CN111239029A - Time-resolved flow type fluorescence detection analysis device and use method thereof - Google Patents
Time-resolved flow type fluorescence detection analysis device and use method thereof Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
- G01N15/1434—Optical arrangements
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
- G01N15/1404—Handling flow, e.g. hydrodynamic focusing
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/01—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials specially adapted for biological cells, e.g. blood cells
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N2015/1006—Investigating individual particles for cytology
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
- G01N15/1434—Optical arrangements
- G01N2015/1438—Using two lasers in succession
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Abstract
The application discloses time-resolved flow-type fluorescence detection analytical equipment, including leading system, sample cell, sheath liquid pond, the room that flows, first laser instrument, the second laser instrument, the capillary, the optical collector, second laser instrument time-resolved fluorescence detecting element, first laser instrument fluorescence detector, data acquisition system, wherein, sheath liquid pond with the bottom of the room that flows is equipped with the opening, the capillary is used for guiding the flow of the microballon mixture liquid that awaits measuring, the capillary sets up in the sheath liquid pond, the bottom of capillary is visited the opening of the room that flows to form the detection zone, first laser instrument with the second laser instrument is arranged into and can arouse microballon mixture liquid in the detection zone. The application also provides a using method of the time-resolved flow type fluorescence detection analysis device.
Description
Technical Field
The application relates to the technical field of medical diagnostic equipment, in particular to a novel time-resolved flow type fluorescence detection analyzer.
Background
The Flow Cytometer (Flow Cytometer) measures scattered light and labeled fluorescence intensity of cells and other biological particles to quickly analyze physical or chemical properties of the particles, can simultaneously measure a plurality of characteristic parameters from one cell to perform qualitative or quantitative analysis, and has the characteristics of high speed, high precision and good accuracy. The liquid path system is a basic system of the flow cytometer, and a sample liquid containing a cell or a microparticle stained by fluorescence is transferred to a flow cell together with a sheath liquid, the cell or the microparticle is formed into a sample flow in a laminar flow state by using a liquid flow focusing principle, and the cell or the microparticle sequentially passes through a detection region of the flow cell. The stable sample flow is the guarantee of the subsequent signal detection and is one of the core technologies of the whole instrument. At present, the flow cytometry detection fluorescence signal has many influencing factors, receives the interference of nonspecific fluorescence, and has high background value, low detection sensitivity and low repeatability.
Many complexes and proteins in biological fluids and serum can themselves fluoresce, and the sensitivity of fluorescence detection using conventional chromophores is severely compromised. Most of the background fluorescence signal is present for a short time, the fluorescence lifetime is very short, the excitation light disappears, and the fluorescence also disappears. However, rare earth metals (Eu, Tb, Sm and Dy) or quantum dot substances with very few fluorescent lifetime can reach 1-2 ms, and can meet the measurement requirement, so that a time-resolved fluorescence or quantum dot fluorescence analysis method is generated. The technology is mainly used in clinic and scientific research at present, lanthanide is used for marking antigen or antibody, fluorescence is measured by using a time resolution technology according to the characteristics of lanthanide luminescence, and two parameters of wavelength and time are detected simultaneously for signal analysis, so that nonspecific fluorescence can be effectively eliminated, the sensitivity of analysis is greatly improved, background interference is reduced, and the repeatability is good. At present, the time-resolved fluorescence technology cannot detect multiple indexes at one time in a high flux manner, and batch processing of samples is realized.
Disclosure of Invention
The present application mainly aims to provide a time-resolved flow-type fluorescence detection and analysis apparatus and a method for using the same, so as to solve the problem that the time-resolved fluorescence and the flow-type cell technology cannot be combined in the related art. In order to achieve the above object, in a first aspect, the present application provides a time-resolved flow-type fluorescence detection and analysis apparatus, which is characterized by comprising a front system, a sample tube, a sheath liquid pool, a flow chamber, a first laser, a second laser, a capillary tube, an optical collector, a second laser time-resolved fluorescence detection unit, a first laser fluorescence detector, and a data acquisition system, wherein the sheath liquid pool and the bottom of the flow chamber are provided with an opening, the capillary tube is used for guiding the flow of microsphere mixed liquid to be detected, the capillary tube is disposed in the sheath liquid pool, the bottom of the capillary tube is extended out of the opening of the flow chamber to form a detection zone, and the first laser and the second laser are arranged to excite the microsphere mixed liquid in the detection zone.
Preferably, the excitation wavelength of the first laser is 600-650nm, and the optimal excitation wavelength of the second laser is 290-380 nm.
The optimal excitation wavelength of the first laser is 635nm, the first laser is matched with the fluorescence detector of the first laser for use through the light collector, the optimal excitation wavelength of the second laser is 295nm, 340nm and 365nm, and the time-resolved fluorescence detection unit of the second laser is matched for use through the light collector
Preferably, after the pulse is excited by the second laser, the second laser time-resolved fluorescence detection unit delays for 4ns-60000ms and then detects and records the fluorescence signal excited by the second laser.
Preferably, the delay time interval of the second laser time-resolved fluorescence detection unit is 8ns-2000 ms.
Preferably, the microsphere mixed solution comprises fluorescent dye high-molecular coding microspheres, liquid or solid microspheres of a sample to be detected and rare earth lanthanide or a chelate thereof.
Preferably, the fluorescent substance in the microsphere mixture solution excited by the second laser comprises a quantum dot fluorescent substance or a liquid or solid microsphere of rare earth lanthanide or its chelate, and the lanthanide is preferably one of europium (Eu), terbium (Tb), samarium (Sm) or any combination thereof.
Preferably, the fluorescent substance excited by the first laser comprises at least two fluorescent dye polymer-encoded microspheres with different spectral characteristics, and preferably, the diameter of the polymer-encoded microspheres is 1 micron to 20 microns.
In another aspect of the application, a method for using the time-resolved flow fluorescence detection analysis device is provided, it is characterized in that the covalent crosslinking of the microsphere excited by the first laser and the protein, the antibody or the nucleic acid probe, the covalent crosslinking of the time-resolved fluorescent substance excited by the second laser and the protein, the antibody or the nucleic acid probe, after the sample to be detected is added, a pre-system is used for carrying out incubation and oscillation treatment on a sample to be detected, sample liquid and sheath liquid are conveyed to a flow chamber together, liquid particles to be detected sequentially pass through a capillary detection area of the flow chamber and are excited by a first laser, a fluorescence detector of the first laser collects fluorescence signals through a light collector, simultaneously, the second laser is excited, after a period of time delay, the time-resolved fluorescence detection unit of the second laser is started, the fluorescence signal is recorded and collected by the light collector, and the collected signal is analyzed and processed by the data collection system.
The application has the following advantages: the device disclosed by the application organically and complementarily combines a quantum dot substance or time-resolved fluorescence technology with a flow cytometry technology, and comprises a front-end system, a flow chamber, a flow cell, a sheath liquid tube, a sample tube, a capillary tube, a first laser (used for exciting a fluorescent substance of microspheres), a second laser (used for exciting time-resolved fluorescent microspheres or quantum dot fluorescent microspheres), a light collector, a fluorescence detector, a time-resolved fluorescence detection unit and a data acquisition system. The time-resolved flow-type fluorescence detection analyzer performs signal resolution by using two parameters of wavelength and time, can perform high-speed multi-index joint detection, can analyze cells, proteins and nucleic acids, can effectively eliminate interference of non-specific fluorescence, and greatly improves analysis sensitivity.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this application, serve to provide a further understanding of the application and to enable other features, objects, and advantages of the application to be more apparent. The drawings and their description illustrate the embodiments of the invention and do not limit it. In the drawings:
FIG. 1 is a schematic structural diagram of a time-resolved flow-type fluorescence detection and analysis apparatus according to the present invention;
reference numerals
1-a front-end system, 2-a sample tube, 3-a sheath liquid pool, 4-a flow chamber, 5-a first laser, 6-a second laser, 7-a capillary, 8-a light collector, 9-a second laser time-resolved fluorescence detection unit, 10-a first laser fluorescence detector, 11-a data acquisition system and 12-a microsphere mixed solution.
Detailed Description
In order to make the technical solutions better understood by those skilled in the art, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is obvious that the described embodiments are only partial embodiments of the present application, but not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.
It should be noted that the terms "first," "second," and the like in the description and claims of this application and in the drawings described above are used for distinguishing between similar elements and not necessarily for describing a particular sequential or chronological order. It should be understood that the data so used may be interchanged under appropriate circumstances such that embodiments of the application described herein may be used. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed, but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.
In this application, the terms "upper", "lower", "left", "right", "front", "rear", "top", "bottom", "inner", "outer", "middle", "vertical", "horizontal", "lateral", "longitudinal", and the like indicate orientations or positional relationships based on the orientations or positional relationships shown in the drawings. These terms are used primarily to better describe the present application and its embodiments, and are not used to limit the indicated devices, elements or components to a particular orientation or to be constructed and operated in a particular orientation.
Moreover, some of the above terms may be used to indicate other meanings besides the orientation or positional relationship, for example, the term "on" may also be used to indicate some kind of attachment or connection relationship in some cases. The specific meaning of these terms in this application will be understood by those of ordinary skill in the art as appropriate.
In addition, the term "plurality" shall mean two as well as more than two.
It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present application will be described in detail below with reference to the embodiments with reference to the attached drawings.
The application provides a time-resolved flow-type fluorescence detection and analysis device, which comprises a front-end system 1, a sample tube 2, a sheath fluid pool 3, a flow chamber 4, a first laser 5, a second laser 6, a capillary 7, a light collector 8, a second laser time-resolved fluorescence detection unit 9, a first laser fluorescence detector 10, a data acquisition system 11 and a microsphere mixed solution 12, wherein,
the optimal excitation wavelength of the first laser 5 is 635nm, the first laser fluorescence detector 10 is matched with the light collector 8 for use, the optimal excitation wavelength of the second laser 6 is 295nm, 340nm and 365nm, and the second laser time-resolved fluorescence detection unit 9 is matched with the light collector 8 for use;
after the second laser 6 excites the pulse, the second laser time-resolved fluorescence detection unit 9 delays a certain time and then starts to detect and record the fluorescence signal excited by the second laser 6;
the delay time interval of the second laser time-resolved fluorescence detection unit 9 is from 4 nanoseconds to 60000ms, and the optimal delay time interval is from 8 nanoseconds to 2000 milliseconds;
the microsphere mixed solution 12 comprises fluorescent dye polymer coding microspheres, liquid or solid microspheres of a sample to be detected (or a calibrator) and rare earth lanthanide or a chelate thereof.
The main working flow and the using method of the time-resolved flow type fluorescence detection and analysis device in the application are as follows: the method comprises the steps of covalently crosslinking microspheres excited by a first laser 5 with a protein, antibody or nucleic acid probe, covalently crosslinking time-resolved fluorescent substances excited by a second laser 6 with the protein, antibody or nucleic acid probe, adding a sample to be detected, incubating and oscillating the sample by a front system 1, conveying sample liquid and sheath liquid to a flow chamber 4, allowing liquid particles to be detected to sequentially pass through a detection area of a capillary tube 7 of the flow chamber, exciting the first laser 5, collecting fluorescent signals by a first laser fluorescence detector 10 through a light collector 8, exciting the second laser 6, delaying for a certain time, starting a second laser time-resolved fluorescence detection unit 9 to record and collect the fluorescent signals through the light collector 8, and analyzing and processing the collected signals through a data collection system 11.
Example 1
Different types of high molecular coding microspheres are respectively coupled with 1 to 50 primary antibodies or antigens and then mixed according to a certain proportion to form high molecular coding microsphere suspension A, time-resolved fluorescent microspheres are also respectively crosslinked with 1 to 50 secondary antibodies or antigens to form time-resolved microsphere suspension B according to a certain proportion, and the high molecular coding microspheres can be identified through coding fluorescence thereof, so that the detection of multiple reactions can be simultaneously carried out in a mixed reaction system. The other secondary antibody or antigen is cross-linked with the time-resolved fluorescent microsphere for measuring the amount of biological reaction. When in application, the high-molecular coding microspheres of different detection objects are directly mixed, a sample to be detected is added, then the time-resolved microsphere suspension B is added to form a microsphere mixed solution 12, target molecules in the suspension are specifically combined with molecules crosslinked on the surfaces of the microspheres, and dozens of or hundreds of different biological reactions can be simultaneously completed in one reaction hole without mutual interference. And finally, measuring by using a time-resolved flow type fluorescence detection and analysis device, and analyzing the microsphere mixed solution 12 by two beams of laser. One of the 635nm red laser-excited microspheres' own fluorescent substance is used to distinguish different items of detection, while the other 365nm laser-excited time-resolved fluorescent microsphere (lanthanide such as europium (Eu), terbium (Tb), samarium (Sm) or chelate thereof) is used to detect the amount of reaction binding. The high-speed laser signal processing reads the microsphere codes to classify the microsphere codes and quantify the reaction on the surfaces of the microsphere codes. The difference lies in that a mode of delaying measurement time is adopted after 365nm exciting light pulse passes in detection, after the decay of short-life background fluorescence disappears, a sampling gate instrument is opened to record the specific fluorescence emitted by the long-life time-resolved fluorescent microspheres, so that background fluorescence interference can be avoided, and the detection precision is improved. The process is as follows: covalent crosslinking of microspheres excited by a first laser 5 and a protein, antibody or nucleic acid probe, covalent crosslinking of a time-resolved fluorescent substance excited by a second laser 6 and the protein, antibody or nucleic acid probe, adding a sample to be detected, performing incubation oscillation treatment by a front-end system 1, conveying a microsphere mixed solution 12 and sheath liquid to a flow chamber, sequentially passing the microsphere mixed solution 12 through a capillary 7 detection area of the flow chamber, exciting the first laser 5, collecting a fluorescence signal by a first laser fluorescence detector 10 through a light collector 8, exciting the second laser 6 at the same time, delaying for a certain time, usually from 10 nanoseconds to 1500 milliseconds, starting a second laser time-resolved fluorescence detection unit 9 to record and collect the fluorescence signal through the light collector 8, and analyzing and processing the collected signal by a data collection system 11.
The background signal value measured by the time-resolved fluorescent microspheres is very low, is similar to the background signal of an instrument, and is greatly lower than the signal value of a common flow type fluorescent detection reagent.
Example 2
Polystyrene microspheres with the diameter of 5.6 microns are respectively coupled with 1 to 20 primary antibodies, antigens, enzymes or nucleic acids, then mixed according to a certain proportion to form a high-molecular coding microsphere suspension A, and samarium (Sm) time-resolved fluorescent microspheres with the diameter of 80 to 300nm are also respectively crosslinked with 1 to 20 secondary antibodies, antigens, enzymes or nucleic acids, so as to form a time-resolved microsphere suspension B according to a certain proportion. The following were added sequentially to 96-well reaction plates: and (3) after the sample (or calibrator) to be detected and the polymer coding microsphere suspension A are added, fully and uniformly mixing in the front-end system 1, and placing in an incubator at 30-37 ℃ for reaction for 10-50 minutes in a dark place. And then adding the time-resolved microsphere suspension B into the reaction plate, fully mixing the mixture, placing the mixture at a temperature of between 30 and 37 ℃ and carrying out a light-shielding reaction in an incubator in the front-end system 1 for 10 to 50 minutes. After the reaction is finished, adding the stop solution, and fully and uniformly mixing. The up reading is then taken by a time-resolved flow fluorometric assay. The microsphere mixed liquid 12 and the sheath liquid are conveyed to the flow chamber together, the microsphere mixed liquid 12 sequentially passes through a detection area of a capillary tube 7 of the flow chamber, the first laser 5 is excited, the first laser fluorescence detector 10 collects fluorescence signals through the light collector 8, meanwhile, the second laser 6 is excited, the excitation wavelength is 340nm, the delay time is adjusted, 10 nanoseconds are 10 milliseconds to 800 milliseconds, the second laser time resolution fluorescence detection unit 9 is started to record and collect fluorescence signals through the light collector 8, and the collected signals are analyzed and processed through the data collection system 11.
Example 3
Polystyrene microspheres with the diameter of 5.6 microns are respectively coupled with 1 to 15 primary antibodies, antigens, enzymes or nucleic acids, then mixed according to a certain proportion to form a high-molecular coding microsphere suspension A, and 100-nm europium (Eu) time-resolved fluorescent microspheres are also respectively crosslinked with 1 to 20 secondary antibodies, antigens or enzymes or nucleic acids to form a time-resolved microsphere suspension B according to a certain proportion. The following were added sequentially to 96-well reaction plates: and (3) after the sample (or calibrator) to be detected and the polymer coding microsphere suspension A are added, fully and uniformly mixing in the front-end system 1, and placing in an incubator at 25-37 ℃ for reaction for 10-50 minutes in a dark place. And then adding the time-resolved microsphere suspension B into the reaction plate, fully mixing the mixture, placing the mixture at a temperature of between 30 and 37 ℃ and carrying out a light-shielding reaction in an incubator in the front-end system 1 for 10 to 45 minutes. After the reaction is finished, adding the stop solution, and fully and uniformly mixing. The up reading is then taken by a time-resolved flow fluorometric assay. The microsphere mixed liquid 12 and the sheath liquid are conveyed to the flow chamber together, the microsphere mixed liquid 12 sequentially passes through a detection area of a capillary tube 7 of the flow chamber, the first laser 5 is excited, the first laser fluorescence detector 10 collects fluorescence signals through the light collector 8, meanwhile, the second laser 6 is excited, the excitation wavelength is 365nm, the delay time is adjusted, after 50 nanoseconds to 1000 milliseconds, the second laser time resolution fluorescence detection unit 9 is started to record and collect the fluorescence signals through the light collector 8, and the collected signals are analyzed and processed through the data collection system 11.
Example 4
Polystyrene microspheres with the diameter of 5.6 microns are respectively coupled with 1 to 15 primary antibodies, antigens, enzymes or nucleic acids, then mixed according to a certain proportion to form a high-molecular coding microsphere suspension A, and 100-nm quantum dot fluorescent microspheres are also respectively crosslinked with 1 to 20 secondary antibodies, antigens, enzymes or nucleic acids to form a time-resolved microsphere suspension B according to a certain proportion. The following were added sequentially to 96-well reaction plates: and (3) after the sample (or calibrator) to be detected and the polymer coding microsphere suspension A are added, fully and uniformly mixing in the front-end system 1, and placing in an incubator at 25-37 ℃ for reaction for 10-50 minutes in a dark place. And then adding the time-resolved microsphere suspension B into the reaction plate, fully mixing the mixture, placing the mixture at a temperature of between 30 and 37 ℃ and carrying out a light-shielding reaction in an incubator in the front-end system 1 for 10 to 45 minutes. After the reaction is finished, adding the stop solution, and fully and uniformly mixing. The up reading is then taken by a time-resolved flow fluorometric assay. The microsphere mixed liquid 12 and the sheath liquid are conveyed to the flow chamber together, the microsphere mixed liquid 12 sequentially passes through a detection area of a capillary tube 7 of the flow chamber, the first laser 5 is excited, the first laser fluorescence detector 10 collects fluorescence signals through the light collector 8, meanwhile, the second laser 6 is excited, the excitation wavelength is 365nm, the delay time is adjusted, after 50 nanoseconds to 1000 milliseconds, the second laser time resolution fluorescence detection unit 9 is started to record and collect the fluorescence signals through the light collector 8, and the collected signals are analyzed and processed through the data collection system 11.
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
Claims (8)
1. The utility model provides a time-resolved flow-type fluorescence detection analytical equipment, its characterized in that includes the leading system, the sample cell, the sheath liquid pond, the flow chamber, first laser instrument, the second laser instrument, the capillary, the optical collector, second laser instrument time-resolved fluorescence detecting element, first laser instrument fluorescence detector, data acquisition system, wherein, the sheath liquid pond with the bottom of flow chamber is equipped with the opening, the capillary is used for guiding the flow of the microballon mixed liquid that awaits measuring, the capillary sets up in the sheath liquid pond, the bottom of capillary is popped out the opening of flow chamber to form the detection zone, first laser instrument with the second laser instrument is arranged into and can arouse the microballon mixed liquid in the detection zone.
2. The time-resolved flow-type fluorescence detection device of claim 1, wherein the excitation wavelength of the first laser is 650nm, and the optimal excitation wavelength of the second laser is 290 nm and 380 nm.
3. The time-resolved streaming fluorescence detection analysis device according to claim 1, wherein after the second laser excites the pulse, the second laser time-resolved fluorescence detection unit detects and records the fluorescence signal excited by the second laser after delaying for 4ns-60000 ms.
4. The time-resolved flow-type fluorescence detection and analysis device of claim 3, wherein the delay time interval of the second laser time-resolved fluorescence detection unit is 8ns-2000 ms.
5. The time-resolved flow-type fluorescence detection and analysis device according to claim 1, wherein the microsphere mixed solution comprises fluorochrome polymer coding microspheres, liquid or solid microspheres or quantum dot fluorescent microspheres of a sample to be detected and rare earth lanthanide or a chelate thereof.
6. The time-resolved flow fluorometric detection and analysis device of claim 1, wherein the fluorescent materials in the microsphere mixture excited by the second laser comprise quantum dot fluorescent microspheres and liquid or solid microspheres of rare earth lanthanide or its chelate, and the lanthanide is preferably one of europium (Eu), terbium (Tb), samarium (Sm) or any combination thereof.
7. The time-resolved flow fluorometric detection device of claim 1, wherein the first laser-excited fluorescent species comprises at least two spectrally distinct fluorescent dye polymer-encoded microspheres, preferably having a diameter of 1 to 20 microns.
8. The method for using the time-resolved flow-type fluorescence detection analysis device according to any one of claims 1 to 7, it is characterized in that the covalent crosslinking of the microsphere excited by the first laser and the protein, the antibody or the nucleic acid probe, the covalent crosslinking of the quantum dot or the time-resolved fluorescent substance excited by the second laser and the protein, the antibody or the nucleic acid probe, after the sample to be detected is added, a pre-system is used for carrying out incubation and oscillation treatment on a sample to be detected, sample liquid and sheath liquid are conveyed to a flow chamber together, liquid particles to be detected sequentially pass through a capillary detection area of the flow chamber and are excited by a first laser, a fluorescence detector of the first laser collects fluorescence signals through a light collector, simultaneously, the second laser is excited, after a period of time delay, the time-resolved fluorescence detection unit of the second laser is started, the fluorescence signal is recorded and collected by the light collector, and the collected signal is analyzed and processed by the data collection system.
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