A single-cell sequencing analysis method for circulating tumor cells

Abstract

The present invention discloses a single-cell sequencing analysis method for circulating tumor cells, and relates to the technical field of single-cell sequencing analysis; it is implemented based on a sequencing analysis system, and the system includes: an acoustic sorting module and a droplet generation module; the acoustic sorting module performs CTC sorting and enrichment, and ultrasonic transducers and reflective layers are integrated on both sides of the microfluidic channel of the acoustic sorting module to form a stable standing wave acoustic field, and the acoustic field frequency and power adjustment of the acoustic sorting module are used to adjust the CTC to focus on the side wall of the channel due to the acoustic radiation force due to its large volume and density. The present invention realizes label-free operation and protects cell activity by setting an acoustic sorting module; gentle treatment is suitable for subsequent amplification and sequencing; by setting a droplet generation module: droplet encapsulation realizes single-cell operation and avoids sample cross contamination; barcode labeling provides accurate traceability for subsequent single-cell sequencing.

Description

Technical Field

[0001] The present invention relates to the technical field of single-cell sequencing analysis, and in particular to a single-cell sequencing analysis method for circulating tumor cells. Background Art

[0002] With the continuous development of cell biology and sequencing technology, the detection of circulating tumor cells (CTCs) and single-cell sequencing have become important tools for cancer diagnosis and precision treatment. Currently, the combination of CTC sorting technology and single-cell sequencing technology faces many challenges, including insufficient cell enrichment efficiency, cell damage, sample loss, and difficulty in efficiently integrating multi-step operations.

[0003] Existing CTC sorting and single-cell sequencing technologies mainly include the following:

[0004] 1. Magnetic bead sorting technology: CTCs are captured using specific antibodies bound to the surface of magnetic beads and separated through an external magnetic field. Advantages: High specificity, effective separation of rare CTCs. Disadvantages: Reliance on antibody labeling, which may miss CTCs with low antigen expression; additional purification steps are required after sorting, making it difficult to directly integrate with single-cell sequencing.

[0005] 2. Sorting based on physical properties: including inertial focusing, size screening and density gradient centrifugation. Advantages: Label-free sorting, easy to operate. Disadvantages: It is difficult to achieve high-purity sorting in samples with extremely low CTC content.

[0006] 3. Flow cytometry sorting: CTC specific antigens are fluorescently labeled and sorted by flow cytometry. Advantages: high resolution and high purity. Disadvantages: complex operation and expensive equipment; difficult to directly combine with microfluidics and single-cell sequencing.

[0007] 4. Acoustic sorting technology (one of the innovative directions in existing technologies): different acoustic radiation forces are applied to different cells through the acoustic field to achieve cell separation. Advantages: no labeling, gentle treatment, and avoidance of cell damage. Disadvantages: equipment parameters need to be precisely designed. Acoustic sorting is currently mostly used in the sample enrichment stage and has not been effectively integrated with single-cell sequencing.

[0008] The existing technologies have the following common problems:

[0009] 1. Sample loss: The multi-step operation of CTC sorting and transfer leads to cell loss, especially for rare samples such as CTCs.

[0010] 2. Lack of integration: It is difficult to achieve seamless integration of CTC sorting and single-cell sequencing on the same platform.

[0011] 3. Damage to cell activity: Some sorting techniques (such as magnetic bead sorting and flow cytometry) may cause cell damage and affect subsequent analysis.

[0012] 4. High complexity and cost: Sorting technology and sequencing preparation are mostly independent steps, which increases the complexity and time cost of the experiment. Summary of the invention

[0013] The purpose of the present invention is to solve the shortcomings of the prior art and to propose a single-cell sequencing analysis method for circulating tumor cells.

[0014] In order to achieve the above object, the present invention adopts the following technical solutions:

[0015] A single-cell sequencing analysis method for circulating tumor cells is implemented based on a sequencing analysis system, which includes but is not limited to:

[0016] Acoustic sorting module, droplet generation module;

[0017] Also includes:

[0018] S1 hole: S1 hole is a sample injection hole for injecting samples. S1 hole is connected to channel A and is used to transport blood samples of circulating tumor cells.

[0019] S2 hole: S2 hole is a DNA tag injection hole for DNA tag injection. S2 hole is connected to E channel for transporting gel balls with DNA tags.

[0020] S3 hole: S3 hole is an amplification reagent injection hole, used for amplification reagent injection, and S3 hole is connected to F channel for transporting amplification reagent;

[0021] S4 hole, S4 hole is a cutting oil injection hole, used for cutting oil injection, S4 hole is connected to H channel, used for conveying cutting oil;

[0022] S5 hole, S5 hole is a CTC recovery hole, used for CTC recovery, S5 hole is connected to I channel, I channel is a droplet generation channel;

[0023] S6 hole: S6 hole is a background cell recovery hole, used for background cell recovery, S6 hole is connected to D channel, used for transporting background cells;

[0024] Wherein, the A channel is connected to the C channel and the D channel after passing through the acoustic sorting module, and the C channel is a circulating tumor cell sorting channel; one end of the C channel is connected to the E channel, the F channel and the G channel, one end of the G channel is connected to the I channel, and the H channel is connected to the G channel;

[0025] The acoustic sorting module is used to sort and enrich CTCs. Ultrasonic transducers and reflective layers are integrated on both sides of the microfluidic channel of the acoustic sorting module to form a stable standing wave acoustic field. Based on the acoustic field frequency and power adjustment of the acoustic sorting module, CTCs are focused on the side walls of the channel due to the acoustic radiation force due to their larger volume and density.

[0026] The droplet generation module performs single cell encapsulation, wherein the sorted CTCs are mixed with the DNA barcode gel balls input by the E channel and the amplification reagents input by the F channel through the C channel, and after combining with the G channel, they are cut into droplets by the oil phase input by the H channel.

[0027] Preferably: In the sequencing analysis system:

[0028] The blood sample is injected from channel A;

[0029] In the standing wave field arranged in area B, CTCs are offset to the exit of channel C, and background cells enter channel D and are discharged.

[0030] Preferably: in the sequencing analysis system, each droplet contains a CTC, a DNA barcode gel ball and an amplification reagent;

[0031] The droplet size is controlled at 50-100 μm to ensure single cell isolation.

[0032] Preferably, in the sequencing analysis system, the amplification and sequencing preparation in the droplet are specifically as follows:

[0033] Amplification reagents in the droplets initiate cell lysis, reverse transcription, and amplification reactions;

[0034] The final product is directly used for high-throughput single-cell sequencing.

[0035] Preferably, the method comprises the following steps:

[0036] Step A: sample processing and pretreatment;

[0037] Step B: acoustic sorting;

[0038] Step C: droplet generation;

[0039] Step D: droplet collection and amplification;

[0040] Step E: Sequencing preparation and analysis.

[0041] Preferably: Step A specifically comprises:

[0042] A1 Sample Collection:

[0043] Collect 10 mL of peripheral blood from the patient into an EDTA anticoagulant tube to ensure that no agglutination occurs;

[0044] After sample collection, try to complete processing within 2 hours to avoid cell degradation;

[0045] A2: Sample centrifugation and stratification:

[0046] The blood samples were centrifuged at 1200 × g for 10 min at room temperature;

[0047] Remove the supernatant plasma, retain the white blood cell layer, and dilute to 10 mL of 1× PBS (without calcium and magnesium);

[0048] A3: Cell filtration:

[0049] Use a 70μm cell filter to filter the diluted sample to remove large particles and aggregated cell clumps to ensure the subsequent sorting effect;

[0050] Collect the filtrate for later use to avoid exposing the sample to the air for too long.

[0051] Preferably: the step B specifically comprises:

[0052] B1: System initialization:

[0053] Turn on the acoustic sorting module and set the ultrasonic frequency to 2 MHz and the power to 0.5 W / cm 2 ;

[0054] Calibration flow rate: sample flow rate was set to 20 μL / min, control flow rate was 10 μL / min;

[0055] B2: Sample loading:

[0056] Use a syringe pump to inject the pretreated sample into the S1 well and into the A channel;

[0057] B3: Sorting process:

[0058] In the acoustic wave generating area, the standing wave acoustic field exerts acoustic radiation force on the cells;

[0059] The sorted CTCs are guided to the C channel and enter the subsequent process;

[0060] Background cells are discharged and flow to well S6 for disposal or further analysis;

[0061] B4: Dynamic monitoring and verification:

[0062] Observe the fluid dynamics in the sorting area under a microscope to ensure the sorting efficiency and flow rate stability;

[0063] After the sorting samples were collected, flow cytometry was performed to assess the sorting purity, with a target value of ≥90%.

[0064] Preferably: the step C specifically comprises:

[0065] C1: System initialization:

[0066] Turn on the microfluidic droplet generation module and set the flow rate parameters:

[0067] E channel: 10 μL / min;

[0068] F channel: 10 μL / min;

[0069] H channel: 50 μL / min;

[0070] C2: Droplet generation process:

[0071] C channel: directly imports sorted CTCs from the acoustic sorting channel without intermediate collection;

[0072] E channel: The gel ball containing the DNA barcode is loaded into the E channel through the S2 hole and merged with the liquid in the C channel;

[0073] F channel: inject amplification reagent through S3 hole and enter F channel;

[0074] G channel: The liquids in channels C, E, and F are mixed here to form a uniform solution;

[0075] H channel: Cutting oil enters the H channel through the S4 hole, cutting the mixed liquid in the G channel into uniform droplets controlled at 50-100 μm;

[0076] C3: Droplet quality control:

[0077] Observe the droplet formation under a microscope to ensure that the droplets are uniform in size and free of mixed contamination;

[0078] Each droplet contains 1 cell, 1 DNA barcoded gel ball and amplification reagents.

[0079] Preferably: the step D specifically comprises:

[0080] D1: Droplet collection:

[0081] The droplets flow into the S5 well through the I channel and are directly collected for subsequent reactions;

[0082] D2: Droplet incubation:

[0083] The collected droplets were transferred to a 96-well plate, with 10 μL of the droplet mixture in each well;

[0084] Incubate at 42°C for 2 h for reverse transcription reaction;

[0085] D3: Amplification reaction:

[0086] Incubate at 65°C for 10 min for initial chain extension, followed by transfer to 72°C for PCR amplification;

[0087] D4: Droplet lysis and recovery:

[0088] Add an emulsion breaker to break the droplets and recover the amplification products in the aqueous phase;

[0089] Purify DNA using magnetic beads and elute into 20 μL of nuclease-free water.

[0090] Preferably: the step E specifically comprises:

[0091] E1: Sequencing library construction:

[0092] Construct a standard sequencing library based on the amplified products;

[0093] Determine the quality and concentration of the library to ensure that it meets sequencing requirements;

[0094] E2: High-throughput sequencing:

[0095] Load the library into the sequencer and select the read length;

[0096] E3: Data Analysis:

[0097] Use barcode information to parse single-cell data and generate a gene expression matrix;

[0098] Combined with downstream bioinformatics methods to conduct tumor heterogeneity analysis or personalized treatment plan research.

[0099] The beneficial effects of the present invention are:

[0100] 1. The present invention realizes label-free operation and protects cell activity by setting up an acoustic sorting module; gentle treatment is suitable for subsequent amplification and sequencing; and a droplet generation module is set up: droplet encapsulation realizes single-cell operation and avoids sample cross-contamination; barcode labeling provides accurate traceability for subsequent single-cell sequencing.

[0101] 2. The present invention is easy to operate, and the integrated system integrates sorting and amplification without the need for complicated purification or transfer operations; it has a wide range of applicability and is suitable for CTC detection, tumor heterogeneity analysis, early cancer screening and other fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0102] Figure 1 A schematic diagram of sequencing analysis of a single-cell sequencing analysis method for circulating tumor cells proposed in the present invention. DETAILED DESCRIPTION

[0103] The technical solution of the present invention is further described in detail below in conjunction with specific implementation methods.

[0104] Embodiment 1:

[0105] A single-cell sequencing analysis method for circulating tumor cells is implemented based on a sequencing analysis system, which includes but is not limited to:

[0106] Acoustic sorting module, droplet generation module;

[0107] Also includes:

[0108] S1 hole: S1 hole is a sample injection hole for injecting samples. S1 hole is connected to channel A and is used to transport blood samples of circulating tumor cells.

[0109] S2 hole: S2 hole is a DNA tag injection hole for DNA tag injection. S2 hole is connected to E channel for transporting gel balls with DNA tags.

[0110] S3 hole: S3 hole is an amplification reagent injection hole, used for amplification reagent injection, and S3 hole is connected to F channel for transporting amplification reagent;

[0111] S4 hole, S4 hole is a cutting oil injection hole, used for cutting oil injection, S4 hole is connected to H channel, used for conveying cutting oil;

[0112] S5 hole, S5 hole is a CTC recovery hole, used for CTC recovery, S5 hole is connected to I channel, I channel is a droplet generation channel;

[0113] S6 hole: S6 hole is a background cell recovery hole, used for background cell recovery, S6 hole is connected to D channel, used for transporting background cells;

[0114] Wherein, the A channel is connected to the C channel and the D channel after passing through the acoustic sorting module, and the C channel is a circulating tumor cell sorting channel; one end of the C channel is connected to the E channel, the F channel and the G channel, one end of the G channel is connected to the I channel, and the H channel is connected to the G channel;

[0115] The acoustic sorting module is used to sort and enrich CTCs. Ultrasonic transducers and reflective layers are integrated on both sides of the microfluidic channel of the acoustic sorting module to form a stable standing wave acoustic field. Based on the acoustic field frequency and power adjustment of the acoustic sorting module, CTCs are focused on the side walls of the channel due to the acoustic radiation force due to their larger volume and density.

[0116] In the sequencing analysis system:

[0117] ①Blood sample is injected from channel A;

[0118] ② In the standing wave field arranged in area B, CTC is offset to the exit of channel C, and background cells enter channel D and are discharged.

[0119] By setting up an acoustic sorting module: label-free operation is achieved to protect cell activity; gentle treatment is suitable for subsequent amplification and sequencing.

[0120] The droplet generation module performs single cell encapsulation, and the sorted CTCs are mixed with DNA barcode gel balls (E channel) and amplification reagents (F channel) through the C channel, and are cut into droplets by the oil phase (H channel) after combining with the G channel.

[0121] In the sequencing analysis system:

[0122] ③Each droplet contains a CTC, a DNA barcode ball and amplification reagents.

[0123] ④The droplet size is controlled at 50-100μm to ensure single cell separation.

[0124] By setting up a droplet generation module: droplet encapsulation realizes single-cell operation and avoids sample cross-contamination; barcode labeling provides accurate traceability for subsequent single-cell sequencing.

[0125] In the sequencing analysis system, the amplification and sequencing preparation in the droplet are specifically as follows:

[0126] ⑤ The amplification reagent in the droplet initiates cell lysis, reverse transcription and amplification reactions;

[0127] ⑥The final product is directly used for high-throughput single-cell sequencing.

[0128] Embodiment 2:

[0129] A single-cell sequencing analysis method for circulating tumor cells, this embodiment is based on embodiment 1, and specifically includes:

[0130] Step A: Sample handling and pretreatment

[0131] A1 Sample Collection

[0132] Collect 10 mL of peripheral blood from the patient into an EDTA anticoagulant tube to ensure that no agglutination occurs.

[0133] After sample collection, try to complete processing within 2 hours to avoid cell degradation.

[0134] A2: Sample centrifugation and stratification

[0135] Blood samples were centrifuged at 1200 × g for 10 min at room temperature.

[0136] The supernatant plasma was removed, and the white blood cell layer was retained and diluted to 10 mL of 1× PBS (without calcium and magnesium).

[0137] A3: Cell Filtration

[0138] Use a 70 μm cell strainer to filter the diluted sample to remove large particles and aggregated cell clumps to ensure the subsequent sorting effect.

[0139] Collect the filtrate for later use to avoid exposing the sample to the air for too long.

[0140] Step B: Acoustic Sorting

[0141] B1: System Initialization

[0142] Turn on the acoustic sorting module and set the ultrasonic frequency (recommended 2MHz) and power (recommended 0.5W / cm 2 ).

[0143] Calibration flow rate: The sample flow rate (from S1 hole into channel A) was set to 20 μL / min, and the control flow rate (from S6 hole into the background channel) was set to 10 μL / min.

[0144] B2: Sample loading

[0145] Use a syringe pump to inject the pretreated sample into the S1 hole (sample injection hole) and into the A channel.

[0146] B3: Sorting process

[0147] In the acoustic wave generating area, the standing wave acoustic field exerts acoustic radiation force on the cells.

[0148] CTC sorting channel (C channel): The sorted CTCs are guided to the C channel and enter the subsequent process.

[0149] Background cell channel (D channel): Background cells are discharged and flow to well S6 for disposal or further analysis.

[0150] B4: Dynamic Monitoring and Verification

[0151] Observe the fluid dynamics in the sorting area under a microscope to ensure the sorting efficiency and flow rate stability.

[0152] After the sorting samples were collected, flow cytometry was performed to assess the sorting purity (target value ≥ 90%).

[0153] Step C: Droplet generation

[0154] C1: System Initialization

[0155] Turn on the microfluidic droplet generation module and set the flow rate parameters:

[0156] E channel (DNA barcode gel ball): 10 μL / min.

[0157] F channel (amplification reagent): 10 μL / min.

[0158] H channel (cutting oil): 50 μL / min.

[0159] C2: Droplet generation process

[0160] C channel: directly imports sorted CTCs from the acoustic sorting channel without intermediate collection.

[0161] Channel E: The gel ball containing the DNA barcode is loaded into the E channel through the S2 hole and merged with the liquid in the C channel.

[0162] F channel: inject amplification reagent through S3 hole and enter F channel;

[0163] G channel: The liquids in channels C, E, and F are mixed here to form a uniform solution;

[0164] H channel: Cutting oil enters the H channel through the S4 hole and cuts the mixed liquid in the G channel into uniform droplets (50-100 μm);

[0165] C3: Droplet Quality Control

[0166] Observe the droplet formation under a microscope to ensure that the droplets are uniform in size and free of mixed contamination.

[0167] Each droplet must contain 1 cell, 1 DNA barcoded gel ball and amplification reagents.

[0168] Step D: Droplet collection and expansion

[0169] D1: Droplet collection

[0170] The droplets flow into the S5 well (CTC recovery well) through the I channel and are directly collected for subsequent reactions.

[0171] D2: Droplet incubation

[0172] The collected droplets were transferred to a 96-well plate with 10 μL of the droplet mixture per well.

[0173] Incubate at 42°C for 2 hours to perform the reverse transcription reaction.

[0174] D3: Amplification reaction

[0175] Incubate at 65°C for 10 min for initial chain extension, followed by transfer to 72°C for PCR amplification (15-20 cycles).

[0176] D4: Droplet lysis and recovery

[0177] An emulsion disruptor is added to disrupt the droplets and recover the amplification products in the aqueous phase.

[0178] Purify DNA using magnetic beads and elute into 20 μL of nuclease-free water.

[0179] Step E: Sequencing preparation and analysis

[0180] E1: Sequencing library construction

[0181] A standard sequencing library (such as using the Illumina platform) is constructed based on the amplified products.

[0182] Measure the quality and concentration of the library to ensure it meets sequencing requirements.

[0183] E2: High-throughput sequencing

[0184] Load the library into the sequencer and select an appropriate read length (such as PE150).

[0185] E3: Data Analysis

[0186] Single-cell data were parsed using barcode information to generate a gene expression matrix.

[0187] Combined with downstream bioinformatics methods to conduct tumor heterogeneity analysis or personalized treatment plan research.

[0188] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A single-cell sequencing analysis method for circulating tumor cells, characterized in that: It is implemented based on a sequencing analysis system, which includes: Acoustic sorting module, droplet generation module; Also includes: S1 hole: S1 hole is a sample injection hole for injecting samples. S1 hole is connected to channel A and is used to transport blood samples of circulating tumor cells. S2 hole: S2 hole is a DNA tag injection hole for DNA tag injection. S2 hole is connected to E channel for transporting gel balls with DNA tags. S3 hole: S3 hole is an amplification reagent injection hole, used for amplification reagent injection, and S3 hole is connected to F channel for transporting amplification reagent; S4 hole, S4 hole is a cutting oil injection hole, used for cutting oil injection, S4 hole is connected to H channel, used for conveying cutting oil; S5 hole, S5 hole is a CTC recovery hole, used for CTC recovery, S5 hole is connected to I channel, I channel is a droplet generation channel; S6 hole: S6 hole is a background cell recovery hole, used for background cell recovery, S6 hole is connected to D channel, used for transporting background cells; Wherein, the A channel is connected to the C channel and the D channel after passing through the acoustic sorting module, and the C channel is a circulating tumor cell sorting channel; one end of the C channel is connected to the E channel, the F channel and the G channel, one end of the G channel is connected to the I channel, and the H channel is connected to the G channel; The acoustic sorting module is used to sort and enrich CTCs. Ultrasonic transducers and reflective layers are integrated on both sides of the microfluidic channel of the acoustic sorting module to form a stable standing wave acoustic field. Based on the acoustic field frequency and power adjustment of the acoustic sorting module, CTCs are focused on the side walls of the channel due to the acoustic radiation force due to their larger volume and density. The droplet generation module performs single cell encapsulation, wherein the sorted CTCs are mixed with the DNA barcode gel balls input by the E channel and the amplification reagents input by the F channel through the C channel, and after combining with the G channel, they are cut into droplets by the oil phase input by the H channel.

2. A single-cell sequencing analysis method for circulating tumor cells according to claim 1, characterized in that: In the sequencing analysis system: The blood sample is injected from channel A; In the standing wave field arranged in area B, CTCs are offset to the exit of channel C, and background cells enter channel D and are discharged.

3. A single-cell sequencing analysis method for circulating tumor cells according to claim 2, characterized in that: In the sequencing analysis system, each droplet contains a CTC, a DNA barcode gel ball and an amplification reagent; The droplet size is controlled at 50-100 μm to ensure single cell isolation.

4. A single-cell sequencing analysis method for circulating tumor cells according to claim 3, characterized in that: In the sequencing analysis system, the amplification and sequencing preparation in the droplet are specifically as follows: Amplification reagents in the droplets initiate cell lysis, reverse transcription, and amplification reactions; The final product is directly used for high-throughput single-cell sequencing.

5. A single-cell sequencing analysis method for circulating tumor cells according to claim 1, characterized in that: The steps include: Step A: sample processing and pretreatment; Step B: acoustic sorting; Step C: droplet generation; Step D: droplet collection and amplification; Step E: Sequencing preparation and analysis.

6. A single-cell sequencing analysis method for circulating tumor cells according to claim 5, characterized in that: The step A specifically comprises: A1 Sample Collection: Collect 10 mL of peripheral blood from the patient into an EDTA anticoagulant tube to ensure that no agglutination occurs; After sample collection, try to complete processing within 2 hours to avoid cell degradation; A2: Sample centrifugation and stratification: The blood samples were centrifuged at 1200 × g for 10 min at room temperature; Remove the supernatant plasma, retain the white blood cell layer, and dilute to 10 mL of 1× PBS (without calcium and magnesium); A3: Cell filtration: Filter the diluted sample using a 70 μm cell strainer to remove large particles and aggregated cell clumps; Collect the filtrate for later use to avoid exposing the sample to the air for too long.

7. A single-cell sequencing analysis method for circulating tumor cells according to claim 6, characterized in that: The step B specifically comprises: B1: System initialization: Turn on the acoustic sorting module and set the ultrasonic frequency to 2 MHz and the power to 0.5 W / cm 2 ; Calibration flow rate: sample flow rate was set to 20 μL / min, control flow rate was 10 μL / min; B2: Sample loading: Use a syringe pump to inject the pretreated sample into the S1 well and into the A channel; B3: Sorting process: In the acoustic wave generating area, the standing wave acoustic field exerts acoustic radiation force on the cells; The sorted CTCs are guided to the C channel and enter the subsequent process; Background cells are discharged and flow to well S6 for disposal or further analysis; B4: Dynamic monitoring and verification: Observe the fluid dynamics in the sorting area under a microscope to ensure the sorting efficiency and flow rate stability; After the sorting samples were collected, flow cytometry was performed to assess the sorting purity, with a target value of ≥90%.

8. A single-cell sequencing analysis method for circulating tumor cells according to claim 7, characterized in that: The step C specifically comprises: C1: System initialization: Turn on the microfluidic droplet generation module and set the flow rate parameters: E channel: 10 μL / min; F channel: 10 μL / min; H channel: 50 μL / min; C2: Droplet generation process: C channel: directly imports sorted CTCs from the acoustic sorting channel without intermediate collection; E channel: The gel ball containing the DNA barcode is loaded into the E channel through the S2 hole and merged with the liquid in the C channel; F channel: inject amplification reagent through S3 hole and enter F channel; G channel: The liquids in channels C, E, and F are mixed here to form a uniform solution; H channel: Cutting oil enters the H channel through the S4 hole, cutting the mixed liquid in the G channel into uniform droplets controlled at 50-100 μm; C3: Droplet quality control: Observe the droplet formation under a microscope to ensure that the droplets are uniform in size and free of mixed contamination; Each droplet contains 1 cell, 1 DNA barcoded gel ball and amplification reagents.

9. A single-cell sequencing analysis method for circulating tumor cells according to claim 8, characterized in that: The step D specifically comprises: D1: Droplet collection: The droplets flow into the S5 well through the I channel and are directly collected for subsequent reactions; D2: Droplet incubation: The collected droplets were transferred to a 96-well plate, with 10 μL of the droplet mixture in each well; Incubate at 42°C for 2 h for reverse transcription reaction; D3: Amplification reaction: Incubate at 65°C for 10 min for initial chain extension, followed by transfer to 72°C for PCR amplification; D4: Droplet lysis and recovery: Add an emulsion breaker to break the droplets and recover the amplification products in the aqueous phase; Purify DNA using magnetic beads and elute into 20 μL of nuclease-free water.

10. A single-cell sequencing analysis method for circulating tumor cells according to claim 9, characterized in that: The step E specifically comprises: E1: Sequencing library construction: Construct a standard sequencing library based on the amplified products; Determine the quality and concentration of the library to ensure that it meets sequencing requirements; E2: High-throughput sequencing: Load the library into the sequencer and select the read length; E3: Data Analysis: Use barcode information to parse single-cell data and generate a gene expression matrix; Combined with downstream bioinformatics methods to conduct tumor heterogeneity analysis or personalized treatment plan research.

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