection layer along the width direction thereof.

[0011] In some embodiments, the injection layer is further provided with a cascade ratio mixer; The inlet of the proportioning mixer is communicated with the first inlet, and the outlet of the proportioning mixer is communicated with the first flow channel.

[0012] In some embodiments, the cascade ratio mixer comprises: a multi-stage mixing main flow channel and a plurality of mixing branch flow channels; The plurality of main mixing channels are arranged side by side in the injection layer, and the plurality of branch mixing channels are arranged side by side in the injection layer; and a plurality of branch mixing channels are arranged side by side between two adjacent main mixing channels; The main mixing channel is in communication with the first inlet, and the branch mixing channels are in communication with the corresponding main mixing channel and the first channel.

[0013] In some embodiments, the length of the mixing branch channel is greater than its projected length on the injection layer.

[0014] In some embodiments, the mixing branch channel is arranged in the injection layer in an S-shape.

[0015] In some embodiments, the main mixing channel is provided with switch valves on both sides of the inlet positions corresponding to the branch mixing channels.

[0016] In some embodiments, the organoid culture layer is further provided with a gas inlet and a gas channel, the gas channel having an open end and a closed end, the open end of the gas channel being connected to the gas inlet; and, The gas channel corresponds to the position of the cascade proportion mixer to control the on and off of the proportion mixer.

[0017] In some embodiments, when the cascaded proportioning mixer comprises a switching valve; The closed end of the gas channel corresponds to the position of the switch valve.

[0018] In some embodiments, the orthographic projection of the closed end on the corresponding switch valve falls outside the switch valve.

[0019] In some embodiments, the gas inlets are symmetrically arranged along the length direction of the organoid culture layer.

[0020] In some embodiments, the organoid culture layer is provided with a plurality of groups of the organoid culture chambers arranged side by side; and, Each group includes a plurality of the organoid culture chambers connected in series.

[0021] In some embodiments, the introduction port is disposed close to the first outlet, and the introduction port is located on the inner side of the first outlet.

[0022] In a second aspect, an embodiment of the present disclosure provides a method for preparing a microfluidic chip, wherein the microfluidic chip is the microfluidic chip described above, and the method comprises the following steps: providing a first substrate, a second substrate, and a third substrate; Patterning the first substrate to form a first protrusion on the first substrate, forming a first flexible material layer on the patterned first substrate, removing the first substrate after heating and curing, and drilling holes at the inlet and outlet of the first flexible material layer to prepare an injection layer; forming a second flexible material layer on the second substrate, patterning the second flexible material layer to form a first opening and a second opening, and removing the second substrate after heating and curing to prepare a porous membrane layer; Patterning the third substrate to form a second protrusion on the third substrate, forming a third flexible material layer on the patterned third substrate, removing the third substrate after heating and curing, and punching holes at the inlet and outlet of the third flexible material layer to prepare an organoid culture layer; The injection layer, the porous membrane layer and the organoid culture layer are bonded and packaged in sequence to prepare the microfluidic chip.

[0023] In some embodiments, the temperature range of the heat curing is 80° C. to 90° C.; and / or the time range of the heat curing is 1.5 h to 2.5 h.

[0024] In some embodiments, the injection layer, the porous membrane layer and the organoid culture layer are bonded and packaged in sequence to prepare the microfluidic chip, including: The injection layer, porous membrane layer and organoid culture layer are ultrasonically cleaned with distilled water, alcohol or isopropanol, and then the surface is treated with plasma. The organoid culture layer is first bonded and packaged with the porous membrane layer, and then the injection layer and the porous membrane layer are bonded and packaged. After the microfluidic chip is placed at high temperature and completely sealed, the complete microfluidic chip is finally formed.

[0025] In some embodiments, the first flexible material layer, the second flexible material layer and the third flexible material layer are made of a mixture of polydimethylsiloxane and a curing agent.

[0026] In some embodiments, the mass ratio of the polydimethylsiloxane to the curing agent is in the range of (8-10):1.

[0027] In a third aspect, an embodiment of the present disclosure further provides a detection method based on a microfluidic chip, wherein the microfluidic chip adopts the microfluidic chip described above, and the detection method comprises the following steps: Introducing tumor cells and extracellular matrix gel for in vitro organoid culture into the organoid culture chamber through the introduction port of the injection layer, the connection port on the porous membrane layer, the second inlet port and the second flow channel on the organoid culture layer; Introducing the tumor drug and the ATP nanosensor into the organoid culture chamber on the organoid culture layer through the first inlet and the first flow channel of the injection layer and the injection hole on the porous membrane layer; The fluorescence intensity change value of the ATP nanosensor in the organoid culture chamber is obtained, and the effectiveness of the tumor drug is determined according to the fluorescence intensity change value.

[0028] In some embodiments, the tumor drug is a tyrosine kinase inhibitor TKI; The method of introducing the tumor drug and the ATP nanosensor into the organoid culture chamber on the organoid culture layer through the first inlet and the first flow channel of the injection layer and the injection hole on the porous membrane layer comprises: injecting the ATP nanosensor and different TKIs into different organoid culture chambers on the organoid culture layer respectively; The method of obtaining the fluorescence intensity change value of the ATP nanosensor in the organoid culture chamber and determining the effectiveness of the tumor drug according to the intensity change value includes: obtaining the fluorescence intensity change values ​​of the ATP nanosensor in different organoid culture chambers, and determining the effectiveness of different TKIs according to the fluorescence intensity change values.

[0029] In some embodiments, the different TKIs include: osimertinib and erlotinib.

[0030] In some embodiments, the tumor drug is a chemotherapy drug in combination of different concentrations; The method of introducing the tumor drug and the ATP nanosensor into the organoid culture chamber on the organoid culture layer through the first inlet and the first flow channel of the injection layer and the injection hole on the porous membrane layer comprises: injecting the ATP nanosensor and chemotherapy drugs of different concentrations into different organoid culture chambers on the organoid culture layer respectively; The step of obtaining the fluorescence intensity change value of the ATP nanosensor in the organoid culture chamber and determining the effectiveness of the tumor drug according to the fluorescence intensity change value includes: obtaining the fluorescence intensity change value of the ATP nanosensor in different organoid culture chambers, and determining the effectiveness of the chemotherapy drugs in different concentration combinations according to the fluorescence intensity change value.

[0031] In some embodiments, the chemotherapy drugs in different concentration combinations include: pemetrexed and carboplatin in different concentration combinations.

[0032] In some embodiments, the tumor drug is a combination of TKI and chemotherapy drugs at different concentrations; The method of introducing the tumor drug and the ATP nanosensor into the organoid culture chamber on the organoid culture layer through the first inlet and the first flow channel of the injection layer and the injection hole on the porous membrane layer comprises: injecting the ATP nanosensor and TKI and chemotherapy drugs of different concentrations into different organoid culture chambers on the organoid culture layer respectively; The step of obtaining the fluorescence intensity change value of the ATP nanosensor in the organoid culture chamber and determining the effectiveness of the tumor drug according to the fluorescence intensity change value includes: obtaining the fluorescence intensity change value of the ATP nanosensor in different organoid culture chambers, and determining the effectiveness of the TKI and chemotherapy drugs in different concentration combinations according to the fluorescence intensity change value.

[0033] In some embodiments, the method further comprises: At least one organoid culture chamber on the organoid culture layer is controlled not to have the tumor drug added therein, serving as a control group.

[0034] The microfluidic chip and its preparation method, as well as the testing method based on the microfluidic chip of the embodiments of the present disclosure can perform the entire PDO-based drug reaction testing process in a single microfluidic chip, and through the injected ATP nanosensor, intracellular ATP can be identified without damaging the cells, and the full-process monitoring requirements of ATP changes in PDO can be achieved. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The above and other features, advantages and aspects of the embodiments of the present disclosure will become more apparent with reference to the following detailed description in conjunction with the accompanying drawings. Throughout the accompanying drawings, the same or similar reference numerals represent the same or similar elements. It should be understood that the drawings are schematic and that components and elements are not necessarily drawn to scale.

[0036] Figure 1 This is a schematic diagram of the structure of a microfluidic chip according to an embodiment of the present disclosure; Figure 2 for Figure 1 A front view of the microfluidic chip shown in; Figure 3 for Figure 1 An exploded view of the microfluidic chip shown in; Figure 4 for Figure 1 Schematic diagram of the structure of the injection layer in the microfluidic chip shown in; Figure 5 for Figure 1 Schematic diagram of the structure of the porous membrane layer in the microfluidic chip shown in; Figure 6for Figure 1 Schematic diagram of the structure of the organoid culture layer in the microfluidic chip shown in; Figure 7 for Figure 4 A schematic diagram of the structure of the proportion mixer in the injection layer shown in ; Figure 8 A schematic diagram of the structure and ATP sensing performance of an ATP nanosensor according to another embodiment of the present disclosure; Fig. 9 A schematic diagram of the characterization and functional verification of an ATP nanosensor according to another embodiment of the present disclosure; Fig.10 A schematic diagram of height distribution analysis of an ATP nanosensor in an AFM according to another embodiment of the present disclosure; Fig.11 A schematic diagram of the fluorescence spectrum of a control structure in response to 0-5 mM ATP according to another embodiment of the present disclosure; Fig.12 A linear regression fitting diagram of detecting ATP in H1975 cells by comparing an ATP nanosensor and an ATP nanosensor according to another embodiment of the present disclosure; Fig.13 This is a schematic diagram of detecting the cell viability of H1975 cells under different concentrations of osimertinib using a CCK-8 kit according to another embodiment of the present disclosure; Fig.14 A flow chart of a method for preparing a microfluidic chip according to another embodiment of the present disclosure; Fig.15 is a process flow chart of a method for preparing a microfluidic chip according to another embodiment of the present disclosure; Fig.16 This is a flow chart of a detection method based on a microfluidic chip according to another embodiment of the present disclosure; Fig.17 This is a schematic diagram of organoid culture and monitoring based on a microfluidic chip according to another embodiment of the present disclosure; Fig.18 Schematic diagram of a PDO-based drug reaction test based on a microfluidic chip according to another embodiment of the present disclosure; Fig.19 Schematic diagram of a fluorescence image of PDO treated with IAA+ATP-nanosensor (red) and stained with Lysotracker (green) according to another embodiment of the present disclosure. DETAILED DESCRIPTION

[0037] In order to enable those skilled in the art to better understand the technical solution of the present disclosure, the present disclosure is further described in detail below in conjunction with the accompanying drawings and specific implementation methods. Obviously, the described embodiments are part of the embodiments of the present disclosure, rather than all of the embodiments. Based on the described embodiments of the present disclosure, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present disclosure.

[0038] Unless otherwise specified, the technical terms or scientific terms used in the embodiments of the present disclosure should be understood by people with ordinary skills in the field to which the present disclosure belongs. The "including" or "comprising" used in the embodiments of the present disclosure neither limits the shapes, numbers, steps, actions, operations, components, originals and / or their groups mentioned, nor excludes the appearance or addition of one or more other different shapes, numbers, steps, actions, operations, components, originals and / or their groups, or the addition of these. In addition, the terms "first" and "second" are only used for descriptive purposes, and cannot be understood as indicating or implying relative importance or implicitly indicating the number and order of the indicated technical features. Thus, the features defined as "first" and "second" can explicitly or implicitly include one or more of the features. In the description of the embodiments of the present disclosure, the meaning of "multiple" is two or more, unless otherwise clearly and specifically defined.

[0039] In some descriptions of the embodiments of the present disclosure, unless otherwise clearly specified and limited, the terms "install", "connect", "connected" or "fixed" and the like are not limited to physical or mechanical connections, but may include electrical connections, whether direct or indirectly connected through an intermediate medium, and may be internal connections between two elements or mutual interactions between two elements. Also, the terms "center", "longitudinal", "lateral", "length", "width", "thickness", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside", "clockwise", "counterclockwise", "axial", "radial", "circumferential" and the like indicate positions or positional relationships based on the positions or positional relationships shown in the accompanying drawings, and are only used to indicate relative positional relationships. When the absolute position of the described object changes, the relative positional relationship may also change accordingly.

[0040] In the embodiments of the present disclosure, unless otherwise clearly specified and limited, the first feature being "above" or "below" the second feature may mean that the first and second features are in direct contact, or the first and second features are in indirect contact through an intermediate medium. Moreover, the first feature being "above", "above" and "above" the second feature may mean that the first feature is directly above or obliquely above the second feature, or simply means that the first feature is higher in level than the second feature, and the first feature being "below", "below" and "below" the second feature may mean that the first feature is directly below or obliquely below the second feature, or simply means that the first feature is lower in level than the second feature.

[0041] It should also be understood that when a layer is referred to as being "on" another layer or substrate, it can be directly on the other layer or substrate, or there may be intervening layers; it should also be understood that when an element (such as a layer, region or substrate) is referred to as being "on", "connected to", "electrically connected to" or "electrically coupled to" another element, it can be directly on the other element, directly connected or coupled to the other element, or there may be one or more intervening elements. Conversely, when an element is referred to as being "directly on", "directly connected to" or "directly coupled to" another element or layer, there are no intervening elements or layers. The term "and / or" as used herein includes any and all combinations of one or more of the relevant listed items) Unless otherwise specifically stated, the relative arrangement of the components and steps, numerical expressions and numerical values ​​set forth in these embodiments do not limit the scope of the present disclosure. At the same time, it should be understood that, for ease of description, the dimensions of the various parts shown in the drawings are not drawn according to the actual proportional relationship, and the techniques, methods and devices known to ordinary technicians in the relevant fields may not be discussed in detail, but where appropriate, the techniques, methods and devices shown should be considered as part of the authorized specification. In all examples shown and discussed here, any specific other examples may have different values. It should be noted that similar symbols and letters represent similar items in the following drawings, so once an item is defined in one drawing, it does not need to be further discussed in subsequent drawings.

[0042] In the description of the embodiments of the present disclosure, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present disclosure. In the embodiments of the present disclosure, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in a suitable manner in any one or more embodiments or examples. In addition, those skilled in the art may combine and combine the different embodiments or examples described in the embodiments of the present disclosure and the features of the different embodiments or examples, unless they are contradictory.

[0043] Figure 1 is a schematic structural diagram of a microfluidic chip according to an embodiment of the present disclosure, Figure 2 for Figure 1 The front view of the microfluidic chip shown in Figure 3 for Figure 1 Exploded view of the microfluidic chip shown in . Figure 4 for Figure 1 The schematic diagram of the structure of the injection layer in the microfluidic chip shown in FIG. Figure 5 for Figure 1 The schematic diagram of the structure of the porous membrane layer in the microfluidic chip is shown in FIG. Figure 6 for Figure 1 Schematic diagram of the structure of the organoid culture layer in the microfluidic chip shown in .

[0044] like Figures 1 to 6 As shown, an embodiment of the present disclosure provides a microfluidic chip, which includes: an injection layer A, a porous membrane layer B, and an organoid culture layer C, which are stacked in sequence. The injection layer A is provided with a first flow channel A1 and a first inlet A2 and a first outlet A3 respectively connected to the first flow channel A1, and the injection layer A is also provided with an introduction port A4. The porous membrane layer B is provided with an injection hole B1 and a connecting port B2, and the connecting port B2 corresponds to and is connected to the position of the introduction port A4. The organoid culture layer C is provided with a second flow channel C1 and a second inlet C2, a second outlet C3, and an organoid culture chamber C4 respectively connected to the second flow channel C1, and the organoid culture chamber C4 corresponds to and is connected to the position of the injection hole B1, and the second inlet C2 corresponds to and is connected to the position of the connecting port B2.

[0045] When the microfluidic chip of this embodiment is actually applied, for example, when detecting the effectiveness of different tumor drugs on tumor organoids (this part of the content will be specifically described in the following embodiments of the present disclosure and will not be repeated here), combined with Figures 1 to 6, tumor cells (such as lung tumor cells, stomach tumor cells, pancreatic tumor cells, etc.) and extracellular matrix gel for in vitro organoid culture are injected downward into the porous membrane layer B through the inlet A4 on the injection layer A, and the tumor cells and extracellular matrix gel are injected downward into the organoid culture layer C through the connection port B2 on the porous membrane layer B, and the tumor cells and extracellular matrix gel enter the second flow channel C1 through the second inlet C2 on the organoid culture layer C, and then are injected into the organoid culture chamber C4 through the second flow channel C1. Correspondingly, the tumor drug and ATP nanosensor enter the first flow channel A1 through the first inlet A2 on the injection layer A, and then are injected downward into the porous membrane layer B through the first flow channel A1, and the tumor drug and ATP nanosensor are injected into the organoid culture chamber C4 on the organoid culture layer C through the injection hole B1 on the porous membrane layer B, and then the fluorescence intensity value of the organoid culture chamber C4 is detected, and the effectiveness of the tumor drug is determined according to the change in ATP value.

[0046] After the effectiveness of the tumor drug is tested, the discarded tumor cells and extracellular matrix gel are led out of the organoid culture chamber C4 through the second flow channel C1 on the organoid culture layer C, and are led out to the outside of the microfluidic chip through the second outlet C3 on the organoid culture layer C. Correspondingly, the excess tumor drug and ATP nanosensor are led out of the microfluidic chip through the first flow channel A1 on the injection layer A and the first outlet A3 on the layer.

[0047] In the embodiments of the present disclosure, in order to monitor the dynamic changes of ATP in living cells, an ATP nanosensor is developed that can sensitively identify ATP without damaging cells.

[0048] The ATP nanosensor is composed of four DNA single strands (denoted as SSDNA1-SSDNA4) and four ATP-targeting aptamer chains in a tetrahedral morphology. Specifically, the ATP nanosensor aptamer chain that can target ATP was synthesized by the base complementary coordination of the Cy3-modified chain and the BHQ2-modified short chain. Due to the occurrence of resonance energy transfer (FRET), the ATP nanosensor remains in a quenched state. With the participation of intracellular ATP, the BHQ2-modified short chain is replaced by ATP molecules. Then, the binding of the Cy3-modified chain to ATP leads to an activated state. Therefore, the ATP nanosensor can be detected by red fluorescence signals in the presence of ATP. The detailed sequences of the DNA single strands (SSDNA1-SSDNA4) and ATP-targeting aptamer chains used to construct the ATP nanosensor are listed in Table 1.

[0049] Table 1

[0050] Assembly of ATP-targeted aptamer chains: Cy3-modified chains and BHQ2-modified short chains were combined by the principle of complementary base pairing, and the Cy3-modified chains were extended by 15 polyAs. SSDNA1, SSDNA2, SSDNA3, and SSDNA4 were connected by complementary base pairing to assemble into tetrahedral framework chains. These SSDNAs were extended by 15 polyTs at the termini. Through complementary base pairing of polyT and polyA, SSDNA1-4 and 4 ATP-targeted aptamer chains assembled the ATP nanosensor. The control structure was assembled with Cy3-modified chains, BHQ2-modified long chains, and SSDNA1-4.

[0051] The preparation process of ATP nanosensor will be described in detail below.

[0052] Specifically, the Cy3-modified chain and the BHQ2-modified short chain were dissolved in a buffer (10mM Tris, 1mM EDTA, and 10mM MgCl2) at a molar ratio of 1:1. The mixture was then heated to 70°C and annealed to 25°C at a rate of 3 min / °C. Next, SSDNA1-SSDNA4 were mixed at a molar ratio of 1:1:1:1, heated to 95°C for 5 min, and then annealed to 25°C at a rate of 3 min / °C. Finally, the PCR products obtained in the first two steps were mixed at a concentration ratio of 4:1 and slowly cooled from 37°C to 25°C at a rate of 10 min / °C to obtain the ATP nanosensor required in this example. The structure and ATP sensing performance of the final ATP nanosensor are shown in FIG. Figure 8 As shown, the ATP nanosensor is composed of a single DNA strand (SSDNA1-SSDNA4) and an ATP-targeted aptamer strand, presenting a tetrahedral morphology and a fluorescence quenching state. When the ATP nanosensor recognizes ATP, the BHQ2-modified short strand is replaced by ATP, resulting in the occurrence of the Cy3 fluorescence activation state.

[0053] Before application on PDO, the ATP nanosensor was validated in cell lines for structural integrity, performance, and recognition specificity of ATP monitoring as follows: The detailed sequences of the DNA single strands (SSDNA1-SSDNA4) and ATP-targeting aptamer strands used to construct the ATP nanosensor are listed in Table 1. Fig. 9 As shown in a, atomic force microscopy was used to show the expected tetrahedral morphology of the ATP nanosensor. Then its height distribution was analyzed, as shown in Fig.10As shown, the average height is 3.84±0.75 nm (± SD, n = 156). These results indicate that the ATP nanosensor was successfully assembled. In order to compare the specificity of the ATP nanosensor in recognizing ATP, we also designed a control structure that cannot recognize ATP. The detailed sequence is shown in Table 1 above. In order to evaluate the specific recognition of ATP by the ATP nanosensor, the fluorescence spectra of different emission wavelengths were measured in different ATP concentration groups with an excitation wavelength of 530 nm, as shown in Figure 1. Fig. 9 As shown in b, after adding ATP (0-5 mM), the fluorescence intensity of the ATP nanosensor increased significantly, while after adding ATP, the fluorescence of the control structure did not change significantly, as shown in Fig.11 Then, the fluorescence intensity was measured in the ATP nanosensor / control structure with 0-5 mM ATP added, as shown in Fig. 9 As shown in Figure c, as the ATP concentration increases, the fluorescence intensity of the ATP nanosensor gradually increases, while the control structure has no fluorescence signal. These results indicate that the ATP nanosensor has a high specificity for recognizing ATP. Fig. 9 As shown in Figure d, compared with the group with 5 mM ATP added, the fluorescence intensity emitted by the ATP nanosensor in the group with the same concentration of CTP, UTP or GTP added was very small, indicating the high selectivity of the ATP nanosensor in recognizing ATP. Therefore, the above results show that the ATP nanosensor has a tetrahedral structure and has high selectivity and sensitivity, which can be used to specifically recognize changes in ATP.

[0054] After successfully constructing and characterizing the ATP nanosensor, we chose to verify the intracellular ATP monitoring performance of the ATP nanosensor in H1975 cells (a lung cancer cell line). In order to study the cell membrane permeability of the ATP nanosensor, we co-incubated H1975 cells with the ATP nanosensor, such as Fig. 9 As shown in Figure 5, the co-localization phenomenon between the fluorescence signal of the ATP nanosensor and the lysosomal staining signal indicates that the ATP nanosensor mainly accumulates in the intracellular environment. To further verify the ability of the ATP nanosensor to monitor changes in intracellular ATP, iodoacetic acid (IAA) was used to inhibit the production of ATP in cells. Compared with cells not treated with IAA, cells pretreated with IAA showed lower fluorescence intensity, as shown in Figure 5. Fig. 9 As shown in e, the intracellular ATP concentration has dropped below the detection threshold. These results indicate that the ATP nanosensor has high cell permeability and high specificity in recognizing ATP.

[0055] In order to quantitatively evaluate the monitoring ability of ATP nanosensor on ATP, a comparative analysis was conducted between the commercial ATP detection kit and the ATP nanosensor in detecting ATP levels in H1975 cells. Statistical analysis of the experimental results showed that the correlation coefficient between the ATP nanosensor and the ATP detection kit was 0.9936. Fig.12 As shown in Figure 9f. This high correlation coefficient indicates that the ATP nanosensor and the ATP detection kit exhibit similar performance in detecting ATP. After verifying the accuracy of the ATP nanosensor in detecting ATP, the ATP nanosensor was incubated with living cells to quantify ATP variation. H1975 cells were exposed to osimertinib and then monitored by the ATP nanosensor for more than 48 hours. As shown in Figure 9f, the ATP variation in living cells continued to decrease with the increase in osimertinib concentration, indicating that the ATP nanosensor can be labeled for 48 hours. Fig.13 As shown, monitoring drug response in cell-based assays, the cell viability of H1975 cells detected by CCK-8 kit was consistent with the ATP changes detected by ATP nanosensor. Fig.13 In the figure, H1975 cells were treated with relevant cell culture medium alone as a control group, and the data shown represent the mean ± SD of three independent experiments. These results highlight the ability of ATP nanosensors to monitor ATP changes in living cells. Crucially, unlike commercial ATP detection kits, ATP nanosensors can track ATP changes throughout the drug response process in cell-based assays, rather than being limited to a single time point.

[0056] To obtain the morphological image of the ATP nanosensor mentioned above, the ATP nanosensor (20 nM, 10 μL) was loaded onto a freshly cut mica substrate. After 10 minutes, the mica was washed three times with distilled deionized water and dried in nitrogen. The morphological image of the ATP nanosensor was obtained using an atomic force microscope.

[0057] In order to verify the ability of the above ATP nanosensor to detect cells, the embodiments of the present disclosure also carried out the establishment of related cell culture and related organoids. The details are as follows: Cell culture: H1975 cells (a lung cancer cell line) were purchased from a medical school. H1975 cells were cultured in RoswellPark Memorial Institute 1640 (RPMI 1640), a cell culture medium supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin. Cells were cultured at 37°C in an environment containing 5% CO2.

[0058] Establishment of PDO: The clinical specimens removed by surgery were approved by the ethics committee of a hospital. The tissue samples were dissected into 1 mm 3 The cubes were then digested using an enzyme solution (1 ml collagenase type II, 500 μL dispase type II, 20 μL primocin, 3.33 μL Y-27632 dissolved in 8.5 ml PBS) at 37 °C for 60 min. When the tissue was dissociated, the digestion process was terminated by adding 2 ml of serum. After filtering with a 70 μm filter, the purified cell suspension was incubated with erythrocyte lysis buffer to eliminate erythrocytes (optional step). These pelleted cells were then resuspended in ice-cold PDO medium (1.5-3 × 10 cells per 10 μl medium). 4 After the cell gel was inverted and solidified in a 37°C incubator for 30 minutes, PDO culture medium was introduced.

[0059] In the subsequent assay, H1975 cells were divided into two parts. One part was used for ATP nanosensor + lysosome staining observation, and the other part was used for iodoacetic acid (IAA) + ATP nanosensor + lysosome staining observation. The ATP nanosensor was diluted to a concentration of 10nM and incubated with the cells at 37°C for 2 hours. IAA (100μM) was first incubated with cells at 37°C for 4 hours to inhibit ATP production. Lysosomal stain (50nM) was incubated at 37°C for 30 minutes to mark the location of lysosomes.

[0060] To characterize the successful establishment of PDO, the cultured PDO were equally divided into 2 parts. One part was used for ATP nanosensor + anti-cytokeratin 7 + 4'6-diamidino-2-phenylindole (DAPI) assay, while the other part was used for ATP nanosensor + lysosome staining + IAA assay. PDO were treated with 10% goat serum albumin to block nonspecific binding and then incubated with anti-cytokeratin 7 for 4 h. After that, PDO were incubated with Alexa Fluor® 488-conjugated secondary antibody for 3 h. ATP nanosensor was diluted to a concentration of 10 nM and incubated with cells at 37°C for 2 h. IAA (100 μM) was first incubated with cells at 37°C for 4 h to inhibit ATP production. Lysosomal stain (50 nM) was used to mark the lysosomal location by incubating cells at 37°C for 30 min. DAPI was used to stain the nucleus for 1 h.

[0061] All the images mentioned above were acquired using a confocal microscope and analyzed using relevant image analysis software to determine the ability of the ATP nanosensor to detect cells.

[0062] The microfluidic chip of the embodiment of the present disclosure can perform the entire PDO-based drug reaction test process in a single microfluidic chip, and through the injected ATP nanosensor, it can identify intracellular ATP without damaging the cells, and can realize the full-process monitoring requirements of ATP changes in PDO.

[0063] For example, Figure 1 , Figure 3 and Figure 4 As shown, the first flow channel A1 is arranged on the side of the injection layer A facing the organoid culture layer C, that is, Figure 3 As shown, the first flow channel A1 is arranged on the lower surface of the injection layer A. Accordingly, the first inlet A2 and the first outlet A3 on the injection layer A should penetrate through its thickness, that is, the first inlet A2 and the first outlet A3 are both via structures. It is not difficult to understand that since the inlet A4 on the injection layer A needs to lead the fluid downward, the inlet A4 should also penetrate through its thickness, that is, the inlet A4 is also a via structure.

[0064] It should be noted that the specific shapes and sizes of the first flow channel A1, the first inlet A2, the first outlet A3 and the introduction port A4 are not limited, and those skilled in the art can determine them according to actual needs. In some preferred embodiments, the cross-sections of the first inlet A2, the first outlet A3 and the introduction port A4 are all circular to simplify the preparation process of the microfluidic chip.

[0065] It should be further explained that there is no limitation on the number of the first flow channel A1, the first inlet A2, the first outlet A3 and the introduction port A4. It is not difficult to understand that when using the microfluidic chip of this embodiment to conduct tumor drug trials, a plurality of organoid culture chambers C4 need to be set on the organoid culture layer C. Therefore, the number of the first flow channel A1, the first inlet A2, the first outlet A3 and the introduction port A4 can be determined according to the number of organoid culture chambers C4 on the organoid culture layer C.

[0066] For example, Figure 1 , Figure 3 and Figure 5 As shown, a plurality of injection holes B1 and connection ports B2 are provided on the porous membrane layer B. It is not difficult to understand that the injection holes B1 and the connection ports B2 are required to inject fluid (tumor cells and related tumor drugs, etc.) into the lower organoid culture layer C, so the injection holes B1 and the connection ports B2 are required to penetrate the thickness thereof, that is, the injection holes B1 and the connection ports B2 are both through-hole structures.

[0067] For example, Figure 5As shown, in order to increase the speed of injecting fluid into the organoid culture layer C, a plurality of injection holes B1 can be set at the position corresponding to each organoid culture chamber C4, and the plurality of injection holes B1 can be arranged in an array, so that the relevant fluid can be injected into the corresponding organoid culture chamber C4 together with the help of the injection holes B1 arranged in the array.

[0068] It should be noted that there is no limitation on the specific shape and size of the injection hole B1 and the connection port B2, and those skilled in the art can determine it according to actual needs. In some preferred embodiments, the cross-sections of the injection hole B1 and the connection port B2 are both circular to simplify the preparation process of the microfluidic chip.

[0069] It should be further explained that there is no limitation on the number of injection holes B1 and connection ports B2. It is not difficult to understand that when using the microfluidic chip of this embodiment to conduct tumor drug trials, a plurality of organoid culture chambers C4 need to be set on the organoid culture layer C. Therefore, the number of injection holes B1 and connection ports B2 can be determined according to the number of organoid culture chambers C4 on the organoid culture layer C.

[0070] For example, Figure 1 , Figure 3 and Figure 6 As shown, the second flow channel C1 and the organoid culture chamber C4 are arranged on the side of the organoid culture layer C facing the injection layer A. That is, Figure 3 As shown, the second flow channel C1 and the organoid culture chamber C4 are arranged on the upper surface of the organoid culture layer C. Accordingly, the second outlet C3 on the organoid culture layer C should penetrate its thickness, that is, the second outlet C3 is a through-hole structure, so as to lead the fluid in the organoid culture chamber C4 to the outside of the microfluidic chip through the second outlet C3.

[0071] It should be noted that the specific shapes and sizes of the second flow channel C1, the organoid culture chamber C4, the second inlet C2 and the second outlet C3 are not limited, and those skilled in the art can determine them according to actual needs. In some preferred embodiments, the cross-sections of the organoid culture chamber C4, the second inlet C2 and the second outlet C3 are all circular to simplify the preparation process of the microfluidic chip.

[0072] It should be further explained that there is no limitation on the number of the second flow channel C1, the organoid culture chamber C4, the second inlet C2 and the second outlet C3. It is not difficult to understand that when using the microfluidic chip of this embodiment to conduct tumor drug trials, a plurality of organoid culture chambers C4 need to be set on the organoid culture layer C. Therefore, the number of the second flow channel C1, the second inlet C2 and the second outlet C3 can be determined according to the number of the organoid culture chambers C4.

[0073] For example, Figure 3 and Figure 4 As shown, the first flow channel A1 on the injection layer A extends outward at the position corresponding to the organoid culture chamber C4 on the organoid culture layer C to match the organoid culture chamber C4. The so-called matching means that the shape and size of the first flow channel A1 at the position corresponding to the organoid culture chamber C4 are basically consistent with the shape and size of the organoid culture chamber C4, that is, Figure 4 As shown, the orthographic projection of the first flow channel A1 at this position on the organoid culture layer C substantially coincides with the organoid culture chamber C4. For example, when the cross section of the organoid culture chamber C4 is circular, the cross section at the corresponding position of the first flow channel A1 is also circular. Of course, in some embodiments, the orthographic projection of the first flow channel A1 at this position on the organoid culture layer C may also fall on the outside or inside of the corresponding organoid culture chamber C4.

[0074] In the microfluidic chip of this embodiment, the position of the first flow channel on the injection layer corresponding to the organoid culture chamber on the organoid culture layer is set to match the organoid culture chamber C4. On the one hand, the structure of the microfluidic chip can be made more compact and beautiful; on the other hand, it can be connected with more injection holes on the porous membrane layer, thereby increasing the injection speed of tumor drugs.

[0075] For example, Figure 4 As shown, the first inlet A2 and the first outlet A3 on the injection layer A are symmetrically distributed on both sides of the injection layer A along its width direction. That is, as shown in FIG. Figure 4 As shown, the first inlet A2 is arranged at the upper end of the injection layer A, and the first outlet A3 is arranged at the lower end of the injection layer A, or the first inlet A2 is arranged at the lower end of the injection layer A, and the first outlet A3 is arranged at the upper end of the injection layer A. Such an arrangement can make the overall structure of the microfluidic chip more compact, thereby simplifying the preparation process of the microfluidic chip.

[0076] For example, Figure 3 and Figure 4 As shown, the injection layer A is further provided with a cascaded proportioning mixer A5. The inlet of the proportioning mixer A5 is communicated with the first inlet A2, and the outlet of the proportioning mixer A5 is communicated with the first flow channel A1.

[0077] The microfluidic chip of this embodiment, through the cascade ratio mixer set on the injection layer, can provide tumor drugs of different concentrations (molar or mass) ratios to the organoid culture chambers on the organoid culture layer through the ratio mixer, thereby enriching the detection function of the microfluidic chip and greatly improving the efficiency of tumor drug verification using the microfluidic chip.

[0078] For example, Figure 7 As shown, Figure 7 for Figure 4 The structural diagram of the ratio mixer in the injection layer is shown in FIG. The cascade ratio mixer A5 comprises: a multi-stage mixing main channel A51 and a plurality of mixing branch channels A52. The plurality of mixing main channels A51 are arranged side by side in the injection layer A, that is, Figure 7 As shown, the plurality of main mixing channels A51 are arranged at intervals along the width direction of the injection layer A. It should be understood that the plurality of main mixing channels A51 can be arranged at equal intervals or at unequal intervals on the injection layer A, and this embodiment does not limit this. The plurality of branch mixing channels A52 are arranged in parallel on the injection layer A, that is, Figure 7 As shown, the multiple mixing branch channels A52 are arranged at intervals along the length direction of the injection layer A. It should be understood that the multiple mixing branch channels A52 can be arranged at equal intervals or unequal intervals on the injection layer A, and this embodiment does not limit this. A plurality of the mixing branch channels A52 are arranged side by side between two adjacent mixing main channels A51. The mixing main channel A51 is connected to the first inlet A2, and the mixing branch channels A52 are connected to the corresponding mixing main channel A51 and the first channel A1.

[0079] Specifically, Figure 7 As shown, the cascade proportioning mixer A5 includes two-stage mixing main channels A51 and seven mixing branch channels A52. Three mixing branch channels A52 are arranged side by side between the two-stage mixing main channels A51, and the three mixing branch channels A52 connect the two-stage mixing main channels A51. Four mixing branch channels A52 are arranged side by side below the bottom mixing main channel A51, and the four mixing branch channels A52 connect the mixing main channel A51 with the inlet of the first channel A1.

[0080] It should be noted that there is no specific limit on the number of cascaded proportional mixers, and those skilled in the art can design a cascade number of three or more stages according to actual needs, and this embodiment does not limit this.

[0081] For example, Figure 4 and Figure 7 As shown, in order to improve the effect of tumor drug mixing, the length of the mixing branch channel A52 is greater than its projected length on the injection layer A, that is, the mixing branch channel A52 is not arranged in a straight line, but is arranged in a curved manner between adjacent mixing main channels A51. In this way, the mixing path length of the tumor drug can be effectively increased, and the tumor drug can be fully mixed. As an example, Figure 7As shown, the branch mixing channel A52 is S-shaped and arranged in the injection layer A. Of course, in addition to this, those skilled in the art can also design other shapes of branch mixing channels 52 according to actual needs, such as a folded line structure, etc., and this embodiment is not limited to this.

[0082] For example, Figure 7 As shown, in order to facilitate the control of the ratio of tumor drug injection, the main mixing channel A51 is provided with switch valves A53 on both sides of the inlet position corresponding to the mixing branch channel A52. In this way, by controlling each switch valve A53, the conduction and shutoff of different mixing branch channels A52 and the first channel A1 can be realized, so that the required tumor drug mixing ratio can be achieved.

[0083] For example, in order to realize the control of each switch valve A53 on the mixed main flow channel A51, as shown in FIG. Figure 4 and Figure 6 As shown, the organoid culture layer C is also provided with a gas inlet C5 and a gas channel C6, wherein the gas channel C6 has an open end and a closed end C61, the open end of the gas channel C6 is connected to the gas inlet C5, and the gas channel C6 corresponds to the position of the cascade proportion mixer A5 to control the on and off of the proportion mixer A5.

[0084] Specifically, Figure 4 and Figure 6 As shown, the gas channel C6 is arranged on the side of the organoid culture layer C facing the injection layer A, that is, as shown in FIG. Figure 4 As shown, the gas channel C6 is arranged on the upper surface of the organoid culture layer C. Accordingly, the gas inlet C5 arranged on the organoid culture layer C should penetrate the thickness thereof, that is, the gas inlet C5 is a through-hole structure.

[0085] It should be noted that there is no limitation on the specific shape and size of the gas inlet C5 and the gas channel C6, and those skilled in the art can determine them according to actual needs. In some preferred embodiments, the cross-section of the gas inlet C5 is circular to simplify the preparation process of the microfluidic chip.

[0086] It should be further explained that there is no limitation on the number of gas inlets C5 and gas channels C6. It is easy to understand that the number of the two depends on the structure of the cascade proportioning mixer A5 so as to better control the on and off of the proportioning mixer.

[0087] For example, Figure 6 and Figure 7As shown, the closed end C61 of the gas channel C6 corresponds to the position of the switch valve A53. In this way, when the gas inlet C5 is used to inflate the gas channel C6, the closed end C61 of the gas channel C6 will expand, and the expanded closed end C61 will squeeze the position of the switch valve A53, so that the fluid flow path at this location can be shut off. Conversely, when conduction is required, the gas channel C6 is stopped from being inflated, and the gas in the gas channel C6 will be discharged through the gas inlet C5, so that the closed end C61 of the gas channel C6 can be restored to the initial state, and then the switch valve A53 is restored to the initial position, so that the fluid flow path at this location is conducted.

[0088] Continue to refer Figure 6 and Figure 7 In order to improve the effect of controlling the on-off of the switch valve, in some embodiments, the positive projection of the closed end C61 on the switch valve A53 falls on the outside of the switch valve A53.

[0089] It should be noted that the switch valve A53 mentioned above is not a physical valve in the strict sense when it is turned on and off by means of gas. Figure 7 As shown, the switch valve A53 can be a switch structure extending outward from the main mixing channel A51. When gas is introduced into the gas channel C6, the closed end C61 of the gas channel C6 completely covers the switch valve A53, so that the channel where the switch valve A53 is located can be completely closed by means of the expanded closed end C61.

[0090] For example, Figure 6 As shown, the gas inlet C5 is symmetrically arranged along the length direction of the organoid culture layer C, that is, Figure 6 As shown, the gas inlets C5 are symmetrically distributed along the left-right direction of the organoid culture layer C, and a plurality of gas inlets C5 are arranged on each side of the organoid culture layer C.

[0091] For example, Figure 3 and Figure 6 As shown, the organoid culture layer C is provided with a plurality of groups of organoid culture chambers C4 arranged side by side. Each group includes a plurality of organoid culture chambers C4 connected in series.

[0092] Specifically, Figure 6 As shown, six groups of organoid culture chambers C4 are arranged on the organoid culture layer C, of ​​which the three groups on the left and the three groups on the right are mirror images. Correspondingly, the remaining structures are also mirror images, such as the gas channel C6, the injection hole B1 and the connection port B2 arranged on the porous membrane layer B, the first flow channel A1, the first inlet A2, the first outlet A3, the introduction port A4 and the proportioning mixer A5 arranged on the injection layer A are all symmetrical structures. Figure 6As shown, each group includes six organoid culture chambers C4 connected in series, that is, the six organoid culture chambers C4 share a second inlet C2 and a second outlet C3.

[0093] For example, Figure 3 and Figure 4 As shown, the inlet A4 is arranged close to the first outlet A2, and the inlet A4 is located inside the first outlet A2. Figure 4 As shown, the introduction port A4 is located at the bottom of the injection layer A and above the first outlet A2.

[0094] Based on the same inventive concept, an embodiment of the present disclosure also provides a method for preparing a microfluidic chip. The microfluidic chip is the microfluidic chip described above. The specific structure of the microfluidic chip can be referred to the above description and will not be repeated here. Fig.14 is a flow chart of a method for preparing a microfluidic chip according to another embodiment of the present disclosure, Fig.15 FIG. 1 is a process flow chart of a method for preparing a microfluidic chip according to another embodiment of the present disclosure. Fig.14 As shown, the preparation method includes the following steps S141 to S145.

[0095] Step S141, providing a first substrate, a second substrate and a third substrate.

[0096] Specifically, in this step, refer to Fig.15 , respectively provide a first substrate 151, a second substrate 152 and a third substrate 153. The first substrate 151, the second substrate 152 and the third substrate 153 can all be made of silicon wafer or silicon glass, etc., which is not limited in this embodiment.

[0097] Step S142, patterning the first substrate to form a first protrusion on the first substrate, forming a first flexible material layer on the patterned first substrate, removing the first substrate after heating and curing, and drilling holes at the inlet and outlet of the first flexible material layer to prepare an injection layer.

[0098] Specifically, in this step, refer to Fig.15 , patterning the first substrate 151 to form a first protrusion 151a on the first substrate 151, forming a first flexible material layer 151b on the patterned first substrate 151, removing the first substrate 151 after heating and curing, and punching holes at the inlet and outlet of the first flexible material layer 151b to prepare an injection layer.

[0099] In some embodiments, the step of patterning the first substrate to form a first protrusion on the first substrate specifically includes: Fig.15As shown, a first photoresist layer 151c is formed on the first substrate 151, and the first photoresist layer 151c is exposed and developed to obtain a patterned first photoresist layer, wherein the first photoresist layer that has not been developed is the first protrusion 151a. Afterwards, a first flexible material layer 151b is formed on the patterned first photoresist layer, and the first substrate 151 is removed after heating and curing, and holes are punched at the inlet and outlet of the first flexible material layer 151b to prepare an injection layer. It is not difficult to understand that the place corresponding to the first protrusion 151a is the flow channel structure on the injection layer, and the punched place corresponds to the inlet and outlet structures on the injection layer.

[0100] In some embodiments, in this step, the first substrate 151 formed with the first flexible material layer 151b is placed in a baking device for heating and curing, and the temperature range of heating and curing can be 80°C to 90°C, preferably, the temperature of heating and curing is 85°C. The time range of heating and curing is 1.5h to 2.5h, preferably, the time of heating and curing is 2h, so that the first flexible material layer 151b is cross-linked and cured.

[0101] In some embodiments, in this step, the first flexible material layer 151b is made of a mixture of polydimethylsiloxane and a curing agent. Preferably, the mass ratio of the polydimethylsiloxane to the curing agent is in the range of (8-10):1, and more preferably, the mass ratio of the polydimethylsiloxane to the curing agent is 10:1.

[0102] Step S143, forming a second flexible material layer on the second substrate, patterning the second flexible material layer to form a first opening and a second opening, and removing the second substrate after heating and curing to prepare a porous membrane layer.

[0103] Specifically, in this step, refer to Fig.15 , forming a second flexible material layer 152a on the second substrate 152, patterning the second flexible material layer 152a to form a first opening 152b and a second opening 152c, and removing the second substrate 152 after heating and curing to obtain a porous membrane layer.

[0104] In some embodiments, in this step, Fig.15As shown, a second flexible material layer 152a is formed on a second substrate 152 by spin coating or the like. Afterwards, a second photoresist layer 152d is formed on the second flexible material layer 152a, and the second photoresist layer 152d is exposed and developed to form a first opening and a second opening, and the second flexible material layer 152a is etched with the patterned second photoresist layer 152d as a mask to form the first opening 152b and the second opening 152c. It is not difficult to understand that the first opening corresponds to the first opening 152b, the second opening corresponds to the second opening 152c, and the first opening 152b corresponds to the injection hole on the porous membrane layer, and correspondingly, the second opening corresponds to the connection port on the porous membrane layer.

[0105] In some embodiments, in this step, the second substrate 152 formed with the second flexible material layer 152a is placed in a baking device for heating and curing, and the temperature range of heating and curing can be 80°C to 90°C, preferably, the temperature of heating and curing is 85°C. The time range of heating and curing is 1.5h to 2.5h, preferably, the time of heating and curing is 2h, so that the second flexible material layer 152a is cross-linked and cured.

[0106] In some embodiments, in this step, the second flexible material layer 152a is made of a mixture of polydimethylsiloxane and a curing agent. Preferably, the mass ratio of the polydimethylsiloxane to the curing agent is in the range of (8-10):1, and more preferably, the mass ratio of the polydimethylsiloxane to the curing agent is 10:1.

[0107] Step S144, patterning the third substrate to form a second protrusion on the third substrate, forming a third flexible material layer on the patterned third substrate, removing the third substrate after heating and curing, and punching holes at the inlet and outlet of the third flexible material layer to prepare an organoid culture layer.

[0108] Specifically, in this step, refer to Fig.15 , patterning the third substrate 153 to form a second protrusion 153a on the third substrate 153, forming a third flexible material layer 153b on the patterned third substrate 153, removing the third substrate 153 after heating and curing, and punching holes at the inlet and outlet of the third flexible material layer 153b to prepare an organoid culture layer.

[0109] In some embodiments, the step of patterning the third substrate to form a second protrusion on the third substrate specifically includes: Fig.15As shown, a third photoresist layer 153c is formed on the third substrate 153, and the third photoresist layer 153c is exposed and developed to obtain a patterned third photoresist layer, wherein the third photoresist layer that has not been developed is the second protrusion 153a. Afterwards, a third flexible material layer 153b is formed on the patterned first photoresist layer, and the third substrate 153 is removed after heating and curing, and holes are punched at the inlet and outlet of the third flexible material layer 153b to prepare an injection layer. It is not difficult to understand that the place corresponding to the second protrusion 153a is the flow channel structure on the organoid culture layer, and the punched place corresponds to the inlet and outlet structures on the organoid culture layer.

[0110] In some embodiments, in this step, the third substrate 153 formed with the third flexible material layer 153b is placed in a baking device for heating and curing, and the temperature range of heating and curing can be 80°C to 90°C, preferably, the temperature of heating and curing is 85°C. The time range of heating and curing is 1.5h to 2.5h, preferably, the time of heating and curing is 2h, so that the third flexible material layer 153b is cross-linked and cured.

[0111] In some embodiments, in this step, the third flexible material layer 153b is made of a mixture of polydimethylsiloxane and a curing agent. Preferably, the mass ratio of the polydimethylsiloxane to the curing agent is in the range of (8-10):1, and more preferably, the mass ratio of the polydimethylsiloxane to the curing agent is 10:1.

[0112] Step S145, bonding and packaging the injection layer, the porous membrane layer and the organoid culture layer in sequence to prepare the microfluidic chip.

[0113] Specifically, in this step, the injection layer, porous membrane layer and organoid culture layer prepared in the above steps S141 to S145 are ultrasonically cleaned with distilled water, alcohol or isopropanol, and then the surface is treated with plasma. The organoid culture layer is first bonded and packaged with the porous membrane layer, and then the injection layer and the porous membrane layer are bonded and packaged. After the microfluidic chip is placed at high temperature and is completely sealed, a complete microfluidic chip is finally formed.

[0114] The preparation method of the microfluidic chip of the embodiment of the present disclosure has a simple preparation process. The prepared microfluidic chip can perform the entire PDO-based drug reaction test process in a single microfluidic chip, and through the injected ATP nanosensor, intracellular ATP can be identified without damaging the cells, and the full-process monitoring requirements of ATP changes in PDO can be achieved.

[0115] Based on the same inventive concept, an embodiment of the present disclosure also provides a detection method based on a microfluidic chip, wherein the microfluidic chip is the microfluidic chip described above. The specific structure of the microfluidic chip can be referred to the above description and will not be repeated here. Fig.16 FIG. 1 is a flow chart of a detection method based on a microfluidic chip according to another embodiment of the present disclosure. Fig.16 As shown, the method includes the following steps S161 to S163.

[0116] Step S161, introducing tumor cells and extracellular matrix gel for in vitro organoid culture into the organoid culture chamber through the introduction port of the injection layer, the connection port on the porous membrane layer, the second inlet and the second flow channel on the organoid culture layer.

[0117] Specifically, in this step, refer to Figure 2 , Figures 4 to 6 The tumor cells and extracellular matrix gel used for in vitro organoid culture are introduced into the organoid culture chamber C4 through the introduction port A4 of the injection layer A, the connection port B2 on the porous membrane layer B, the second inlet C2 on the organoid culture layer C, and the second flow channel C1.

[0118] Step S162, introducing the tumor drug and the ATP nanosensor into the organoid culture chamber on the organoid culture layer through the first inlet and the first flow channel of the injection layer and the injection hole on the porous membrane layer.

[0119] Specifically, in this step, refer to Figure 2 , Figures 4 to 6 The tumor drug and the ATP nanosensor are introduced into the organoid culture chamber C4 on the organoid culture layer C through the first inlet A2 and the first flow channel A1 of the injection layer A and the injection hole B1 on the porous membrane layer B.

[0120] Step S163, obtaining the fluorescence intensity change value represented by the ATP nanosensor in the organoid culture chamber, and determining the effectiveness of the tumor drug according to the detected fluorescence intensity change value.

[0121] Specifically, in this step, refer to Figure 6 and Fig.17 , Fig.17 h shows a cross-sectional view of the organoid culture. Tumor drugs and ATP nanosensors diffuse from the injection layer A to the organoid culture chamber C4 through the porous membrane. When the ATP nanosensor enters the cell interior of the organoid, the ATP-mediated molecular replacement process is activated, emitting a strong red fluorescence signal, as shown in Figure 4. Fig.17As shown in Figure g, the organoid incubated with the ATP nanosensor shows red fluorescence. Therefore, by obtaining the fluorescence intensity change value of the organoid culture chamber C4, the effectiveness of the tumor drug can be determined according to the numerical change. In some embodiments, a microscope can be used to obtain a fluorescent image characterized by the ATP nanosensor in the microfluidic chip, and an ELISA instrument can be used to obtain the fluorescence intensity change value of the organoid incubated with the ATP nanosensor. After obtaining the numerical change, the operator can obtain the inhibitory effect of the current tumor drug on tumor cells, so that it can be convenient for subsequent treatment of different tumors with the tumor drug with the best inhibitory effect.

[0122] In some embodiments, the tumor drug is a tyrosine kinase inhibitor (TKIs); The step of introducing the tumor drug and the ATP nanosensor into the organoid culture chamber on the organoid culture layer through the first inlet and the first flow channel of the injection layer and the injection hole on the porous membrane layer comprises: injecting the ATP nanosensor and different TKIs into different organoid culture chambers on the organoid culture layer, respectively. The step of obtaining the fluorescence intensity change value detected by the ATP nanosensor in the organoid culture chamber and determining the effectiveness of the tumor drug according to the fluorescence intensity change value comprises: obtaining the fluorescence intensity change value detected by the ATP nanosensor in different organoid culture chambers and determining the effectiveness of the different TKIs according to the fluorescence intensity change value.

[0123] In some embodiments, the different TKIs include: osimertinib and erlotinib.

[0124] In some embodiments, the tumor drug is a chemotherapy drug in combination of different concentrations; The step of introducing the tumor drug and the ATP nanosensor into the organoid culture chamber on the organoid culture layer through the first inlet and the first flow channel of the injection layer and the injection hole on the porous membrane layer comprises: injecting the ATP nanosensor and chemotherapy drugs of different concentrations into different organoid culture chambers on the organoid culture layer respectively. The step of obtaining the fluorescence intensity change value of the ATP nanosensor in the organoid culture chamber and determining the effectiveness of the tumor drug according to the fluorescence intensity change value comprises: obtaining the fluorescence intensity change value of the ATP nanosensor in different organoid culture chambers and determining the effectiveness of the chemotherapy drugs of different concentrations according to the fluorescence intensity change value.

[0125] In some embodiments, the chemotherapy drugs in different concentration combinations include: pemetrexed and carboplatin in different concentration combinations.

[0126] In some embodiments, the tumor drugs are TKIs and chemotherapy drugs in different combinations; The step of introducing the tumor drug and the ATP nanosensor into the organoid culture chamber on the organoid culture layer through the first inlet and the first flow channel of the injection layer and the injection hole on the porous membrane layer comprises: injecting the ATP nanosensor and TKI and chemotherapy drugs of different concentrations into different organoid culture chambers on the organoid culture layer respectively. The step of obtaining the fluorescence intensity change value of the ATP nanosensor in the organoid culture chamber and determining the effectiveness of the tumor drug according to the fluorescence intensity change value comprises: obtaining the fluorescence intensity change value of the ATP nanosensor in different organoid culture chambers and determining the effectiveness of TKI and chemotherapy drugs of different concentrations according to the fluorescence intensity change value.

[0127] In some embodiments, the method further comprises: At least one organoid culture chamber on the organoid culture layer is controlled not to have the tumor drug added therein, serving as a control group.

[0128] In order to explain the detection process based on the above-mentioned microfluidic chip in more detail, several specific embodiments will be described below.

[0129] Example 1 Clinical samples from two lung cancer patients were cultured in a microfluidic chip for 10 days, and the cellular components of PDO were stained, e.g. Fig.18 As shown in a. The green fluorescence staining of cytokeratin 7 indicates the establishment of PDO, while the red fluorescence staining of ATP nanosensor indicates the activation of ATP nanosensor after recognizing ATP molecules in PDO. No fluorescence was observed in the group pre-incubated with IAA. Fig.19 As shown, it shows that the ATP nanosensor is specific for detecting intracellular ATP in PDO. To demonstrate the capabilities of the microfluidic chip, the drug injection channel of the microfluidic chip was set up for testing with different TKIs. The time point when PDO was successfully established was designated as day 0, and the drug was injected on day 0. Starting from day 0, the ATP nanosensor was injected every 3 days to measure intracellular ATP. Different kinds of TKIs were drug tested in 18 organoid culture chambers, and PDOs derived from two patients were tested. When testing TKIs, 6 organoid culture chambers were filled with osimertinib (20 nM), 6 organoid culture chambers were filled with erlotinib (30 nM), and the remaining 6 organoid culture chambers were not filled with drugs (as a control group).

[0130] FIG18b shows the drug response of PDO of patient 1. Fig.18As shown in (b), both osimertinib (20 nM) and erlotinib (30 nM) effectively inhibited PDO growth from day 3 to day 15. However, osimertinib showed a more significant inhibitory effect on day 15. The results showed that osimertinib had a better effect than erlotinib for patient 1.

[0131] Fig.18 d shows the drug response of the PDO of patient 2, who was treated with different types of TKIs using the same treatment strategy as patient 1. Fig.18 As shown in d, osimertinib (20 nM) and erlotinib (30 nM) showed comparable PDO inhibitory effects over 15 days.

[0132] Example 2 Cancer cells derived from clinical samples of two lung cancer patients were cultured in a microfluidic chip for 10 days, and the cellular components of PDO were stained, such as Fig.18 As shown in a. The green fluorescence staining of cytokeratin 7 indicates the establishment of PDO, while the red fluorescence staining of ATP nanosensor indicates the activation of ATP nanosensor that recognizes ATP molecules in PDO. No fluorescence was observed in the group pre-incubated with IAA, as shown in Fig.19 As shown, it was shown that the ATP nanosensor was specific for detecting intracellular ATP in PDO. To demonstrate the capabilities of the microfluidic chip, the drug injection channel of the microfluidic chip was set up to test different concentration combinations of chemotherapeutic drugs. The time point when PDO was successfully established was designated as day 0, and the drug was injected on day 0. Starting from day 0, the ATP nanosensor was injected every 3 days to measure intracellular ATP. Chemotherapeutic drugs were tested in 18 organoid culture chambers, and PDOs derived from the above two patients were tested. When testing chemotherapeutic drugs, 12 organoid culture chambers were filled with pemetrexed and carboplatin at different concentration ratios (P:C=0:3, 1:3, 3:1 and 3:0), and 6 organoid culture chambers without drugs were used as controls.

[0133] FIG18c shows the drug response of PDO of patient 1. Fig.18 As shown in (c), all four drug combinations showed comparable PDO inhibition effects over 15 days, indicating that it is not possible to select the best chemotherapy drug combination for patients.

[0134] Fig.18 e shows the drug response of the PDO of patient 2, who was treated with chemotherapy drugs using the same treatment strategy as patient 1. Fig.18As shown in Figure 5e, the drug combination with a P:C = 3:1 ratio was superior to the other 3 combinations. These findings suggest that patient 2 showed higher sensitivity to chemotherapy drugs and that the drug combination (P:C = 3:1) represented the best treatment option.

[0135] In summary, the microfluidic chip-based assay generated a comprehensive curve describing the overall drug response of PDO. Compared with commercial ATP kits that provide single time point detection, the O-ATP system can more accurately assess drug response. Fig.18 As shown in b, taking patient 1 as an example, before day 6, erlotinib showed better inhibitory effect than osimertinib, and after day 6, osimertinib showed stronger inhibitory effect than erlotinib ( Fig.18 b). The comparative results observed at different time points indicate that full-course monitoring can provide more comprehensive information for treatment planning and avoid possible misdiagnosis. The number of organoid culture chambers and the design of the proportional mixer on the microfluidic chip can be easily adjusted to meet the clinical requirements of different tumor types.

[0136] It is to be understood that the above embodiments are merely exemplary embodiments used to illustrate the principles of the present disclosure, but the present disclosure is not limited thereto. For those of ordinary skill in the art, various modifications and improvements can be made without departing from the spirit and substance of the present disclosure, and these modifications and improvements are also considered to be within the scope of protection of the present disclosure.

Claims

1. A microfluidic chip, characterized in that: The microfluidic chip comprises: an injection layer, a porous membrane layer and an organoid culture layer which are stacked in sequence; The injection layer is provided with a first flow channel and a first inlet and a first outlet respectively connected with the first flow channel, and the injection layer is also provided with an introduction port; The porous membrane layer is provided with an injection hole and a connection port, and the connection port corresponds to the position of the introduction port and is connected; The organoid culture layer is provided with a second flow channel and a second inlet, a second outlet and an organoid culture chamber respectively connected to the second flow channel, the organoid culture chamber corresponds to and is connected to the injection hole, and the second inlet corresponds to and is connected to the connection port; wherein, The first inlet is used to provide tumor drugs and ATP nanosensors to the organoid culture chamber through the first flow channel and the injection hole; The introduction port is used to provide tumor cells for in vitro culturing of organoids to the organoid culture chamber through the connection port, the second inlet and the second flow channel.

2. The microfluidic chip according to claim 1, characterized in that: The first flow channel is arranged on a side of the injection layer facing the organoid culture layer; and / or the second flow channel and the organoid culture chamber are arranged on a side of the organoid culture layer facing the injection layer.

3. The microfluidic chip according to claim 1, characterized in that: The first flow channel extends outward at a position corresponding to the organoid culture chamber to match the organoid culture chamber.

4. The microfluidic chip according to claim 1, characterized in that: The first inlet and the first outlet are symmetrically distributed on both sides of the injection layer along the width direction thereof.

5. The microfluidic chip according to claim 1, characterized in that: The injection layer is also provided with a cascade proportioning mixer; The inlet of the proportioning mixer is communicated with the first inlet, and the outlet of the proportioning mixer is communicated with the first flow channel.

6. The microfluidic chip according to claim 5, characterized in that: The cascade proportional mixer comprises: a multi-stage mixing main flow channel and a plurality of mixing branch flow channels; The plurality of main mixing channels are arranged side by side in the injection layer, and the plurality of branch mixing channels are arranged side by side in the injection layer; and a plurality of branch mixing channels are arranged side by side between two adjacent main mixing channels; The main mixing channel is in communication with the first inlet, and the branch mixing channels are in communication with the corresponding main mixing channel and the first channel.

7. The microfluidic chip according to claim 6, characterized in that: The length of the mixing branch channel is greater than its projection length on the injection layer.

8. The microfluidic chip according to claim 7, characterized in that: The mixing branch channel is arranged in the injection layer in an S shape.

9. The microfluidic chip according to claim 6, characterized in that: The main mixing channel is provided with switch valves on both sides of the inlet positions corresponding to the branch mixing channels.

10. The microfluidic chip according to any one of claims 5 to 9, characterized in that: The organoid culture layer is further provided with a gas inlet and a gas channel, wherein the gas channel has an open end and a closed end, and the open end of the gas channel is connected to the gas inlet; and The gas channel corresponds to the position of the cascade proportion mixer to control the on and off of the proportion mixer.

11. The microfluidic chip according to claim 10, characterized in that: When the cascaded proportioning mixer comprises a switching valve; The closed end of the gas channel corresponds to the position of the switch valve.

12. The microfluidic chip according to claim 11, characterized in that: The orthographic projection of the closed end on the corresponding switch valve falls outside the switch valve.

13. The microfluidic chip according to claim 10, characterized in that: The gas inlets are symmetrically arranged along the length direction of the organoid culture layer.

14. The microfluidic chip according to any one of claims 1 to 9, characterized in that: The organoid culture layer is provided with a plurality of groups of organoid culture chambers arranged side by side; and, Each group includes a plurality of the organoid culture chambers connected in series.

15. The microfluidic chip according to any one of claims 1 to 9, characterized in that: The introduction port is arranged close to the first outlet, and the introduction port is located at the inner side of the first outlet.

16. A method for preparing a microfluidic chip, characterized in that: The microfluidic chip is the microfluidic chip according to any one of claims 1 to 15, and the method comprises the following steps: providing a first substrate, a second substrate, and a third substrate; Patterning the first substrate to form a first protrusion on the first substrate, forming a first flexible material layer on the patterned first substrate, removing the first substrate after heating and curing, and drilling holes at the inlet and outlet of the first flexible material layer to prepare an injection layer; forming a second flexible material layer on the second substrate, patterning the second flexible material layer to form a first opening and a second opening, and removing the second substrate after heating and curing to prepare a porous membrane layer; Patterning the third substrate to form a second protrusion on the third substrate, forming a third flexible material layer on the patterned third substrate, removing the third substrate after heating and curing, and punching holes at the inlet and outlet of the third flexible material layer to prepare an organoid culture layer; The injection layer, the porous membrane layer and the organoid culture layer are bonded and packaged in sequence to prepare the microfluidic chip.

17. The preparation method according to claim 16, characterized in that: The temperature range of the heating curing is 80° C. to 90° C.; and / or the time range of the heating curing is 1.5 h to 2.5 h.

18. The preparation method according to claim 16, characterized in that: The injection layer, the porous membrane layer and the organoid culture layer are bonded and packaged in sequence to prepare the microfluidic chip, comprising: The injection layer, porous membrane layer and organoid culture layer are ultrasonically cleaned with distilled water, alcohol or isopropanol, and then the surface is treated with plasma. The organoid culture layer is first bonded and packaged with the porous membrane layer, and then the injection layer and the porous membrane layer are bonded and packaged. After the microfluidic chip is placed at high temperature and completely sealed, the complete microfluidic chip is finally formed.

19. The preparation method according to any one of claims 16 to 18, characterized in that: The first flexible material layer, the second flexible material layer and the third flexible material layer are made of a mixture of polydimethylsiloxane and a curing agent.

20. The preparation method according to claim 19, characterized in that: The mass ratio of the polydimethylsiloxane to the curing agent is in the range of (8-10):

1.

21. A detection method based on a microfluidic chip, characterized in that: The microfluidic chip adopts the microfluidic chip according to any one of claims 1 to 15, and the detection method comprises the following steps: Introducing tumor cells and extracellular matrix gel for in vitro organoid culture into the organoid culture chamber through the introduction port of the injection layer, the connection port on the porous membrane layer, the second inlet port and the second flow channel on the organoid culture layer; Introducing the tumor drug and the ATP nanosensor into the organoid culture chamber on the organoid culture layer through the first inlet and the first flow channel of the injection layer and the injection hole on the porous membrane layer; The fluorescence intensity change value of the ATP nanosensor in the organoid culture chamber is obtained, and the effectiveness of the tumor drug is determined according to the fluorescence intensity change value.

22. The detection method according to claim 21, characterized in that: The tumor drug is a tyrosine kinase inhibitor TKI; The method of introducing the tumor drug and the ATP nanosensor into the organoid culture chamber on the organoid culture layer through the first inlet and the first flow channel of the injection layer and the injection hole on the porous membrane layer comprises: injecting the ATP nanosensor and different TKIs into different organoid culture chambers on the organoid culture layer respectively; The method of obtaining the fluorescence intensity change value of the ATP nanosensor in the organoid culture chamber and determining the effectiveness of the tumor drug according to the fluorescence intensity change value includes: obtaining the fluorescence intensity change values ​​of the ATP nanosensor in different organoid culture chambers, and determining the effectiveness of the different TKIs according to the fluorescence intensity change values.

23. The detection method according to claim 22, characterized in that: The different TKIs include: osimertinib and erlotinib.

24. The detection method according to claim 21, characterized in that: The tumor drugs are chemotherapy drugs in different concentration combinations; The method of introducing the tumor drug and the ATP nanosensor into the organoid culture chamber on the organoid culture layer through the first inlet and the first flow channel of the injection layer and the injection hole on the porous membrane layer comprises: injecting the ATP nanosensor and chemotherapy drugs of different concentrations into different organoid culture chambers on the organoid culture layer respectively; The step of obtaining the fluorescence intensity change value of the ATP nanosensor in the organoid culture chamber and determining the effectiveness of the tumor drug according to the fluorescence intensity change value includes: obtaining the fluorescence intensity change value of the ATP nanosensor in different organoid culture chambers, and determining the effectiveness of the chemotherapy drugs in different concentration combinations according to the fluorescence intensity change value.

25. The detection method according to claim 24, characterized in that: The chemotherapy drugs in different concentration combinations include: pemetrexed and carboplatin in different concentration combinations.

26. The detection method according to claim 21, characterized in that: The tumor drugs are TKI and chemotherapy drugs in different concentration combinations; The method of introducing the tumor drug and the ATP nanosensor into the organoid culture chamber on the organoid culture layer through the first inlet and the first flow channel of the injection layer and the injection hole on the porous membrane layer comprises: injecting the ATP nanosensor and TKI and chemotherapy drugs of different concentrations into different organoid culture chambers on the organoid culture layer respectively; The step of obtaining the fluorescence intensity change value of the ATP nanosensor in the organoid culture chamber and determining the effectiveness of the tumor drug according to the fluorescence intensity change value includes: obtaining the fluorescence intensity change value of the ATP nanosensor in different organoid culture chambers, and determining the effectiveness of the TKI and chemotherapy drugs in different concentration combinations according to the fluorescence intensity change value.

27. The detection method according to any one of claims 21 to 26, characterized in that: The method further comprises: At least one organoid culture chamber on the organoid culture layer is controlled not to have the tumor drug added therein, serving as a control group.

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