A new method and device for single-cell fiber-integrated scatter fluorescence detection

Abstract

This invention provides a novel fiber-optic integrated backscattered fluorescence detection method and device for single cells. Its features include: an outer sheath, a coreless optical fiber, a first capillary optical fiber, a second capillary optical fiber, a multi-core optical fiber, the analyte particles, a microfluidic pump module, a waste liquid collection tube, a light source module, a photoelectric detection module, and a computer. The analyte cell solution is injected into the first capillary optical fiber through the microfluidic pump module and flows out through the second capillary optical fiber. A portion of the multi-core optical fiber core is used to transmit laser light, while another portion is used to collect the scattered light and fluorescence from the cells. This invention can be used for the simultaneous detection of backscattered light and sidescattered fluorescence from single cells and can be widely applied in fields such as single-cell fluorescence detection and mass spectrometry analysis.

Description

(I) Technical Field

[0001] This invention relates to a novel fiber-optic integrated scattering fluorescence detection method and device for single cells, which can be used for simultaneous measurement of multiple parameters of scattered light in cell spectral analysis, and belongs to the field of biological cell analysis technology. (II) Background Technology

[0002] Flow cytometry is a novel analytical and sorting technique developed in the 1970s that enables rapid measurement of cellular or subcellular structures. Flow cytometry is a comprehensive and interdisciplinary product of modern scientific and technological development, representing the culmination of multiple technological advancements.

[0003] Traditional flow cytometers use spatial light sources to excite particles and employ spatial optical components to detect, collect, and analyze scattered light and fluorescence. However, they have limitations such as large system size and high cost. Therefore, improvements to flow cytometers are urgently needed.

[0004] In recent years, the widespread adoption and development of microfluidic technology has provided new directions for the advancement of flow cytometry. Devices based on microfluidic technology can rapidly complete sample detection. Furthermore, experiments that previously could only be performed in biological or chemical laboratories can now be completed on a chip measuring only a few square centimeters. Microfluidic chips offer advantages such as high portability, low reagent consumption, high automation, and parallel processing capabilities. Therefore, this presents a significant opportunity to provide portable, accurate, and sensitive flow cytometers for resource-scarce regions and some developing countries.

[0005] In 2019, Li Qingling et al. disclosed an optical fluidic flow cytometer for circulating tumor cell isolation, analysis, and typing (publication number CN110186836B), which combines the advantages of microfluidic chips, fluid dynamics, and flow cytometry. This achieves automated, continuous blood sample injection, efficient separation of circulating tumor cells, 3D focusing, and highly sensitive, multi-parameter, real-time in-situ single-cell analysis, along with sensitive and high-throughput typing detection. In the same year, Dai Li et al. (application number CN201910181056.4) disclosed a flow cytometer based on microfluidic three-dimensional focusing technology, featuring a lensless sensor below the microfluidic chip for image acquisition. These inventions utilize microfluidic chips to form single-cell flows, while the collection of excitation and scattered light is achieved through optical devices. Although this reduces the overall volume of the flow cytometer, the spatial optical devices still occupy a significant portion of the volume, and the collection of scattered light and fluorescence by these devices incurs substantial losses.

[0006] In 2020, Yuan Libo et al. disclosed an improved flow cytometer based on an integrated microfluidic chip using optical fibers (application number CN202010931452.7), integrating optical fibers with a microfluidic chip to achieve single-cell analysis. In the same year, they also disclosed a flow cytometer based on an integrated microfluidic chip using optical fibers (application number: CN202010770059.4), integrating optical fibers and electrodes onto the microfluidic chip to achieve single-cell analysis and sorting. These inventions replace bulky spatial optical devices with optical fibers, integrating them into the microfluidic chip. However, the drawbacks are that currently, if the microfluidic chip is fabricated using PDMS casting, a series of complex mask fabrication processes are required, which are cumbersome and costly; the optical fiber size is comparable to the microfluidic chip, making it difficult to insert the fiber into the microfluidic chip.

[0007] This invention discloses a novel fiber-optic integrated method and apparatus for detecting backscattered and side-scattered fluorescence in single-cell flow cytometers. Hollow-core, coreless, and multi-core optical fibers are arranged in a specific order and fixed together using a sleeve to form a microfluidic cavity, replacing the traditional method of forming a single-cell flow using a microfluidic chip. The hollow-core fibers on both sides are used to transmit the cells to be tested, the edge cores of the multi-core fibers are used to excite cell fluorescence and collect fluorescence simultaneously, and the middle core is used to collect side-scattered light. This invention further reduces the size of traditional flow cytometers, further integrates functions, further reduces costs, and simplifies fabrication. (III) Summary of the Invention

[0008] The purpose of this invention is to provide a novel fiber-optic integrated backscattering and side scattering fluorescence detection method and device for single-cell flow that is simple in structure, easy to operate and integrate.

[0009] The objective of this invention is achieved as follows:

[0010] A novel fiber-optic integrated scattering fluorescence detection method and device for single cells is characterized by comprising an outer sheath 1, a coreless optical fiber 2, a first capillary optical fiber 3, a second capillary optical fiber 4, a multi-core optical fiber 5, a sample 6, a microfluidic pump module 7, a waste liquid collection tube 8, a light source module 9, a photoelectric detection module 10, and a computer 11. In this system, the coreless optical fiber 2 is positioned opposite the first capillary optical fiber 3, and the second capillary optical fiber 4 is positioned opposite the multi-core optical fiber 5. The coreless optical fiber 2, the first capillary optical fiber 3, the second capillary optical fiber 4, and the multi-core optical fiber 5 are fixed by the outer sheath 1. The sample cell solution is injected through the microfluidic pump module 7 from the first capillary optical fiber 3 and flows out from the second capillary optical fiber 4. Waste liquid flows out to the waste liquid collection tube 8, and the coreless optical fiber 2 is used to seal any excess outlet. One end of the multi-core optical fiber 5 is processed with a frustum through grinding to converge the light beam. The light source module 9 injects excitation light through a portion of the edge core of the multi-core optical fiber 5 to excite fluorescence. At the same time, the excited fluorescence is collected by another portion of the edge core of the multi-core optical fiber 5, and the backscattered light is collected by the middle core of the multi-core optical fiber 5. The fluorescence and scattered light collected by the multi-core optical fiber are converted into electrical signals by the photoelectric detection module 10 and then transmitted to the computer 11 for recording.

[0011] The first capillary fiber is connected to a pneumatic microfluidic pump via a flexible tube. After the cell suspension enters the first capillary fiber, the cells converge to the center of the first capillary fiber due to inertial forces, thus generating a single-cell flow. Because a coreless fiber is blocking the position directly opposite the first capillary fiber, the single-cell flow changes direction and flows into the second capillary fiber.

[0012] The second capillary fiber is placed opposite the multi-core fiber, so when a single cell flows into the second capillary fiber, it will pass through the end of the multi-core fiber.

[0013] One end of the multi-core optical fiber is processed with a frustum by grinding to converge the beam.

[0014] Multi-core optical fibers can be four-core optical fibers, seven-core optical fibers, or fiber core ring distribution optical fibers.

[0015] Multi-core optical fiber can be an array-distributed fiber with an outer circle and an inner square, containing two types of cores with different numerical apertures, arranged in a square pattern. The cores with larger numerical apertures are used to collect fluorescence and scattered light, while those with smaller numerical apertures are used to transmit laser light.

[0016] A schematic diagram of the outer circle and inner square array distributed fiber end face structure is shown below. Figure 7 As shown, there are two types of fiber cores: the thinner core is used to transmit single-mode excitation light, while the thicker core has a larger numerical aperture for better collection of fluorescence and scattered light.

[0017] The excitation source is transmitted from a portion of the edge core of a multi-core optical fiber, converges at the fiber end onto the single-cell stream, and the fluorescence signal flowing through the particles is excited. Since the fluorescence signal is non-directional, the remaining edge core can be used to collect the fluorescence signal.

[0018] A light source with no wavelength interference is used for fluorescence detection, which illuminates the middle core of a multi-core optical fiber and can receive backscattered light from cells through a circulator.

[0019] The received fluorescence and backscattered light are converted into electrical signals by a photodetector, then collected by a data acquisition card and sent to a computer for storage.

[0020] Compared with the prior art, the outstanding advantages of the present invention are:

[0021] (1) A novel multi-core fiber optic device for backscattering and sidescattering fluorescence detection of single-cell flow was proposed, realizing the integration of a compact single-cell optical analysis path, laying the technical foundation for the miniaturization of flow cytometers.

[0022] (2) Easy to implement, low cost, and easy to assemble. (iv) Description of the attached drawings

[0023] Figure 1 This is a structural diagram of the entire device. 1 is the outer jacket, 2 is the coreless optical fiber, 3 is the first capillary optical fiber, 4 is the second capillary optical fiber, 5 is the multi-core optical fiber, 6 is the particle to be measured, 7 is the microfluidic pump module, providing power to the fluid, 8 is the waste liquid collection tube, 9 is the light source module, 10 is the photoelectric detection module, and 11 is the computer.

[0024] Figure 2 This is a diagram of a microfluidic chip structure. 1 is the outer sheath, 2 is the coreless optical fiber, 3 is the first capillary optical fiber, 4 is the second capillary optical fiber, 5 is the multi-core optical fiber, and 6 is the particle to be measured.

[0025] Figure 3 This diagram shows the connection of the peripheral devices at the fiber end, using a seven-core optical fiber as an example. 3-1 is a multi-core optical fiber with a tapered fiber end; 3-2 is the first laser; 3-3 is the second laser; 3-4 is the third laser; 3-5 is the first photodetector; 3-6 is the second photodetector; 3-7 is the third photodetector; 3-8 is the fourth photodetector; 3-9 is the data acquisition card; 3-10 is the computer; 3-11 is the backscattering light source; and 3-12 is the circulator.

[0026] Figure 4 This is a schematic diagram of the end face of a seven-core optical fiber. 4-1 is the fiber core, and 4-2 is the cladding.

[0027] Figure 5 This is a schematic diagram of a seven-core fiber optic light source transmission. 5-1 shows the particle to be measured, and 5-2 shows the transmission of the light source within the fiber core.

[0028] Figure 6 This is a schematic diagram of fluorescence and backscattered light in a seven-core optical fiber. 6-1 is the particle to be tested, 6-2 is a schematic diagram of fluorescence transmission in the fiber core, and 6-3 is a schematic diagram of backscattered light transmission in the fiber core.

[0029] Figure 7 This is a schematic diagram of an optical fiber end face with an outer circle and an inner square shape. 7-1 is the cladding, 7-2 is the single-mode fiber core, and 7-3 is the large numerical aperture fiber core. (V) Detailed Implementation

[0030] The present invention will be further illustrated below with reference to specific embodiments.

[0031] Take a coreless optical fiber with a diameter of 125 μm and remove the coating.

[0032] Prepare the first and second capillary optical fibers, both with a diameter of 125um and a capillary diameter of 90um. Remove the coating and cut the end face flat.

[0033] Take a multi-core optical fiber with a diameter of 125um, remove the coating layer, and cut the end face flat; first grind the end of the optical fiber into a truncated cone shape with coarse sandpaper, and then polish it with fine sandpaper.

[0034] Take a 2cm long capillary tube with an inner diameter of 250um for packaging.

[0035] Coreless optical fiber, capillary optical fiber, multi-core optical fiber, and outer sheath are classified according to... Figure 2 The optical fibers are assembled in this manner. The gaps between the optical fibers and the sleeves are then sealed with UV-cured adhesive to prevent liquid leakage.

[0036] Multi-core fiber optic tail connector method as follows Figure 3 As shown in the diagram. The first laser 3-2 has a wavelength of 488nm, 3-3 has a wavelength of 532nm, and the third laser 3-4 has a wavelength of 635nm. These three lasers are used to excite fluorescence. The backscattering light source 3-11 has a wavelength of 980nm, and this light source is used to detect backscattered light. All four light sources are simultaneously transmitting light. Because the multi-core fiber has frustums at the ends, the beams converge after passing through the frustums. A schematic diagram of the beam transmission is shown below. Figure 5 As shown.

[0037] Cell fluid is injected into the first capillary optical fiber via a pneumatic pump. Due to inertial forces, the cells converge towards the center of the capillary optical fiber, forming a single-cell stream. When a cell passes the intersection of the light beams, its fluorescence is excited.

[0038] Fluorescence and backscattered light collection optical paths are as follows Figure 6As shown, the remaining core of the multi-core optical fiber is used for fluorescence collection. The first photodetector 3-5, the second photodetector 3-6, and the third photodetector 3-8 collect fluorescence excited at wavelengths of 488nm, 532nm, and 635nm, respectively. Each photodetector receiver is equipped with a filter of a specific wavelength to eliminate stray light interference with the fluorescence.

[0039] Backscattering light source 3-11 injects light through the intermediate core, and the scattered light that hits the cell returns to the photodetector through the circulator.

[0040] All photodetectors convert the received light into electrical signals, which are received by the data acquisition card. The acquired data is then transmitted to a computer for analysis and storage.

Claims

1. A fiber-optically integrated scattered fluorescence detection device for single cells, characterized by: The detection device consists of an outer sheath (1), a coreless optical fiber (2), a first capillary optical fiber (3), a second capillary optical fiber (4), a multi-core optical fiber (5), a particle to be tested (6), a microfluidic pump module (7), a waste liquid collection tube (8), a light source module (9), a photoelectric detection module (10), and a computer (11). The coreless optical fiber (2) is positioned opposite the first capillary optical fiber (3), and the second capillary optical fiber (4) is positioned opposite the multi-core optical fiber (5). The coreless optical fiber (2), the first capillary optical fiber (3), the second capillary optical fiber (4), and the multi-core optical fiber (5) are fixed by an outer sheath (1). The cell fluid to be tested is injected from the first capillary optical fiber (3) through the microfluidic pump module (7) and flows out from the second capillary optical fiber (4). The waste liquid flows out to the waste liquid collection tube (8). The coreless optical fiber (2) is used to seal the excess outlet. The multi-core fiber (5) has a truncated cone at one end through grinding to focus the light beam. The light source module (9) injects the excitation light through part of the edge core of the multi-core fiber (5) to excite fluorescence. At the same time, the excited fluorescence is collected by another part of the edge core of the multi-core fiber (5), and the backscattered light is collected by the middle core of the multi-core fiber (5). The fluorescence and scattered light collected by the multi-core fiber are converted into electrical signals by the photoelectric detection module (10) and transmitted to the computer (11) for recording. The multi-core fiber (5) contains two types of fiber cores with different numerical apertures, and the fiber cores are arranged in a square. The fiber core with a large numerical aperture is used to collect fluorescence and scattered light, and the fiber core with a small numerical aperture is used to transmit laser light.

2. The fiber-optic integrated scattering fluorescence detection device for single cells according to claim 1, characterized in that: One end of the multi-core optical fiber (5) used is processed with a truncated cone to converge the beam.

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