Micro-fluidic liquid drop platform for single cell RNA sequencing and application of micro-fluidic liquid drop platform

Abstract

The invention relates to the technical field of RNA (Ribonucleic Acid) sequencing, in particular to a micro-fluidic liquid drop platform for single-cell RNA sequencing and application of the micro-fluidic liquid drop platform. The invention provides a microfluidic liquid drop platform for single cell RNA sequencing, the platform is based on a PDMS chip, and the platform comprises a cell specificity separation and enrichment module, a single cell liquid drop generation module and a pairing fusion module which are arranged in sequence, the pairing fusion module is a module for pairing and fusing single-cell liquid drops and magnetic nanoparticle liquid drops with DNA tag sequences one by one. The high-quality to-be-detected sample library can be prepared in a short time in a high-purity and high-throughput manner by only needing a small number of to-be-detected samples, so that rapid and high-quality sequencing prediction steps are achieved, finally single-cell transcriptome analysis of the to-be-detected samples is achieved, the requirement for the single-cell sample size is met, analysis of large-scale single-cell genetic information is facilitated, and the application prospect is wide. And a foundation is laid for researching heterogeneity and clone evolution of the sample.

Description

Technical Field

[0001] This application belongs to the field of RNA sequencing technology, and in particular relates to a microfluidic droplet platform for single-cell RNA sequencing and its application. Background Technology

[0002] Neuroblastoma is a leading cause of cancer death in infants and young children, characterized by its high progressiveness and aggressiveness, with a strong tendency to invade other organs. This metastatic tendency often results in initial diagnosis at an intermediate or advanced stage of cancer progression. In these cases, tumor heterogeneity is a major obstacle to developing new early detection and treatment methods for childhood neuroblastoma. Tumor heterogeneity exists not only at the cell cluster level but also at the single-cell level. Single-cell analysis can overcome the neglect of intercellular heterogeneity and clonal evolution in the most common clinical diagnostic method, which focuses on cell clusters. Therefore, single-cell analysis is significant in analyzing the oncological characteristics of patients and shows translational potential in clinical applications. Although emerging methods such as RNA sequencing (RNA-seq) have been used to obtain genetic information from tumor cell clusters, the underlying origins of childhood neuroblastoma and the phenotypic diversity at single-cell resolution remain unclear.

[0003] In recent years, RNA-seq has become an important strategy for gene expression and transcriptome analysis. However, current analysis samples typically focus on the multicellular level, neglecting the genetic characteristics of single cells. Furthermore, the scarcity of target samples such as circulating tumor cells (CTCs) results in insufficient sample sizes for sequencing analysis.

[0004] Therefore, there is an urgent need to provide a new strategy for analyzing pediatric neuroblastoma at single-cell resolution to achieve high-throughput single-cell RNA sequencing. Summary of the Invention

[0005] The purpose of this application is to provide a microfluidic droplet platform for single-cell RNA sequencing and its application, aiming to solve the problems of unbiased single-cell separation, low RNA capture efficiency and purification difficulties in existing RNA sequencing platforms, which make it impossible to perform effective sequencing analysis of cells at single-cell resolution.

[0006] To achieve the above-mentioned objectives, the technical solution adopted in this application is as follows:

[0007] In a first aspect, this application provides a microfluidic droplet platform for single-cell RNA sequencing. The microfluidic droplet platform is based on a PDMS chip and includes a cell-specific separation and enrichment module, a single-cell droplet generation module, and a pairing and fusion module arranged sequentially. The pairing and fusion module is a module that pairs and fuses single-cell droplets with magnetic nanoparticle droplets carrying DNA tag sequences one by one.

[0008] In some embodiments, the single-cell droplet generation module includes a "water-in-oil" single-cell droplet generation module and a droplet generation module encapsulating encoded nanoparticles, wherein the "water-in-oil" single-cell droplet generation module includes a cross channel, which includes separately arranged aqueous phase channels and oil phase channels.

[0009] In some embodiments, the single-cell droplet generation module utilizes a cross-channel pressure pump to control the driving pressure of the aqueous phase channel and the driving pressure of the oil phase channel, and adjusts the driving pressure ratio between the water and oil phases to generate "water-in-oil" single-cell droplets.

[0010] In some embodiments, the ratio of cell number to droplet number in the aqueous phase channel is 0.5 to 0.6.

[0011] In some embodiments, the magnetic nanoparticles with a DNA tag sequence have the DNA tag sequence as shown in Seq. No. 1, and the magnetic nanoparticles include a magnetic inner shell structure and a gold coating layer disposed on the outer surface of the magnetic inner shell structure.

[0012] In some embodiments, the pairing and fusion module includes a Y-shaped channel and a droplet deceleration region connected to the outlet of the Y-shaped channel in sequence; wherein the droplet deceleration region includes an integrated electrode.

[0013] In some embodiments, the external electric field generated by the integrated electrode is an alternating electric field with a sinusoidal signal, a frequency of 1.5 to 1.6 MHz, and a voltage of 5 to 6 V; and the alternating electric field is connected to a power amplifier, which amplifies the power by 50% to 70%.

[0014] In some embodiments, the pressure of the Y-channel is 50–100 mbar.

[0015] In some embodiments, the single-cell magnetic nanoparticle complex with DNA tag sequence obtained in the pairing fusion module is sequenced by a sequencing system that is independent of the microfluidic droplet platform.

[0016] In some embodiments, a method for RNA sequencing of single cells using a microfluidic droplet platform includes the following steps:

[0017] The sample to be tested was added to the cell-specific separation and enrichment module for sequential cell separation and cell enrichment to obtain a cell suspension.

[0018] The cell suspension and oil phase solution to be tested are added to the single-cell droplet generation module. The driving pressure is controlled by the multi-channel pressure pump in the single-cell droplet generation module to generate "water-in-oil" single-cell droplets.

[0019] Magnetic nanoparticles with DNA tag sequences are provided. Water-in-oil single-cell droplets and magnetic nanoparticle droplets with DNA tag sequences are added to the pairing and fusion module respectively. The pressure of each channel drives the flow to pair them one by one. Then, the fusion is achieved by the external electric field generated by the integrated electrode to obtain the single-cell-magnetic nanoparticle complex with DNA tag sequence to be tested.

[0020] The RNA of the single-cell-to-analyte magnetic nanoparticle complex with DNA tag sequence was sequenced using a sequencing system.

[0021] The first aspect of this application provides a microfluidic droplet platform for single-cell RNA sequencing, comprising a specific cell-specific separation and enrichment module capable of achieving high-specificity and high-throughput separation of the target cells; a single-cell droplet generation module capable of generating single-cell encapsulated droplets in high throughput; and a pairing and fusion module for pairing and fusion. The platform includes magnetic nanoparticle droplets with DNA tag sequences for pairing. On one hand, the DNA tag sequences can pair one-to-one with the RNA strands in the single cells for RNA capture and library preparation, and improve the barcode encoding efficiency of single cells. On the other hand, it provides... The nanoparticles possess a magnetic structure, which enables magnetic separation of RNA from cell lysis mixtures by applying an external magnetic field, significantly improving separation purity. Therefore, the microfluidic droplet platform for single-cell RNA sequencing provided in this application requires only a small amount of sample to achieve high-quality sample library preparation in a short time, with high purity and high throughput, enabling rapid and high-quality prediction sequencing steps. Ultimately, it realizes single-cell transcriptome analysis of the sample, meeting the requirements for single-cell sample size and facilitating large-scale analysis of single-cell genetic information, laying the foundation for studying sample heterogeneity and clonal evolution. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 This is a schematic diagram of a microfluidic droplet platform for single-cell RNA sequencing provided in an embodiment of this application; wherein, a is a cell-specific separation and enrichment module; b is a single-cell droplet generation module and a pairing and fusion module.

[0024] Figure 2These are diagrams illustrating the steps and results of the single-cell droplet generation module provided in this application embodiment; wherein, a is a schematic diagram of the microfluidic chip used for droplet generation, including the oil phase inlet on the left (white arrow), the aqueous phase inlet in the middle (gray arrow), and the droplet collection port on the right; purple inset: a snapshot of the emulsification of BCO nanoparticles (left) and isolated circulating tumor cells (right); orange inset: a fluorescence image showing the streamlines in the asymmetric channel used for single-cell focusing; b is a microscope image of single-cell droplet encapsulation, bright field (top) and fluorescence (bottom); c is the relationship between the oil-water two-phase pressure difference and the droplet size; d is the encapsulation efficiency of single cells at different cell densities.

[0025] Figure 3 This is a schematic diagram of the BCO-Fe3O4@Au nanoparticle synthesis process and single-cell mRNA extraction workflow provided in the embodiments of this application.

[0026] Figure 4 These are analytical diagrams of the steps in the pairing and fusion module provided in this application embodiment; a is a microscopic image of the droplet pairing process, with type A droplets in the upper channel, spacer oil in the middle channel, and type B droplets in the lower channel; b is a microscopic image of the time-lapse process of the paired droplet merging. The paired droplets enter the fusion region at a reduced flow rate, and an external electric field triggers the fusion process in the fusion region, followed by the outflow of a larger fused droplet.

[0027] Figure 5 Characterization of the synthesized gold-coated magnetic nanoparticles provided in the embodiments of this application; a is a transmission electron microscope (TEM) image of Fe3O4 nanoparticles, with the inset showing the size distribution of the nanoparticles; b is a TEM image of Fe3O4@Au nanoparticles, with the inset showing the size distribution of the nanoparticles; c is the zeta potential of Fe3O4 nanoparticles and Fe3O4@Au nanoparticles; d is the hydrodynamic size distribution of Fe3O4 nanoparticles and Fe3O4@Au nanoparticles; e is the ultraviolet-visible (UV-vis) spectrum of Fe3O4 nanoparticles and Fe3O4@Au nanoparticles.

[0028] Figure 6 The following are characterizations of the BCO-Fe3O4@Au nanoparticles provided in the embodiments of this application: a) is the binding ratio of BCO-Fe3O4@Au nanoparticles under different reaction ratios; b) is the weight ratio of BCO to nanoparticles in BCO-Fe3O4@Au nanoparticles under different reaction ratios; c) is the normalized ultraviolet-visible spectrum of BCO-Fe3O4@Au nanoparticles under different reaction ratios; d) is the transmission electron microscope (TEM) image of BCO(1:2)-Fe3O4@Au nanoparticles.

[0029] Figure 7The capture rates of BCO(1:2)-Fe3O4@Au nanoparticles and BCO(1:5)-Fe3O4@Au nanoparticles provided in the embodiments of this application at different nanoparticle:poly(A) ratios are given. Detailed Implementation

[0030] To make the technical problems, technical solutions, and beneficial effects of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0031] In this application, the term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. A and B can be singular or plural. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0032] In this application, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single or multiple items. For example, "at least one of a, b, or c", or "at least one of a, b, and c", can both mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, and c can be single or multiple.

[0033] It should be understood that in the various embodiments of this application, the order of the above processes does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0034] The terminology used in the embodiments of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of this application. The singular forms "a" and "the" as used in the embodiments of this application and the appended claims are also intended to include the plural forms, unless the context clearly indicates otherwise.

[0035] The weights of the relevant components mentioned in the embodiments of this application can refer not only to the specific content of each component, but also to the proportional relationship between the weights of the components. Therefore, any scaling up or down of the content of the relevant components according to the embodiments of this application is within the scope disclosed in the embodiments of this application. Specifically, the mass in the embodiments of this application can be a well-known unit of mass in the chemical industry, such as μg, mg, g, or kg.

[0036] The terms "first" and "second" are used for descriptive purposes only, to distinguish objects, such as substances, from one another, and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. For example, without departing from the scope of the embodiments of this application, "first XX" may also be referred to as "second XX," and similarly, "second XX" may also be referred to as "first XX." Thus, features defined with "first" and "second" may explicitly or implicitly include one or more of that feature.

[0037] The first aspect of this application provides a microfluidic droplet platform for single-cell RNA sequencing. The microfluidic droplet platform is based on a PDMS chip and includes a cell-specific separation and enrichment module, a single-cell droplet generation module, and a pairing and fusion module arranged sequentially. The pairing and fusion module is a module that pairs and fuses single-cell droplets with magnetic nanoparticle droplets carrying DNA tag sequences one by one.

[0038] The first aspect of this application provides a microfluidic droplet platform for single-cell RNA sequencing, comprising a specific cell-specific separation and enrichment module capable of achieving high-specificity and high-throughput separation of the target cells; a single-cell droplet generation module capable of generating single-cell encapsulated droplets in high throughput; and a pairing and fusion module for pairing and fusion. Magnetic nanoparticle droplets with DNA tag sequences are provided for pairing. On the one hand, the DNA tag sequences can pair one-to-one with the RNA chains in the single cells for RNA capture and library preparation, and improve the barcoding efficiency of single cells; on the other hand, it improves... The provided nanoparticles have a magnetic structure. When RNA is separated from the cell lysis mixture, the magnetic structure can achieve magnetic separation by applying an external magnetic field, which significantly improves the separation purity. Therefore, the microfluidic droplet platform for single-cell RNA sequencing provided in this application only requires a small amount of sample to prepare a high-quality sample library in a short time with high purity and high throughput, so as to achieve a rapid and high-quality prediction sequence step, and finally realize single-cell transcriptome analysis of the sample. This meets the requirements of single-cell sample size and helps to analyze large-scale single-cell genetic information, laying the foundation for studying sample heterogeneity and clonal evolution.

[0039] Single-cell RNA sequencing typically involves three steps: single-cell isolation, library construction, and sequencing. Traditional single-cell isolation methods include limiting dilution of target cells and manual separation under a microscope using micropipettes. However, these methods are time-consuming and have low throughput, making them unsuitable for large-scale screening. Emerging high-throughput strategies, such as fluorescence-activated cell sorting (FACS) and magnetically activated cell sorting (MACS), can specifically isolate single cells based on labeled cell surface markers. However, these strategies require expensive equipment and additional steps for RNA extraction, reverse transcription, and library construction. Therefore, the microfluidic platform provided in this application integrates multiple functional modules, offering advantages such as high throughput, specific single-cell isolation, and droplet generation.

[0040] The embodiments of this application are mainly aimed at obtaining circulating tumor cells from blood samples of children with neuroblastoma. The first step of the microfluidic droplet platform for single-cell RNA sequencing is to set up a cell-specific separation and enrichment module, which is mainly used to separate and enrich circulating tumor cells from blood samples.

[0041] In some embodiments, the cell-specific separation and enrichment module includes a cell enrichment zone, a cell separation zone, and a cell capture zone. One end of the cell enrichment zone has one or more inlets, and the other end has a waste liquid outlet and an enrichment liquid outlet. One end of the cell separation zone has a buffer inlet and an enrichment liquid inlet connected to the enrichment liquid outlet of the cell enrichment zone, and the other end has an outlet. One end of the cell capture zone has an inlet connected to the outlet of the cell separation zone, and the other end has a separation liquid outlet. The cell fluid to be separated flows into the cell enrichment zone through the inlet, increasing the concentration of target cells in the cell fluid. The enrichment liquid flowing out of the cell enrichment zone flows into the cell separation zone through the enrichment liquid inlet and the buffer solution through the buffer solution inlet, allowing the incoming cells to be separated according to size. The cells separated by size enter the cell capture zone, which captures the target cells.

[0042] Furthermore, the enriched circulating tumor cell fluid is processed through a single-cell droplet generation module to prepare "water-in-oil" single-cell droplets. The single-cell encapsulation process utilizes the water-in-oil process (i.e., emulsification) at the cross-shaped micron structure. Specifically, it uses the oil phase liquid to shear the water phase liquid to achieve droplet formation and encapsulate the cells in the water phase.

[0043] In some embodiments, the single-cell droplet generation module includes a "water-in-oil" single-cell droplet generation module and a droplet generation module encapsulating encoded nanoparticles, wherein the "water-in-oil" single-cell droplet generation module includes a cross channel, which includes separately arranged aqueous phase channels and oil phase channels.

[0044] The flow of fluid in the water phase channel and the oil phase channel is controlled by a multi-channel pressure pump.

[0045] In some embodiments, the "water-in-oil" single-cell droplet generation module includes a cross channel. In the cross channel, a pressure pump is used to drive the driving pressure of the aqueous phase channel and the driving pressure of the oil phase channel respectively, and to regulate the ratio of the driving pressures of the water and oil phases to generate "water-in-oil" single-cell droplets.

[0046] Furthermore, the encapsulation efficiency of a single cell is determined by the cell density in the suspension. To improve the encapsulation efficiency, cells are further focused through asymmetric channels before entering the emulsion region. Their distribution roughly follows a Poisson distribution:

[0047] f(n,λ)=λ^ke^(-λ) / k!

[0048] Where n is the number of cells in a single droplet, and λ is the cell density in the suspension.

[0049] In some embodiments, the droplet size can be precisely controlled by the pressure ratio between the water and oil phases. By adjusting the pressure between the two phases, single cells enter the interface between the two phases to form single-cell droplets. Furthermore, within a certain pressure ratio range, the droplet size exhibits a negative linear relationship with the pressure difference between the two phases.

[0050] In some embodiments, the aqueous phase comprises the enriched circulating tumor cell fluid. The oil phase is 3M NOVEC 7500 fluorinated oil containing 2% dSURF surfactant.

[0051] Unlike chemical reactions, droplet formation and encapsulation are physical processes, and there are no specific requirements for their proportions. Generally, it consists of 1 mL of oil phase in a pressure storage tank and 1 mL-2 mL of liquid phase in a pressure storage tank.

[0052] In some embodiments, the ratio of cell number to droplet number in the aqueous channel is 0.5 to 0.6. When the ratio of cell number to droplet number is 0.5, the single-cell encapsulation efficiency reaches a relatively high level.

[0053] In some embodiments, in the droplet generation module that encapsulates encoded nanoparticles, droplets of magnetic nanoparticles with DNA tag sequences are generated.

[0054] Current single-cell RNA sequencing technologies mostly use DNA-tagged sequence microbeads for RNA extraction. However, due to the limited specific surface area of ​​these microbeads, the RNA extraction efficiency and single-cell barcoding efficiency are unsatisfactory. Furthermore, after extraction, current technologies still face difficulties in separating the DNA-tagged sequence microbeads from free proteins and nucleic acids. In this application, magnetic nanoparticles carrying DNA tags provide a barcode sequence comprising a universal sequence, followed by a cell barcode (BCO), then a unique molecular identifier (UMI), and finally a dT sequence for capturing mRNA. The provided DNA tag sequence has a high density, and the nanoparticles have a large specific surface area, enabling one-to-one pairing with RNA chains in single cells, resulting in efficient RNA capture and library preparation, and improving the barcoding efficiency of single cells.

[0055] In some embodiments, in the magnetic nanoparticles with the DNA tag sequence, the DNA tag sequence is as shown in Seq. No. 1, specifically, Seq. No. 1 is shown below:

[0056] 5'- / SH / -TTTTTTTAAGCAGTGGTATCAACGCAGAGTACnnnnnNNNNNNNTTTTTTTTTTTTTTTTTTTTTTTTTTT-3';

[0057] In this code, nnnnn represents a 5-base cell barcode, and NNNNNNNN represents an 8-base UMI; there is a -SH group at the 5' end to facilitate labeling on gold-plated nanoparticles.

[0058] In some embodiments, the magnetic nanoparticles carrying the DNA tag sequence include a magnetic inner shell structure and a gold coating layer disposed on the outer surface of the magnetic inner shell structure. The provided nanoparticles have a magnetic structure, which enables magnetic separation of RNA from a cell lysis mixture by applying an external magnetic field, significantly improving separation purity.

[0059] In some embodiments, the magnetic nanoparticles with DNA tag sequences have a core of iron(III) oxide (Fe3O4) and a shell of gold. This core-shell structure enhances the dispersibility and antioxidant properties of Fe3O4; when separating RNA from cell lysis mixtures, the magnetic core can achieve magnetic separation by applying an external magnetic field; and a gold coating layer is incorporated. Gold (Au) was chosen as the shell due to its excellent low toxicity, biocompatibility, suitability for various surface modifications, and better dispersibility and antioxidant properties, ultimately leading to the development of various nanoparticles. The designed magnetic nanoparticles with cellular DNA tag sequences solve the problem of low RNA extraction, separation, and enrichment efficiency, and exhibit superior biochemical performance.

[0060] In some implementations, the average size of Fe3O4 nanoparticles ranges from 7.8 to 13.4 nanometers. Excessive particle size reduces the surface area, hindering subsequent mRNA capture; conversely, smaller particle size leads to decreased magnetism. With the addition of a gold coating, the overall average particle size ranges from 15.9 to 26.3 nanometers.

[0061] Furthermore, the DNA tag sequence was modified onto the surface of Fe3O4@Au nanoparticles. The preparation method included mixing the DNA tag sequence with tris(2-carboxyethyl)phosphine (TCEP) at room temperature for one hour, mainly to activate the thiol groups on the DNA tag sequence.

[0062] The DNA tag sequence was mixed with Fe3O4@Au nanoparticles, and sodium dodecyl sulfate (SDS) and TAE buffer were added to convert the system to 0.01% SDS and 1×TAE buffer.

[0063] Add sodium chloride (NaCl) solution and react for 30 minutes. Repeat this step ten times to make the NaCl concentration in the system reach 0.5M. Wash the nanoparticles to obtain barcoded Fe3O4@Au nanoparticles (BCO-Fe3O4@Au nanoparticles).

[0064] Furthermore, droplet pairing and fusion were performed on BCO Fe3O4@Au nanoparticles for single-cell mRNA extraction.

[0065] Furthermore, the obtained single-cell droplets to be tested need to pass through a pairing and fusion module, which is a module that pairs and fuses single-cell droplets with magnetic nanoparticle droplets carrying DNA tag sequences one by one.

[0066] In some embodiments, the pairing and fusion module includes a Y-shaped channel arranged sequentially and a droplet deceleration region connected to the outlet of the Y-shaped channel; wherein the droplet deceleration region includes an integrated electrode. Two generated and collected droplets pass sequentially through the Y-shaped channel to form droplet pairs, and the distance between each pair of droplets is controlled by a corresponding interval channel before entering the Y-shaped channel; the droplets pass sequentially through the Y-shaped channel and are controlled by a pressure pump in their respective flow channels; the fusion between droplets is achieved by an external electric field generated by the integrated electrode in the deceleration region.

[0067] In some embodiments, the external electric field generated by the integrated electrode is an alternating electric field with a sinusoidal signal, a frequency of 1.5 to 1.6 MHz, and a voltage of 5 to 6 V; and the alternating electric field is connected to a power amplifier, which amplifies the power by 50% to 70%.

[0068] In some embodiments, the pressure of the Y-channel is 50–100 mbar.

[0069] In some embodiments, the single-cell magnetic nanoparticle complex with DNA tag sequence obtained in the pairing fusion module is sequenced by a sequencing system that is independent of the microfluidic droplet platform.

[0070] In some embodiments, a method for RNA sequencing of single cells using a microfluidic droplet platform includes the following steps:

[0071] S01. Add the sample to be tested into the cell-specific separation and enrichment module to perform cell separation and cell enrichment treatment in sequence to obtain a cell suspension.

[0072] S02. The cell suspension and oil phase solution to be tested are added to the single-cell droplet generation module. The driving pressure is controlled by the multi-channel pressure pump in the single-cell droplet generation module to generate "water-in-oil" single-cell droplets.

[0073] S03. Provide magnetic nanoparticles with DNA tag sequences, add "water-in-oil" single-cell droplets and magnetic nanoparticle droplets with DNA tag sequences to the pairing and fusion module respectively, use the pressure of their respective channels to drive the flow to pair them one by one, and then achieve fusion through the external electric field generated by the integrated electrode to obtain the single-cell-magnetic nanoparticle complex with DNA tag sequence to be tested.

[0074] S04. The single-cell-to-be-tested magnetic nanoparticle complex with DNA tag sequence is subjected to RNA sequencing using a sequencing system.

[0075] The following description is based on specific embodiments.

[0076] Example 1

[0077] Microfluidic Droplet Platform for Single-Cell RNA Sequencing and Its Applications

[0078] like Figure 1 As shown, the microfluidic droplet platform is based on a PDMS chip and includes a cell-specific separation and enrichment module, a single-cell droplet generation module, and a pairing and fusion module arranged sequentially. The pairing and fusion module is a module that pairs and fuses single-cell droplets with magnetic nanoparticle droplets carrying DNA tag sequences one by one.

[0079] A method for RNA sequencing of single cells using a microfluidic droplet platform includes the following steps:

[0080] (1) The sample to be tested is added to the cell-specific separation and enrichment module for sequential cell separation and enrichment treatment to obtain a cell suspension to be tested. Specifically, the cell-specific separation and enrichment module includes a cell enrichment zone, a cell separation zone, and a cell capture zone. One end of the cell enrichment zone is provided with one or more inlets, and the other end is provided with a waste liquid outlet and an enrichment liquid outlet. One end of the cell separation zone is provided with a buffer inlet and an enrichment liquid inlet connected to the enrichment liquid outlet of the cell enrichment zone, and the other end is provided with an outlet. One end of the cell capture zone is provided with an inlet connected to the outlet of the cell separation zone, and the other end is provided with a separation liquid outlet. The cell fluid to be separated flows in from the inlet of the cell enrichment zone and enters the cell enrichment zone. The cell enrichment zone can increase the concentration of target cells in the cell fluid. The enrichment liquid flowing out from the cell enrichment zone flows into the cell separation zone through the enrichment liquid inlet and the buffer solution flows into the cell separation zone through the buffer solution inlet. The cell separation zone can separate the incoming cells according to their size. The cells separated according to their size enter the cell capture zone, which can capture the target cells.

[0081] (2) The cell suspension and oil phase solution to be tested are added to the single cell droplet generation module. The driving pressure is controlled by the multi-channel pressure pump in the single cell droplet generation module to generate "water-in-oil" single cell droplets.

[0082] This module consists of a set of cross-shaped channels, including a single-cell suspension channel (aqueous phase) and a pair of channels for droplet generation (oil phase). The flow of the two-phase fluid is controlled by a multi-channel pressure pump, and the encapsulation efficiency of a single cell is determined by the cell density in the suspension. To improve the encapsulation efficiency, cells are further focused through asymmetric channels before entering the emulsion region. Their distribution roughly follows a Poisson distribution: f(n,λ)=λ^ke^(-λ) / k!, where n is the number of cells in a single droplet and λ is the cell density in the suspension. The droplet size can be precisely controlled by the pressure ratio between the water and oil phases. By adjusting the pressure between the two phases, single cells enter the junction of the two phases to form single-cell droplets. Figure 2 (a) Within a certain pressure ratio range, the droplet size has a negative linear relationship with the pressure difference between the two phases. Figure 2(c). After optimization, when the cell density is 0.5 ( Figure 2 (d) The single-cell encapsulation efficiency reached a high level. Fluorescence microscopy images showed that most droplets were empty or single-cell (d). Figure 2 (b).

[0083] (3) Provide magnetic nanoparticles with DNA tag sequences, add water-in-oil single cell droplets and magnetic nanoparticle droplets with DNA tag sequences to the pairing and fusion module respectively, use the pressure of their respective channels to drive the flow to pair them one by one, and then achieve fusion through the external electric field generated by the integrated electrode to obtain the single cell-magnetic nanoparticle complex with DNA tag sequence to be tested.

[0084] ① The process involves first synthesizing Fe3O4@Au nanoparticles, including the following steps: 1.79 g of FeCl3·6H2O and 1.10 g of FeCl2·4H2O were dissolved in 40 mL of deionized water (ddH2O). Then, 5.5 mL of 28% NH3·H2O was added while stirring. After 10 minutes, 4.4 g of trisodium citrate dihydrate was added, and the temperature was raised to 90℃ and maintained for 30 minutes. The Fe3O4 nanoparticles (Fe3O4 Nps) were washed twice with acetone and once with deionized water, and then separated using a magnet. These particles were freeze-dried to obtain a powdered product.

[0085] ② To deposit an Au layer on the surface of Fe3O4 Nps, the following steps were taken: 0.2315 mg of Fe3O4 Nps was dispersed in 1 mL of 0.1 M tetramethylammonium hydroxide (TMAOH); then, 0.1 mL of the Fe3O4 / TMAOH solution was added to 100 mL of ddH2O. Subsequently, 3 mL of 0.2 M trisodium citrate dihydrate was added, and the mixture was sonicated for 5 minutes. After stirring for 10 minutes, 0.5 mL of 1 wt% HAuCl4 was slowly added. After 10 seconds, 0.2 mL of 0.2 M NH2OH·HCl was rapidly added, and the reaction was allowed to continue for 20 minutes. The above steps were repeated five times. The gold-coated Fe3O4 Nps (Fe3O4@Au Nps) were washed three times with deionized water (6000 rpm, 30 minutes) and collected using a magnet; after freeze-drying, Fe3O4@Au nanoparticle powder was obtained.

[0086] ③ Further, the DNA tag sequence is modified onto the surface of Fe3O4@Au nanoparticles (e.g., Figure 3 As shown), the DNA tag sequence (BCO) is:

[0087] 5'- / SH / -TTTTTTTAAGCAGTGGTATCAACGCAGAGTACnnnnnNNNNNNNNTTTTTTTTTTTTTTTTTTTTTTTTTTTT-3'. Here, nnnnn represents a 5-base cell barcode, and NNNNNNNN represents an 8-base UMI; the 5' end of BCO has a -SH group to facilitate labeling on gold-plated nanoparticles.

[0088] The preparation method is as follows: BCO was pretreated with tris(2-carboxyethyl)phosphine (TCEP) for one hour to activate its thiol groups. Then, BCO was mixed with Fe3O4@Au nanoparticles. Next, sodium dodecyl sulfate (SDS) and TAE buffer were added to convert the system to 0.01% SDS and 1×TAE buffer. Afterward, sodium chloride (NaCl) solution was added and the reaction was carried out for 30 minutes. This step was repeated ten times to ensure the NaCl concentration in the system reached 0.5M. Finally, the nanoparticles were washed to obtain barcoded Fe3O4@Au nanoparticles (BCO-Fe3O4@Au nanoparticles).

[0089] ④ Add water-in-oil single-cell droplets and magnetic nanoparticle droplets with DNA tag sequences to the pairing and fusion module (e.g., Figure 4 (As shown).

[0090] The pairing and fusion module includes a Y-shaped channel arranged sequentially and a droplet deceleration region connected to the outlet of the Y-shaped channel; the droplet deceleration region includes an integrated electrode. Two generated and collected droplets pass sequentially through the Y-shaped channel, forming droplet pairs. The distance between each pair of droplets is controlled by a corresponding interval channel before entering the Y-shaped channel. The droplets pass sequentially through the Y-shaped channel, controlled by pressure pumps in their respective channels, with pressures ranging from 50 mbar to 100 mbar. The fusion of droplets is achieved through an external electric field generated by the integrated electrode in the deceleration region; this external electric field is an AC electric field with a sinusoidal signal, a frequency of 1.5–1.6 MHz, and a voltage of 5–6 V; furthermore, the AC electric field is connected to a power amplifier, which amplifies the power by 50%–70%.

[0091] (4) The single-cell-to-be-tested magnetic nanoparticle complex with DNA tag sequence was sequenced for RNA using a sequencing system.

[0092] Performance Testing and Result Analysis

[0093] (I) Characterization of the properties of the synthesized gold-coated magnetic nanoparticles.

[0094] The synthesized gold-coated magnetic nanoparticles underwent a series of characterizations. The morphology of the nanoparticles was characterized using transmission electron microscopy (TEM). Figure 5 As shown in figure a, the average size of Fe3O4 nanoparticles is 7.8–13.4 nm. After gold coating, as... Figure 5 As shown in b, the average size increased to 15.9–26.3 nanometers. Meanwhile, as... Figure 5 As shown in d, dynamic light scattering (DLS) further confirms the increased hydration size of Fe3O4@Au nanoparticles. Furthermore, as... Figure 5 As shown in c, the negative charge of the gold-coated magnetic nanoparticles decreases. This can be explained by the fact that the surface of the prepared Fe3O4 nanoparticles was modified with sodium citrate, thus possessing a strong negative charge. Upon addition of HAuCl4, due to electrostatic interactions, positively charged gold ions are reduced on the surface of the Fe3O4 nanoparticles, thereby increasing the zeta potential of the particles, indicating the successful synthesis of Fe3O4@Au nanoparticles. To further verify the synthesis of Fe3O4@Au nanoparticles, the nanoparticles were characterized by ultraviolet-visible spectroscopy (UV-vis). Figure 5 (e) Obviously, after gold coating, the gold nanoparticles exhibit a characteristic absorption peak near 530 nm, indicating that the gold coating on the surface of the magnetic nanoparticles was successful.

[0095] (II) Property Analysis of BCO-Fe3O4@Au Nanoparticles

[0096] The synthesized BCO-Fe3O4@Au nanoparticles were incubated with 240 mM dithiothreitol (DTT) to break the disulfide bonds between the Fe3O4@Au nanoparticles and BCO. The concentration of ssDNA in the supernatant was measured, such as... Figure 6 As shown in figure a, the binding rate can be obtained. Figure 6 As shown in b, the weight ratio of BCO on the nanoparticles to the weight of the nanoparticles (BCO / NPs weight ratio) is also considered. Both the binding rate and the BCO / NPs weight ratio reach their highest values ​​when the reaction ratio is 1:2. Furthermore, as shown in... Figure 6 As shown in Figure c, the normalized UV-Vis spectrum reveals a peak around 260 nm, indicating that BCO was successfully modified onto the surface of Fe3O4@Au nanoparticles, with the highest peak observed in BCO(1:2)-Fe3O4@Au nanoparticles. Therefore, BCO(1:2)-Fe3O4@Au nanoparticles may be a better choice for further experiments. To further confirm the modification, the BCO(1:2)-Fe3O4@Au nanoparticles were negatively stained and observed using transmission electron microscopy (TEM). Figure 6 As shown in d, from the TEM image, we found a DNA shell on the surface of the Fe3O4@Au nanoparticles, indicating that the modification was successful.

[0097] (III) Testing the capture of mRNA by BCO-Fe3O4@Au nanoparticles

[0098] In this experiment, SY5Y cells were used, and total RNA was extracted using the RNAiso Plus kit. To further clarify the optimal ratio of BCO-Fe3O4@Au nanoparticles for the experiment, the mRNA capture rates of BCO(1:2)-Fe3O4@Au and BCO(1:5)-Fe3O4@Au nanoparticles were tested. Although BCO(1:2)-Fe3O4@Au nanoparticles had a higher binding ratio and a higher weight ratio of BCO to nanoparticles, excessively high BCO density may cause steric hindrance, leading to a decrease in mRNA capture rate. To calculate the mRNA capture rate of BCO-Fe3O4@Au nanoparticles, 30 μL of 50 μg / mL BCO-Fe3O4@Au nanoparticles were mixed with poly(A) single-stranded DNA at weight ratios of 5:1, 10:1, 25:1, 50:1, and 75:1 in lysis buffer on ice. The mixture was then incubated at 37°C for 30 minutes. The concentration of ssDNA in the supernatant was detected using a nano-tipping analyzer, and the capture rate was calculated as follows:

[0099] Capture rate = (input poly(A) single-stranded DNA - poly(A) single-stranded DNA in supernatant) / (input poly(A) single-stranded DNA) × 100%.

[0100] From the results ( Figure 7 As can be seen, the capture rate increases with the increase of the ratio between BCO-Fe3O4@Au nanoparticles and poly(A) single-stranded DNA, up to a ratio of 50:1. Furthermore, the performance of BCO(1:2)-Fe3O4@Au nanoparticles is superior to that of BCO(1:5)-Fe3O4@Au nanoparticles. Therefore, BCO(1:2)-Fe3O4@Au nanoparticles were chosen for subsequent experiments.

[0101] In summary, the provided microfluidic droplet platform for single-cell RNA sequencing includes a specific cell-specific separation and enrichment module that enables high-specificity and high-throughput separation of the target cells; a single-cell droplet generation module that enables high-throughput generation of single-cell encapsulated droplets; and a pairing and fusion module for pairing and fusion. This module utilizes magnetic nanoparticle droplets with DNA-tagged sequences for pairing. On one hand, the DNA sequence can pair one-to-one with the RNA strands in the single cell, efficiently capturing RNA and preparing the library, and improving the barcoding efficiency of single cells. On the other hand, the provided... Nanoparticles possess magnetic structures, which can achieve magnetic separation by applying an external magnetic field when separating RNA from a cell lysis mixture, significantly improving separation purity. Therefore, the microfluidic droplet platform for single-cell RNA sequencing provided in this application requires only a small amount of sample to prepare high-quality sample libraries in a short time with high purity and high throughput, enabling rapid and high-quality prediction sequencing steps. Ultimately, it achieves single-cell transcriptome analysis of the sample, meeting the requirements for single-cell sample size and facilitating the analysis of large-scale single-cell genetic information, laying the foundation for studying sample heterogeneity and clonal evolution.

[0102] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A microfluidic droplet platform for single-cell RNA sequencing, characterized in that, The microfluidic droplet platform is based on a PDMS chip and includes a cell-specific separation and enrichment module, a single-cell droplet generation module, and a pairing and fusion module arranged sequentially. The pairing and fusion module is a module that pairs and fuses single-cell droplets with magnetic nanoparticle droplets carrying DNA tag sequences one by one.

2. The microfluidic droplet platform for single-cell RNA sequencing according to claim 1, characterized in that, The single-cell droplet generation module includes a "water-in-oil" single-cell droplet generation module and a droplet generation module that encapsulates encoded nanoparticles. The "water-in-oil" single-cell droplet generation module includes a cross channel, which comprises a separately arranged aqueous phase channel and an oil phase channel.

3. The microfluidic droplet platform for single-cell RNA sequencing according to claim 2, characterized in that, In the single-cell droplet generation module, the driving pressure of the water phase channel and the driving pressure of the oil phase channel are controlled by the pressure pump of the cross channel, and the driving pressure ratio of the water and oil phases is adjusted to generate "water-in-oil" single-cell droplets.

4. The microfluidic droplet platform for single-cell RNA sequencing according to claim 2, characterized in that, In the aqueous channel, the ratio of cell number to droplet number is 0.5 to 0.

6.

5. The microfluidic droplet platform for single-cell RNA sequencing according to claim 1, characterized in that, The magnetic nanoparticles with the DNA tag sequence have the DNA tag sequence as shown in Seq. No. 1, and the magnetic nanoparticles include a magnetic inner shell structure and a gold coating layer disposed on the outer surface of the magnetic inner shell structure.

6. The microfluidic droplet platform for single-cell RNA sequencing according to claim 1, characterized in that, The pairing and fusion module includes a Y-shaped channel arranged in sequence and a droplet deceleration region connected to the outlet of the Y-shaped channel; wherein the droplet deceleration region includes an integrated electrode.

7. The microfluidic droplet platform for single-cell RNA sequencing according to claim 6, characterized in that, The external electric field generated by the integrated electrode is an AC electric field with a sinusoidal signal, a frequency of 1.5 to 1.6 MHz, and a voltage of 5 to 6 V; and the AC electric field is connected to a power amplifier, which amplifies the power by 50% to 70%.

8. The microfluidic droplet platform for single-cell RNA sequencing according to claim 6, characterized in that, The pressure of the Y-shaped channel is 50-100 mbar.

9. The microfluidic droplet platform for single-cell RNA sequencing according to claim 1, characterized in that, The single-cell magnetic nanoparticle complex with DNA tag sequence obtained in the pairing and fusion module is sequenced by a sequencing system that is independent of the microfluidic droplet platform.

10. The microfluidic droplet platform for single-cell RNA sequencing according to claim 1, characterized in that, The method for RNA sequencing of single cells using the microfluidic droplet platform includes the following steps: The sample to be tested is added to the cell-specific separation and enrichment module for sequential cell separation and cell enrichment to obtain a cell suspension. The cell suspension and oil phase solution to be tested are added to the single-cell droplet generation module, and the driving pressure is controlled by the multi-channel pressure pump in the single-cell droplet generation module to generate "water-in-oil" single-cell droplets. Magnetic nanoparticles with DNA tag sequences are provided. The "water-in-oil" single-cell droplets and the magnetic nanoparticle droplets with DNA tag sequences are respectively added to the pairing and fusion module. The pressure of each channel drives the flow to pair them one by one. Then, the fusion is achieved by the external electric field generated by the integrated electrode to obtain the single-cell-magnetic nanoparticle complex with DNA tag sequence to be tested. The RNA of the single-cell-DNA-tagged magnetic nanoparticle complex was sequenced using a sequencing system.

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