Single cell sorting system and sorting method based on laser-induced jet flow
By combining laser-induced jet and microfluidic control technology, a special plating sorting chip and PDMS microfluidic control chip are used to achieve efficient and lossless single-cell sorting, solving the contradiction between sorting accuracy-fluid-cell activity in the existing technology, and supporting high-throughput screening and sequencing.
Patent Information
- Application Number
- CN202510699471.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-28
- Publication Date
- 2025-08-15
AI Technical Summary
There is a contradiction between the sorting accuracy-throughput-cell activity in the existing single-cell sorting technology, and it is difficult for the existing technology to achieve high accuracy, high throughput and not damage cells.
Combining laser-induced jet and microfluidic control technology, a special plating sorting chip and PDMS microfluidic chip are used to generate bubbles and push the jet through laser induction, realizing contactless sorting, and screening cells with multi-parameter information.
It achieves efficient single-cell sorting efficiency of 50 per minute and has a survival rate of 94%. It supports multi-parameter screening of label-free rare cells, and is suitable for high-throughput screening and sequencing.
Smart Images
Figure CN120484953A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biomedical engineering and precision instrument technology, and in particular, is a single cell sorting system and a sorting method based on laser-induced jet, which are suitable for sorting, culturing and sequencing of cells and bacteria. Background Art
[0002] Current single-cell sorting technologies mainly rely on flow cytometry (FACS), optical tweezers, magnetic sorting, and microfluidic chips, but there are still bottlenecks in the following key technologies, such as:
[0003] ① Limitations of flow cytometry (FACS);
[0004] Fluorescence dependence and cell damage: Fluorescent labeling is required to identify cells, which may interfere with the natural activity of cells. In addition, the sorting process involves high-voltage sheath fluid, high-frequency oscillation, and electric field deflection, which leads to a decrease in cell survival rate.
[0005] The contradiction between low precision and throughput: the sorting accuracy is limited by the droplet generation frequency, and the throughput is significantly reduced during high-precision sorting.
[0006] ② Shortcomings of optical tweezers technology;
[0007] Extremely low throughput: Optical tweezers manipulate cells one by one through optical traps, and the sorting speed is usually <10 cells / min, which is difficult to meet high-throughput requirements.
[0008] Risk of thermal damage: Prolonged irradiation with high-power lasers may cause local heating, leading to cell membrane rupture or protein denaturation.
[0009] ③ Challenges of microfluidic sorting technology;
[0010] Fluid dynamics limitations: Relying on passive fluid control (such as inertial focusing and dielectrophoresis), sorting accuracy is greatly affected by channel design and flow rate, making it difficult to achieve submicron positioning.
[0011] Weak multi-parameter sorting capabilities: Existing microfluidic systems usually only support a single marker (such as size or surface antigen), and it is difficult to simultaneously integrate multi-dimensional characteristics such as mechanics and electrophysiology.
[0012] ④ Development bottleneck of laser induced sorting technology;
[0013] Energy control problems: For example, in laser-induced forward transfer (LIFT) technology, improper matching of laser energy density and pulse width can easily lead to irreversible cell damage (such as membrane perforation or DNA breakage).
[0014] Existing technologies generally face the contradiction of "sorting accuracy-throughput-cell activity": high-precision technologies (such as optical tweezers) sacrifice throughput; high-throughput technologies (such as flow cytometry) damage cell activity; non-labeling technologies (such as Raman spectroscopy) are expensive and have limited sorting speed. Summary of the Invention
[0015] In order to solve the above technical problems, the present invention provides a single-cell sorting system and sorting method based on laser-induced jet, which combines laser-induced forward transfer with a microfluidic scheme, and bonds a sorting chip with a special coating and a PDMS microfluidic chip with micro-pit capture to form a jet microfluidic sorting chip, ensuring that the liquid environment of the cells does not change. By focusing a 532nm pulsed laser on the coating, bubbles can be generated to push the liquid to form a jet, and the target cells are identified using bright field or fluorescence. The cells captured in the micro-pits are selectively flushed out, and the cells are flushed to the outlet of the flow channel and received by the receiver in a drop-by-tube manner for cell culture and downstream analysis.
[0016] A single cell sorting system and sorting method based on laser-induced fluidics, wherein:
[0017] A single cell sorting system based on laser-induced fluidics, comprising:
[0018] Laser induction module, jet microfluidic sorting chip and microscopic imaging module;
[0019] Furthermore, the laser induction module includes, in order from left to right: a pulsed laser, a first lens, a second lens, a beam modulation module, a first reflector, and an objective lens disposed directly below the first reflector;
[0020] As an example, the pulse laser specification is: 532nm.
[0021] As an example, the first lens and the second lens are used as a lens combination to expand the pulsed laser beam.
[0022] As an example, the beam modulation module is used to modulate the light beam into various mode beams.
[0023] As an example, the objective lens is used to focus a light beam onto a specially coated surface, inducing bubble formation and driving a jet.
[0024] Furthermore, the jet microfluidic sorting chip refers to: a sorting chip with a special coating bonded to a microfluidic chip, wherein: the microfluidic chip contains a micro-pit capture structure and a fluid channel.
[0025] As an example, the microfluidic chip is a PDMS microfluidic chip, which is made of PDMS by soft lithography or photocuring 3D printing, and contains a micropit array with a diameter of 10-50 μm, the depth of which matches the cell size, supporting single cell capture and dynamic manipulation.
[0026] As an example, the special coating sorting chip refers to a sorting chip using a glass substrate and subjected to metal coating.
[0027] As an example, the metal coating includes: a gold film or a titanium film; thickness: 50-200 nm.
[0028] Furthermore, the microscopic imaging module includes, in order from left to right: a camera, a first focusing lens, a fluorescent filter turntable, a second reflector, and also includes a second focusing lens and a light source arranged in sequence directly above the fluorescent filter turntable, and an imaging objective lens arranged directly above the second reflector.
[0029] As an example, the microscopy imaging module can realize multimodal imaging, integrate bright field and fluorescence modules, and support label-free cell identification (such as morphology, size) and labeled cell screening (such as fluorescent labeling).
[0030] As an example, the camera is a high-speed camera.
[0031] A sorting method based on a laser-induced fluidics single-cell sorting system, comprising:
[0032] Step 1: Cell capture;
[0033] ① Prepare cell suspension;
[0034] ② The prepared cell suspension is injected into the microfluidic chip at a flow rate of 1–5 μL / min, and single cells are captured through its own micro-pit capture structure;
[0035] Step 2: Target identification and jet triggering;
[0036] The microscopic imaging module collects target cell information. If the target cell information meets the screening conditions, the laser induction module is triggered. Laser pulses act on the special coating to generate micron-sized bubbles. The bubbles expand to produce jets, which flush the target cells out of the micro-pits and into the exit channel.
[0037] As an example, the screening condition refers to: specific fluorescence intensity or morphological parameters.
[0038] As an example, after the laser pulse (pulse width <2ns) is absorbed by the coating, high temperature is instantly generated, causing the material to liquefy, vaporize or evaporate. The liquid in contact with the coating generates micron-sized bubbles under the action of heat and force.
[0039] As an example, when the laser pulse width is much shorter than the thermal diffusion time (a 10 ns pulse corresponds to a thermal diffusion depth of approximately 1 μm), the heat is confined to a very small volume near the coating and will not cause thermal damage to the cells.
[0040] Step 3: Single cell collection;
[0041] The sorted target cells are dropped into the receiving device in a "one drop per tube" format for subsequent culture, sequencing or drug treatment.
[0042] Beneficial effects of the present invention:
[0043] The single-cell sorting system achieves a single-cell sorting efficiency of 50 cells per minute, a 100% success rate, and a 94% survival rate for received single cells. Single cells can be sorted directly after being introduced into the microfluidic chip, or they can be cultured to form monoclonal clones before sorting. Alternatively, cells can be immunofluorescently stained, drug-treated, or transfected within the microfluidic chip before sorting.
[0044] This system is easy to use and has potential applications in a variety of high-throughput screening assays, including cell analysis, drug screening, and phenotype-based cell sorting.
[0045] This invention adopts a non-contact sorting design concept: the laser only acts on the special coating material, avoiding direct irradiation of cells and reducing phototoxicity; the jet impact time is <1ms, the cells are evenly stressed, and the risk of membrane damage is low.
[0046] The present invention adopts a multi-parameter sorting strategy: combining multi-dimensional information such as morphology and fluorescence, it can screen unlabeled rare cells (such as circulating tumor cells) or cells in a specific metabolic state (such as drug-resistant populations) BRIEF DESCRIPTION OF THE DRAWINGS
[0047] Figure 1 This is a schematic diagram of the overall structure of a single-cell sorting system based on laser-induced fluidics of the present invention.
[0048] Figure 2.1 This is a schematic diagram of the structure of a jet microfluidic sorting chip for a single cell sorting system based on laser-induced jets. (Wherein: Figure 2.1 A is a three-dimensional schematic diagram of the chip. Figure 2.1 B is a schematic diagram of the cross section of the chip)
[0049] Figure 2.2 This is a physical picture of the jet microfluidic sorting chip of the single cell sorting system based on laser-induced jet in the present invention.
[0050] Figure 2.3This is a schematic diagram of the jet sorting principle of a jet microfluidic sorting chip in a single cell sorting system based on laser-induced jet according to the present invention.
[0051] Figure 3 This is a schematic diagram of the overall process of a single cell sorting method based on a laser-induced fluidics system of the present invention. (Wherein: Figure 3 A: Single cell loading is captured by micropits. Figure 3 B is the single cell identification and sorting process, Figure 3 C is the process of single cells being washed away and received in the flow channel.)
[0052] Figure 4 This is an example diagram of the experimental effect of a single cell sorting method based on a laser-induced fluidics system of the present invention. (Wherein: Figure 4 A is the image of a single cell captured by a micropit before sorting. Figure 4 B is the image after single cell sorting. Figure 4 C is the image of a single cell received by the receiver, Figure 4 D. Figure 4 E. Figure 4 F Figure 4 A. Figure 4 B. Figure 4 Fluorescence imaging of C.
[0053] Figure 5 This is a high-speed camera recording of the sorting process of a single-cell sorting system based on laser-induced fluidics of the present invention. (Wherein: Figure 5 A. Figure 5 B. Figure 5 C. Figure 5 D are images at 1ms, 2ms, 3ms, and 32ms respectively)
[0054] Figure 6 This is a schematic diagram of the staining effect of confirming cell activity in a sorting method of a single-cell sorting system based on laser-induced fluidics of the present invention. DETAILED DESCRIPTION
[0055] The technical solutions in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work shall fall within the scope of protection of the present application. Figures 1 to 6 shown.
[0056] A single cell sorting system and sorting method based on laser-induced fluidics, wherein:
[0057] A single cell sorting system based on laser-induced fluidics, comprising:
[0058] Laser induction module 101, jet microfluidic sorting chip 102 and microscopic imaging module 103;
[0059] Furthermore, the laser induction module includes, in order from left to right: a pulse laser 201, a first lens 202, a second lens 203, a beam modulation module 204, a first reflector 205, and an objective lens 206 disposed directly below the first reflector;
[0060] As an example, the pulse laser specification is: 532nm.
[0061] As an example, the first lens 202 and the second lens 203 are used as a lens combination to expand the pulsed laser beam.
[0062] As an example, the beam modulation module 204 is used to modulate the light beam into various mode beams.
[0063] As an example, the objective lens 206 is used to focus the light beam on a special coating surface to induce bubble generation and drive the jet.
[0064] Furthermore, the jet microfluidic sorting chip 102 refers to: a sorting chip with a special coating bonded to a microfluidic chip, wherein: the microfluidic chip contains a micro-pit capture structure and a fluid channel.
[0065] As an example, the microfluidic chip is a PDMS microfluidic chip, which is made of PDMS by soft lithography or photocuring 3D printing, and contains a micropit array with a diameter of 10-50 μm, the depth of which matches the cell size, supporting single cell capture and dynamic manipulation.
[0066] As an example, the special coating sorting chip refers to a sorting chip using a glass substrate and subjected to metal coating.
[0067] As an example, the metal coating includes: a gold film or a titanium film; thickness: 50-200 nm.
[0068] Furthermore, the microscopic imaging module 103 includes, in order from left to right: a camera 301, a first focusing lens 302, a fluorescent filter wheel 303, a second reflector 306, and also includes a second focusing lens 304 and a light source 305 arranged in sequence directly above the fluorescent filter wheel 303, and an imaging objective lens 307 arranged directly above the second reflector 306.
[0069] As an example, the microscopic imaging module 103 can realize multimodal imaging, integrate bright field and fluorescence modules, and support label-free cell identification (such as morphology, size) and labeled cell screening (such as fluorescent labeling).
[0070] A sorting method based on a laser-induced fluidics single-cell sorting system, comprising:
[0071] Step 1: Cell capture;
[0072] ① Prepare cell suspension;
[0073] ② The prepared cell suspension is injected into the microfluidic chip at a flow rate of 1–5 μL / min, and single cells are captured through its own micro-pit capture structure;
[0074] Step 2: Target identification and jet triggering;
[0075] The microscopic imaging module collects target cell information. If the target cell information meets the screening criteria, the laser induction module is triggered. Laser pulses act on the special coating to generate micron-sized bubbles. The bubbles expand to produce jets, which flush the target cells out of the micro-pits and into the exit channel.
[0076] As an example, the screening condition refers to: specific fluorescence intensity or morphological parameters.
[0077] Step 3: Single cell collection;
[0078] The sorted target cells are dropped into the receiving device in a "one drop per tube" format for subsequent culture, sequencing or drug treatment.
[0079] In order to better illustrate the design advantages of the present invention, the present invention is now compared with the prior art in sorting operations, and the comparative data are as follows:
[0080] 1. Sorting efficiency and throughput:
[0081] The sorting efficiency of the present invention reaches 50 cells / minute, far exceeding traditional optical tweezers (<10 cells / minute) and dielectrophoresis technology (≈20 cells / minute).
[0082] 2. Cell activity and compatibility:
[0083] The sorting survival rate of the present invention is 94%, which is significantly higher than that of optical tweezers, flow cytometry and piezoelectric impact printing.
[0084] 3. The present invention supports in situ cell manipulation: the microfluidic chip can integrate a culture chamber (supporting 3D cell culture), a drug loading module (such as a microvalve to control concentration gradients), and a transfection reagent channel (such as an electroporation area) to achieve staining and processing before sorting or direct analysis after sorting.
[0085] This invention achieves non-destructive sorting through a short laser action time (<2ns), a heat diffusion distance of <2μm, and a cell survival rate of 94%. The jet momentum acts only on the target cell micropits and has no effect on neighboring cells. The sorting throughput is 50 cells / minute, and the single-cell sorting success rate is 100%. The entire process is closed-loop: sorted cells can be directly used for culture or downstream single-cell sequencing. It supports combination with in situ analytical technologies (such as Raman spectroscopy and impedance sensing) to achieve "sorting-analysis" integration.
[0086] The above are only preferred embodiments of the present invention. It should be understood that the description of the above embodiments is only used to help understand the method and core ideas of the present invention, and is not used to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, etc. made within the ideas and principles of the present invention should be included in the scope of protection of the present invention.
Claims
1. A single cell sorting system based on laser-induced fluidics, characterized in that: include: Laser induction module, jet microfluidic sorting chip and microscopic imaging module; The laser induction module includes, in order from left to right: a pulse laser, a first lens, a second lens, a beam modulation module, a first reflector, and an objective lens disposed directly below the first reflector; The jet microfluidic sorting chip refers to: a sorting chip with a special coating bonded to a microfluidic chip, wherein: the microfluidic chip contains a micro-pit capture structure and a fluid channel; The microscopic imaging module includes, from left to right, a camera, a first focusing lens, a fluorescent filter wheel, a second reflector, a second focusing lens and a light source arranged in sequence directly above the fluorescent filter wheel, and an imaging objective lens arranged directly above the second reflector.
2. A single cell sorting system based on laser-induced fluidics according to claim 1, characterized in that: The pulse laser specification is: 532nm.
3. The single cell sorting system based on laser-induced fluidics according to claim 1, characterized in that: The first lens and the second lens are combined as a lens to expand the pulsed laser beam.
4. The single cell sorting system based on laser-induced fluidics according to claim 1, characterized in that: The light beam modulation module is used to modulate the light beam into light beams of various modes.
5. The single cell sorting system based on laser-induced fluidics according to claim 1, characterized in that: The objective lens is used to focus the light beam on the special coating surface, induce bubble generation and drive the jet.
6. The single cell sorting system based on laser-induced fluidics according to claim 1, characterized in that: The microfluidic chip is a PDMS microfluidic chip, which is made of PDMS by soft lithography or photocuring 3D printing, and contains a micropit array with a diameter of 10-50 μm, supporting single cell capture and dynamic manipulation.
7. The single cell sorting system based on laser-induced fluidics according to claim 1, characterized in that: The special coating sorting chip refers to a sorting chip that uses a glass substrate and is metal-coated.
8. The single cell sorting system based on laser-induced fluidics according to claim 7, characterized in that: The metal coating includes: gold film or titanium film; thickness: 50-200nm.
9. The single cell sorting system based on laser-induced fluidics according to claim 1, characterized in that: The camera is a high-speed camera.
10. A sorting method based on a laser-induced fluidics single-cell sorting system, comprising: Step 1: Cell capture; ① Prepare cell suspension; ② The prepared cell suspension is injected into the microfluidic chip at a flow rate of 1–5 μL / min, and single cells are captured through its own micro-pit capture structure; Step 2: Target identification and jet triggering; The microscopic imaging module collects target cell information. If the target cell information meets the screening criteria, the laser induction module is triggered. Laser pulses act on the special coating to generate micron-sized bubbles. The bubbles expand to produce jets, which flush the target cells out of the micro-pits and into the exit channel. Step 3: Single cell collection; The sorted target cells are dropped into the receiving device in a "one drop per tube" format for subsequent culture, sequencing or drug treatment.