A label-free single-cell analysis system and method based on droplet photofluidization

Abstract

This invention discloses a label-free single-cell analysis system and method based on droplet photofluidization. Its aqueous phase flow control module includes an aqueous material sample inlet and a mixing structure microchannel. Two aqueous materials pass through the mixing structure microchannel to form a uniformly mixed aqueous flow, and the optical properties of the aqueous flow are controlled by changing the flow rate ratio of the two aqueous materials. In the droplet generation module, the aqueous and oil phase flows through a T-shaped microchannel to form water-in-oil droplets. The incident light module includes a single-mode fiber microchannel, an incident light collimating lens, and a monochromatic laser source to form parallel light for droplet detection. The scattered light receiving module includes a set of fiber microchannels at different angles to collect the spatial scattering signal of the droplet sample. The photoelectric conversion and signal display module includes a photomultiplier tube and an oscilloscope for signal collection, analysis, and feedback. This system provides accurate capture and analysis and is easy to operate.

Description

Technical Field

[0001] This invention relates to the field of single-cell analysis technology, and in particular to a label-free single-cell analysis system and method based on droplet photofluidization. Background Technology

[0002] Single-cell analysis has provided crucial knowledge for modern biology and clinical medicine. An accurate understanding of cell properties and behavior offers a scientific basis for cell biology, disease diagnosis, and drug development. Compared to traditional cell analysis, single-cell analysis can reveal information such as morphology, gene expression, and growth characteristics that can be used to identify individual cells, further elucidating the heterogeneity of cells in physiological processes such as metabolism, reproduction, and signal transduction. However, the tiny size of individual cells and the diversity of their constituent materials mean that single-cell analysis still faces significant challenges.

[0003] Microdroplet microfluidics is a miniaturized experimental analysis platform that utilizes two immiscible fluids (two-phase flow) to generate microdroplets within a specially designed structure on a microfluidic chip. By adjusting the flow rate, the droplet production frequency (100 Hz to 10 kHz) and droplet size (a few microliters to a few picoliters) can be controlled. These droplets act as liquid chambers, separating individual cells, making it an ideal platform for single-cell analysis. However, due to the differences in the properties of the two-phase flow materials, the most commonly used method for single-cell analysis based on droplet microfluidics is fluorescence labeling, which suffers from problems such as sample preparation complexity, cell invasiveness, and low fluorescence stability. Therefore, there is an urgent need to develop novel, label-free single-cell analysis techniques based on microdroplets.

[0004] Optofluidics is a rapidly developing emerging technology in recent years. It is an effective means of combining modern optics and microfluidics to study the interaction between light and fluids or biochemical samples within fluids, and to explore new phenomena, mechanisms, and applications. It has advantages such as high sensitivity, high precision, high integration, and ease of modulation. In recent years, microdroplets have received widespread attention in optofluidics, especially for real-time control of the light field. However, label-free single-cell analysis based on microdroplets in optofluidics still faces significant challenges and difficulties, particularly in controlling the optical properties of non-biotoxic microdroplets.

[0005] For example, Chinese patent CN104677877A discloses a quartz microfluidic chip and method for continuously acquiring Raman spectra of cells / particles under flowing conditions by dielectric single-cell capture / release. It utilizes the principle of dielectric capture of cells / particles, integrating electrode pairs on the microchannels of the microfluidic chip and periodically applying high-frequency voltage signals to capture / release single cells / particles, acquiring their Raman spectral signals during the capture period. However, its sorting, capture, and analysis accuracy is relatively low. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a label-free single-cell analysis system and method based on droplet photofluidization, aiming to achieve accurate capture and analysis while being easy to operate.

[0007] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0008] A label-free single-cell analysis system based on droplet photofluidization includes an aqueous phase flow control module, a droplet generation module, an incident light module, a scattered light receiving module, and a photoelectric conversion and signal display module;

[0009] The aqueous liquid flow control module includes two aqueous material sample inlets and a mixed structure microchannel, through which two aqueous liquid materials flow from the sample inlets into the mixed structure microchannel.

[0010] The droplet generation module includes an oil phase material sample inlet, a sample outlet, a microfluidic channel, and a T-shaped microchannel for droplet generation; the oil phase material sample inlet is connected to the mixed structure microchannel and one end of the microfluidic channel through the T-shaped microchannel, and the other end of the microfluidic channel is connected to the sample outlet;

[0011] The incident light module includes a corresponding incident light single-mode fiber microchannel, an incident light collimating microlens, and a monochromatic laser source;

[0012] The scattered light receiving module includes a set of fiber optic microchannels at different angles. The set of fiber optic microchannels are arranged circumferentially and form a circle at the center. The microfluidic channel passes through the circular area.

[0013] The photoelectric conversion and signal display module includes a connected oscilloscope and a set of photomultiplier tubes, with the photomultiplier tubes connected to the outer end of the corresponding fiber optic microchannel.

[0014] Further or preferred:

[0015] The two aqueous material sample inlets, the mixed structure microchannel, the oil phase material sample inlet, the sample outlet, the microfluidic channel, the T-shaped microchannel, the incident light single-mode fiber microchannel, and a set of fiber microchannels are all integrated on the microfluidic chip.

[0016] The angle range of the set of fiber optic microchannels is -145° to 140°, where the angle is defined as 0° with respect to the direction of incident light, with clockwise direction being negative and counterclockwise direction being positive.

[0017] The microstructure in the microfluidic chip includes two heights: the sample inlet, sample outlet, and microfluidic channel are shorter than the incident light single-mode fiber channel, the incident light collimating lens, and a set of fiber microchannels.

[0018] Both the sample inlet and the sample outlet are circular channels.

[0019] The hybrid structure microchannel is a bent microchannel structure.

[0020] The oil phase material sample inlet is connected to the contraction microchannel through the outer microchannel and the contraction microchannel, forming a T-shaped microchannel structure.

[0021] It also includes a glass slide, on which the microfluidic chip is fixed.

[0022] It also includes single-mode fiber and multimode fiber, with the fiber end with the cladding removed leading into the microfluidic chip, and the other end connected to the incident laser source and photomultiplier tube respectively, for transmitting the incident light and receiving the scattered light signal.

[0023] A label-free single-cell multi-angle light scattering analysis method based on droplet photofluidization includes the following steps:

[0024] S1. Prepare oil-phase materials, aqueous-phase materials, and cell samples with a certain concentration;

[0025] S2. The sample is pushed into the microfluidic channel by a micropump. The two aqueous phase materials form a uniformly mixed aqueous liquid flow with adjustable refractive index at the mixed structure microchannel. The oil liquid flow and the aqueous liquid flow flow through the T-shaped microchannel to form water-in-oil microdroplets. Individual cells are randomly wrapped in the droplets.

[0026] S3. The incident light is introduced by a single-mode optical fiber and then collimated by a microlens to form parallel light for sample scattered light detection.

[0027] S4. Collect the scattered light from the sample using a set of optical fibers at different angles. Use a photomultiplier tube and an oscilloscope to acquire and analyze the scattered light signal of the sample, including the scattered light signal of droplets and the scattered light signal of single cell samples.

[0028] S5. Through data analysis and feedback, the flow rate ratio of the aqueous phase material is adjusted in real time to further reduce or eliminate the scattered light signal of the droplets, thereby obtaining the scattered light signal of unlabeled single cells in the droplets.

[0029] Compared with the prior art, the present invention has the following advantages:

[0030] The label-free single-cell analysis system and method based on droplet photofluidization is reasonably designed. By controlling the flow rate of two aqueous phase materials, a uniformly mixed and refractive index-adjustable aqueous phase flow is formed, which reduces or eliminates the background scattering light signal brought by the droplet, and realizes the light scattering analysis of label-free single cells in the droplet. The capture and analysis are accurate and easy to operate. In addition, the structure is simple, easy to implement, and relatively low in cost. Attached Figure Description

[0031] The following is a brief explanation of the contents of each of the accompanying drawings and the markings in the drawings:

[0032] Figure 1 This is a schematic diagram of the microfluidic chip structure of the present invention.

[0033] Figure 2 This is a micrograph of the hybrid structure microchannel of the present invention.

[0034] Figure 3 This is a diagram of the T-shaped microchannel structure and droplet generation of the present invention.

[0035] Figure 4 This is a microscopic image of the incident light module and the scattered light receiving module of the present invention.

[0036] Figure 5 Microscopic images of microdroplets formed by aqueous phase materials with different refractive indices according to the present invention.

[0037] Figure 6 This is a label-free single-cell light scattering signal diagram of the present invention.

[0038] In the picture:

[0039] 1-Microfluidic chip; 2-Aqueous material sample inlet I; 3-Aqueous material sample inlet II; 4-Oil phase material sample inlet; 5-Hybrid structure microchannel; 6-T-shaped microchannel; 7-Sample outlet; 8-Incident fiber microchannel; 9-Incident light collimating microlens; 10-Fiber microchannel with an inclination angle of 65°; 11-Fiber microchannel with an inclination angle of 45°; 12-Fiber microchannel with an inclination angle of 25°; 13-Fiber microchannel with an inclination angle of 0°; 14-Fiber microchannel with an inclination angle of -25°; 15-Fiber microchannel with an inclination angle of -45°; 16-Fiber microchannel with an inclination angle of -65°; 17-Fiber microchannel with an inclination angle of -125°; 18-Fiber microchannel with an inclination angle of -145°; 19-Fiber microchannel with an inclination angle of 140°; 20-Fiber microchannel with an inclination angle of 120°. Detailed Implementation

[0040] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings and through the description of the examples.

[0041] like Figures 1 to 6 As shown, the present invention is a label-free single-cell multi-angle light scattering analysis system based on droplet photofluidization, including an aqueous phase liquid flow control module, a droplet generation module, an incident light module, a scattered light receiving module, and a photoelectric conversion and signal display module.

[0042] The aqueous liquid flow control module includes two aqueous material sample inlets and a mixing structure microchannel. The two aqueous material sample inlets are aqueous material sample inlet I2 and aqueous material sample inlet II3, which are arranged side by side. Two kinds of aqueous liquid materials flow into the mixing structure microchannel 5 from the sample inlets to form a uniformly mixed aqueous liquid flow with adjustable refractive index.

[0043] The droplet generation module includes an oil phase material sample inlet 4, a sample outlet 7, a microfluidic channel, and a T-shaped microchannel for droplet generation. The oil phase material sample inlet is connected to the mixed structure microchannel and one end of the microfluidic channel via the T-shaped microchannel, while the other end of the microfluidic channel is connected to the sample outlet. Samples, including oil phase, aqueous phase liquid materials, and pretreated cells, enter the microfluidic channel through the sample inlet. The oil phase and aqueous phase liquid flows through the T-shaped microchannel to form water-in-oil droplets, in which individual cells are randomly encapsulated within the droplets.

[0044] The incident light module includes a corresponding incident light single-mode fiber microchannel 8, an incident light collimating microlens 9, and a monochromatic laser source.

[0045] The scattered light receiving module includes a set of fiber optic microchannels at different angles. The set of fiber optic microchannels are arranged circumferentially and form a circle at the center. The microfluidic channel passes through the circular area. The angle range of the set of fiber optic microchannels is -145° to 140°, where the angle is 0° with respect to the direction of incident light, clockwise is negative, and counterclockwise is positive.

[0046] The photoelectric conversion and signal display module includes a connected oscilloscope and a set of photomultiplier tubes, with the photomultiplier tubes connected to the outer end of the corresponding fiber optic microchannel.

[0047] like Figure 1 As shown, two aqueous material sample inlets, a mixed structure microchannel, an oil phase material sample inlet, a sample outlet, a microfluidic channel, a T-shaped microchannel, an incident single-mode fiber microchannel, and a set of fiber microchannels are all integrated on the microfluidic chip 1, forming an integral microfluidic chip 1 structure. The structure is simple and the operation is convenient. The oil phase material sample inlet is connected to the contraction microchannel through the outer microchannel, and the outer microchannel and the contraction microchannel form a T-shaped microchannel 6.

[0048] Furthermore, the microstructure in the microfluidic chip includes two heights: a 50μm sample inlet / outlet and microfluidic channel, and a 100μm high incident light single-mode fiber channel, incident light collimating lens, and a set of fiber microchannels.

[0049] Both the sample inlet and outlet are 800 μm circular channels. The microfluidic channel is 50 μm wide, and the incident light single-mode fiber channel and a set of fiber microchannels are 120 μm wide. One end of the sample inlet is connected to the microfluidic channel, and the other end is connected to a micropump. The hybrid structure microchannel is a zigzag structure with a width of 50 μm. The T-shaped microchannel used for droplet generation has a 30 μm width at the confluence of the two-phase liquid flows.

[0050] The incident light collimating microlens is a plano-concave air lens, with a radius of curvature of 200 μm at the concave surface, a lens thickness of 90 μm, a lens width of 250 μm, and a distance of 700 μm from the incident light single-mode fiber microchannel and 350 μm from the center of the microfluidic channel.

[0051] A set of fiber optic microchannels for receiving scattered light are arranged sequentially on a ring with a radius of 350 μm centered on the microfluidic channel. There are a total of 11 multimode fiber optic microchannels. The center angles of the multimode fiber optic microchannels are 0°, 25°, 45°, 65°, 120°, 140°, -25°, -45°, -65°, -125°, and -145°, respectively. The direction of incident light is taken as 0°, counterclockwise is positive, and clockwise is negative.

[0052] According to the above scheme, the system also includes a glass slide, on which the microfluidic chip is fixed. The system also includes single-mode and multimode optical fibers, wherein the fiber with its cladding removed has one end inserted into the microfluidic chip, and the other end connected to the incident laser source and a photomultiplier tube, respectively, for transmitting the incident light and receiving the scattered light signal. The photomultiplier tube is connected to an oscilloscope via a transmission line, and the oscilloscope records the scattered light signal.

[0053] In this invention, by controlling the flow rates of two aqueous phase materials, a uniformly mixed aqueous liquid flow with adjustable refractive index is formed, containing pretreated cell samples. The aqueous and oil phase liquid flows form water-in-oil droplets at a T-shaped microchannel, in which cell samples are randomly encapsulated. The incident light module emits incident light through a monochromatic laser source, which is collimated into parallel light by a single-mode fiber and an incident light collimating microlens and then incident into the microfluidic channel. The scattered light receiving module receives the light signals scattered by the droplets in the microfluidic channel through multimode fibers at different angles and connects to a photomultiplier tube and an oscilloscope to acquire data.

[0054] By controlling the flow rates of two aqueous materials through real-time data feedback, an aqueous liquid flow with a suitable refractive index is obtained, reducing or eliminating background scattering light signals from the droplets and enabling light scattering analysis of label-free single cells within the droplets. This system has a simple structure and is easy to operate, achieving 100% efficiency in capturing light scattering signals from single-cell samples within the droplets.

[0055] A preferred embodiment of the present invention is as follows:

[0056] like Figure 1 As shown, the system for label-free single-cell multi-angle light scattering analysis based on droplet photofluidization is as follows:

[0057] In this embodiment, the microfluidic chip 1 is made of the organic material polydimethylsiloxane (PDMS). The fabrication method is as follows: the shapes of microchannels with a height of 50μm and 100μm are drawn using software, and photomasks are made according to the shapes. Then, through ultraviolet lithography, a silicon wafer mold with microchannels of two heights is obtained. PDMS is then used for casting, followed by heating, curing, casting, and fabrication to finally obtain the microfluidic chip 1.

[0058] The aqueous phase flow control module includes an aqueous material sample inlet I2 and an aqueous material inlet II3, as well as a mixing structure microchannel 5. The mixing structure is a zigzag-shaped microchannel. The two sample inlets are connected to the mixing structure microchannel 5 via microfluidic channels. The two aqueous materials enter the mixing structure microchannel 5 through sample inlets I2 and II3. By controlling the flow rate of the two inlet aqueous materials, a uniformly mixed and refractive-index-adjustable aqueous phase flow is obtained. See [link to module description]. Figure 2 The aqueous phase material includes pretreated cells, phosphate-buffered saline (PBS solution, refractive index 1.334), and density gradient culture medium (DGM solution, refractive index 1.429); in this embodiment, the cells are human breast cancer cells.

[0059] The droplet generation module includes an oil phase material sample inlet 4 and a T-shaped microchannel 6; the oil phase material forms an oil phase liquid flow through the oil phase material sample inlet 4; the oil phase liquid flow and the water phase liquid flow form water-in-oil microdroplets at the T-shaped microchannel 6, as shown in [reference needed]. Figure 3 In this embodiment, cell samples are randomly encapsulated within droplets; the oil phase material is n-hexadecane and the surfactant Span 80.

[0060] The incident light module includes a 488nm monochromatic solid-state laser, a single-mode fiber microchannel 8, and an incident light collimating microlens 9. The 488nm monochromatic solid-state laser serves as the incident light source. The single-mode fiber microchannel acts as a channel for the single-mode fiber, introducing the incident light into the microfluidic chip. The incident light collimating microlens 9 collimates the incident light emitted from the single-mode fiber, ensuring perpendicular incidence into the microfluidic channel for sample detection. In this embodiment, the single-mode fiber has a core diameter of 9μm and an outer diameter of 125μm.

[0061] The scattered light receiving module includes 11 multimode fiber microchannels at different angles, arranged sequentially on a ring with a radius of 350 μm centered on the microfluidic channel. The center angles of the multimode fiber microchannels are 0°, 25°, 45°, 65°, 125°, 145°, -25°, -45°, -65°, -125°, and -140°, respectively. The incident light direction is considered 0°, counterclockwise is positive, and clockwise is negative. Figure 1 and 4 As shown, there are fiber microchannels with tilt angles of 65° (10), 45°, 25°, 0°, -25°, -45°, -65°, -125°, -145°, -140°, and 120°. In this embodiment, the multimode fiber has a core diameter of 100 μm and an outer diameter of 125 μm.

[0062] The photoelectric conversion and signal display module includes a photomultiplier tube (PMT) and an oscilloscope; one end of the PMT is connected to a multimode optical fiber to collect the scattered light from the sample and convert the optical signal into an electrical signal; the other end of the PMT is connected to the oscilloscope to display the electrical signal in real time, which is used to record and analyze the scattered signal of the sample.

[0063] The microfluidic chip comprises two heights: a 50 μm sample inlet / outlet and a microfluidic channel, and a 100 μm high single-mode fiber channel 8, a collimating lens 9, and a multimode fiber microchannel. Both the sample inlet and outlet are 800 μm circular channels, and the microfluidic channel is 50 μm wide. One end of the sample inlet is connected to the microfluidic channel, and the other end is connected to a micropump.

[0064] The hybrid structure microchannel 5 is zigzag-shaped and 50 μm wide. The T-shaped microchannel 6, used for droplet generation, has an inlet width of 30 μm at the channel junction.

[0065] The incident light collimating microlens is a plano-concave air lens, with a radius of curvature of 200 μm at the concave surface, a lens thickness of 60 μm, a lens width of 200 μm, and a distance of 700 μm from the incident light single-mode fiber microchannel and 350 μm from the center of the microfluidic channel.

[0066] Furthermore, the system also includes a glass slide, on which the microfluidic chip is fixed. The system also includes single-mode and multimode optical fibers, wherein the fiber with its cladding removed has one end inserted into the microfluidic chip, and the other end is connected to the incident laser source and the PMT, respectively, for transmitting the incident light and receiving the scattered light signal.

[0067] A method for label-free single-cell multi-angle light scattering analysis based on droplet photofluidization includes the following steps:

[0068] S1. Prepare two aqueous phase materials: pure PBS solution and pure DGM solution, each containing 4×10⁻⁶ DGM. 6 Cell samples per mL; prepare oil phase material as n-hexadecane (refractive index 1.434) containing 3 wt% Span 80;

[0069] S2. The sample is injected into the microchannel using a microfluidic pump. The total flow rate of the two aqueous phase materials is 70 μL / h. By adjusting the flow rate ratio, aqueous phase flows with different refractive indices are obtained, while the oil phase flow rate is 100 μL / h. Stable water-in-oil droplets are formed at the T-shaped microchannel. The optical properties of the droplets, i.e., the refractive index, can be controlled by the flow rate of the aqueous phase materials. See [link to relevant documentation]. Figure 2 In this process, individual cells are randomly encapsulated within droplets, as shown in the image. Figure 5 ;

[0070] S3. A 488nm monochromatic solid-state laser is introduced into the microfluidic chip through a single-mode fiber and collimated into parallel light by an incident light collimating microlens and incident into the microfluidic channel for sample scattered light detection.

[0071] S4. Multimode optical fibers at different angles are fed into the microfluidic chip, with the other end connected to a PMT and an oscilloscope to collect and analyze the scattered light signals from the samples. These signals include the scattered light signals from droplets and the scattered light signals from cell samples. (See...) Figure 6 ;

[0072] S5. By changing the flow rate parameters of the microfluidic pump through data feedback, the flow rate ratio of the aqueous phase material is adjusted in real time to further reduce or eliminate the droplet scattering signal, thereby obtaining the scattering signal of the unlabeled single cell in the droplet.

[0073] In summary, this invention proposes a label-free single-cell multi-angle light scattering analysis system based on droplet photofluidization. A microfluidic pump delivers the sample from the sample inlet to the microfluidic channel. The aqueous phase material is formed into a uniformly mixed and refractive-index-adjustable aqueous liquid flow through a mixed-structure microchannel, then passes through a T-shaped microchannel to form water-in-oil microdroplets, which randomly encapsulate label-free single cells. The sample passes through a collimated incident light detection area, and the scattered light signal is collected by single-cell light scattering at different angles via multimode fiber. The multimode fiber connects to a PMT and an oscilloscope to acquire and analyze the sample light scattering signal in real time. Furthermore, through data feedback, the flow rate ratio of the aqueous phase material is adjusted to change the droplet optical properties, eliminating the droplet scattered light signal, thereby achieving label-free analysis within the droplet.

[0074] The above description is merely an illustration of preferred embodiments of the present invention, and the above technical features can be arbitrarily combined to form multiple embodiments of the present invention.

[0075] The present invention has been described above by way of example with reference to the accompanying drawings. Obviously, the specific implementation of the present invention is not limited to the above-described manner. Any non-substantial improvements made using the concept and technical solution of the present invention, or the direct application of the concept and technical solution of the present invention to other occasions without modification, are all within the protection scope of the present invention.

Claims

1. A label-free single-cell analysis system based on droplet photofluidization, characterized in that: include: The aqueous liquid flow control module includes two aqueous material sample inlets and a mixing structure microchannel. Two aqueous liquid materials flow into the mixing structure microchannel from the sample inlets to form a uniformly mixed aqueous liquid flow with adjustable refractive index. The oil liquid flow and the aqueous liquid flow flow through the T-shaped microchannel to form water-in-oil microdroplets. Individual cells are randomly encapsulated within droplets; The droplet generation module includes an oil phase material sample inlet, a sample outlet, a microfluidic channel, and a T-shaped microchannel for droplet generation; the oil phase material sample inlet is connected to the mixed structure microchannel and one end of the microfluidic channel through the T-shaped microchannel, and the other end of the microfluidic channel is connected to the sample outlet; The incident light module includes a corresponding incident light single-mode fiber microchannel, an incident light collimating microlens, and a monochromatic laser source; The scattered light receiving module includes a set of fiber optic microchannels at different angles. The set of fiber optic microchannels are arranged circumferentially and form a circle at the center. The microfluidic channel passes through the circular area. The photoelectric conversion and signal display module includes a connected oscilloscope and a set of photomultiplier tubes, with the photomultiplier tubes connected to the outer end of the corresponding fiber optic microchannel.

2. The label-free single-cell analysis system based on droplet photofluidization as described in claim 1, characterized in that: The microfluidic chip includes two aqueous material sample inlets, a mixed structure microchannel, an oil phase material sample inlet, a sample outlet, a microfluidic channel, a T-shaped microchannel, an incident light single-mode fiber microchannel, and a set of fiber microchannels, all of which are integrated on the microfluidic chip.

3. The label-free single-cell analysis system based on droplet photofluidization as described in claim 1, characterized in that: The angle range of the set of fiber optic microchannels is -145° to 140°, where the angle is defined as 0° with respect to the direction of incident light, with clockwise direction being negative and counterclockwise direction being positive.

4. The label-free single-cell analysis system based on droplet photofluidization as described in claim 2, characterized in that: The microstructure in the microfluidic chip includes two heights: the sample inlet, sample outlet, and microfluidic channel are shorter than the incident light single-mode fiber channel, the incident light collimating lens, and a set of fiber microchannels.

5. The label-free single-cell analysis system based on droplet photofluidization as described in claim 1, characterized in that: Both the sample inlet and the sample outlet are circular channels.

6. The label-free single-cell analysis system based on droplet photofluidization as described in claim 1, characterized in that: The hybrid structure microchannel is a zigzag-shaped bent microchannel structure.

7. The label-free single-cell analysis system based on droplet photofluidization as described in claim 1, characterized in that: The oil phase material sample inlet is connected to the contraction microchannel through the outer microchannel and the contraction microchannel, forming a T-shaped microchannel structure.

8. The label-free single-cell analysis system based on droplet photofluidization as described in claim 2, characterized in that: It also includes a glass slide, on which the microfluidic chip is fixed.

9. The label-free single-cell analysis system based on droplet photofluidization as described in claim 2, characterized in that: It also includes single-mode fiber and multimode fiber, with the fiber end with the cladding removed leading into the microfluidic chip, and the other end connected to the incident laser source and photomultiplier tube respectively, for transmitting the incident light and receiving the scattered light signal.

10. A label-free single-cell multi-angle light scattering analysis method based on droplet photofluidization, characterized in that: The analytical method includes the following steps: S1. Prepare oil-phase materials, aqueous-phase materials, and cell samples with a certain concentration; S2. The sample is pushed into the microfluidic channel by a micropump. The two aqueous phase materials form a uniformly mixed aqueous liquid flow with adjustable refractive index at the mixed structure microchannel. The oil liquid flow and the aqueous liquid flow flow through the T-shaped microchannel to form water-in-oil microdroplets. Individual cells are randomly wrapped in the droplets. S3. The incident light is introduced by a single-mode optical fiber and then collimated by a microlens to form parallel light for sample scattered light detection. S4. Collect the scattered light from the sample using a set of optical fibers at different angles. Use a photomultiplier tube and an oscilloscope to acquire and analyze the scattered light signal of the sample, including the scattered light signal of droplets and the scattered light signal of single cell samples. S5. Through data analysis and feedback, the flow rate ratio of the aqueous phase material is adjusted in real time to further reduce or eliminate the scattered light signal of the droplets, thereby obtaining the scattered light signal of unlabeled single cells in the droplets.

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