Methods for determining the relationship of a plurality of substances to cell interaction and micro-well array chips

Abstract

A method for determining the relationship between a plurality of substances and cells, comprising: providing a first droplet, a second droplet, a third droplet, and a micro-well array chip, the micro-well array chip having a plurality of micro-well combinations, each micro-well combination comprising a large micro-well (110) and a plurality of small micro-wells (120) adjacent to and in communication with the large micro-well (110), the pore size of the large micro-well (110) being larger than the pore size of the small micro-well (120) (S100); first adding the second droplet into the micro-well array chip and into the large micro-well (110), and then adding the first droplet into the micro-well array chip and into the plurality of small micro-wells (120) (S200); allowing the first droplet and the second droplet to fuse, culturing cells in the micro-well array chip (S300); after the cell culture is completed, adding the third droplet into the empty small micro-well (120), the third droplet fusing with the first fused droplet after cell culture to obtain a second fused droplet (S400); demulsifying the second fused droplet, collecting magnetic beads, constructing a library and sequencing the nucleic acid carried on the magnetic beads, and determining the relationship between the plurality of substances and the cells based on the sequencing results (S500).

Description

Technical Field

[0001] This invention relates to the field of medicine. Specifically, this invention proposes a method for determining the interaction between various substances and cells, and a microwell array chip. Background Technology

[0002] Precision medicine refers to designing the best treatment plan for each patient based on individual differences, aiming to achieve the best therapeutic effect and minimize side effects. With the increasing prevalence of cancer, precise and personalized treatment has become one of the strategies to address cancer heterogeneity. Thanks to the rapid development of sequencing technology over the past decade, scientists have discovered many genes related to cancer in different tissues, such as Myc and Ras. By sequencing the genes in a patient's biopsy sample, information on potential oncogenic gene mutations can be obtained, allowing for the development of corresponding treatment plans. In the long run, personalized medicine, through more accurate diagnosis, provides more effective and targeted treatments, preventing the occurrence of certain diseases and saving on treatment costs compared to current methods. However, our understanding of the relationship between sequencing results and cancer cell responses to various exogenous substances (including compounds, hormones, antibodies, and other substances that can affect cells) remains very limited. Therefore, the guiding role of sequencing results in the development of personalized treatment plans is currently relatively limited.

[0003] To address this issue, in vitro experiments can be conducted on patient biopsy samples to assess the response of cancer cells to different substances (including compounds, hormones, antibodies, and other substances that can affect cells). This data can then be used to assist in the development of personalized treatment plans. For treatment effectiveness, a large number of potentially effective substances need to be screened, making high-throughput screening even more crucial in multi-drug screening.

[0004] In recent years, with the continuous development of DNA encoding and sequencing technologies, DNA barcoding has been introduced into single-cell sequencing, enabling high-throughput sequencing at the single-cell level. The realization of this high-throughput single-cell sequencing is also inseparable from the development of microfluidic technology. Microfluidics is a technology capable of precisely controlling and manipulating microscale fluids, capable of miniaturizing laboratory experiments onto a chip measuring only a few square centimeters. It is also an interdisciplinary field encompassing physics, engineering, chemistry, and biology. Because the microstructure of microfluidic chips is comparable in size to a single cell, microfluidic chips are considered the most promising high-throughput single-cell analysis platform in the field of biology, especially in single-cell related research. Analysis at the single-cell level has significant research implications for the early diagnosis and treatment of major diseases and drug screening, and has become a research hotspot in recent years. Currently reported methods for drug screening at the single-cell level cannot achieve high-throughput drug screening; therefore, a method is needed to study the effects of different drugs on single cells at the transcriptome level, enabling large-scale in vitro drug screening. This would help identify synergistic drugs, providing assistance for personalized precision medicine and combination therapy. Summary of the Invention

[0005] This invention aims to at least partially address the technical problems existing in the prior art. To this end, this invention proposes a method and a microwell array chip for determining the interaction relationships between multiple substances and cells. This method allows for the study of the interaction relationships of multiple substances at the single-cell level, facilitating high-throughput screening of combined substances and holding significant importance for the study of combined substances at the transcriptome level and guiding combination drug therapy.

[0006] In one aspect of the invention, a method for determining the interaction relationships between multiple substances and cells is provided. According to an embodiment of the invention, the method includes:

[0007] (1) Provides a first droplet, a second droplet, a third droplet and a microwell array chip; wherein, the first droplet is a mixed droplet containing multiple different molecular coding droplets, the second droplet is a droplet containing a single cell, the third droplet is a droplet containing a single sequencing magnetic bead, cell lysis buffer and cleavage reagent, each of the molecular coding droplets contains a substance and a matching coding nucleic acid molecule and indexing magnetic bead, the microwell array chip has multiple microwell combinations, each of the microwell combinations includes a large microwell and multiple small microwells adjacent to and connected to the large microwell, the pore size of the large microwell is larger than the pore size of the small microwells;

[0008] (2) First, add the second droplet into the micro-well array chip and let it fall into the large micro-well; then add the first droplet into the micro-well array chip and let it fall into multiple small micro-wells.

[0009] (3) The first droplet and the second droplet are fused together to obtain a first fused droplet. The first fused droplet occupies the large micro well and the small micro well is vacated. The micro well array chip is then used for cell culture.

[0010] (4) After the cell culture is completed, the third droplet is added to the empty micro well and the third droplet is fused with the first fusion droplet after cell culture. The cell lysis solution in the third droplet causes cell rupture. The nucleic acid molecules in the cell and the coding nucleic acid molecules are captured by the sequencing magnetic beads. At the same time, the index sequence on the index magnetic beads is broken off by the cleavage reagent in the third droplet and is also captured by the sequencing magnetic beads to obtain the second fusion droplet. The second fusion droplet is collected.

[0011] (5) Demulsify the second fusion droplet, collect sequencing magnetic beads, construct a library and sequence the nucleic acid and index sequence carried on the sequencing magnetic beads, and determine the interaction relationship between various substances and cells based on the sequencing results.

[0012] In the method according to embodiments of the present invention, different substances are encoded and labeled using nucleic acid molecules (also referred to as "molecular coding") to facilitate subsequent sequencing result analysis. Substance droplets and magnetic bead droplets are small droplets, while cell droplets are large droplets. The microwell array chip contains interconnected microwells with different pore sizes; small-pore microwells can capture small droplets, while large-pore microwells can capture large droplets.

[0013] First, a second droplet with a larger pore size (also called a "cell droplet") is added to the chip and falls into a large microwell. Then, a first droplet with a smaller pore size (also called a "substance droplet") is added to the chip, with each substance droplet randomly falling into a small microwell. Next, one cell droplet and multiple substance droplets are fused together to complete the addition process. The chip is then incubated in an incubator for a certain period before being removed. Due to interfacial tension, the fused large droplet occupies the large microwell, leaving the small microwell empty for subsequent loading of sequencing magnetic bead droplets. After substance processing, a third droplet (a small droplet containing sequencing magnetic beads, cell lysis buffer, and fragmentation reagent, also called a "magnetic bead droplet") is added to a small microwell within the chip and fused with the large droplet, thereby completing cell lysis and capturing mRNA, molecular coding, and index sequences. The fused droplets are collected, the magnetic beads are demulsified and recovered, and the nucleic acid and index sequence information carried by the magnetic beads is used for subsequent single-cell library construction.

[0014] Because multiple substance droplets act on a single cell droplet, and multiple sequencing magnetic beads (the same number as the substance droplets) capture nucleic acid information after cell culture, index magnetic beads carrying index sequences are added to each substance droplet to determine whether different sequencing magnetic beads act on the same cell droplet. Sequencing magnetic beads can capture index sequences, and based on the type of index sequence, it can be determined whether they originate from the same cell droplet. This allows for the identification of multiple substances acting on the same cell, facilitating high-throughput combination substance screening. This is of great significance for research on combination substances at the transcriptome level and for guiding combined drug therapy.

[0015] According to embodiments of the present invention, the method for determining the interaction relationship between multiple substances and cells may further have the following additional technical features:

[0016] According to an embodiment of the present invention, each of the molecularly encoded droplets contains 3 to 8 indexed magnetic beads, and each of the indexed magnetic beads contains a different index sequence.

[0017] According to an embodiment of the present invention, the cleaving agent is selected from those suitable for cleaving disulfide bonds.

[0018] According to embodiments of the present invention, the cleavage agent is selected from dithiothreitol, tris(2-carbonylethyl)phosphohydrochloride, tris(3-hydroxypropyl)phosphine and / or β-thioethanol.

[0019] According to an embodiment of the present invention, the second droplet and the third droplet are obtained by sorting using a sorting chip.

[0020] According to an embodiment of the present invention, the fusion is an electrofusion or a chemical fusion.

[0021] According to an embodiment of the present invention, the pore size of the large micro well is 80-100 micrometers and the depth is 60-80 micrometers; the pore size of the small micro well is 40-60 micrometers and the depth is 60-80 micrometers.

[0022] According to an embodiment of the present invention, the sequencing magnetic beads are adapted to capture nucleic acid molecules and index sequences.

[0023] According to an embodiment of the present invention, before performing step (2), the micro-well array chip provided in step (1) is subjected to surface plasma treatment so that the grooves in the micro-well array chip for droplet flow are bonded to the large and small micro-wells to facilitate droplet capture.

[0024] According to an embodiment of the present invention, the method for collecting the second fusion droplet includes: flipping the microwell array chip by 180° so that the openings of the large microwell and the small microwell face upward, adding oil into the microwell array chip so that the second fusion droplet flows out from the large microwell into the collection container.

[0025] In another aspect, the present invention proposes a microwell array chip. According to an embodiment of the present invention, the microwell array chip includes: a microwell array layer, wherein a plurality of microwell combinations are disposed on the microwell array layer, each microwell combination including a large microwell and a plurality of small microwells adjacent to and connected to the large microwell, the pore size of the large microwell being larger than the pore size of the small microwells; and a channel layer, wherein the channel layer is stacked on top of the microwell array layer, and grooves are disposed on the channel layer, the openings of the large and small microwells facing the grooves. As mentioned above, the microwell array chip according to the embodiments of the present invention can be used to study the interaction relationships of compound substances at the single-cell level, which is helpful for achieving high-throughput compound substance screening and is of great significance for the study of compound substances at the transcriptome level.

[0026] According to an embodiment of the present invention, the pore size of the large micro well is 80-100 micrometers and the depth is 60-80 micrometers; the pore size of the small micro well is 40-60 micrometers and the depth is 60-80 micrometers.

[0027] According to an embodiment of the present invention, the groove is connected to the large or small micro well by chemical bonds.

[0028] According to an embodiment of the present invention, the microwell array chip is used to implement the previously described method for screening composite materials.

[0029] The method designed in this invention utilizes molecular coding to encode substances under different conditions. Combined with microfluidic technology, it encapsulates single cells, nucleic acid capture magnetic beads, substances, and their corresponding codes in the same droplet. Through single-cell sequencing technology, it can achieve high-throughput composite material screening at the single-cell level, improving composite material screening efficiency and reducing the consumption of manpower and material resources.

[0030] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0031] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0032] Figure 1 This diagram illustrates a method flow chart for determining the interaction relationship between multiple substances and cells according to an embodiment of the present invention.

[0033] Figure 2 A top view of a microwell array chip structure according to an embodiment of the present invention is shown;

[0034] Figure 3 A schematic diagram of a channel layer structure according to an embodiment of the present invention is shown;

[0035] Figure 4 A side view of a microwell array chip structure according to an embodiment of the present invention is shown;

[0036] Figure 5 A schematic diagram of a droplet preparation process according to an embodiment of the present invention is shown;

[0037] Figure 6 A schematic flowchart of droplet capture and fusion according to an embodiment of the present invention is shown;

[0038] Figure 7 The images show a physical diagram (a) of a droplet capturing a large droplet and a physical diagram (b) of a droplet capturing a small droplet according to an embodiment of the present invention, with a scale bar of 100 micrometers.

[0039] Figure 8 A schematic diagram illustrating the structure of sequencing magnetic beads capturing sequence information and material encoding according to an embodiment of the present invention is shown.

[0040] Figure 9 The image shows the fragment distribution after index sequence-specific amplification according to an embodiment of the present invention, with a distinct characteristic peak appearing at approximately 170 bp;

[0041] Figure 10 The image shows the fragment distribution after specific amplification of the substance coding sequence according to an embodiment of the present invention, with a distinct characteristic peak appearing at around 130 bp. Detailed Implementation

[0042] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0043] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0044] This invention proposes a method for determining the interaction between multiple substances and cells, and a microwell array chip, which will be described in detail below.

[0045] Methods for determining the interaction between multiple substances and cells

[0046] In one aspect of this invention, a method for determining the interaction between various substances and cells is provided. This invention does not strictly limit the specific type of the term "substance," which can include compounds, hormones, antibodies, and other substances that may or may not have an effect on the body. For ease of understanding and description, this invention typically specifies "substance" as "drug."

[0047] According to an embodiment of the present invention, see Figure 1 The method for determining the interaction between multiple substances and cells includes:

[0048] S100 provides first droplet, second droplet, third droplet, and microwell array chips.

[0049] In this embodiment, a first droplet, a second droplet, a third droplet, and a microwell array chip are provided. The first droplet is a mixed droplet containing multiple droplets with different molecular coding, the second droplet is a droplet containing a single cell, and the third droplet is a droplet containing a single sequencing magnetic bead (for capturing nucleic acids), cell lysis buffer, and cleavage reagent. Each molecular coding droplet contains a substance and a matching coding nucleic acid molecule (also referred to as "molecular coding"). The microwell array chip has multiple microwell combinations, and each microwell combination includes a large microwell and multiple small microwells adjacent to and connected to the large microwell.

[0050] Fluorescent coding technology uses the color of fluorescent dyes to encode solutions of different drugs and generate droplets, which are then mixed with droplets containing single cells to achieve single-cell-based drug screening. However, this method is limited by the types of fluorescent dyes and the detection devices, making it difficult to screen large numbers of drugs and lacking information on intracellular gene expression.

[0051] In this application, a molecular coding design is adopted, based on the exogenous sequence information introduced in international patent WO2021147069A1, with a total of M×N coding schemes (M=4). 10 N=4 10 By synthesizing this sequence as a drug code, a specific code corresponds to a specific drug under a given condition. For example, the molecular code may contain a fixed sequence UMI, a specific sequence, and a magnetic bead trapping sequence.

[0052] According to an embodiment of the present invention, the method for preparing the first droplet includes: mixing a certain amount of molecular code with the corresponding substance and index magnetic beads, and then injecting the mixture into a droplet generation chip to generate molecular code droplets of uniform size, and so on to generate droplets with different molecular codes. Finally, the generated droplets are collected and mixed in the same collection tube to complete the preparation of the substance droplets. The purpose of adding molecular codes is to distinguish different substances. The reason for introducing index magnetic beads is that multiple sequencing magnetic beads will be captured when capturing the sequencing magnetic bead droplets. This results in the final fused droplet containing multiple sequencing magnetic beads. Without the index sequence, the final sequencing cannot determine which two sequencing magnetic beads come from the same droplet, and the mRNA information captured by the sequencing magnetic beads cannot be grouped together. That is, it is impossible to determine which drugs act on the same cell, which makes the single-cell sequencing data incomplete and unusable. To address this issue, index beads carrying index sequences were introduced. Under the action of a fragmentation reagent, the index beads are fragmented to release their index sequences. Since each index bead is unique, each final large droplet contains multiple different index sequences. Sequencing analysis determines which sequencing beads capture the same type of index sequence, indicating that these beads originated from the same droplet. An algorithm then merges the captured mRNA and molecular coding information from these beads into a single sequence, enabling the sequencing of a complete single cell. Specifically, each molecular coding droplet contains 3–8 index beads, each with a different index sequence.

[0053] The method for preparing the second droplet includes: injecting a certain amount of cell suspension into the droplet generation chip, adjusting the cell concentration to ensure a high ratio of single cells encapsulated in the droplet while maintaining a low double-encapsulation rate, and then sorting the generated droplets using methods such as dielectrophoresis through a sorting chip to obtain droplets containing single cells.

[0054] The preparation method of the third droplet includes: injecting a certain amount of sequencing magnetic bead suspension (obtained by resuspending sequencing magnetic beads using cell lysis buffer and fragmentation reagent) into the droplet generation chip; adjusting the concentration of sequencing magnetic beads to ensure a high ratio of single sequencing magnetic beads encapsulated in the droplet while maintaining a low double encapsulation rate; and then sorting the generated droplets using a sorting chip and methods such as dielectrophoresis to obtain droplets containing single sequencing magnetic beads.

[0055] It should be noted that the terms "large" and "small" in the descriptions of "large microwell" and "small microwell" in this invention refer to the pore size of the microwell; the pore size of a large microwell is larger than that of a small microwell. According to embodiments of the present invention, the pore size of a large microwell is 80–100 micrometers, and its depth is 60–80 micrometers; the pore size of a small microwell is 40–60 micrometers, and its depth is 60–80 micrometers. Thus, large microwells can capture single-cell droplets, while small microwells can capture material droplets and magnetic bead droplets.

[0056] According to embodiments of the present invention, the cleavage reagent is selected from those suitable for cleaving disulfide bonds. The index sequence is linked to the magnetic beads via disulfide bonds. The cleavage reagent breaks these disulfide bonds, causing the index sequence carried on the index beads to detach from the beads. Since the sequencing beads contain sequences matching the index sequence, the detached index sequence can be captured, facilitating subsequent sequencing and classification to identify multiple substances acting on the same cell. In specific embodiments of the present invention, the cleavage reagent is selected from dithiothreitol (DTT), tris(2-carbonylethyl)phosphohydrochloride (TCEP), tris(3-hydroxypropyl)phosphine (THPP), and / or β-thioethanol. This allows for specific cleavage of disulfide bonds without affecting the nucleic acid molecular structure.

[0057] S200 first adds the second droplet into the chip and it falls into the large microwell, then adds the first droplet into the chip and it falls into multiple small microwells.

[0058] In this embodiment, the second droplet is first added to the micro-well array chip and falls into the large micro-well, and then the first droplet is added to the micro-well array chip and falls into multiple small micro-wells.

[0059] According to an embodiment of the present invention, before performing step S200, the micro-well array chip provided in step S100 is subjected to surface plasma treatment so that the grooves in the micro-well array chip for droplet flow are bonded to the large and small micro-wells to facilitate droplet capture.

[0060] The term "bonding" as used in this invention refers to the technique of directly combining two homogeneous or heterogeneous semiconductor materials with clean, atomically flat surfaces after surface cleaning and activation treatment, under certain conditions, by bonding the wafers together through van der Waals forces, molecular forces, or even atomic forces.

[0061] The cured chip (also known as a "PDMS substrate") has a certain degree of adhesion. A pair of molded PDMS substrates can bond naturally due to intermolecular attraction without any further treatment. However, this adhesion strength is limited and leakage is prone to occur. Plasma treatment introduces hydrophilic -OH groups into the surface of PDMS, replacing the -CH groups, thus giving the PDMS surface extremely strong hydrophilic properties. When two layers of treated PDMS are bonded together, the Si-OH groups on both surfaces undergo the following reaction: 2Si- -O-Si+2H2O. A strong Si-O bond is formed between the two PDMS layers, thus completing the irreversible bonding between them.

[0062] The first and second droplets of S300 merge and occupy the large microwell, leaving the small microwell empty for cell culture.

[0063] In this embodiment, the first droplet and the second droplet are fused to obtain a first fused droplet. The first fused droplet occupies the large micro-well, leaving the small micro-well empty, and the micro-well array chip is used for cell culture.

[0064] Due to interfacial tension, the first fusion droplet after fusion mainly occupies the large microwells, leaving the positions of the small microwells vacant for subsequent loading of sequencing magnetic bead droplets.

[0065] It should be noted that the present invention does not strictly limit the fusion method of the two droplets. For example, it can be achieved by using an electric field to disrupt the stability of the interface, or by using chemical reagents such as perfluorobutanol. The specific method can be flexibly selected according to actual needs.

[0066] S400 adds the third droplet into the micro well, where it merges with the first fusion droplet, and the resulting second fusion droplet is collected.

[0067] In this embodiment, after cell culture is completed, a third droplet is added to the empty microwell. The third droplet will fuse with the first fusion droplet after cell culture. Under the action of the cell lysis solution in the third droplet, the cells rupture. The nucleic acid molecules in the cells and the coding nucleic acid molecules are captured by the sequencing magnetic beads. At the same time, under the action of the cleavage reagent in the third droplet, the index sequence on the index magnetic beads will be broken off and also captured by the sequencing magnetic beads, resulting in a second fusion droplet. The second fusion droplet is collected.

[0068] After drug processing, small droplets containing sequencing magnetic beads, cell lysis buffer, and cleavage reagent are added into the chip and fused with the first fusion droplet to obtain the second fusion droplet, thereby completing cell lysis and capture of mRNA, molecular coding, and index sequences.

[0069] S500 demulsification, library construction, and sequencing

[0070] In this embodiment, the second fusion droplet is demulsified, sequencing magnetic beads are collected, and the nucleic acids and index sequences carried on the sequencing magnetic beads are used for library construction and sequencing to determine the interaction relationships between multiple substances and cells.

[0071] According to an embodiment of the present invention, the method for collecting the second fusion droplet includes: flipping the microwell array chip by 180° so that the openings of the large microwell and the small microwell face upward, adding oil into the microwell array chip so that the second fusion droplet flows out from the large microwell into the collection container.

[0072] The microwell array chip includes a stacked microwell array layer and a channel layer. Large microwells and small microwells are disposed on the microwell array layer, and the openings of the microwells face the grooves on the channel layer for liquid flow.

[0073] Because the aqueous phase has a lower density than the oil phase, the second fusion droplet floats above the grooves and cannot be carried away by adding oil into the grooves. Therefore, the chip needs to be rotated 180° so that the openings of the large and small microwells face upwards. This allows the second fusion droplet to float inside the grooves, and oil can be used to push out the droplets detached from the microwells and collect them in centrifuge tubes. The magnetic beads are then recovered after demulsification for subsequent single-cell library construction. Sequencing information is used to determine the correspondence between single cells and drugs, thus achieving high-throughput drug screening at the single-cell level.

[0074] The drug screening method of the present invention also has the following advantages:

[0075] 1) High throughput: By introducing additional molecular coding, multiple drug conditions (including drug type, drug concentration, etc.) can be encoded; by introducing additional index magnetic beads, multiple substances acting on the same cell can be distinguished, which facilitates the study of the effects of multiple substances on cells and helps with combination drug therapy.

[0076] 2) Saves time and effort: By utilizing droplet microfluidics, not only can the amount of reagents used be reduced, but also tedious manual operations can be avoided, thereby improving screening efficiency;

[0077] 3) High accuracy: Since this invention targets single-cell analysis, by introducing molecular coding and combining it with single-cell sequencing, the correspondence between the transcriptome of a single cell and the drug can be obtained, thereby more accurately showing the effect of the drug on the single-cell transcriptome under different conditions, which is more valuable for research.

[0078] Microwell array chip

[0079] In another aspect of the invention, a microwell array chip is proposed. See embodiments of the invention. Figure 2 The microwell array chip includes a microwell array layer 100 and a channel layer 200.

[0080] The microwell array layer 100 has multiple microwell combinations. Each microwell combination includes a large microwell 110 and multiple small microwells 120 adjacent to and connected to the large microwell 110. The pore size of the large microwell 110 is larger than that of the small microwells 120. Drug droplets and magnetic bead droplets are small droplets, while cell droplets are large droplets. The microwell array layer has interconnected microwells with different pore sizes. Microwells with smaller pore sizes can capture small droplets, while microwells with larger pore sizes can capture large droplets.

[0081] According to embodiments of the present invention, the pore size of the large microwell is 80–100 micrometers, and the depth is 60–80 micrometers; the pore size of the small microwell is 40–60 micrometers, and the depth is 60–80 micrometers. Thus, the large microwell can capture single-cell droplets, while the small microwell can capture drug droplets and magnetic bead droplets.

[0082] See Figure 3 and 4 The channel layer 200 and the microwell array layer 100 are stacked together. The channel layer has grooves 210, and the openings of the large microwells 110 and the small microwells 120 face the grooves 210. Since the microwells and grooves of the microwell array layer have undergone surface plasma treatment beforehand, they can be bonded together as one, thereby preventing leakage when liquid is added. When droplets are added into the grooves, since the droplets are lighter than oil, the microwells can capture the droplets under the action of buoyancy.

[0083] According to an embodiment of the present invention, the groove 210 is connected to the large microwell 110 or the small microwell 120 by chemical bonds. This allows droplets flowing into the groove to be captured by the microwell.

[0084] Specifically, before adding droplets, two holes are drilled on the microwell array layer, namely the liquid inlet and the liquid outlet, both of which are opposite to the groove. Droplets are added into the groove through the liquid inlet, and the liquid will be captured by the microwell. Then, oil is added into the groove through the liquid inlet to flush away the droplets that are not captured in the groove, and the droplets are sucked out from the liquid outlet.

[0085] According to embodiments of the present invention, a microwell array chip is used to implement the drug screening method described above. The features and advantages described above for the drug screening method also apply to this microwell array chip, and will not be repeated here.

[0086] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0087] Example 1

[0088] Step 1: This embodiment uses a droplet generation device similar to CN209144161U, wherein the droplet generation chip is replaced with the chip involved in patent WO2020063864A1, and the syringe is replaced with a BD 30ml syringe. A schematic diagram of droplet generation is shown below. Figure 5 As shown, cell droplets, drug droplets, and sequencing magnetic bead droplets are generated by following these steps.

[0089] 1. Design of molecular coding

[0090] The molecular coding used in this invention is based on exogenous sequence information introduced in international patent WO2021147069A1, and there are a total of M×N codes (M=410, N=410). By synthesizing this sequence as a drug code, a drug under a certain condition corresponds to a certain code.

[0091] 2. Preparation of drug droplets

[0092] A certain amount of molecular code, the corresponding drug, and index magnetic beads carrying index sequences are mixed and injected into a droplet generation chip to generate molecularly encoded droplets of uniform size. This process is repeated to generate droplets with different molecular codes. Finally, the generated droplets are collected and mixed in the same collection tube to complete the preparation of drug droplets.

[0093] 3. Preparation of single-cell droplets

[0094] A certain amount of cell suspension is injected into a droplet generation chip. By adjusting the cell concentration, the ratio of single cells encapsulated in the droplets is made high while the double-encapsulation rate is kept low. Then, the generated droplets are sorted by a cell sorting chip using methods such as dielectrophoresis to obtain droplets containing single cells.

[0095] 4. Preparation of sequencing magnetic bead droplets

[0096] A certain amount of sequencing magnetic bead suspension (obtained by resuspending sequencing magnetic beads using cell lysis buffer) is injected into a droplet generation chip. By adjusting the concentration of sequencing magnetic beads, the ratio of single sequencing magnetic beads encapsulated in the droplets is made high while the double encapsulation rate is kept low. Then, the generated droplets are sorted by a sorting chip using methods such as dielectrophoresis to obtain droplets containing single sequencing magnetic beads.

[0097] Step 2: This embodiment utilizes, for example... Figures 1 to 4The microwell chip shown captures droplets. This chip comprises a microwell array layer and a channel layer. The microwell array layer has 28,800 interconnected microwells (with apertures of 90 micrometers and 50 micrometers respectively), and a depth of 70 micrometers. This ensures that a large microwell can only capture a large droplet, and a small microwell can only capture a small droplet, achieving a 1:1 droplet pairing. Since the density of the aqueous phase is lower than that of the oil phase, the droplets float on the surface of the oil phase. To capture the droplets, the chip needs to be rotated 180° in practical use, with the microwell openings facing downwards. Surface plasma treatment is then used to connect the droplets to the microwell. Figure 2 The channel layer shown is bonded, and the channel contains more than 25,000 effective microwells. Two holes are drilled on the microwell array layer using a 15 mm aperture punch, one for liquid inlet and one for liquid outlet.

[0098] The specific operating steps are as follows:

[0099] First, large droplets (90 micrometers in diameter) encapsulating single cells are generated, and then the cell droplets are loaded into a microwell array chip. Figure 6 a) Allow the large microwells to capture cell droplets, such as Figure 6 b and Figure 7 As shown in Figure a.

[0100] Then, small droplets (50 micrometers in diameter) containing various types of mixed encapsulated materials, molecularly encoded, and indexed magnetic beads are loaded into the microwell array chip. Different types of droplets are randomly captured by the microwells. Each group of microwells randomly captures two material droplets and one large single-cell droplet. After the microwells have captured the droplets, oil is injected to push away excess droplets. Figure 6 c and Figure 7 As shown in Figure b, the droplet capture efficiency is above 95%.

[0101] The chip is placed on a shaker and gently shaken to allow droplets from the same group to collide. Simultaneously, a corona treatment or the chemical reagent perfluorobutanol is used to fuse the droplets. Figure 6 As shown in d, the fused droplets at this time randomly contain one or two substances and their corresponding molecular codes, as well as indexed magnetic beads (the index sequence is not broken down because no breaking reagent is added). The fusion leaves the positions of the two small droplets empty, and the fusion efficiency is over 80%.

[0102] After incubating the droplets for a period of time, a small droplet containing one sequencing magnetic bead is added. This way, each microwell will capture two sequencing magnetic bead droplets. Excess droplets are then pushed out with oil. The droplet state at this point is as follows: Figure 6 As shown in e.

[0103] Finally, after the droplets are fused, the sequencing magnetic bead droplets contain cell lysis buffer and index sequence breaking reagent, which causes cell lysis. At the same time, the breaking reagent breaks the index sequence on the index magnetic beads. In this way, the sequencing magnetic beads capture the mRNA produced by cell lysis, the index sequence broken from the index magnetic beads, and the corresponding molecular codes.

[0104] Step 3: After droplet incubation is complete, remove the microfluidic chip and inject the sequencing magnetic bead droplet. The droplet will then be captured again by the vacated microwells. Excess droplets are then pushed out with oil. The droplet's state at this point is as follows: Figure 6 As shown in e. Finally, repeat step two of the droplet fusion process; at this point, the state of the droplets is as shown in e. Figure 6 As shown in f.

[0105] Step 4: To recover the droplets after secondary fusion, the microwell array chip needs to be rotated 180°. Then, oil is used to push the droplets detached from the microwells out of the chip, and the droplets are collected in a centrifuge tube. For example... Figure 8 As shown, because the sequencing magnetic bead droplets contain cell lysis buffer, once the droplets fuse a second time, the cells will be lysed, releasing intracellular mRNA. At this point, the sequencing magnetic beads will capture all nucleic acid information, including mRNA, molecular coding, and index sequences. Finally, the BGI single-cell sequencing pipeline is used, and bioinformatics analysis is used to classify magnetic beads labeled with the same type of index sequence as originating from the same droplet. The reaction solutions for purifying the secondary specific amplification index sequence and the secondary specific amplification material encoding were separately tested using gel electrophoresis, and the experimental results are shown below. Figure 9 and Figure 10 As shown, there are obvious peaks at around 170bp and 130bp, respectively, proving that the invention can perform composite molecular coding detection through single-cell sequencing technology. The introduction of the index sequence is to mark multiple sequencing magnetic beads in the same droplet.

[0106] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0107] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for determining the interaction between multiple substances and cells, characterized in that, include: (1) Provide a first droplet, a second droplet, a third droplet, and a microwell array chip; The first droplet is a mixed droplet containing multiple different molecular coding droplets, the second droplet is a droplet containing a single cell, and the third droplet is a droplet containing a single sequencing magnetic bead, cell lysis buffer, and cleavage reagent. Each molecular coding droplet contains a substance and a matching coding nucleic acid molecule and indexing magnetic bead. The microwell array chip has multiple microwell combinations, and each microwell combination includes a large microwell and multiple small microwells adjacent to and connected to the large microwell. The pore size of the large microwell is larger than that of the small microwells. (2) First, add the second droplet into the micro-well array chip and let it fall into the large micro-well; then add the first droplet into the micro-well array chip and let it fall into multiple small micro-wells. (3) The first droplet and the second droplet are fused to obtain a first fused droplet. The first fused droplet occupies the large micro well and the small micro well is vacated. The micro well array chip is then used for cell culture. (4) After the cell culture is completed, the third droplet is added to the empty micro well and the third droplet is fused with the first fusion droplet after cell culture. The cell lysis solution in the third droplet causes cell rupture. The nucleic acid molecules in the cell and the coding nucleic acid molecules are captured by the sequencing magnetic beads. At the same time, the index sequence on the index magnetic beads is broken off by the cleavage reagent in the third droplet and is also captured by the sequencing magnetic beads to obtain the second fusion droplet. The second fusion droplet is collected. (5) Demulsify the second fusion droplet, collect the sequencing magnetic beads, construct a library and sequence the nucleic acid and index sequence carried on the sequencing magnetic beads, and determine the interaction relationship between various substances and cells based on the sequencing results.

2. The method according to claim 1, characterized in that, Each of the molecularly encoded droplets contains 3 to 8 indexed magnetic beads, and each of the indexed magnetic beads contains a different index sequence.

3. The method according to claim 1 or 2, characterized in that, The cleavage reagent is suitable for cleaving disulfide bonds.

4. The method according to claim 1 or 2, characterized in that, The cleavage reagent is selected from dithiothreitol, tris(2-carbonylethyl)phosphohydrochloride, tris(3-hydroxypropyl)phosphine and / or β-thioethanol.

5. The method according to claim 1 or 2, characterized in that, The second and third droplets are obtained by sorting using a sorting chip.

6. The method according to claim 1 or 2, characterized in that, The fusion is either electrofusion or chemical fusion.

7. The method according to claim 1 or 2, characterized in that, The pore size of the micro-well is 80-100 micrometers, and the depth is 60-80 micrometers; The micro well has a pore size of 40-60 micrometers and a depth of 60-80 micrometers.

8. The method according to claim 1 or 2, characterized in that, The sequencing magnetic beads are suitable for capturing nucleic acid molecules and index sequences.

9. The method according to claim 1 or 2, characterized in that, Before step (2), the micro-well array chip provided in step (1) is subjected to surface plasma treatment so that the grooves in the micro-well array chip for droplet flow are bonded to the large and small micro-wells to facilitate droplet capture.

10. The method according to claim 1 or 2, characterized in that, The method for collecting the second fusion droplet includes: The microwell array chip is flipped 180° so that the openings of the large and small microwells face upwards. Oil is added to the microwell array chip so that the second fusion droplet flows out from the large microwell into the collection container.

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