阅读说明：本技术 一种微流控激光免疫时域光谱检测方法 (Microfluidic laser immunity time domain spectrum detection method ) 是由 万雄 王泓鹏 袁汝俊 于 2019-09-10 设计创作，主要内容包括：本发明的目的在于提供一种微流控激光免疫时域分辨荧光的检测方法,该方法是在微流控激光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、免疫时域分辨荧光光谱信息获取、时域荧光光谱数据后处理三个步骤。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；多幅时域荧光提供时变光谱信息,提高物种血液鉴别能力。(The invention aims to provide a method for detecting micro-fluidic laser immune time domain resolved fluorescence, which is realized on a micro-fluidic laser immune time domain resolved fluorescence detector. The method comprises three steps of initialization, self-focusing, immune time domain resolution fluorescence spectrum information acquisition and time domain fluorescence spectrum data post-processing. 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 multiple time domain fluorescence provides time-varying spectral information, and the blood identification capability of species is improved.)

1. A microfluidic laser immune time domain resolution fluorescence spectrum blood detection method is realized on a microfluidic laser immune time domain resolution fluorescence spectrum blood detector, wherein the detector consists of a three-dimensional electric platform (1), a microscope objective (2), a bicolor patch (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) and an automatic sample feeding platform (19); the detection 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; an electric propeller, a detection thin tube, a reagent tube, a blood tube, a Y-shaped joint and a waste liquid tank of the automatic sample feeding platform are assembled and arranged on a three-dimensional electric platform;

the main controller sends out an instruction to start the electric propeller, pushes the chelate marker in the reagent tube and the blood to be detected in the blood tube to be mixed in the Y-shaped joint at a certain speed, generates a fluorescent marker point after an immune reaction, obtains a marked blood, and flows into the detection tubule;

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 a detection tubule area, and a generated backward signal enters a fluorescence spectrometer through the coupling of an optical fiber coupling mirror 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 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 detection tubule;

2) 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;

3) time domain fluorescence spectroscopy data post-processing

The main controller extracts and analyzes parameters such as curve form, curve time domain change rate and the like of B/delta t fluorescence spectra of blood to be detected, constructs a characteristic database of the characteristic database, 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 laser immunity 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.

A 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, and the sensitivity is high, so that the time-resolved immunofluorescence analysis tool 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 immune time domain resolution fluorescence fine spectrum means is combined with micro-fluidic sample injection, and the requirements of blood analysis and identification of rare animals can be met.

In summary, the invention provides a blood detection method adopting micro-fluidic laser 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 the micro-fluidic laser immune time domain resolved fluorescence, which can accurately obtain immune time domain resolved fluorescence signals of fluorescent specific substance components such as hemoglobin, cytoplasm, biomacromolecules and the like in rare blood and meet the requirements of detection, identification, traceability, protection and the like of the rare blood.

The invention provides a microfluidic laser immune time domain resolution fluorescence spectrum blood detection method which is realized on a microfluidic laser immune time domain resolution fluorescence spectrum blood 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 and an automatic sample inlet platform;

wherein the automatic sample feeding platform consists of an electric propeller, a detection tubule, a reagent tube, a blood tube and a Y-shaped joint; the Y-shaped joint has two inlets and one outlet, and connects the reagent tube, the blood tube and the detection tubule; the electric propeller pushes the chelate marker in the reagent tube and the blood to be detected in the blood tube to be mixed in the Y-shaped joint in a meeting way, after an immune reaction is generated, a fluorescence marker point is generated, marked blood is obtained and flows into the detection tubule, 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 a detection tubule 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 transmitting optical axis is vertical to the receiving optical axis; the aperture of the optical fiber coupling mirror is equal to that of the ultraviolet laser beam expanding mirror, and the distances from the optical fiber coupling mirror and the ultraviolet laser beam expanding mirror to the bicolor patches are equal, 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 control program of the input/output port of the main controller can realize the control of the three-dimensional electric platform, the electric propeller, the digital delay generator and the stripe camera sensor; immune time domain resolution fluorescence spectrum information output by the streak camera sensor can be received, a 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 laser immune time domain resolution fluorescence spectrum 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; an electric propeller, a detection thin tube, a reagent tube, a blood tube, a Y-shaped joint and a waste liquid tank of the automatic sample feeding platform are assembled and arranged on a three-dimensional electric platform;

the main controller sends out an instruction to start the electric propeller, pushes the chelate marker in the reagent tube and the blood to be detected in the blood tube to be mixed in the Y-shaped joint at a certain speed, generates a fluorescent marker point after an immune reaction, obtains a marked blood, and flows into the detection tubule;

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 a detection tubule area, and a generated backward signal enters a fluorescence spectrometer through the coupling of an optical fiber coupling mirror 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 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 detection tubule;

(2) 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;

(3) time domain fluorescence spectroscopy data post-processing

The main controller extracts and analyzes parameters such as curve form, curve time domain change rate and the like of B/delta t fluorescence spectra of blood to be detected, constructs a characteristic database of the characteristic database, 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 multiple time domain fluorescence provides time-varying spectral 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-Y-shaped joint; 19-autosampler station; 20-a blood vessel; 21-blood to be tested; 22-reagent tube; 23-a chelating label; 24-electric propeller.

Detailed Description

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

The invention provides a microfluidic laser immune time domain resolution fluorescence spectrum blood detection method which is realized on a microfluidic laser immune time domain resolution fluorescence spectrum blood detector, wherein the detector consists of a three-dimensional electric platform 1, a microscope objective 2, a bicolor patch 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 expanding mirror 10, an ultraviolet low-repetition frequency pulse laser 11, a main controller 12, a wireless network transceiver 13, a waste liquid tank 16 and an automatic sample feeding platform 19;

wherein the automatic sample feeding table 19 consists of an electric propeller 24, a detection tubule 17, a reagent tube 22, a blood tube 20 and a Y-shaped joint 18; the Y-shaped joint 18 has two inlets and one outlet, and connects the reagent tube 22, the blood tube 20 and the detection tubule 17; the electric propeller 24 pushes the chelate marker 23 in the reagent tube 22 to be mixed with the blood 21 to be detected in the blood tube 20 in the Y-shaped joint 18, after immune reaction is generated, a fluorescence marker site is generated, the marked blood 15 is obtained and flows into the detection tubule 17, and after 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 bicolor sheet 3, and is focused to labeled blood 15 in a detection tubule 17 through a microscope 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 transmission optical axis 14 is perpendicular to the reception optical axis 4; 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 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 propeller 24, the digital delay generator 9 and the stripe camera sensor 8; immune time domain resolution fluorescence spectrum information output by the streak camera sensor 8 can be received, a 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 13 to realize the uploading and downloading of a database and cloud inquiry;

the invention provides a microfluidic laser immune time domain resolution fluorescence spectrum 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 reagent tube 22, the blood tube 20, the Y-shaped joint 18 and the waste liquid tank 16 of the automatic sample feeding platform 19 are assembled and installed on the three-dimensional electric platform 1;

the main controller 12 sends an instruction to start the electric propeller 24, and pushes the chelate marker 23 in the reagent tube 22 and the blood 21 to be detected in the blood tube 20 to meet and mix in the Y-shaped joint 18 at a certain speed, and after an immune reaction is generated, a fluorescence marker site is generated, so that the marked blood 15 is obtained and flows into the detection tubule 17;

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 an ultraviolet low-repetition-frequency pulse laser 11 expands and focuses to the area of a detection tubule 17, and a generated backward 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 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, and at this time, the ultraviolet laser is accurately focused to the labeled blood 15 in the detection tubule 17;

(2) 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;

(3) time domain fluorescence spectroscopy data post-processing

The main controller 12 extracts and analyzes parameters such as curve form, curve time domain change rate and the like of B/delta t fluorescence spectra of the blood 21 to be detected, constructs a characteristic database of the characteristic database, 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.