Inertial microfluidic system for focusing and sorting tumor cells

Abstract

The invention discloses an inertial microfluidic system for focusing and sorting tumor cells, and relates to the field of circulating tumor cell detection. Comprising an inertial focusing sorting module used for CTC sorting and enrichment; the liquid drop generation module is used for wrapping single cells; in-droplet amplification and sequencing, the generated single-cell droplets are subjected to splitting decomposition, reverse transcription and amplification reactions under the guidance of bar code marks, and then high-throughput single-cell sequencing is directly carried out. The inertial focusing module can realize CTC sorting at a relatively high sample flow rate, is suitable for clinical and large-scale sample screening, ensures that each liquid drop only contains one CTC through a liquid drop wrapping technology, realizes labeling and analysis of a single cell level, integrates inertial focusing and liquid drop generation through an integrated micro-fluidic system, remarkably simplifies CTC sorting and sequencing sample preparation processes, and improves the detection efficiency. The inertial focusing technology does not need marking operation and can adapt to CTC samples with different antigen expression levels.

Description

Technical Field

[0001] The present invention relates to the field of circulating tumor cell detection, and particularly to an inertial microfluidic system for tumor cell focusing and sorting. Background Art

[0002] The sorting and single-cell sequencing technologies of circulating tumor cells (CTCs) are important tools for cancer diagnosis and precision medicine research. The rarity of CTCs and the complex blood sample background pose extremely high requirements for sorting technologies. In traditional technologies, the sorting methods mainly rely on the physical properties of cells (such as size, density) or surface-specific antigens (such as EpCAM).

[0003] The existing technologies mainly include magnetic bead sorting technology, flow cytometry, acoustic sorting technology, and inertial focusing sorting technology:

[0004] Magnetic bead sorting technology:

[0005] · Principle: Capture CTCs by binding magnetic beads with antibodies and separate them from blood samples using a magnetic field.

[0006] · Advantage: High specificity, suitable for sorting rare cells.

[0007] · Disadvantage:

[0008] · Dependent on antibody labeling, may not be able to capture CTCs with low antigen expression.

[0009] · Multiple washing steps increase the operation complexity and may cause cell loss at the same time.

[0010] Flow cytometry:

[0011] · Principle: Fluorescently label the surface-specific antigens of CTCs and perform sorting in combination with a flow cytometer.

[0012] · Advantage: High resolution, can separate multiple types of cells.

[0013] · Disadvantage:

[0014] · The sorting process may cause damage to cells and is not suitable for subsequent single-cell sequencing.

[0015] · The operation is complex and the equipment is expensive.

[0016] Acoustic sorting technology:

[0017] · Principle: Achieve cell separation by applying acoustic radiation force through an acoustic field.

[0018] · Advantage: Label-free, gentle treatment, protecting cell viability.

[0019] · Disadvantage:

[0020] · The parameter optimization is difficult, and the sorting efficiency is sensitive to the background of blood samples.

[0021] Inertial focusing sorting technology:

[0022] · Principle: Utilize the differences in inertial force and shear force of cells in a high-speed flowing microfluidic channel to autonomously focus on specific positions.

[0023] · Advantages:

[0024] · No labeling is required, there is no chemical treatment during the sorting process, and the cells are not damaged.

[0025] · The operation is simple and suitable for high-throughput sample analysis.

[0026] · Disadvantages:

[0027] · When there are many white blood cells, red blood cells and other impurities in the sample, the sorting purity is relatively low.

[0028] · It is difficult to be seamlessly connected with traditional sequencing operations.

[0029] Therefore, the present invention proposes an inertial microfluidic system for tumor cell focusing sorting Summary of the Invention

[0030] The purpose of the present invention is to solve the deficiencies existing in the prior art, and to propose an inertial microfluidic system for tumor cell focusing sorting.

[0031] To achieve the above purpose, the present invention adopts the following technical solutions:

[0032] An inertial microfluidic system for tumor cell focusing sorting, comprising:

[0033] An inertial focusing sorting module for CTC sorting and enrichment;

[0034] A droplet generation module for single-cell encapsulation;

[0035] Amplification and sequencing within the droplet, which completes lysis, reverse transcription and amplification reactions for the generated single-cell droplets under the guidance of barcode labeling, and then directly performs high-throughput single-cell sequencing.

[0036] Preferably: The inertial focusing sorting module adopts a spiral channel with a rectangular cross-section, which includes an inlet A channel, a focusing area B channel, a CTC outlet C channel and an impurity outlet D channel.

[0037] Preferably: In the inertial focusing sorting module, the material of the spiral channel with a rectangular cross-section is PDMS or glass.

[0038] Preferably, in the inertial focusing sorting module, the width of the spiral channel with a rectangular cross-section is 100 - 300 μm, the height is 50 - 150 μm, and the flow rate is 1 - 10 μL / min.

[0039] Preferably, the droplet generation module has a three-channel confluence structure, specifically including an E channel containing barcoded microspheres, an F channel containing amplification reagents, and an oil phase H channel that orthogonally converges with the F channel.

[0040] Preferably, the viscosity of the oil phase in the oil phase H channel is 5 - 15 mPa·s, and the interfacial tension coefficient is γ = 25 - 35 mN / m.

[0041] Preferably, the diameter of the droplets generated by the droplet generation module is 50 - 100 μm, and the single-cell encapsulation rate > 95%.

[0042] Preferably, in the inertial focusing sorting module and the droplet generation module, the ratio of the C channel, E channel, and H channel is 1:0.8:2.5.

[0043] Preferably, the working logic of the inertial microfluidic system for tumor cell focusing and sorting is as follows:

[0044] S1: The blood sample is injected from the A hole, passes through the B area, that is, the inertial focusing area composed of multiple circular microchannels for sorting. The CTCs in the sample are pushed towards the channel wall under the action of inertial force and fluid gradient force, and focused into the C channel, while white blood cells, red blood cells, and other impurities flow into the D channel along the center.

[0045] S2: The sorted CTCs enter from the C channel, converge with the E channel containing barcoded microspheres and the F channel containing amplification reagents, and are then cut into droplets by the oil phase H channel. Each droplet contains 1 CTC, 1 barcoded microsphere, and amplification reagents.

[0046] The beneficial effects of the present invention are as follows:

[0047] 1. The inertial focusing module of the present invention can achieve CTC sorting at a relatively high sample flow rate, is suitable for clinical and large-scale sample screening. The droplet encapsulation technology ensures that each droplet contains only one CTC, realizing single-cell level labeling and analysis. The integrated microfluidic system integrates inertial focusing and droplet generation, significantly simplifying the CTC sorting and sequencing sample preparation process. The inertial focusing technology does not require labeling operations and can adapt to CTC samples with different antigen expression levels. The cost of this microfluidic device is low, and the inertial focusing module has no consumable requirements, making it suitable for large-scale applications. Description of the Drawings

[0048] Figure 1 It is a structural diagram of an inertial microfluidic system for tumor cell focusing and sorting proposed by the present invention. Detailed implementation manners

[0049] The technical solutions of the present invention will be further described in detail below in conjunction with the specific implementation manners.

[0050] In the description of the present invention, it should be noted that unless otherwise clearly specified and limited, the terms "installation", "connection", "connection", and "setting" should be understood in a broad sense. For example, it can be fixedly connected and set, or detachably connected and set, or integrally connected and set. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific situations.

[0051] An inertial microfluidic system for tumor cell focusing and sorting, which includes:

[0052] An inertial focusing and sorting module for CTC sorting and enrichment;

[0053] A droplet generation module for single cell encapsulation;

[0054] Amplification and sequencing within droplets, which completes lysis, reverse transcription and amplification reactions for the generated single cell droplets under the guidance of barcode labeling, and then directly performs high-throughput single cell sequencing.

[0055] The inertial focusing and sorting module adopts a spiral channel with a rectangular cross-section, which includes an inlet A channel, a focusing area B channel, a CTC outlet C channel and an impurity outlet D channel.

[0056] In the inertial focusing and sorting module, the material of the spiral channel with a rectangular cross-section is PDMS or glass.

[0057] In the inertial focusing and sorting module, the width of the spiral channel with a rectangular cross-section is 100 - 300 μm, the height is 50 - 150 μm, and the flow rate is 1 - 10 μL / min.

[0058] The droplet generation module has a three-channel intersection structure, specifically including an E channel containing barcode hydrogel beads, an F channel containing amplification reagents, and an oil phase H channel orthogonally intersecting with the F channel.

[0059] The viscosity of the oil phase in the oil phase H channel is 5 - 15 mPa·s, and the interfacial tension coefficient is γ = 25 - 35 mN / m.

[0060] The droplets generated by the droplet generation module have a diameter of 50 - 100 μm, and the single cell encapsulation rate > 95%.

[0061] In the inertial focusing and sorting module and the droplet generation module, the ratio of the C channel, the E channel, and the H channel is 1:0.8:2.5

[0062] The working logic of the inertial microfluidic system for tumor cell focused sorting is as follows:

[0063] S1: The blood sample is injected from hole A, passes through area B, i.e., the inertial focusing area composed of multiple circular microchannels for sorting. In the sample, CTCs are pushed towards the channel wall under the action of inertial force and fluid gradient force due to their larger volume, and are focused into channel C. White blood cells, red blood cells and other impurities flow into channel D along the center.

[0064] S2: The sorted CTCs enter from channel C, converge with channel E containing barcode hydrogels and channel F containing amplification reagents, and are then cut by the oil phase H channel to form droplets. Each droplet contains 1 CTC, 1 barcode hydrogel and amplification reagents.

[0065] Example 1:

[0066] Detection of blood samples from breast cancer patients:

[0067] Steps: Sample injection → Inertial focusing sorting (flow rate 5 μL / min) → Droplet generation → Amplification → Sequencing.

[0068] Example 2:

[0069] Verification of samples from prostate cancer patients:

[0070] Sample pretreatment: 4 mL of whole blood is lysed with red blood cells (treated with ACK buffer) and resuspended in PBS + 0.5% BSA;

[0071] Sorting parameters: Flow rate 7 μL / min, Dean number De = 2.3, shear stress τ = 0.8 Pa;

[0072] Results:

[0073] CTC capture rate: 89.7% (n = 15);

[0074] Effective droplet generation rate: 96.2% (N = 10^5 droplets);

[0075] Single-cell transcriptome data: Average number of detected genes > 4500 / cell.

[0076] Example 3:

[0077] System robustness test:

[0078] Interference conditions: Hematocrit change (30% - 50%), temperature fluctuation (4 - 37 °C);

[0079] Performance stability: Capture rate fluctuation < ±2.1%, droplet diameter CV remains < 4.5%.

[0080] The present invention: The inertial focusing module can achieve CTC sorting at a relatively high sample flow rate, and is suitable for clinical and large-scale sample screening.

[0081] The droplet encapsulation technology ensures that each droplet contains only one CTC, enabling single-cell level labeling and analysis.

[0082] The integrated microfluidic system integrates inertial focusing and droplet generation, significantly simplifying the CTC sorting and sequencing sample preparation process.

[0083] The inertial focusing technology does not require labeling operations and can adapt to CTC samples with different antigen expression levels.

[0084] The cost of manufacturing this microfluidic device is low. The inertial focusing module has no consumable requirements and is suitable for large-scale applications.

[0085] The above are only the preferred specific embodiments of the invention, but the protection scope of the invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the invention, according to the technical solution of the invention and its inventive concept, making equivalent substitutions or changes, shall be covered by the protection scope of the invention.

Claims

1. An inertial microfluidic system for focusing and sorting tumor cells, characterized in that: include: Inertial focusing sorting module, which is used for CTC sorting and enrichment; A droplet generation module for single-cell encapsulation; In-droplet amplification and sequencing, the generated single-cell droplets complete lysis, reverse transcription and amplification reactions under the guidance of barcode labels, and then directly perform high-throughput single-cell sequencing.

2. The inertial microfluidic system for focusing and sorting tumor cells according to claim 1, characterized in that: The inertial focusing sorting module adopts a spiral channel with a rectangular cross-section, which includes an inlet A channel, a focusing area B channel, a CTC outlet C channel and an impurity outlet D channel.

3. The inertial microfluidic system for focusing and sorting tumor cells according to claim 2, characterized in that: In the inertial focusing sorting module, the spiral channel with a rectangular cross section is made of PDMS or glass.

4. The inertial microfluidic system for focusing and sorting tumor cells according to claim 3, characterized in that: In the inertial focusing sorting module, the width of the spiral channel with a rectangular cross section is 100-300 μm, the height is 50-150 μm, and the flow rate is 1-10 μL / min.

5. The inertial microfluidic system for focusing and sorting tumor cells according to claim 4, characterized in that: The droplet generation module is a three-channel intersection structure, specifically including an E channel containing a barcode gel ball, an F channel containing an amplification reagent, and an oil phase H channel orthogonally intersecting with the F channel.

6. The inertial microfluidic system for focusing and sorting tumor cells according to claim 5, characterized in that: The oil phase viscosity of the oil phase H channel is 5-15 mPa·s, and the interfacial tension coefficient is γ=25-35 mN / m.

7. The inertial microfluidic system for focusing and sorting tumor cells according to claim 6, characterized in that: The droplet diameter generated by the droplet generation module is 50-100 μm, and the single cell encapsulation rate is greater than 95%.

8. The inertial microfluidic system for focusing and sorting tumor cells according to claim 7, characterized in that: In the inertial focusing sorting module and the droplet generation module, the ratio of the C channel, the E channel, and the H channel is 1:0.8:2.

5.

9. The inertial microfluidic system for focusing and sorting tumor cells according to claim 8, characterized in that: The working logic of the inertial microfluidic system for tumor cell focusing and sorting is: S1: The blood sample is injected from hole A and sorted through area B, which is an inertial focusing area composed of multiple circular microchannels. Due to its large volume, the CTC in the sample is pushed to the channel wall under the action of inertial force and fluid gradient force and focused into channel C. White blood cells, red blood cells and other impurities flow into channel D along the center; S2: The sorted CTCs enter from the C channel, merge with the E channel containing the barcode gel ball and the F channel containing the amplification reagent, and are cut by the oil phase H channel to form droplets. Each droplet contains 1 CTC, 1 barcode gel ball and amplification reagent.

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