阅读说明：本技术 一种基于共焦拉曼免疫时域分辨荧光的动物血液检测方法 (Animal blood detection method based on confocal Raman immune time domain resolved fluorescence ) 是由 万雄 王泓鹏 袁汝俊 于 2019-09-10 设计创作，主要内容包括：本发明公开了一种基于共焦拉曼免疫时域分辨荧光的动物血液检测方法,该方法是在微流控共焦拉曼免疫时域分辨荧光检测仪上实现的,该方法包括初始化及自聚焦、共焦拉曼光谱信息获取、免疫时域分辨荧光光谱信息获取、综合光谱数据后处理四个步骤。本发明的有益效果是,光路的共焦设计可有效提高空间分辨率,可对血液中的微小区域分子进行探测；长荧光寿命免疫标记为多次时域分辨采样提供了基础；条纹相机传感器光谱仪实现单次激光诱导荧光多次采集；共焦拉曼与多幅时域荧光提供综合光谱信息,提高物种血液鉴别能力。(The invention discloses an animal blood detection method based on confocal Raman time domain resolved fluorescence, which is realized on a microfluidic confocal Raman time domain resolved fluorescence detector. The invention has the advantages that the confocal design of the light path can effectively improve the spatial resolution and can detect the molecules in the micro area in the blood; the long-fluorescence lifetime immunolabeling provides a basis for multiple time domain resolution sampling; the stripe camera sensor spectrometer realizes multiple acquisition of single laser induced fluorescence; the confocal Raman and the multiple time domain fluorescence provide comprehensive spectrum information, and the blood identification capability of species is improved.)

1. The animal blood detection method based on confocal Raman immune time domain resolved fluorescence is realized on a microfluidic confocal Raman immune time domain resolved fluorescence spectrum blood detector which is composed of a three-dimensional electric platform (1), a microscope objective (2), a bicolor sheet (3), an optical fiber coupling mirror (5), a fluorescence receiving optical fiber (6), a fluorescence spectrometer (7), a stripe camera sensor (8), a digital delay generator (9), an ultraviolet laser beam expander (10), an ultraviolet low-repetition-frequency pulse laser (11), a main controller (12), a wireless network transceiver (13), a waste liquid tank (16), a Raman objective (31), a Raman spectrometer (33), a Raman optical fiber (34), a Raman coupling mirror (35), a Raman bicolor sheet (37), a Raman beam expander (38), a Raman laser (39), a Rayleigh filter (40), A self-focusing module (41) and an automatic sample introduction system; the method is characterized by comprising the following steps:

1) initialization and self-focusing

Injecting blood to be tested of a rare animal into a blood tube, and injecting a chelate marker into a reagent tube; assembling an electric propeller, a detection tubule, a blood tube and a blood joint of the automatic sample feeding table; assembling a reagent propeller, a reagent pipe, a reagent joint and a Y-shaped pipe of the reagent sample injection platform; connecting the middle joint with a detection thin tube and a Y-shaped tube; connecting the Y-shaped pipe with a waste liquid tank; then the whole automatic sampling system is arranged on a three-dimensional electric platform;

the main controller sends out an instruction to start the electric propeller, and pushes the blood to be detected in the blood tube to flow into the detection tubule through the blood joint at a certain speed and flow into the Y-shaped tube through the middle joint; the main controller sends an instruction to start the reagent propeller, pushes the chelate marker in the reagent tube and the blood to be detected in the detection tubule to be mixed in the Y-shaped tube at a certain speed, generates a fluorescence marker locus after an immunoreaction, obtains a marker blood, and flows out from the outlet of the Y-shaped tube;

the main controller sends out an instruction to start the digital delay generator; the digital delay generator sends out two trigger pulses according to the set delay T, and the ultraviolet low repetition frequency pulse laser and the stripe camera sensor are started in sequence; the main controller sends out an instruction to enable the stripe camera sensor to work in a single-frame exposure mode, and the exposure time Es is set;

ultraviolet pulse laser emitted by an ultraviolet low-repetition-frequency pulse laser expands and focuses to an outlet area of a Y-shaped tube, and a generated backward signal is coupled by an optical fiber coupling mirror to enter a fluorescence spectrometer and then to a stripe camera sensor for photoelectric conversion; the stripe camera sensor transmits the acquired spectrum signal to the main controller; the main controller calculates the total intensity I of the spectrum signal, namely the total area enclosed by the spectrum curve;

the main controller sends out an instruction to control the three-dimensional electric platform to perform stepping micro-motion adjustment along three XYZ axes, at each position, the digital delay generator sends out two trigger pulses according to the set delay T, and the ultraviolet low-repetition-frequency pulse laser and the stripe camera sensor are started successively; the main controller calculates the total intensity I of the echo spectrum signal at the position until the total intensity I reaches the maximum value, and at the moment, the ultraviolet laser is accurately focused to the marked blood in the outlet of the Y-shaped tube;

2) confocal raman spectral information acquisition

The main controller sends out an instruction to start the self-focusing module, the Raman laser and the Raman spectrometer and set the exposure time of the Raman spectrometer; raman pump laser emitted by the Raman laser expands and focuses to a detection tubule area, a generated backward signal is coupled into the Raman spectrometer through the Raman coupling mirror, and the Raman spectrometer transmits a collected spectrum signal to the main controller; the main controller calculates the total intensity IR of the spectrum signal, wherein the total intensity IR is the total area enclosed by the spectrum curve;

the main controller sends out an instruction, the self-focusing module drives the Raman objective lens to move up and down along the Raman emission axis, at each position, the main controller calculates the total intensity IR of the echo spectrum signal at the position until the IR reaches the maximum value, and at the moment, the Raman pump laser is accurately focused to the blood to be detected in the detection tubule; in the tight focusing state, the Raman spectrometer transmits a Raman spectrum signal of the blood to be detected at the Raman focusing point to the main controller;

3) immune time domain resolved fluorescence spectrum information acquisition

Under the tight focusing state, the main controller sends out an instruction to enable the stripe camera sensor to work in a continuous multi-frame acquisition mode; setting single frame exposure time Em, sampling period delta t and total acquisition time B; the digital delay generator sends out two trigger pulses according to the set delay T, and the ultraviolet low repetition frequency pulse laser and the stripe camera sensor are started in sequence;

ultraviolet pulse laser emitted by an ultraviolet low-repetition-frequency pulse laser expands and focuses to mark blood, and a generated backward immunofluorescence signal is coupled by an optical fiber coupling mirror to enter a fluorescence spectrometer and is subjected to photoelectric conversion on a stripe camera sensor;

the stripe camera sensor transmits the acquired spectrum signal to the main controller; the stripe camera sensor carries out time domain resolution high-speed continuous exposure according to the set single-frame exposure time Em, the sampling period delta t and the total acquisition time B, records B/delta t fluorescence spectra which are attenuated along with time and are excited by single-emitting laser pulses and sends the fluorescence spectra to the main controller;

4) integrated spectral data post-processing

The main controller extracts and analyzes parameters such as a confocal Raman spectral line and B/delta t fluorescence spectra of blood to be detected, such as curve form, curve time domain change rate and the like, constructs a comprehensive spectral characteristic database of the blood to be detected, and sends database information to a cloud system of an entry and exit supervision department through a wireless network transceiver; the method can be used for rapidly detecting, establishing a warehouse and identifying the blood of the rare animal, and is convenient for the customs import and export inspection and quarantine departments to trace the source, identify and protect the rare animal.

Technical Field

The invention relates to a blood detection method, in particular to a blood detection method adopting microfluidic confocal Raman time domain resolved fluorescence spectroscopy, which is suitable for customs to detect and identify the blood of exported rare animals and belongs to the field of photoelectric detection.

Background

In various commodities at the entrance and exit of customs, strict control measures are mostly adopted for the entrance and exit of blood products in various countries. Since the blood components of animals, especially important rare animals, contain important biological information such as genetic characteristics of rare species, once the blood components are outflowed, the national biosafety is affected, and export is prohibited. However, lawbreakers often steal precious animal blood in common animal blood products, so special instruments and equipment are urgently needed for detection to distinguish common animals from precious animals and the categories of precious animals, so that blood smuggling and illegal criminal behaviors are prevented, and national biological safety is guaranteed.

The rapid detection and identification of blood of rare animals is difficult because the genetic difference of blood of some animals is very small, the external optical characteristics are very similar, and the interspecies difference and the intraspecies difference are always in the same order of magnitude. Therefore, a feasible method is urgently needed to be found.

An effective analysis tool is a confocal raman technique, which can focus raman pump laser to a very small area, cover several biomacromolecules, and pick up raman frequency shift caused by molecular vibration, thereby effectively identifying the molecular composition of the detected object; another powerful analysis tool is time-resolved immunofluorescence analysis, lanthanide series rare earth element chelate is used as a marker, the characteristics of long service life and large Stokes shift of the fluorescent substances are utilized, non-specific background fluorescence interference is effectively eliminated through time resolution, the sensitivity is high, and the method becomes a powerful tool for biomedical ultramicro-analysis. Because the blood of rare animals is extremely rare and small in amount, small-amount and microanalysis is required. Microfluidics (Microfluidics) refers to systems that use microscale tubing to process or manipulate micro-fluids, which can meet the requirements of micro-biological analysis. The micro-fluidic device has the characteristics of miniaturization, integration and the like, and is generally called a micro-fluidic Chip, also called a Lab-on-a-Chip (Lab-on-a-Chip) and a micro-Total Analytical System (micro-Total Analytical System). The confocal Raman and immune time domain resolution fluorescence fine spectrum means are combined with microfluidic sample injection, and the requirements of rare animal blood analysis and identification can be met.

In summary, the invention provides a blood detection method adopting microfluidic confocal raman immune time domain resolved fluorescence, which is suitable for rapid detection, library construction and identification of rare animals and is convenient for the customs import and export inspection and quarantine departments to trace the source, identify and protect the rare animals.

Disclosure of Invention

The invention aims to provide a method for detecting micro-fluidic confocal Raman immune time domain resolved fluorescence, which can accurately obtain confocal Raman and immune time domain resolved fluorescence signals of hemoglobin, cytoplasm, biomacromolecules and other substance components in rare blood, and meet the requirements of detection, identification, traceability, protection and the like of the rare blood.

The invention provides a detection method of microfluidic confocal Raman immune time domain resolved fluorescence, which is realized on a microfluidic confocal Raman immune time domain resolved fluorescence detector, wherein the detector consists of a three-dimensional electric platform, a microscope objective, a bicolor patch, an optical fiber coupling mirror, a fluorescence receiving optical fiber, a fluorescence spectrometer, a stripe camera sensor, a digital delay generator, an ultraviolet laser beam expander, an ultraviolet low-repetition-frequency pulse laser, a main controller, a wireless network transceiver, a waste liquid tank, a Raman objective, a Raman spectrometer, a Raman optical fiber, a Raman coupling mirror, a Raman bicolor patch, a Raman beam expander, a Raman laser, a Rayleigh filter, a self-focusing module and an automatic sample introduction system;

the automatic sample introduction system consists of an automatic sample introduction table and a reagent sample introduction table; the automatic sample feeding platform consists of an electric propeller, a detection tubule, a blood tube and a blood joint; the reagent sample feeding table consists of a reagent propeller, a reagent pipe, a reagent joint and a Y-shaped pipe;

the blood joint is connected with the blood tube and the detection tubule; the electric propeller pushes the blood to be detected in the blood tube to flow into the detection tubule through the blood joint and flow into the Y-shaped tube through the middle joint; the Y-shaped pipe has two inlets and one outlet, wherein one inlet is connected with the detection tubule through an intermediate joint, and the other inlet is connected with the reagent pipe through a reagent joint; the reagent propeller pushes the chelate marker in the reagent tube to be mixed with the blood to be detected in the Y-shaped tube in a meeting manner, after an immune reaction is generated, a fluorescence marker site is generated to obtain marked blood, the marked blood flows out from the outlet of the Y-shaped tube, and after the detection is finished, the marked blood is collected by a waste liquid tank;

ultraviolet pulse laser emitted by an ultraviolet low-repetition-frequency pulse laser along an emission optical axis is expanded and collimated by an ultraviolet laser beam expander, passes through a bicolor sheet and is focused to marked blood in an outlet of a Y-shaped tube by a microscope objective; the generated backward fluorescence signal passes through a microscope objective along an emission optical axis, travels along a receiving optical axis after being reflected by a dichromatic film, is coupled into a fluorescence receiving optical fiber through an optical fiber coupling mirror, and then enters a fluorescence spectrometer; a light splitting element in the fluorescence spectrometer splits the fluorescence signal and projects the split fluorescence signal to a stripe camera sensor for photoelectric conversion; the stripe camera sensor is provided with a high-speed continuous shutter with adjustable gate width (namely exposure time), time domain resolution high-speed continuous exposure can be carried out at a fixed sampling period delta t (namely a time domain sampling interval), and a plurality of fluorescence spectra attenuated along with time are recorded for subsequent analysis;

the self-focusing module can drive the Raman objective lens to move along the Raman emission axis, so that the movement of a focus is realized; raman pump laser emitted by a Raman laser along a Raman emission shaft is expanded and collimated by a Raman beam expander, passes through a Raman double-color sheet and is focused to blood to be detected at a Raman focusing point in a detection tubule by a Raman objective lens; the generated backward Raman scattering signal passes through a Raman objective lens along a Raman emission axis, travels along a Raman receiving axis after being reflected by a Raman dichromatic chip, is coupled into a Raman optical fiber, a Rayleigh filter is arranged in the Raman coupling lens, can filter a Rayleigh scattering signal with the same wavelength as the pumping light in a Raman echo signal, and then enters a Raman spectrometer, and a Raman spectrum is obtained after light splitting and photoelectric conversion for subsequent analysis;

the Raman transmitting shaft is vertical to the Raman receiving shaft, the transmitting optical axis is vertical to the receiving optical axis, and the Raman transmitting shaft is parallel to the transmitting optical axis; the aperture of the optical fiber coupling mirror is equal to that of the ultraviolet laser beam expander, and the distances from the optical fiber coupling mirror to the bicolor patch, namely L2 and L1, are equal, so that the confocal symmetry requirement is basically met; the Raman coupling lens and the Raman beam expanding lens have the same aperture, and the distances from the Raman coupling lens and the Raman beam expanding lens to the Raman dichroic filter are equal to L3 and L4, so that the confocal symmetry requirement is basically met;

the digital delay generator starts an ultraviolet low-repetition-frequency pulse laser and a stripe camera sensor in a pulse external triggering mode, and sets a time delay T between two external triggering pulses to obtain a time domain resolution fluorescence spectrum with an optimal noise ratio;

the input/output port control program of the main controller can realize the control of the three-dimensional electric platform, the electric propeller, the reagent propeller, the digital delay generator, the stripe camera sensor, the Raman laser, the Raman spectrometer and the self-focusing module; the confocal Raman spectrum information output by the Raman spectrometer and the immune time domain resolution fluorescence spectrum information output by the stripe camera sensor can be received, a comprehensive spectrum database corresponding to rare animal blood is constructed, blood analysis and classification identification are carried out, and query and remote transmission of the database are realized; the system can also be connected with a customhouse cloud system through a wireless network transceiver to realize the uploading and downloading of a database and cloud inquiry;

the invention provides a microfluidic confocal Raman immune time domain resolved fluorescence spectroscopy blood detection method which comprises the following steps:

(1) initialization and self-focusing

Injecting blood to be tested of a rare animal into a blood tube, and injecting a chelate marker into a reagent tube; assembling an electric propeller, a detection tubule, a blood tube and a blood joint of the automatic sample feeding table; assembling a reagent propeller, a reagent pipe, a reagent joint and a Y-shaped pipe of the reagent sample injection platform; connecting the middle joint with a detection thin tube and a Y-shaped tube; connecting the Y-shaped pipe with a waste liquid tank; then the whole automatic sampling system is arranged on a three-dimensional electric platform;

the main controller sends out an instruction to start the electric propeller, and pushes the blood to be detected in the blood tube to flow into the detection tubule through the blood joint at a certain speed and flow into the Y-shaped tube through the middle joint; the main controller sends an instruction to start the reagent propeller, pushes the chelate marker in the reagent tube and the blood to be detected in the detection tubule to be mixed in the Y-shaped tube at a certain speed, generates a fluorescence marker locus after an immunoreaction, obtains a marker blood, and flows out from the outlet of the Y-shaped tube;

the main controller sends out an instruction to start the digital delay generator; the digital delay generator sends out two trigger pulses according to the set delay T, and the ultraviolet low repetition frequency pulse laser and the stripe camera sensor are started in sequence; the main controller sends out an instruction to enable the stripe camera sensor to work in a single-frame exposure mode, and the exposure time Es is set;

ultraviolet pulse laser emitted by an ultraviolet low-repetition-frequency pulse laser expands and focuses to an outlet area of a Y-shaped tube, and a generated backward signal is coupled by an optical fiber coupling mirror to enter a fluorescence spectrometer and then to a stripe camera sensor for photoelectric conversion; the stripe camera sensor transmits the acquired spectrum signal to the main controller; the master controller calculates the total intensity I of the spectral signal (note: total area enclosed by the spectral curve);

the main controller sends out an instruction to control the three-dimensional electric platform to perform stepping micro-motion adjustment along three XYZ axes, at each position, the digital delay generator sends out two trigger pulses according to the set delay T, and the ultraviolet low-repetition-frequency pulse laser and the stripe camera sensor are started successively; the main controller calculates the total intensity I of the echo spectrum signal at the position until the total intensity I reaches the maximum value, and at the moment, the ultraviolet laser is accurately focused to the marked blood in the outlet of the Y-shaped tube;

(2) confocal raman spectral information acquisition

The main controller sends out an instruction to start the self-focusing module, the Raman laser and the Raman spectrometer and set the exposure time of the Raman spectrometer; raman pump laser emitted by the Raman laser expands and focuses to a detection tubule area, a generated backward signal is coupled into the Raman spectrometer through the Raman coupling mirror, and the Raman spectrometer transmits a collected spectrum signal to the main controller; the master controller calculates the total intensity IR of the spectral signal (note: total area enclosed by the spectral curve);

the main controller sends out an instruction, the self-focusing module drives the Raman objective lens to move up and down along the Raman emission axis, at each position, the main controller calculates the total intensity IR of the echo spectrum signal at the position until the IR reaches the maximum value, and at the moment, the Raman pump laser is accurately focused to the blood to be detected in the detection tubule; in the tight focusing state, the Raman spectrometer transmits a Raman spectrum signal of the blood to be detected at the Raman focusing point to the main controller;

(3) immune time domain resolved fluorescence spectrum information acquisition

Under the tight focusing state, the main controller sends out an instruction to enable the stripe camera sensor to work in a continuous multi-frame acquisition mode; setting single frame exposure time Em, sampling period delta t and total acquisition time B; the digital delay generator sends out two trigger pulses according to the set delay T, and the ultraviolet low repetition frequency pulse laser and the stripe camera sensor are started in sequence;

ultraviolet pulse laser emitted by an ultraviolet low-repetition-frequency pulse laser expands and focuses to mark blood, and a generated backward immunofluorescence signal is coupled by an optical fiber coupling mirror to enter a fluorescence spectrometer and is subjected to photoelectric conversion on a stripe camera sensor;

the stripe camera sensor transmits the acquired spectrum signal to the main controller; the stripe camera sensor carries out time domain resolution high-speed continuous exposure according to the set single-frame exposure time Em, the sampling period delta t and the total acquisition time B, records B/delta t fluorescence spectra which are attenuated along with time and are excited by single-emitting laser pulses and sends the fluorescence spectra to the main controller;

(4) integrated spectral data post-processing

The main controller extracts and analyzes parameters such as a confocal Raman spectral line and B/delta t fluorescence spectra of blood to be detected, such as curve form, curve time domain change rate and the like, constructs a comprehensive spectral characteristic database of the blood to be detected, and sends database information to a cloud system of an entry and exit supervision department through a wireless network transceiver; the method can be used for rapidly detecting, establishing a warehouse and identifying the blood of the rare animal, and is convenient for the customs import and export inspection and quarantine departments to trace the source, identify and protect the rare animal.

The invention has the advantages that the confocal design of the light path can effectively improve the spatial resolution and can detect the molecules in the micro area in the blood; the long-fluorescence lifetime immunolabeling provides a basis for multiple time domain resolution sampling; the stripe camera sensor spectrometer realizes multiple acquisition of single laser induced fluorescence; the confocal Raman and the multiple time domain fluorescence provide comprehensive spectrum information, and the blood identification capability of species is improved.

Drawings

FIG. 1 is a schematic diagram of the system of the present invention, in which: 1-three-dimensional electric platform; 2-microscope objective; 3-two-color chip; 4-receive optical axis; 5-fiber coupled mirror; 6-fluorescence receiving fiber; 7-fluorescence spectrometer; 8-streak camera sensor; 9-digital delay generator; 10-ultraviolet laser beam expander; 11-ultraviolet low repetition rate pulsed laser; 12-main controller; 13-Wireless network Transceiver; 14-emission optical axis; 15-labeled blood; 16-waste liquid tank; 17-detecting tubules; 18-blood connection; 19-autosampler station; 20-a blood vessel; 21-blood to be tested; 22-reagent tube; 23-a chelating label; 24-electric thruster; 25-reagent mover; 26-Y-shaped tube; 27-intermediate joint; 28-reagent sample introduction station; 29-reagent linker; 30-Raman focusing point; 31-Raman objective; 32-Raman emission axis; 33-Raman spectrometer; 34-Raman fiber; 35-Raman coupled mirror; 36-Raman receive axis; 37-raman dichromatic tablets; 38-Raman beam expander; 39-Raman laser; 40-Rayleigh filter; 41-self-focusing module.

Detailed Description

The specific embodiment of the present invention is shown in fig. 1.

The invention provides a detection method of microfluidic confocal Raman immune time domain resolved fluorescence, which is realized on a microfluidic confocal Raman immune time domain resolved fluorescence detector, wherein the detector consists of a three-dimensional electric platform 1, a microscope objective 2, a dichromatic lens 3, an optical fiber coupling mirror 5, a fluorescence receiving optical fiber 6, a fluorescence spectrometer 7, a stripe camera sensor 8, a digital delay generator 9, an ultraviolet laser beam expander 10, an ultraviolet low-heavy-frequency pulse laser 11, a main controller 12, a wireless network transceiver 13, a waste liquid tank 16, a Raman objective 31, a Raman spectrometer 33, a Raman optical fiber 34, a Raman coupling mirror 35, a Raman dichromatic lens 37, a Raman beam expander 38, a Raman laser 39, a Rayleigh filter 40, a self-focusing module 41 and an automatic sample introduction system;

wherein the automatic sample introduction system consists of an automatic sample introduction table 19 and a reagent sample introduction table 28; the automatic sample feeding table 19 consists of an electric propeller 24, a detection tubule 17, a blood tube 20 and a blood joint 18; the reagent sample feeding table 28 consists of a reagent propeller 25, a reagent tube 22, a reagent joint 29 and a Y-shaped tube 26;

the blood joint 18 is connected with the blood tube 20 and the detection tubule 17; the electric propeller 24 pushes the blood 21 to be detected in the blood tube 20 to flow into the detection tubule 17 through the blood joint 18 and flow into the Y-shaped tube 26 through the middle joint 27; the Y-shaped pipe 26 has two inlets and one outlet, wherein one inlet is connected with the detection tubule 17 through an intermediate joint 27, and the other inlet is connected with the reagent pipe 22 through a reagent joint 29; the reagent propeller 25 pushes the chelate marker 23 in the reagent tube 22 to be mixed with the blood 21 to be detected in the Y-shaped tube 26 in a contact manner, after an immune reaction is generated, a fluorescence marker site is generated, the marked blood 15 is obtained and flows out from the outlet of the Y-shaped tube 26, and after the detection is finished, the marked blood is collected by the waste liquid tank 16;

ultraviolet pulse laser light emitted by an ultraviolet low-repetition-frequency pulse laser 11 (in this embodiment, the wavelength is 266nm, the repetition frequency is less than 1Hz, the single pulse energy is 0.5mJ, the pulse width is 6ns, and light emission is controlled by external triggering) along a transmission optical axis 14 is expanded and collimated by an ultraviolet laser beam expander 10, passes through a dichroic filter 3, and is focused to marking blood 15 in an outlet of a Y-shaped tube 26 by a micro objective 2; the generated backward fluorescence signal (in this embodiment, a fluorescence signal with a wavelength of more than 266 nm) passes through the microscope objective 2 along the emission optical axis 14, travels along the receiving optical axis 4 after being reflected by the dichroic filter 3, is coupled into the fluorescence receiving optical fiber 6 through the optical fiber coupling mirror 5, and then enters the fluorescence spectrometer 7; a light splitting element in the fluorescence spectrometer 7 splits the fluorescence signal and projects the split fluorescence signal to a stripe camera sensor 8 for photoelectric conversion; the stripe camera sensor 8 is provided with a high-speed continuous shutter with adjustable gate width (namely exposure time), can perform time domain resolution high-speed continuous exposure with a fixed sampling period delta t (namely a time domain sampling interval), and records a plurality of fluorescence spectra attenuated along with time for subsequent analysis;

the self-focusing module 41 can drive the raman objective lens 31 to move along the raman emission axis 32, thereby realizing the movement of the focus; raman pump laser emitted by a raman laser 39 (in this embodiment, a 532nm single longitudinal mode narrow linewidth continuous laser) along a raman emission axis 32 is expanded and collimated by a raman beam expander 38, passes through a raman dichroic filter 37, and is focused to the blood 21 to be detected at a raman focusing point 30 in the detection tubule 17 by a raman objective lens 31; the generated backward Raman scattering signal passes through the Raman objective 31 along the Raman emission axis 32, is reflected by the Raman dichromatic sheet 37 and then travels along the Raman receiving axis 36, the Raman coupling mirror 35 is coupled into the Raman optical fiber 34, the Rayleigh filter 40 is arranged in the Raman coupling mirror 35, the Rayleigh scattering signal with the same wavelength as the pumping light in the Raman echo signal can be filtered, the Raman signal entering the Raman optical fiber 34 enters the Raman spectrometer 33, and the Raman spectrum is obtained after light splitting and photoelectric conversion for subsequent analysis;

the Raman emission axis 32 and the Raman receiving axis 36 are vertical, and the four axes of the emission optical axis 14 and the receiving optical axis 4 are coplanar, wherein the Raman emission axis 32 is vertical to the Raman receiving axis 36, the emission optical axis 14 is vertical to the receiving optical axis 4, and the Raman emission axis 32 is parallel to the emission optical axis 14; the aperture of the optical fiber coupling mirror 5 is equal to that of the ultraviolet laser beam expander 10, and the distances L2 from the optical fiber coupling mirror and the ultraviolet laser beam expander to the bicolor patch 3 are equal to that of L1, so that the confocal symmetry requirement is basically met; the Raman coupling mirror 35 and the Raman beam expanding mirror 38 have the same aperture, and the distances L3 from the Raman coupling mirror 35 to the Raman dichroic filter 37 are equal to L4, so that the confocal symmetry requirement is basically met;

the digital delay generator 9 starts the ultraviolet low-repetition-frequency pulse laser 11 and the stripe camera sensor 8 in a pulse external triggering mode, and sets a time delay T (100 ns in the embodiment) between two external triggering pulses to obtain a time domain resolution fluorescence spectrum with an optimal noise ratio;

the input/output port control program of the main controller 12 can realize the control of the three-dimensional electric platform 1, the electric thruster 24, the reagent thruster 25, the digital delay generator 9, the stripe camera sensor 8, the Raman laser 39, the Raman spectrometer 33 and the self-focusing module 41; the confocal Raman spectrum information output by the Raman spectrometer 33 and the immune time domain resolution fluorescence spectrum information output by the streak camera sensor 8 can be received, a comprehensive spectrum database corresponding to the blood of the rare animal is constructed, the blood is analyzed and classified and identified, and the query and remote transmission of the database are realized; the system can also be connected with a customhouse cloud system through a wireless network transceiver 13 to realize the uploading and downloading of a database and cloud inquiry;

the invention provides a microfluidic confocal Raman time domain resolved fluorescence spectroscopy blood detection method which comprises the following steps:

(1) initialization and self-focusing

Injecting blood 21 to be tested of a rare animal into a blood tube 20, and injecting a chelate marker 23 into a reagent tube 22; the electric propeller 24, the detection tubule 17, the blood tube 20 and the blood joint 18 of the automatic sample feeding table 19 are assembled; assembling the reagent pusher 25, the reagent tube 22, the reagent joint 29 and the Y-shaped tube 26 of the reagent sample introduction table 28; the middle joint 27 is connected with the detection tubule 17 and the Y-shaped pipe 26; connecting the Y-shaped pipe 26 with the waste liquid tank 16; then the whole automatic sampling system is arranged on the three-dimensional electric platform 1;

the main controller 12 sends a command to start the electric propeller 24, and pushes the blood 21 to be detected in the blood tube 20 to flow into the detection tubule 17 through the blood joint 18 and flow into the Y-shaped tube 26 through the intermediate joint 27 at a certain speed; the main controller 12 sends an instruction to start the reagent pusher 25, pushes the chelating label 23 in the reagent tube 22 and the blood 21 to be detected in the detection tubule 17 to meet and mix in the Y-shaped tube 26 at a certain speed, generates a fluorescent label site after an immunoreaction, obtains a labeled blood 15, and flows out from the outlet of the Y-shaped tube 26;

the main controller 12 sends out an instruction to start the digital delay generator 9; the digital delay generator 9 sends out two trigger pulses according to the set delay T, and starts the ultraviolet low repetition frequency pulse laser 11 and the stripe camera sensor 8 in sequence; the main controller 12 sends out an instruction to make the streak camera sensor 8 work in the single frame exposure mode, and sets the exposure time Es (10 ms in this embodiment);

ultraviolet pulse laser emitted by the ultraviolet low-repetition-frequency pulse laser 11 expands and focuses to an outlet area of the Y-shaped tube 26, and a generated backward signal is coupled by the optical fiber coupling mirror 5 to enter the fluorescence spectrometer 7 and is subjected to photoelectric conversion on the stripe camera sensor 8; the stripe camera sensor 8 transmits the collected spectrum signal (the spectrum range of 266-540nm in the embodiment) to the main controller 12; the master controller 12 calculates the total intensity I of the spectral signal (note: total area enclosed by the spectral curve);

the main controller 12 sends out an instruction to control the three-dimensional electric platform 1 to perform stepping micro-motion adjustment along three XYZ axes, at each position, the digital delay generator 9 sends out two trigger pulses according to the set delay T, and the ultraviolet low-repetition-frequency pulse laser 11 and the stripe camera sensor 8 are started successively; the main controller 12 calculates the total intensity I of the echo spectrum signal at the position until I reaches the maximum value, at this time, the ultraviolet laser has been accurately focused to the labeled blood 15 at the outlet of the Y-shaped tube 26;

(2) confocal raman spectral information acquisition

The main controller 12 sends out an instruction to start the self-focusing module 41, the Raman laser 39 and the Raman spectrometer 33, and sets the exposure time of the Raman spectrometer 33; raman pump laser emitted by the Raman laser 39 expands and focuses to the area of the detection tubule 17, generated backward signals are coupled into the Raman spectrometer 33 through the Raman coupling mirror 35, and the Raman spectrometer 33 transmits collected spectrum signals to the main controller 12; the master controller 12 calculates the total intensity IR of the spectral signal (note: total area enclosed by the spectral curve);

the main controller 12 sends out an instruction, the self-focusing module 41 drives the raman objective 31 to move up and down along the raman emission axis 32, at each position, the main controller 12 calculates the total intensity IR of the echo spectrum signal at the position until the IR reaches the maximum value, and at the moment, the raman pump laser is accurately focused to the blood 21 to be detected in the detection tubule 17; in this tight focusing state, the raman spectrometer 33 transmits the raman spectrum signal of the blood 21 to be measured at the raman focusing point 30 to the main controller 12;

(3) immune time domain resolved fluorescence spectrum information acquisition

In this tight focus state, the main controller 12 issues an instruction to cause the streak camera sensor 8 to operate in a continuous multi-frame acquisition mode; setting a single-frame exposure time Em (8 ms in this embodiment), a sampling period Δ t (10 ms in this embodiment), and a total acquisition time B (500 ms in this embodiment); the digital delay generator 9 sends out two trigger pulses according to the set delay T, and starts the ultraviolet low repetition frequency pulse laser 11 and the stripe camera sensor 8 in sequence;

ultraviolet pulse laser emitted by an ultraviolet low-repetition-frequency pulse laser 11 expands and focuses to labeled blood 15, and a generated backward immunofluorescence signal is coupled by an optical fiber coupling mirror 5 to enter a fluorescence spectrometer 7 and is subjected to photoelectric conversion by a stripe camera sensor 8;

the stripe camera sensor 8 transmits the collected spectrum signal (the spectrum range of 266-540nm in the embodiment) to the main controller 12; the streak camera sensor 8 performs time-domain-resolved high-speed continuous exposure according to the set single-frame exposure time Em (8 ms in this embodiment), sampling period Δ t (10 ms in this embodiment) and total acquisition time B (500 ms in this embodiment), records B/Δ t (50 in this embodiment, 500ms/10 ms) fluorescence spectra excited by a single laser pulse, which decays with time, and sends the recorded fluorescence spectra to the main controller 12;

(4) integrated spectral data post-processing

The main controller 12 extracts and analyzes parameters such as a confocal Raman spectral line and B/delta t fluorescence spectra of the blood 21 to be detected, such as curve form, curve time domain change rate and the like, constructs a comprehensive spectral characteristic database of the blood, and sends database information to a cloud system of an entry and exit supervision department through the wireless network transceiver 13; the method can be used for rapidly detecting, establishing a warehouse and identifying the blood of the rare animal, and is convenient for the customs import and export inspection and quarantine departments to trace the source, identify and protect the rare animal.