CN116908149A - Novel optical fiber integrated scattering fluorescence detection method and device for single cells - Google Patents
Novel optical fiber integrated scattering fluorescence detection method and device for single cells Download PDFInfo
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- CN116908149A CN116908149A CN202310238278.1A CN202310238278A CN116908149A CN 116908149 A CN116908149 A CN 116908149A CN 202310238278 A CN202310238278 A CN 202310238278A CN 116908149 A CN116908149 A CN 116908149A
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- 239000002245 particle Substances 0.000 claims abstract description 9
- 238000001514 detection method Methods 0.000 claims abstract description 8
- 239000002699 waste material Substances 0.000 claims abstract description 8
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- 210000004027 cell Anatomy 0.000 description 29
- 238000010586 diagram Methods 0.000 description 8
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/01—Arrangements or apparatus for facilitating the optical investigation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/47—Scattering, i.e. diffuse reflection
- G01N21/49—Scattering, i.e. diffuse reflection within a body or fluid
- G01N21/53—Scattering, i.e. diffuse reflection within a body or fluid within a flowing fluid, e.g. smoke
Abstract
The invention provides a novel method and a device for single-cell optical fiber integrated scattering fluorescence detection. The method is characterized in that: the device consists of an outer sleeve, a coreless optical fiber, a first capillary optical fiber, a second capillary optical fiber, a multi-core optical fiber, particles to be tested, a microfluidic pump module, a waste liquid collecting pipe, a light source module, a photoelectric detection module and a computer. The cell fluid to be tested is injected from the first capillary optical fiber through the microfluidic pump module, flows out from the second capillary optical fiber, and one part of the fiber cores of the multi-core optical fiber are used for transmitting laser, and the other part of the fiber cores are used for collecting scattered light and fluorescence of cells. The invention can be used for simultaneous detection of the back scattered light and the side scattered fluorescence of single cells, and can be widely used in the fields of single cell fluorescence detection, mass spectrometry and the like.
Description
Field of the art
The invention relates to a novel optical fiber integrated scattering fluorescence detection method and device for single cells, which can be used for simultaneous measurement of multiple parameters of cell spectrum analysis and scattered light, and belongs to the technical field of biological cell analysis.
(II) background art
The flow analysis technology is a novel analysis technology and a sorting technology which can rapidly test the cell or subcellular structure developed in the 70 s of the 20 th century. Flow cytometry is a comprehensive cross product of modern scientific and technical development, and is a 'adult' integrating multiple technical developments.
The traditional flow cytometer uses a space light source to excite particles, uses a space optical element to detect, collect and analyze scattered light and fluorescence, has the limitations of huge system volume, high price and the like, and therefore, the improvement of the flow cytometer is urgent.
In recent years, the popularization and development of microfluidic technology provide a new direction for the progress of flow cytometry. The device based on the development of the microfluidic technology can rapidly complete the detection of the sample, and meanwhile, the previous experiment which can only be completed in a biological or chemical laboratory can be completed only by a chip with a size of a few square centimeters. The microfluidic chip has the advantages of high portability, small reagent consumption, strong automation, parallelization treatment and the like, so that the microfluidic chip is a good opportunity for providing a portable, accurate and sensitive flow cytometer for resource-deficient areas and some developing countries.
In 2019, li Qingling et al disclose an optofluidic flow cytometer (publication number CN 110186836B) for separating, analyzing and typing counting circulating tumor cells, which combines the advantages of microfluidic chips, hydrodynamics and flow cytometry, and realizes automatic and continuous blood sample injection, efficient separation of circulating tumor cells, 3D focusing, high sensitivity, multi-parameter, real-time in-situ single cell analysis and other operations and sensitive and high-throughput typing counting detection. In the same year Dai Li et al (application number CN 201910181056.4) disclose a flow cytometer based on microfluidic three-dimensional focusing technology, and a lens-free sensor is arranged below a microfluidic chip, which is beneficial to image acquisition. The single cell flow is formed by a microfluidic chip, the collection of excitation light and scattered light is realized by an optical device, the volume is reduced in the flow type single cell flow, but the space optical device still occupies a large part of the volume, and the space optical device can generate great loss when collecting the scattered light, fluorescence and the like.
In 2020, yuan Liang et al disclose an improved flow cytometer (application number CN 202010931452.7) based on an integrated microfluidic chip with optical fibers integrated with the microfluidic chip to perform single cell analysis. In the same year, they disclose a flow cytometer (application number: CN 202010770059.4) based on an optical fiber integrated microfluidic chip, wherein an optical fiber and an electrode are integrated on the microfluidic chip, so as to realize the functions of analyzing and sorting single cells. These inventions replace the otherwise bulky spatial optics with optical fibers, integrating the fibers into microfluidic chips. However, the method has the defects that if the current microfluidic chip is realized by adopting a PDMS pouring mode, a series of complex procedures are needed for processing the mask, the procedures are complicated, and the processing cost is too high; the optical fiber size is equivalent to that of the microfluidic chip, and the optical fiber is difficult to be inserted into the microfluidic chip.
The invention discloses a novel optical fiber integrated backward and side scattering fluorescence detection method and a device thereof for single cell flow, which have the advantages of simple and exquisite structure, easy operation and integration. The hollow optical fiber, the coreless optical fiber and the multi-core optical fiber are arranged in a certain order, and a plurality of optical fibers are fixed together in a sleeve mode to form a micro-flow cavity, so that the traditional method of forming single cell flow by using a micro-flow chip is replaced. The hollow optical fibers on two sides are used for transmitting cells to be detected, the side cores of the multi-core optical fibers are used for exciting cell fluorescence and collecting fluorescence at the same time, and the middle core is used for collecting side scattering light. The invention further reduces the volume of the traditional flow cytometer, further integrates functions, further reduces the cost and is simpler to manufacture.
(III) summary of the invention
The invention aims to provide a novel optical fiber integrated backward and side scattering fluorescence detection method and a device thereof for single cell flow, which have simple and exquisite structure and are easy to operate and integrate.
The purpose of the invention is realized in the following way:
a novel method and a device for single-cell optical fiber integrated scattering fluorescence detection are characterized in that: the device consists of an outer sleeve 1, a coreless optical fiber 2, a first capillary optical fiber 3, a second capillary optical fiber 4, a multi-core optical fiber 5, particles 6 to be detected, a microfluidic pump module 7, a waste liquid collecting pipe 8, a light source module 9, a photoelectric detection module 10 and a computer 11. In the system, the coreless optical fiber 2 is opposite to the first capillary optical fiber 3, and the second capillary optical fiber 4 is opposite to 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 sleeve 1. The cell liquid to be tested is injected from the first capillary optical fiber 3 through the microfluidic pump module 7, flows out from the second capillary optical fiber 4, and the waste liquid flows out to the waste liquid collecting pipe 8, and the coreless optical fiber 2 is used for sealing redundant outlets. One end of the multi-core optical fiber 5 is processed with a frustum in a grinding mode and is used for converging light beams, the light source module 9 injects excitation light through part of side cores of the multi-core optical fiber 5 and is used for exciting fluorescence, and meanwhile the other part of side cores of the multi-core optical fiber 5 is used for collecting the excited fluorescence, and the middle core of the multi-core optical fiber 5 is used for collecting back scattered light. The fluorescence and scattered light collected by the multi-core optical fiber are converted into electric signals by the photoelectric detector module 10 and then transmitted to the computer 11 for recording.
The first capillary optical fiber is connected with the air pressure microfluidic pump through a hose, and after the cell suspension enters the first capillary optical fiber, cells can be converged to the center of the first capillary optical fiber due to the action of inertia force, so that single cell flow is generated. Because there is no core fiber blocking in the position opposite to the first capillary fiber, the single cell flow will change direction and flow into the second capillary fiber.
The second capillary fiber is positioned opposite the multicore fiber so that a single cell passes through the fiber end of the multicore fiber before flowing into the second capillary fiber.
One end of the multi-core optical fiber is processed with a frustum in a grinding mode and is used for converging light beams.
The multi-core optical fiber can be four-core optical fiber, seven-core optical fiber and fiber core annular distribution optical fiber.
The multi-core optical fiber can be a round outer square inner square array distribution optical fiber, and comprises fiber cores with two numerical apertures, wherein the fiber cores are arranged in a square shape, the fiber cores with large numerical apertures are used for collecting fluorescence and scattered light, and the fiber cores with small numerical apertures are used for transmitting laser.
The end face structure of the outer circle and inner square array distribution optical fiber is shown in fig. 7, the fiber core has two sizes, the thin fiber core is used for transmitting single-mode excitation light, the numerical aperture of the thick fiber core is large, and the thin fiber core is used for better collecting fluorescence and scattered light.
The excitation light source is transmitted from part of the side core of the multi-core optical fiber, and is converged to the single cell flow through the fiber end, and fluorescent signals flowing through the particles can be excited. Since the fluorescent signal is unoriented, the fluorescent signal can be collected with the remaining side core.
Light from the intermediate core of the multi-core fiber is transmitted by a light source that does not interfere with fluorescence detection at the wavelength, and the backscattered light from the cell can be received by the circulator.
The received fluorescence and the back scattered light are converted into electric signals through the photoelectric detector, then are collected by the data collection card, and are sent to a computer for storage.
Compared with the prior art, the invention has the outstanding advantages that:
(1) The novel device for detecting the multi-core optical fiber by using the backward and side scattering fluorescence of the single cell flow is provided, the integration of a compact single cell optical analysis light path is realized, and a technical foundation is laid for microminiaturization of a flow cytometer.
(2) Easy to realize, low in cost and convenient to assemble.
(IV) description of the drawings
Fig. 1 is a structural diagram of the whole apparatus. Wherein 1 is the outer tube, 2 is coreless fiber, 3 is first capillary fiber, 4 is second capillary fiber, 5 is multicore fiber, 6 is the particle that awaits measuring, 7 is the micropump module, provides power for fluid, 8 is the waste liquid collecting pipe, 9 is the light source module, 10 is the photoelectric detection module, 11 is the computer.
Fig. 2 is a block diagram of a microfluidic chip. Wherein 1 is an outer sleeve, 2 is a coreless optical fiber, 3 is a first capillary optical fiber, 4 is a second capillary optical fiber, 5 is a multi-core optical fiber, and 6 is a particle to be measured.
Fig. 3 is a connection diagram of a peripheral device at the fiber end, taking a seven-core optical fiber as an example. 3-1 is a multi-core optical fiber of a fiber end grinding cone, 3-2 is a first laser, 3-3 is a second laser, 3-4 is a third laser, 3-5 is a first photoelectric detector, 3-6 is a second photoelectric detector, 3-7 is a third photoelectric detector, 3-8 is a fourth photoelectric detector, 3-9 is a data acquisition card, 3-10 is a computer, 3-11 is a back scattering light source, and 3-12 is a circulator.
Fig. 4 is a schematic diagram of a seven-core fiber end face. 4-1 is the core and 4-2 is the cladding.
Fig. 5 is a schematic diagram of seven-core fiber optic light source transmission. 5-1 is the particle to be measured and 5-2 is the schematic of the light source transmission in the core.
Fig. 6 is a schematic diagram of seven-core fiber fluorescence and back-scattered light. 6-1 is the particle to be measured, 6-2 is the transmission of fluorescence in the core, and 6-3 is the transmission of backscattered light in the core.
FIG. 7 is a schematic diagram of an end face of an outer-round inner-square optical fiber. 7-1 is the cladding, 7-2 is the single mode core, and 7-3 is the large numerical aperture core.
(fifth) detailed description of the invention
The invention is further illustrated below in conjunction with specific examples.
Taking a coreless optical fiber with the diameter of 125um, and removing the coating layer.
Preparing a first capillary optical fiber and a second capillary optical fiber, wherein the diameters of the optical fibers are 125um, the sizes of the intermediate capillaries are 90um, removing the coating layer, and cutting and flattening the end face.
Taking a multi-core optical fiber with the diameter of 125um, stripping a coating layer, and cutting the end face to be smooth; the fiber ends were first ground to a frustum with coarse sandpaper and then polished with fine sandpaper.
A capillary tube 2cm long and 250 μm in inner diameter was used for packaging.
The coreless fiber, capillary fiber, multicore fiber, and outer jacket were assembled as in fig. 2. And the gaps between the optical fibers and the sleeve are sealed by ultraviolet curing adhesive, so that liquid leakage is prevented.
The tail end connection mode of the multi-core optical fiber is shown in fig. 3. The wavelength of the first laser 3-2 is 488nm, the wavelength of the third laser 3-3 is 532nm, the wavelength of the third laser 3-4 is 635nm, and the three lasers are used for exciting fluorescence; the wavelength of the back-scattered light source 3-11 is 980nm, which is used for the detection of back-scattered light. The four light sources are simultaneously electrified, and as the multi-core optical fiber end is processed with the frustum, the light beam can form a converging effect through the frustum, and the light beam transmission schematic diagram is shown in fig. 5.
Cell fluid is injected from the first capillary fiber by a pneumatic pump. Due to the action of inertia force, cells can be converged towards the middle of the capillary optical fiber to form single cell flow. When the cell passes through the beam intersection point, the cell fluorescence will be excited.
The fluorescence, back-scattered light collection path is shown in fig. 6. Fluorescence collection was performed with the remaining cores of the multicore fiber. The first photoelectric detector 3-5, the second photoelectric detector 3-6 and the third photoelectric detector 3-8 respectively collect fluorescence excited by wavelength of 488nm,532nm and 635nm, and the receiving end of the photoelectric detector is provided with a filter with specific wavelength for eliminating interference of stray light on fluorescence.
The back-scattered light sources 3-11 inject light through the intermediate core and the scattered light impinging on the cells is returned to the photodetector through the circulator.
All photoelectric detectors convert received light into electric signals, the electric signals are received by a data acquisition card, and acquired data are transmitted to a computer for analysis and storage.
Claims (3)
1. A novel method and a device for single-cell optical fiber integrated scattering fluorescence detection are characterized in that: the system comprises an outer sleeve (1), a coreless optical fiber (2), a first capillary optical fiber (3), a second capillary optical fiber (4), a multi-core optical fiber (5), particles to be detected (6), a micro-flow pump module (7), a waste liquid collecting pipe (8), a light source module (9), a photoelectric detection module (10) and a computer (11), wherein in the system, the coreless optical fiber (2) is opposite to the first capillary optical fiber (3), and the second capillary optical fiber (4) is opposite to the multi-core optical fiber (5); the device comprises a coreless optical fiber (2), a first capillary optical fiber (3), a second capillary optical fiber (4) and a multi-core optical fiber (5), wherein an outer sleeve (1) is arranged outside the coreless optical fiber (2), the second capillary optical fiber (4) and the multi-core optical fiber (5), cell liquid to be detected is injected from the first capillary optical fiber (3) through a microfluidic pump module (7), waste liquid flows out of the second capillary optical fiber (4) to a waste liquid collecting pipe (8), the coreless optical fiber (2) is used for sealing an excessive outlet, one end of the multi-core optical fiber (5) is processed with a frustum in a grinding mode and is used for converging light beams, a light source module (9) injects excitation light into part of side cores of the multi-core optical fiber (5) and is used for exciting fluorescence, fluorescence excited by the other part of side cores of the multi-core optical fiber (5) is collected by the middle cores of the multi-core optical fiber (5), and the fluorescence and scattered light collected by the multi-core optical fiber is converted into an electrical signal through a photoelectric detector module (10) and then transmitted to a computer (11) for recording.
2. The novel method and device for single-cell optical fiber integrated scattering fluorescence detection, as claimed in claim 1, characterized in that: one end of the adopted multi-core optical fiber (5) is provided with a frustum for converging light beams.
3. The novel method and device for single-cell optical fiber integrated scattering fluorescence detection, as claimed in claim 1, characterized in that: the multi-core optical fiber (5) simultaneously comprises fiber cores with two numerical apertures, the fiber cores are arranged in a square shape, the fiber cores with large numerical apertures are used for collecting fluorescence and scattered light, and the fiber cores with small numerical apertures are used for transmitting laser.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310238278.1A CN116908149A (en) | 2023-03-13 | 2023-03-13 | Novel optical fiber integrated scattering fluorescence detection method and device for single cells |
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| CN202310238278.1A CN116908149A (en) | 2023-03-13 | 2023-03-13 | Novel optical fiber integrated scattering fluorescence detection method and device for single cells |
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| CN202310238278.1A Pending CN116908149A (en) | 2023-03-13 | 2023-03-13 | Novel optical fiber integrated scattering fluorescence detection method and device for single cells |
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- 2023-03-13 CN CN202310238278.1A patent/CN116908149A/en active Pending
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