Rare animal blood detector of micro-fluidic laser immunity time domain spectrum
阅读说明:本技术 一种微流控激光免疫时域光谱珍稀动物血液检测仪 (Rare animal blood detector of micro-fluidic laser immunity time domain spectrum ) 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光谱珍稀动物血液检测仪,由三维电动平台、显微物镜、双色片、光纤耦合镜、荧光接收光纤、荧光光谱仪、条纹相机传感器、数字延迟发生器、紫外激光扩束镜、紫外低重频脉冲激光器、主控制器、无线网络收发器、废液罐、自动进样台组成。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 microfluidic laser immunity time domain spectroscopy rare animal blood detector which 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 expanding mirror, an ultraviolet low-repetition-frequency pulse laser, a main controller, a wireless network transceiver, a waste liquid tank and an automatic sample feeding platform. 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 rare animal blood detector of micro-fluidic laser immunity time domain spectrum, the instrument is by three-dimensional electronic platform (1), micro objective (2), double-colored piece (3), optical fiber coupling mirror (5), fluorescence receive optic fibre (6), fluorescence spectrometer (7), stripe camera sensor (8), digital delay generator (9), ultraviolet laser beam expander lens (10), ultraviolet low repetition frequency pulse laser (11), master controller (12), wireless network transceiver (13), waste liquid tank (16), enter the appearance platform automatically (19) to make up; the method is characterized in that:
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); an electric propeller (24) pushes a chelating marker (23) in a reagent tube (22) and blood (21) to be detected in a blood tube (20) to meet and mix in a Y-shaped joint (18), after immune reaction is generated, a fluorescent marker site is generated, marked blood (15) is obtained and flows into a detection tubule (17), and after detection is finished, the marked blood is collected by a waste liquid tank (16);
ultraviolet pulse laser emitted by an ultraviolet low repetition frequency pulse laser (11) along an emission optical axis (14) is expanded and collimated by an ultraviolet laser beam expanding lens (10), passes through a bicolor patch (3), and is focused to marked blood (15) in a detection tubule (17) through a microscope objective (2); the generated backward fluorescence signal passes through the microscope objective (2) along an emission optical axis (14), is reflected by the bicolor sheet (3), then travels along a receiving optical axis (4), is coupled into a fluorescence receiving optical fiber (6) through an optical fiber coupling mirror (5), and then enters a 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; a strip camera sensor (8) is provided with a high-speed continuous shutter with adjustable door 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 emission optical axis (14) is perpendicular to the reception optical axis (4); the aperture of the optical fiber coupling lens (5) is equal to that of the ultraviolet laser beam expanding lens (10), and the distance between the optical fiber coupling lens and the ultraviolet laser beam expanding lens to the bicolor sheet (3) is equal, so that the confocal symmetry requirement is basically met;
a digital delay generator (9) starts an ultraviolet low-repetition-frequency pulse laser (11) and a stripe camera sensor (8) 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 performance-to-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 the database and cloud inquiry.
Technical Field
The invention relates to a blood detection instrument, in particular to a blood detection instrument 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 instrument adopting micro-fluidic laser immunity 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 micro-fluidic laser immune time domain resolved fluorescence detection instrument, 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 micro-fluidic laser immune time domain resolution fluorescence spectrum blood detector which consists of a three-dimensional electric platform, a microscope objective, a double-color chip, 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 feeding 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 micro-fluidic laser immune time domain resolution fluorescence spectrum blood detector which consists of a three-dimensional
wherein the automatic sample feeding table 19 consists of an
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
the
the input/output port control program of the
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
the
the
ultraviolet pulse laser emitted by an ultraviolet low-repetition-
the
(2) immune time domain resolved fluorescence spectrum information acquisition
In this tight focus state, the
ultraviolet pulse laser emitted by an ultraviolet low-repetition-
the
(3) time domain fluorescence spectroscopy data post-processing
The
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