Novel microfluidic cell focusing and analysis method based on special optical fiber

Abstract

The invention provides a novel microfluidic cell focusing and analysis method based on a special optical fiber. The device is characterized by comprising a microfluidic channel, a seven-core optical fiber and a peripheral device, a frustum is processed at the optical fiber end of the seven-core optical fiber, light beams can form a convergent light field after passing through the frustum, a spherical capture area can be formed at a convergent point, and particles can form single cell flow after passing through the capture area; a middle core of the seven-core optical fiber transmits exciting light to irradiate a single cell flow, scattered light and fluorescent light can be generated, back scattered light passes through the middle core and is collected by a circulator, and fluorescent light signals are collected through side cores. The method can be used for generating single cell flow and can be widely applied to the fields of single cell analysis technology and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to a novel microfluidic chip for single-cell focusing and analysis of special optical fibers, which can be used for single-cell flow generation and analysis, and belongs to the technical field of microfluidic analysis. BACKGROUND

[0002] Cell analysis is one of the core technologies of modern life sciences, clinical diagnosis and drug research. Traditional cell analysis methods, such as flow cytometry and microscope imaging technology, are powerful and widely used, but they rely on large and expensive optical systems, complex sample pretreatment processes, and relatively large sample consumption, which limits their application in bedside diagnosis, on-site rapid detection, and long-term dynamic monitoring.

[0003] In recent years, microfluidic technology, also known as "chip laboratory", has brought revolutionary changes to cell analysis. This technology precisely manipulates extremely small amounts of fluid in micron-scale chip channels, integrating sample processing, reaction, separation and detection into a micro-platform, with the advantages of low reagent consumption, fast analysis speed, high integration, and great automation potential. The combination of microfluidic technology and cell analysis forms an important branch of microfluidic cell analysis, which can achieve high-throughput and high-precision manipulation and detection of single cells.

[0004] However, existing microfluidic cell analysis methods still face challenges in the detection process. The mainstream on-chip optical detection usually relies on external microscopes and photodetectors built on optical platforms, which to some extent deviates from the original intention of miniaturization and integration of microfluidic technology. Although some technologies attempt to integrate micro-LEDs and photodiodes onto the chip, their detection sensitivity, resolution and versatility often cannot match traditional optical systems.

[0005] On the other hand, optical fiber sensing technology has been widely used in biological sensing due to its small size, high sensitivity, and ease of integration. In particular, the emergence of special optical fibers provides a new way to solve the bottleneck of microfluidic cell analysis.

[0006] Special optical fibers (such as microstructured optical fibers, photonic crystal fibers, multi-core optical fibers, tapered optical fibers, etc.) have unique optical properties that traditional optical fibers do not have, such as:

[0007] Enhanced light-matter interaction: The air holes of microstructured optical fibers can serve as natural microfluidic channels, confining the cells to be tested in the core area of the light field propagation, greatly enhancing the interaction between light and cells, and significantly improving the detection sensitivity.

[0008] Flexible field distribution regulation: Multi-core optical fiber, tapered optical fiber, etc. can produce special evanescent field or interference field, which is extremely sensitive to the changes of refractive index, size, morphology, etc. of cells flowing through its surface or inside.

[0009] Multifunctional integration potential: A special optical fiber can realize multiple functions such as optical transmission, signal excitation and collection, and even cell manipulation (such as optical tweezers) at the same time, greatly simplifying the system structure.

[0010] The special optical fiber is used as the core sensing element to deeply and organically integrate with the microfluidic chip, and a highly integrated, sensitive and multifunctional cell analysis platform is constructed, which has become a clear technical development trend in this field. At present, some researches have reported the preliminary exploration of special optical fiber and microfluidic channel for cell detection, but these methods still need to be improved in terms of precise manipulation of cells, synchronous analysis of multiple parameters and system robustness.

[0011] In 2019, Wang Xiaoli et al. disclosed a forward scattering light detection system, a flow cytometer and a method for measuring cell diameter (application number: CN201910780140.8). In 2023, Li Kenhang et al. disclosed a fluid system of a flow cytometer (application number: CN202310219241.4). In the same year, Chen Qizhong et al. disclosed an optical system and a flow cytometer (application number: CN202310309231.X). In the above patents, complex optical elements are used to generate three-dimensional single cell flow, which is complex to assemble and has high cost.

[0012] In order to solve the problems of the prior art, the present application discloses a new microfluidic cell analysis method of special optical fiber. It can be used for generating single cell flow in flow cytometer, and can be widely used in flow cytometry, single cell spectral analysis and flow cytometry imaging fields. By processing a frustum on the end of the multi-core optical fiber, the light beam is converged, and a potential well for capturing particles is formed at the light beam convergence. The particles form a single cell flow when passing through the potential well area. The middle core of the multi-core optical fiber can transmit excitation light to irradiate on the cells, and the scattered light can be received from the middle core through the circulator, and the fluorescence signal can be received from the edge core of the multi-core optical fiber. SUMMARY

[0013] The purpose of the present application is to provide a new method for focusing and analyzing single cells in a microfluidic chip.

[0014] The purpose of the present application is achieved as follows:

[0015] A new type of microfluidic cell focusing and analysis method based on special optical fiber, which is composed of microfluidic channel, seven-core fiber and peripheral devices. The end of the seven-core fiber is processed with a frustum, and the light beam will form a converging light field after passing through the frustum. There will be a spherical capture area at the convergence point, and particles will form a single cell flow after passing through the capture area. The middle core of the seven-core fiber transmits excitation light to irradiate on the single cell flow, which will produce scattered light and fluorescence. The backscattered light is collected by the circulator through the middle core, and the fluorescence signal is collected by the edge core.

[0016] The end face distribution of the seven-core fiber is shown in Figure 5 , which contains 7 independent single-mode cores, with 1 single-mode core in the middle and 6 single-mode cores symmetrically distributed around it.

[0017] The single cell flow generation method based on the seven-core fiber is shown in Figure 1 . Unfocused cells (particles) flow into the channel from the left. The end of the seven-core fiber is processed with a frustum structure. Among the 7 independent cores of the seven-core fiber, the second edge core 4-6, the fourth edge core 4-8, and the sixth edge core 4-10 of the seven-core fiber are connected to a 980 nm wavelength laser. After passing through the fiber end frustum structure, it will converge to form a spherical capture area that can capture particles. When the flowing particles pass through the capture area, they will be captured to the center of the area. Due to the action of fluid thrust, the particles will continue to move forward, forming a single cell flow.

[0018] As shown in Figure 1 , the x-axis is perpendicular to the particle flow direction, and the y-axis is along the particle flow direction. The forces in the x and y directions are shown in Figure 3 and Figure 2 , respectively. As can be seen from the figure, the capture range is about 40 um. That is, in the spherical area with the convergence point as the center and 40 um as the radius, all particles can be captured to the convergence point.

[0019] The angle of the seven-core fiber end frustum is α°. Generally, α is 19.

[0020] The middle core of the seven-core fiber is used to transmit excitation light to excite the scattered light and fluorescence of the cells. The middle core receives the backscattered light signal, and the three edge cores receive the fluorescence signal. The specific connection diagram is shown in Figure 6 . The 488 nm laser 6-1, 532 nm laser 6-2, and 635 nm laser 6-3 are coupled into the middle core of the seven-core fiber through the fiber combiner 6-4. The seven-core fiber can independently control each core through the fan-in fan-out device 6-9.

[0021] For the middle core of the seven-core fiber, a circulator 6-5 is added between the fiber combiner 6-4 and the fan-in fan-out device 6-9, which is used to transmit excitation light while receiving backscattered light signals from cells.

[0022] The first 980 nm laser 6-6, the second 980 nm laser 6-7, and the third 980 nm laser 6-8 are connected with the second edge core 4-6 of the seven-core fiber, the fourth edge core 4-8 of the seven-core fiber, and the sixth edge core 4-10 of the seven-core fiber respectively through the seven-core fiber fan-in fan-out 6-9, and are used for transmitting the trapping light.

[0023] The fluorescent signal is received by the first edge core 4-5, the third edge core 4-7, and the fifth edge core 4-9, and is transmitted to the fluorescent photodetector 6-13 for receiving, and there are filters 6-12 corresponding to respective wavelengths between the first edge core 4-5, the third edge core 4-7, and the fifth edge core 4-9, and the filters 6-12 are used for filtering out stray light.

[0024] The middle core of the seven-core fiber has a core diameter of A um, and the edge core has a core diameter of B um, and generally, A is 4.5 and B is 9.

[0025] The middle core of the seven-core fiber has a numerical aperture of 0.12, and the edge core has a numerical aperture of 0.22.

[0026] The seven-core fiber can also be a four-core fiber, a nineteen-core fiber, and other structures in which a middle core is a single-mode fiber core and four peripheral cores are arranged in a symmetrical ring shape.

[0027] The width of the micro flow channel is A um, and the depth is B um, and generally, A is 125 and B is 125.

[0028] Through calculation, it is known that the trapping range of the seven-core fiber converging light beam is about 40 um, and therefore, if the channel depth is 125 um, a part of particles is not in the trapping range, and therefore, the diameter of the particle flow needs to be preliminarily reduced when the particles are injected.

[0029] The diameter of the particle flow is preliminarily reduced in a manner of capillary fiber auxiliary sheath liquid extrusion, and the micro flow chip structure is as shown in Figure 7 The capillary fiber is placed in the capillary fiber channel 7-6, the sample solution containing particles is injected from the air hole of the capillary fiber, the sheath liquid is injected through the first sheath liquid channel 7-1 and the second sheath liquid channel 7-5, and the sample solution is extruded so as to reduce the diameter of the sample solution.

[0030] The end surface of the capillary fiber is as shown in Figure 8 The cladding diameter is A um, the outer diameter of the annular core is B um, and the diameter of the middle air hole is C um, and generally, A is 125, B is 68, and C is 54.

[0031] Compared with the prior art, the present application has the following advantages:

[0032] (1) High integration, structure simplification: The core innovation of this design is to integrate all the key functional modules such as single-cell flow focusing, optical excitation (scattered light and fluorescence) and signal detection into a special optical fiber. This highly integrated design fundamentally replaces the complex lenses, spatial light paths and discrete detector arrays in traditional flow cytometers, greatly simplifying the overall structure and significantly reducing the size

[0033] (2) Precise alignment, high stability: Since all optical functions are achieved through the built-in waveguide structure of the same optical fiber, the excitation light path and the detection light path are permanently and precisely aligned at the factory. This "solidified light path" effectively avoids the problems of lens misalignment and light path misalignment caused by vibration, temperature drift or long-term use in traditional instruments, greatly improving the long-term stability and reliability of the system and reducing maintenance requirements.

[0034] (3) Easy to package, cost controllable: The simplified optical structure results in fewer mechanical fixtures and adjustment mechanisms, which not only reduces the difficulty and time of production and assembly, but also makes the packaging of the entire device more convenient. Thanks to the simplified structure and the use of standard optical fiber components, this solution has a significant cost advantage in mass production, laying a solid foundation for the miniaturization and popularization of flow cytometry devices. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 is a working schematic of a new type of microfluidic cell analysis method based on special optical fiber. 1-1 is a microfluidic channel, 1-2 is an optical potential well region, 1-3 is a particle forming a single-cell flow, 1-4 is a seven-core optical fiber, and 1-5 is an unfocused particle.

[0036] Figure 2 is a curve of the force change of the particle along the y direction.

[0037] Figure 3 is a curve of the force change of the particle along the x direction.

[0038] Figure 4 is a schematic diagram of particle scattered light, fluorescence excitation and detection based on a seven-core optical fiber. 4-1 is a particle passing through the middle core, 4-2 is an optical fiber end taper structure, 4-3 is the first fluorescence receiving light trajectory, 4-4 is the middle core of the seven-core optical fiber, 4-5 is the first edge core of the seven-core optical fiber, 4-6 is the second edge core of the seven-core optical fiber, 4-7 is the third edge core of the seven-core optical fiber, 4-8 is the fourth edge core of the seven-core optical fiber, 4-9 is the fifth edge core of the seven-core optical fiber, 4-10 is the sixth edge core of the seven-core optical fiber, 4-11 is the particle trapping optical fiber trajectory, and 4-12 is the excitation light trajectory.

[0039] Figure 5is a schematic diagram of the end face distribution of a seven-core optical fiber. 4-0 is the cladding of the seven-core optical fiber, 4-4 is the middle core of the seven-core optical fiber, 4-5 is the first edge core of the seven-core optical fiber, 4-6 is the second edge core of the seven-core optical fiber, 4-7 is the third edge core of the seven-core optical fiber, 4-8 is the fourth edge core of the seven-core optical fiber, 4-9 is the fifth edge core of the seven-core optical fiber, and 4-10 is the sixth edge core of the seven-core optical fiber.

[0040] Figure 6 is a schematic diagram of a scattered light and fluorescence detection peripheral device. 6-1 is a 488 nm laser, 6-2 is a 532 nm laser, 6-3 is a 635 nm laser, 6-4 is a fiber combiner, 6-5 is a circulator, 6-6 is a first 980 nm laser, 6-7 is a second 980 nm laser, 6-8 is a third 980 nm laser, 6-9 is a seven-core fiber fan-in fan-out device, 6-10 is a seven-core fiber, 6-11 is a computer, 6-12 is a filter, 6-13 is a fluorescence photodetector, 6-14 is a data acquisition card, and 6-15 is a scattered light detector.

[0041] Figure 7 is a schematic diagram of a microfluidic chip structure. 7-1 is a first sheath liquid channel, 7-2 is a microfluidic chip, 7-3 is a waste liquid outflow channel, 7-4 is a seven-core fiber channel, 7-5 is a second sheath liquid channel, and 7-6 is a capillary fiber channel.

[0042] Figure 8 is a schematic diagram of the end face of a capillary fiber. 8-1 is a cladding, 8-2 is an annular core, and 8-3 is an air hole. DETAILED DESCRIPTION

[0043] The application will be further described below in conjunction with specific examples.

[0044] Microfluidic chip processing process:

[0045] (1) Draw the designed microfluidic chip (as shown in Figure 7 ) into a three-dimensional graph in STL format, and the width and depth of the channel are both 125 um.

[0046] (2) Prepare a clean quartz wafer, place it on the processing platform in the femtosecond laser microprocessing system and fix it. Replace the objective lens with a 50X lens.

[0047] (3) Import the three-dimensional graph in STL format drawn into the slicing software, set the Z-direction slicing interval to 1 um, and the path spacing to 500 nm.

[0048] (4) Select the wavelength of the femtosecond laser to be 515 nm, and set the laser processing power to 2 mw.

[0049] (5) Start the processing process and wait for the processing to be completed.

[0050] (6) Put the processed quartz piece into 10% hydrofluoric acid for 30 minutes.

[0051] (7) Mix PDMS and curing agent in a ratio of 10:1, and use a vacuum pump to remove air bubbles. Put the PDMS into a heating furnace for curing, with a heating temperature of 80°C.

[0052] (8) Put the cured PDMS and microfluidic channel into a plasma cleaning device for processing.

[0053] (9) Put the PDMS and microfluidic channel processed by the plasma cleaning device together, and put them into a vacuum drying box. Vacuumize and heat, and the microfluidic chip is completed.

[0054] Take a piece of seven-core optical fiber with a length of 1 m, remove the coating layer of 3 cm, grind the end face flat, put it into the optical fiber grinder, and grind the optical fiber end taper angle to 19°. Wash the residual substances in the ultrasonic cleaning machine. Insert it into the seven-core optical fiber channel 7-4, and weld the other end of the optical fiber with the multi-core optical fiber fan-in fan-out device 6-9.

[0055] Take a piece of capillary optical fiber with a length of 1 m, remove the coating layer of 3 cm, grind the end face flat, and put it into the capillary optical fiber channel 7-6.

[0056] 488 nm laser 6-1, 532 nm laser 6-2 and 635 nm laser 6-3 are connected to port 1 of circulator 6-5 through fiber combiner 6-4, port 2 of circulator 6-5 is connected to middle core 4-4 of seven-core optical fiber through seven-core optical fiber fan-in fan-out device, port 3 of circulator 6-5 is connected to scattered light detector 6-15. First 980 nm laser 6-6, second 980 nm laser 6-7 and third 980 nm laser 6-8 are connected to second edge core 4-6, fourth edge core 4-8 and sixth edge core 4-10 of seven-core optical fiber through seven-core optical fiber fan-in fan-out device 6-9 respectively. Fluorescence photodetector 6-13 is connected to first edge core 4-5, third edge core 4-7 and fifth edge core 4-9 of seven-core optical fiber through seven-core optical fiber fan-in fan-out device 6-9. Fluorescence photodetector 6-13 and scattered light detector 6-15 are connected to data acquisition card 6-14, and data acquisition card 6-14 is connected to computer 6-11.

[0057] Inject sheath liquid into first sheath liquid channel 7-1 and second sheath liquid channel 7-5 through pressure pump, and inject sample solution containing particles into air hole of capillary optical fiber through pressure pump. The pressure ratio of sheath liquid channel to sample solution is set to 1:1, and the particle flow diagram at this time is shown in Figures 1-5 .

[0058] The fiber end output power of the first 980 nm laser 6-6, the second 980 nm laser 6-7 and the third 980 nm laser 6-8 is set to 100 mW. After the laser passes through the fiber end taper, it converges to form a potential well 1-2 that can capture particles.

[0059] When the unfocused particles 1-5 pass through the optical potential well area 1-2, they will be captured to the center of the potential well due to the action of the optical force, and at the same time, the single cell flow is realized due to the fluid thrust to the right.

[0060] The power of the 488 nm laser 6-1, the 532 nm laser 6-2 and the 635 nm laser 6-3 is set to 20 mW. When the laser irradiates the particles on the single cell flow, scattered light and fluorescent signals will be generated. The backscattered light is collected by the middle core of the seven-core fiber through the circulator, and the corresponding fluorescent signals generated by different excitation lights are collected by the first edge core 4-5, the third edge core 4-7 and the fifth edge core 4-9 of the seven-core fiber.

Claims

1. A novel microfluidic cell focusing and analysis method based on specialty optical fiber, characterized by: It is composed of microfluidic channel, seven-core optical fiber and peripheral devices; the fiber end of the seven-core optical fiber is processed with a frustum, and the light beam will form a converging light field after passing through the frustum, and there will be a spherical capture area at the converging point, and the particles will form a single cell flow after passing through the capture area; the middle core of the seven-core optical fiber transmits excitation light to irradiate on the single cell flow, which will produce scattered light and fluorescence, and the backscattered light is collected by the ring-shaped device through the middle core, and the fluorescence signal is collected through the edge core.

2. The novel microfluidic cell focusing and analysis method based on special optical fiber according to claim 1, characterized in that, The fiber end taper angle is 17°-19°.

3. The novel microfluidic cell focusing and analysis method based on special optical fiber according to claim 1, characterized in that, The seven-core optical fiber can be a four-core optical fiber or a nineteen-core optical fiber.

4. The novel microfluidic cell focusing and analysis method based on special optical fiber according to claim 1, characterized in that, The middle core diameter of the seven-core optical fiber is 4.5 um, and the edge core diameter is 9 um.

5. The novel microfluidic cell focusing and analysis method based on special optical fiber according to claim 1, characterized in that, The numerical aperture of the middle core of the seven-core optical fiber is 0.12, and the numerical aperture of the edge core is 0.22.

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