CN118103521A

CN118103521A - Method for screening drugs

Info

Publication number: CN118103521A
Application number: CN202180103265.2A
Authority: CN
Inventors: 刘杨; 刘亚; 刘龙奇; 徐讯
Original assignee: BGI Shenzhen Co Ltd
Current assignee: BGI Shenzhen Co Ltd
Priority date: 2021-10-20
Filing date: 2021-10-20
Publication date: 2024-05-28
Also published as: WO2023065174A1

Abstract

The invention discloses a method for screening medicines, which comprises the following steps: (1) Providing a first droplet, a second droplet, a third droplet and a micro-well array chip; (2) Firstly, adding a second liquid drop into a micro-well array chip and falling into a large micro-well, and then adding a first liquid drop into the micro-well array chip and falling into a small micro-well; (3) Fusing the first liquid drop and the second liquid drop, and culturing cells of the micro-well array chip; (4) After the cell culture is completed, adding a third liquid drop into the small empty micro-well, and fusing the third liquid drop with the first fused liquid drop after the cell culture to obtain a second fused liquid drop; (5) And demulsifying the second fusion liquid drop, collecting the magnetic beads, carrying out library construction and sequencing on nucleic acid carried on the magnetic beads, and screening out target drugs based on a sequencing result.

Description

Method for screening drugs

Technical Field

The invention relates to the field of medicine. Specifically, the invention provides a method for screening medicines.

Background

The traditional cell level drug screening is mainly to conduct drug screening under various conditions after culturing a large number of cells in an orifice plate, the operation steps are complicated, a large amount of manpower and material resources are required to be consumed, and finally, only apparent level changes can be obtained. Due to the heterogeneity of cells, conventional methods have difficulty in performing high throughput drug screening at the single cell level and obtaining large amounts of intracellular gene expression differences.

In recent years, DNA encoding techniques and sequencing techniques have been developed, and DNA barcodes have been introduced into single cell sequencing, thereby achieving high throughput sequencing at the single cell level. Meanwhile, the realization of the high-throughput single-cell sequencing is not separated from the development of microfluidic technology. Microfluidic technology is a technology that enables precise control and manipulation of microscale fluids, with the ability to scale experiments performed in the laboratory to a chip on the order of a few square centimeters, while also being a cross-discipline involving physics, engineering, chemistry, biology, and the like. Since the microstructure of the microfluidic chip is equivalent to the size of a single cell, the microfluidic chip is considered as the most potential high-throughput single cell analysis platform in the field of biology, particularly single cell related research. The analysis of single cells has important research significance in early diagnosis and treatment of serious diseases, drug screening and other aspects, and has become a hot spot of research in recent years. The currently reported method for carrying out drug screening on the single cell level cannot realize high-throughput drug screening, so that a method capable of researching the action of different drugs on single cells from the transcriptome level is needed, large-scale in-vitro drug screening is realized, and assistance is provided for personalized accurate medical treatment.

Disclosure of Invention

The present invention aims to solve, at least to some extent, the technical problems existing in the prior art. Therefore, the invention provides a method for screening medicines and a micro-well array chip, and the method can be used for researching the action relation of medicines at a single cell level, is beneficial to realizing high-flux medicine screening and has important significance for medicine research at a transcriptome level.

In one aspect of the invention, a method of screening for a drug is provided. According to an embodiment of the invention, the method comprises: (1) Providing a first liquid drop, a second liquid drop, a third liquid drop and a micro-well array chip, wherein the first liquid drop is a mixed liquid drop containing a plurality of different molecule coding liquid drops, the second liquid drop is a liquid drop containing single cells, the third liquid drop is a liquid drop containing single sequencing magnetic beads and cell lysate, each molecule coding liquid drop contains a drug and coding nucleic acid molecules matched with the drug, the micro-well array chip is provided with a plurality of micro-well combinations formed by adjacent and communicated large micro-wells and small micro-wells, and the aperture of the large micro-wells is larger than that of the small micro-wells; (2) Firstly adding the second liquid drop into the micro-well array chip and falling into a large micro-well, and then adding the first liquid drop into the micro-well array chip and falling into a small micro-well; (3) Fusing the first liquid drop and the second liquid drop to obtain a first fused liquid drop, wherein the first fused liquid drop occupies the large micro-well, the small micro-well is vacated, and the micro-well array chip is subjected to cell culture; (4) After the cell culture is completed, adding the third liquid drop into the vacated small micro well, fusing the third liquid drop with the first fused liquid drop after the cell culture, breaking cells under the action of a cell lysate in the third liquid drop, capturing nucleic acid molecules in the cells and the encoding nucleic acid molecules together by a sequencing magnetic bead to obtain a second fused liquid drop, and collecting the second fused liquid drop; (5) And demulsifying the second fusion liquid drop, collecting magnetic beads, constructing a library and sequencing nucleic acid carried on the magnetic beads, and screening out a target medicament based on a sequencing result.

In methods according to embodiments of the invention, different drugs are coded and labeled with coding nucleic acid molecules (also referred to as "molecular coding") to facilitate subsequent analysis of sequencing results. The drug droplets and the magnetic bead droplets are small droplets, and the cell droplets are large droplets. The micro-well array chip is provided with communicated micro-wells with different apertures, the micro-wells with small apertures can capture small liquid drops, and the micro-wells with large apertures can capture large liquid drops. First, a second droplet with a larger pore size (also called a "cell droplet") is first added to the chip and falls into the large micro-well. The first droplet with small pore size (also called "drug droplet") is then added to the chip, the drug droplets randomly fall into the small micro-wells, then 2 droplets are fused to complete the dosing process, and the chip is taken out after being cultured in an incubator for a certain time. Due to the action of interfacial tension, the fused large droplet occupies the large micro-well, and the position of the small micro-well is vacated for loading of the magnetic bead droplet for subsequent sequencing. After the drug treatment is completed, a third droplet (a small droplet encapsulating the sequencing magnetic beads and cell lysate, also referred to as a "magnetic bead droplet") is added to a small microwell within the chip and fused with the large droplet, thereby completing the cell lysis and capture of mRNA and molecular encoding. And collecting fused liquid drops, demulsifying and recovering the magnetic beads, carrying out subsequent single-cell library building flow on nucleic acid information carried on the magnetic beads, and splitting sequencing information to obtain the corresponding relationship between single cells and medicines, thereby being beneficial to realizing high-flux medicine screening and having important significance on medicine research on transcriptome level.

According to an embodiment of the present invention, the above method for screening drugs may further have the following additional technical features:

According to the embodiment of the invention, the second liquid drop and the third liquid drop are obtained by sorting through a sorting chip.

According to an embodiment of the invention, the fusion is electrofusion or chemical fusion.

According to the embodiment of the invention, the aperture of the large micro-well is 80-100 micrometers, and the depth is 60-80 micrometers; the aperture of the small micro-well is 40-60 micrometers, and the depth is 60-80 micrometers.

According to an embodiment of the invention, the sequencing magnetic beads are adapted for capturing nucleic acids.

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 grooves in the micro-well array chip for droplet flow are bonded with the large micro-wells and the small micro-wells, so as to capture droplets.

According to an embodiment of the invention, the method of collecting the second fused droplets comprises: and turning the micro-well array chip by 180 degrees, enabling the openings of the large micro-well and the small micro-well to be upward, and adding oil into the micro-well array chip so that the second fused liquid drops flow out of the large micro-well to a collecting container.

In yet another aspect of the present invention, a micro-well array chip is provided. According to an embodiment of the present invention, the micro well array chip includes: the micro-well array layer is provided with a plurality of micro-well combinations of adjacent and communicated large micro-wells and small micro-wells, and the aperture of the large micro-wells is larger than that of the small micro-wells; the channel layer is arranged in a lamination mode with the micro-well array layer, grooves are formed in the channel layer, and openings of the large micro-wells and the small micro-wells face the grooves. As described above, the micro-well array chip according to the embodiment of the present invention can be used to study the action relationship of drugs at the single cell level, which is helpful to achieve high-throughput drug screening, and has important significance for drug study at the transcriptome level.

According to an embodiment of the invention, the grooves are connected with the large or small micro-wells by chemical bonds.

According to an embodiment of the present invention, the micro-well array chip is used for implementing the method for screening medicines described above.

The method designed by the invention utilizes molecular codes to code the drugs under different conditions, combines a microfluidic technology to wrap single cells, nucleic acid capture magnetic beads, the drugs and corresponding codes in the same liquid drop, and can realize single-cell-level high-flux drug screening by single-cell sequencing technology, thereby improving the drug screening efficiency and reducing the consumption of manpower and material resources.

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.

Drawings

The foregoing and/or additional aspects and advantages of the invention will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:

FIG. 1 shows a schematic flow diagram of a method for screening drugs according to one embodiment of the invention;

FIG. 2 shows a top view of a micro-well array chip structure according to one embodiment of the invention;

FIG. 3 shows a schematic view of a channel layer structure according to one embodiment of the invention;

FIG. 4 shows a side view of a micro-well array chip structure according to one embodiment of the invention;

FIG. 5 shows a schematic flow diagram of droplet preparation according to an embodiment of the invention;

FIG. 6 shows a schematic flow diagram of droplet capture and fusion according to one embodiment of the invention;

FIG. 7 shows an electron micrograph after droplet capture (left) and fusion (right), scale 100 μm, according to one embodiment of the invention;

FIG. 8 shows a schematic representation of the sequencing magnetic bead capture sequence information and drug encoding configuration according to one embodiment of the invention;

FIG. 9 shows a fragment distribution after drug encoding specific amplification according to one embodiment of the present invention.

Detailed Description

Embodiments of the present invention are described in detail below. The following examples are illustrative only and are not to be construed as limiting the invention.

It should be noted that the terms "first," "second," and "second" are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implying a number of technical features being indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include one or more such feature. Further, in the description of the present invention, unless otherwise indicated, the meaning of "a plurality" is two or more.

The present invention provides a method for screening drugs and a micro-well array chip, which will be described in detail below, respectively.

Method for screening drugs

In one aspect of the invention, a method of screening for a drug is provided. According to an embodiment of the present invention, referring to fig. 1, the method for screening a drug includes:

s100 provides a first droplet, a second droplet, a third droplet, and a micro-well array chip.

In this embodiment, a first droplet, a second droplet, a third droplet and a micro-well array chip are provided, wherein the first droplet is a mixed droplet containing a plurality of different molecule encoding droplets, the second droplet is a droplet containing a single cell, the third droplet is a droplet containing a single sequencing magnetic bead (for capturing nucleic acid) and a cell lysate, each molecule encoding droplet contains a drug and an encoding nucleic acid molecule (also referred to as "molecule encoding") matched with the drug, and a plurality of micro-well combinations composed of adjacent and communicated large micro-wells and small micro-wells are arranged on the micro-well array chip.

The fluorescent coding technology is to code solutions of different drugs by using the color of fluorescent dye, generate droplets, and then mix the droplets with the droplets coated with single cells, thereby realizing single-cell-based drug screening. However, this method is limited by the kind of fluorescence and the detection means, making it difficult to screen a large amount of drugs with this technique, and lacking information about gene expression inside the cell.

In the present application, a molecular coding design is adopted, and m×n codes (m= ¹⁰,N＝4 ¹⁰) are shared based on the exogenous sequence information introduced in international patent WO2021147069 A1. By synthesizing the sequence as a drug code, one drug under one condition corresponds to one code. For example, the molecular code contains the immobilized sequence UMI, the specific sequence and the magnetic bead capture sequence.

According to an embodiment of the present invention, a method of preparing a first droplet includes: and mixing a certain amount of molecular codes with corresponding medicines, injecting the mixture into a liquid drop generating chip to generate liquid drops with uniform molecular codes, and the like to generate liquid drops with different molecular codes. Finally, the generated liquid drops are collected and mixed in the same collecting pipe, and the preparation of the liquid drops of the medicine is completed.

The preparation method of the second liquid drop comprises the following steps: and injecting a certain amount of cell suspension into the liquid drop generating chip, regulating the cell concentration to ensure that the ratio of wrapping single cells in the liquid drops is higher, ensuring that the double-wrapping ratio is at a lower level, and then separating the generated liquid drops by using a dielectrophoresis method through the separating chip to obtain the liquid drops containing the single cells.

The preparation method of the third liquid drop comprises the following steps: injecting a certain amount of sequencing magnetic bead suspension (obtained by resuspending sequencing magnetic beads by using cell lysate) into a droplet generation chip, adjusting the concentration of the sequencing magnetic beads to ensure that the ratio of single sequencing magnetic beads packed in the droplets is higher, ensuring that the double packing rate is at a lower level, and sorting the generated droplets by using a dielectrophoresis method by using a sorting chip to obtain droplets containing single sequencing magnetic beads.

It should be noted that, the "large" and "small" referred to in the "large micro-well" and the "small micro-well" described in the present invention refer to the pore size of the micro-well, and the pore size of the large micro-well is larger than the pore size of the small micro-well. According to the embodiment of the invention, the aperture of the large micro-well is 80-100 micrometers, and the depth is 60-80 micrometers; the aperture of the small micro-well is 40-60 micrometers, and the depth is 60-80 micrometers. Thus, large micro-wells can capture single cell droplets, and small micro-wells can capture drug droplets and magnetic bead droplets.

S200, adding the second liquid drop into the chip and falling into the large micro-well, and then adding the first liquid drop into the chip and falling into the small micro-well.

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 the small micro-well.

According to an embodiment of the present invention, before step S200 is performed, the micro-well array chip provided in step S100 is subjected to surface plasma treatment, so that grooves for flowing droplets in the micro-well array chip are bonded with large micro-wells and small micro-wells, so as to capture the droplets.

The term bonding used in the invention refers to a technology of bonding two pieces of homogeneous or heterogeneous semiconductor materials with clean surfaces and flat atomic levels into a whole through Van der Waals force, molecular force and even atomic force by directly combining the two pieces of homogeneous or heterogeneous semiconductor materials under certain conditions through surface cleaning and activating treatment.

The surface of the cured chip (also called as 'PDMS substrate') has certain adhesive force, and a pair of formed PDMS substrates can be naturally adhered by means of intermolecular attraction without any treatment, but the adhesive strength is limited, and liquid leakage is easy to occur. The surface of the PDMS after plasma treatment is introduced with-OH groups with hydrophilic property and replaces-CH groups, so that the PDMS surface shows extremely strong hydrophilic property. Attaching the treated two layers of PDMS, wherein the following reaction occurs between Si-OH on the two surfaces: A firm Si-O bond was formed between the two PDMS layers, thus completing the irreversible bond between the two.

S300, the first liquid drop and the second liquid drop are fused and occupy a large micro-well, a small micro-well is vacated, and the cells are cultured.

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, the small micro-well is emptied, and the micro-well array chip is subjected to cell culture.

Due to the action of interfacial tension, the fused first fused liquid drop mainly occupies a large micro-well, and the position of a small micro-well is vacated for loading of a subsequent sequencing magnetic bead liquid drop.

It should be noted that the fusion mode of the two droplets is not strictly limited, for example, the fusion mode can be realized by using the stability of an electric field to break the interface, or can be realized by using chemical reagents such as perfluorobutanol, and the like, and the fusion mode can be flexibly selected according to actual needs.

And S400, adding the third liquid drop into the small micro-well, fusing with the first fused liquid drop, and collecting the obtained second fused liquid drop.

In this example, after the cell culture is completed, a third droplet is added into the small well, the third droplet fuses with the first fused droplet after the cell culture, cell disruption occurs under the action of the cell lysate in the third droplet, the nucleic acid molecules in the cells and the encoding nucleic acid molecules are captured together by the sequencing magnetic beads, a second fused droplet is obtained, and the second fused droplet is collected.

After the drug treatment is completed, small liquid drops which encapsulate the sequencing magnetic beads and the cell lysate are added into the chip, and are fused with the first fused liquid drops to obtain second fused liquid drops, so that the cell lysis and the capture of mRNA and molecular codes are completed.

S500 demulsification, library establishment and sequencing

In this example, the second fusion droplet is demulsified, the magnetic beads are collected, nucleic acids carried on the magnetic beads are subjected to library construction and sequencing, and the target drug is screened based on the sequencing result.

According to an embodiment of the invention, a method of collecting a second fused droplet comprises: the micro-well array chip is turned over by 180 degrees, so that the openings of the large micro-well and the small micro-well are upward, and oil is added into the micro-well array chip, so that the second fused liquid drops flow out of the large micro-well into the collecting container.

The micro-well array chip comprises a micro-well array layer and a channel layer which are arranged in a stacked mode, wherein large micro-wells and small micro-wells are arranged on the micro-well array layer, and micro-well openings face to grooves which are arranged on the channel layer and are used for flowing liquid.

Because the density of the aqueous phase is lower than that of the oil phase, the second fused droplets float above the grooves and cannot be carried away by oiling into the grooves. Therefore, the chip needs to be flipped 180 ° so that the openings of the large and small micro-wells are up so that the second fused droplet will float in the recess and the droplet detached from the micro-well can be pushed out with oil and collected in a centrifuge tube. And then demulsification recovery magnetic beads are subjected to a subsequent single-cell library building process, and the corresponding relationship between single cells and medicines is obtained through resolution of sequencing information, so that high-throughput screening of medicines on the single cell level is realized.

The method for screening drugs of the present invention may also have the following advantages:

1) High flux: by additionally introducing molecular codes, the codes of various drug conditions (including drug types, drug concentrations and the like) can be realized;

2) Time and labor saving: by utilizing the droplet microfluidic technology, the reagent dosage can be reduced, complicated manual operation can be avoided, and the drug screening efficiency is improved;

3) The accuracy is high: the invention aims at single cell analysis, and the corresponding relation between the single cell transcriptome and the medicine can be obtained by introducing molecular codes and combining single cell sequencing means, so that the influence of the medicine on the single cell transcriptome under different conditions is displayed more accurately, and the research value is higher.

Micro-well array chip

In another aspect of the invention, the invention provides a micro-well array chip. According to an embodiment of the present invention, referring to fig. 2, the micro well array chip includes: a micro well array layer 100 and a channel layer 200.

The micro-well array layer is provided with a plurality of micro-well combinations formed by adjacent large micro-wells 110 and small micro-wells 120, and the aperture of the large micro-wells 110 is larger than that of the small micro-wells 120. The drug droplets and the magnetic bead droplets are small droplets, and the cell droplets are large droplets. The micro-well array layer is provided with communicated micro-wells with different apertures, the micro-wells with small apertures can capture small liquid drops, and the micro-wells with large apertures can capture large liquid drops.

According to the embodiment of the invention, the aperture of the large micro-well is 80-100 micrometers, and the depth is 60-80 micrometers; the aperture of the small micro-well is 40-60 micrometers, and the depth is 60-80 micrometers. Thus, large micro-wells can capture single cell droplets, and small micro-wells can capture drug droplets and magnetic bead droplets.

Referring to fig. 3 and 4, a channel layer 200 is stacked with the micro well array layer 100, and grooves 210 are provided on the channel layer, and openings of the large micro well 110 and the small micro well 120 face the grooves 210. The micro wells and the grooves of the micro well array layer are subjected to surface plasma treatment in advance and can be bonded into a whole, so that leakage of added liquid is avoided, liquid drops are added into the grooves, and the micro wells can capture the liquid drops under the action of buoyancy due to the fact that the liquid drops are lighter than oil.

According to an embodiment of the present invention, the groove 210 is connected to the large micro well 110 or the small micro well 120 by a chemical bond. Thus, so that the droplets flowing into the grooves can be captured by the micro-wells.

Specifically, before the liquid drop is required to be added, two holes, namely a liquid adding port and a liquid outlet, are drilled on the micro-well array layer, are opposite to the groove, the liquid drop is added into the groove through the liquid adding port, the liquid is captured by the micro-well, and then oil is added into the groove through the liquid adding port, so that the liquid drop which is not captured in the groove is washed away, and the liquid drop is sucked out from the liquid outlet.

According to an embodiment of the present invention, a micro-well array chip is used to perform the method of screening drugs described above. The features and advantages described above for the method of screening drugs are equally applicable to the micro-well array chip and are not described here.

The scheme of the present invention will be explained below with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

Example 1

Step one, this embodiment employs a droplet generation device similar to CN209144161U, in which the droplet generation chip is replaced with the chip of patent WO2020063864A1, the syringe is replaced with a BD 30ml syringe, the droplet generation schematic diagram is shown in fig. 5, and the following steps are referred to generate cell droplets, drug droplets, and sequencing magnetic bead droplets.

1. Design of molecular coding

The molecular codes used in the invention are based on exogenous sequence information introduced in the invention patent of International patent WO2021147069A1, and M multiplied by N codes (M=410 and N=410) are used in total. By synthesizing the sequence as a drug code, one drug under one condition corresponds to one code.

2. Preparation of drug droplets

And mixing a certain amount of molecular codes with corresponding medicines, injecting the mixture into a liquid drop generating chip to generate liquid drops with uniform molecular codes, and the like to generate liquid drops with different molecular codes. Finally, the generated liquid drops are collected and mixed in the same collecting pipe, and the preparation of the liquid drops of the medicine is completed.

3. Preparation of single cell droplets

And injecting a certain amount of cell suspension into the liquid drop generating chip, regulating the cell concentration to ensure that the ratio of wrapping single cells in the liquid drops is high and the double-wrapping rate is ensured to be at a lower level, and then separating the generated liquid drops by using a dielectrophoresis method and other methods through the cell separation chip to obtain the liquid drops containing the single cells.

4. Preparation of sequencing magnetic bead droplets

Injecting a certain amount of sequencing magnetic bead suspension (obtained by resuspending sequencing magnetic beads by using cell lysate) into a droplet generation chip, regulating the concentration of the sequencing magnetic beads to ensure that the ratio of single sequencing magnetic beads packed in the droplets is higher and simultaneously ensure that the double packing rate is at a lower level, and then sorting the generated droplets by using a dielectrophoresis method by using a sorting chip to obtain the droplets containing the single sequencing magnetic beads.

Step two, the present embodiment captures droplets using a micro-well chip as shown in fig. 1 to 4, where the micro-well chip includes a micro-well array layer and a channel layer, and the micro-well array layer has 28800 connected micro-wells (with apertures of 90 micrometers and 50 micrometers, respectively), and the micro-well depth is 70 micrometers, so that it can be ensured that one large micro-well can capture only one large droplet, and one small micro-well can capture only one small droplet, thereby realizing droplet 1: 1. Because the density of the water phase is lower than that of the oil phase, the liquid drops float on the upper layer of the oil phase, the chip is turned over by 180 degrees in actual use for capturing the liquid drops, the micro-well heads are flushed down, the micro-well heads are bonded with the channel layer shown in fig. 2 through surface plasma treatment, and at the moment, more than 25000 effective micro-wells are contained in the channel. Two holes, namely a liquid filling hole and a liquid outlet, are punched on the micro-well array layer by using a puncher with the aperture of 1.5 mm.

First, single cell droplets are slowly injected into the micro-well array chip (a of fig. 6), and then the injection oil pushes away the excess droplets, and the droplet state at this time is shown as b of fig. 6. Then slowly injecting the encoded liquid drops of the medicine, injecting oil to push away redundant liquid drops after the liquid drops are captured by the micro-wells, wherein the liquid drop states at the moment are shown in the c of fig. 6 and the left diagram of fig. 7, and the liquid drop capturing efficiency is more than 95%. And finally, placing the chip on a shaking table to slightly shake so as to enable the same group of liquid drops to collide, and at the same time, using a corona machine or a chemical reagent perfluorobutanol to perform liquid drop fusion, wherein the state of the liquid drops is shown in the right graph of fig. 6 d and fig. 7, and the small micro-wells can be emptied due to the liquid drop fusion, so that the fusion efficiency is more than 80%. After droplet fusion, the chip was placed in an incubator for incubation.

And thirdly, after the incubation of the liquid drops is completed, taking out the microfluidic chip, then injecting the liquid drops into the sequencing magnetic bead liquid drops, capturing the liquid drops again by the small empty micro-wells, pushing out the redundant liquid drops by using oil, and enabling the state of the liquid drops to be shown as e in fig. 6. And finally, repeating the fusion process of the second liquid drops, wherein the state of the liquid drops is shown as f in fig. 6.

And fourthly, in order to recover the droplets after the secondary fusion, the micro-well array chip is required to be turned over for 180 degrees, then the droplets separated from the micro-wells are pushed out of the chip by using oil, and the droplets are collected in a centrifuge tube. Since the droplets of the sequencing beads contain cell lysate, once the droplets are fused twice, the cells are lysed to release intracellular mRNA, as shown in FIG. 8, and the sequencing beads capture all the nucleic acid information including mRNA and molecular codes. Finally, a Huada single cell sequencing flow is carried out, wherein the reaction liquid of the purified secondary specificity amplification drug code is subjected to gel running verification, the experimental result is shown in figure 9, and an obvious peak is formed at 168bp, namely a sequence corresponding to the molecular code, so that the invention can carry out drug molecular code detection through a single cell sequencing technology.

In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.

While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.

Claims

A method of screening for a drug comprising:

(1) Providing a first droplet, a second droplet, a third droplet and a micro-well array chip;

The first liquid drop is a mixed liquid drop containing a plurality of different molecule coding liquid drops, the second liquid drop is a liquid drop containing single cells, the third liquid drop is a liquid drop containing single sequencing magnetic beads and cell lysate, each molecule coding liquid drop contains a medicine and coding nucleic acid molecules matched with the medicine, a plurality of micro-well combinations formed by adjacent and communicated large micro-wells and small micro-wells are arranged on a micro-well array core, and the aperture of the large micro-well is larger than that of the small micro-well;

(2) Firstly adding the second liquid drop into the micro-well array chip and falling into a large micro-well, and then adding the first liquid drop into the micro-well array chip and falling into a small micro-well;

(3) Fusing the first liquid drop and the second liquid drop to obtain a first fused liquid drop, wherein the first fused liquid drop occupies the large micro-well, the small micro-well is vacated, and the micro-well array chip is subjected to cell culture;

(4) After the cell culture is completed, adding the third liquid drop into the vacated small micro well, fusing the third liquid drop with the first fused liquid drop after the cell culture, breaking cells under the action of a cell lysate in the third liquid drop, capturing nucleic acid molecules in the cells and the encoding nucleic acid molecules together by a sequencing magnetic bead to obtain a second fused liquid drop, and collecting the second fused liquid drop;

(5) And demulsifying the second fusion liquid drop, collecting magnetic beads, constructing a library and sequencing nucleic acid carried on the magnetic beads, and screening out a target medicament based on a sequencing result.
The method of claim 1, wherein the second and third droplets are sorted by a sorting chip.
The method of claim 1, wherein the fusion is electrofusion or chemical fusion.
The method of claim 1, wherein the large micro-wells have a pore size of 80-100 microns and a depth of 60-80 microns;

The aperture of the small micro-well is 40-60 micrometers, and the depth is 60-80 micrometers.
The method of claim 1, wherein the sequencing magnetic beads are adapted to capture nucleic acids.
The method of claim 1, wherein prior to step (2), the micro-well array chip provided in step (1) is subjected to a surface plasma treatment to bond grooves in the micro-well array chip for droplet flow with the large and small micro-wells so as to capture droplets.
The method of claim 1, wherein the method of collecting the second fused droplets comprises:

And turning the micro-well array chip by 180 degrees so that the openings of the large micro-well and the small micro-well are upward, and adding oil into the micro-well array chip so that the second fused liquid drops flow out of the large micro-well into a collecting container.
A micro-well array chip, comprising:

the micro-well array layer is provided with a plurality of micro-well combinations formed by adjacent and communicated large micro-wells and small micro-wells, and the aperture of the large micro-wells is larger than that of the small micro-wells;

The channel layer is arranged in a lamination mode with the micro-well array layer, grooves are formed in the channel layer, and openings of the large micro-wells and the small micro-wells face the grooves.
The micro well array chip of claim 8, wherein the large micro well has a pore size of 80-100 microns and a depth of 60-80 microns.
The micro well array chip of claim 8, wherein the small micro wells have a pore size of 40-60 microns and a depth of 60-80 microns.
The micro well array chip of claim 8, wherein the groove is connected to the large micro well or the small micro well by a chemical bond.
The micro well array chip of claim 8, wherein the micro well array chip is used to perform the method of screening for a drug of any one of claims 1 to 7.