CN101825570B - Quantum dot coding fluorescence immunity analyzer - Google Patents
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- CN101825570B CN101825570B CN 201010165974 CN201010165974A CN101825570B CN 101825570 B CN101825570 B CN 101825570B CN 201010165974 CN201010165974 CN 201010165974 CN 201010165974 A CN201010165974 A CN 201010165974A CN 101825570 B CN101825570 B CN 101825570B
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Abstract
The invention discloses a quantum dot coding fluorescence immunity analyzer, which comprises an analysis workstation, a liquid phase chip, a microflow control flow path control device, a fluorescence excitation light source, an optical imaging device, a fluorescence signal detection device and a code microsphere image locating and conjoint fault tolerance detection device. The invention adopts a fiber spectrometer as a fluorescence detector which can detect the wavelength and intensity information of fluorescence generated by the excited code microsphere at the same time and quantitatively detects a multicolor code microsphere as well as decodes the multicolor code; and therefore the operation is easier and more convenient.
Description
Technical field
The present invention relates to nanometer technology, liquid phase biochip technology, low-light detection power technology, laser technology, biological immune labelling technique, computer technology etc. and be the biomedical engineering technology field of one; Specifically, be a kind of quantum dot coding fluorescence immunity analyzer.
Background technology
The development of Quantum Dot Labeling technology is advanced by leaps and bounds, and becomes a kind of novel fluorescence label.Quantum dot is compared with the organic conventional fluorescer of tradition, and have special optical characteristics: 1. quantum dot has very wide excitation spectrum and narrower emission spectrum.The excitation spectrum of quantum dot is continuous and wide, and its fluorescence can be excited by any light source of wavelength less than its quantum confinement peak.Simultaneously, the emission spectrum of quantum dot is very narrow and be the Gaussian distribution of symmetry, and spectral width is between 20～40nm, and the yardstick through changing quantum dot or the component that changes the quantum dot kernel can be come the wavelength of the emission spectrum of regulated quantity point.2. quantum dot has bigger Stokes shift.Excite between the wavelength of light wavelength and emission photopeak value difference big, can avoid the overlapping of emission spectrum and PLE.3. quantum dot has strong anti-photobleaching ability.Under long-time irradiation, the fluorescence intensity of quantum dot has no decay.4. quantum dot has long fluorescence lifetime.The fluorescence lifetime of quantum dot is sustainable to reach several 10ns.After nanosecond was counted in optical excitation, most autofluorescence background decayed, and quantum dot fluorescence still exists, and can obtain not have the fluorescence signal of background interference this moment.The unique optical properties of this novel nano-material of quantum dot for the sign of cell and biomacromolecule provides a kind of strong tools, makes fluorescence-encoded technology can access practical application.
Quantum point coding microball is according to the preparation of Quantum Dot Labeling technology; Present stage is to its check and analysis; Generally be fluorescence liquid-phase chip system, flow cytometer (Flow Cytometry through Luminex; FCM) or laser confocal scanning microscope (laser scanning confocal microscope LSCM) carries out.But no matter be Luminex, FCM or LSCM; The information of its detection all is that fluorescence passes through the fluorescence intensity that detected by photomultiplier behind the color filter; Any wavelength information that does not have fluorescence; Can not carry out detection by quantitative to many color codings microballoon through these equipment, and be difficult for realizing the decoding of many color codings microballoon.For this reason, must there be special checkout equipment that single encoded microballoon is carried out check and analysis.
Summary of the invention
The present invention is exactly the defective that exists to existing quantum point coding microball analytic system; Adopt fiber spectrometer as the fluoroscopic examination device; Can detect coding microball the be excited wavelength and the strength information of the fluorescence that the back produces simultaneously; Can carry out detection by quantitative to many color codings microballoon, and realize how color-coded decoding, it is easier to operate.
Quantum dot coding fluorescence immunity analyzer of the present invention; Be achieved through following technical proposals, said analyser comprises analytical work station, liquid-phase chip, micro-fluidic stream control device, fluorescence excitation light source, optical imaging device, fluorescence signal pick-up unit and coding microball framing and conjuncted fault-tolerant pick-up unit;
Said analytical work station is to contain the computing machine of analyzing with control program, and the analytical work station connects micro-fluidic stream control device, fluorescence excitation light source, fluorescence signal pick-up unit and coding microball framing and conjuncted fault-tolerant pick-up unit respectively;
Said liquid-phase chip is made up of substrate and cover plate, and substrate surface is provided with two Buffer Pools, sample cell and waste liquid pools, and the liquid bath that connects two Buffer Pools is crosswise with the liquid bath that is connected sample cell and waste liquid pool and intersects;
Said micro-fluidic stream control device comprises syringe pump, damping fluid syringe, sample liquid syringe, pump line and communication interface; Syringe pump is connected with the sample liquid syringe with the damping fluid syringe respectively through pump line; The damping fluid syringe connects two Buffer Pools respectively, and the sample liquid syringe connects sample cell; Syringe pump is connected with the communication of analytical work station through communication interface;
Said fluorescence excitation light source is a semiconductor laser;
Said optical imaging device comprises objective table, lens barrel, object lens, laser semi-transparent semi-reflecting lens, fluorescence semi-transparent semi-reflecting lens, filter plate and optical fiber; The liquid-phase chip below is followed successively by objective table and lens barrel, sets gradually laser semi-transparent semi-reflecting lens, fluorescence semi-transparent semi-reflecting lens, filter plate and optical fiber in the lens barrel;
Said fluorescence signal pick-up unit comprises coding microball decoding device and antibody labeling thing low-light pick-up unit, and the coding microball decoding device is a fiber spectrometer, and antibody labeling thing low-light pick-up unit is the side window photomultiplier;
Said coding microball framing and conjuncted fault-tolerant pick-up unit are the CCD camera, and CCD camera and liquid-phase chip are adjacent.
Technology such as nanometer technology, liquid phase biochip technology, low-light monitoring power technology, laser technology, biological immune labelling technique, computer technology have been merged in the present invention, have integrated the quantum dot coding fluorescence immunity analyzer of one such as analytical work station, micro-fluidic stream control device, liquid-phase chip, fluorescence excitation light source, optical imaging device, fluorescence signal pick-up unit, coding microball framing and conjuncted fault-tolerant pick-up unit.The invention can detect coding microball the be excited wavelength and the strength information of the fluorescence that the back produces simultaneously; Can carry out detection by quantitative to many color codings microballoon; And realize how color-coded decoding, and make that detection and analysis result are more accurate, it is easier to operate.
Description of drawings
Fig. 1 is a structured flowchart of the present invention;
Fig. 2 is a liquid-phase chip structural representation of the present invention;
Fig. 3 is analytical work of the present invention station inner analysis and control program process flow diagram.
Embodiment
Below in conjunction with accompanying drawing the present invention is done and to further describe.
As shown in Figure 1; The present invention mainly is made up of seven parts such as analytical work station, micro-fluidic stream control device, liquid-phase chip, fluorescence excitation light source, optical imaging device, fluorescence signal pick-up unit, coding microball framing and conjuncted fault-tolerant pick-up unit, and the analytical work station connects micro-fluidic stream control device, fluorescence excitation light source, fluorescence signal pick-up unit and coding microball framing and conjuncted fault-tolerant pick-up unit respectively.
The analytical work station comprises one of computing machine and analysis thereof and control program, and it is configured to: more than the CPU 2G, use double-core and above CPU best; More than the internal memory 1G, 2G is best; Video card video memory 128M, 512M is best; 17 inches of screens.Workstation operating system is: Windows XP, Windows 2000 or Windows2003, suggestion Windows Server 2003R2; Database is Microsoft SQL Server 2000; Analyze with Control Software and write by Visual C++6.0, coded message input database and the coded message of mainly accomplishing quantum point coding microball extracted, the analytical calculation of the collection of the collection of the PC control of stream, spectral information and analysis, coding microball fluoroscopic image and analysis, detection information and draw diagnostic result.
Liquid-phase chip: size: 20mm * 50mm, substrate are quartz glass, and cover plate is PDMS, and optical characteristics is preferably arranged.This chip is the detection platform of coding microball.As shown in Figure 2, the liquid bath that connects two Buffer Pools is crosswise with the liquid bath that is connected sample cell and waste liquid pool and intersects.Damping fluid and sample liquid flow get into waste liquid pool through liquid bath behind zone to be checked, after waste liquid (mixed liquor of damping fluid and sample liquid) is full of waste liquid pool, will be injected in the waste liquid cup through emulsion tube.
Micro-fluidic stream control device comprises syringe pump, syringe, pump line and communication interface etc.; Pump line and communication interface; Syringe pump is connected with the sample liquid syringe with the damping fluid syringe respectively through pump line, and the damping fluid syringe connects two Buffer Pools respectively, and the sample liquid syringe connects sample cell; Syringe pump is connected with the communication of analytical work station through communication interface.Syringe pump is the KD200 syringe pump system of U.S. KD Scientific, and parameter is: syringe capacity 10uL-140mL, flow velocity 0.001uL/h-145mL/min; Withstand voltage 90N; Per step reach length 0.165um, accuracy≤1%, reappearance≤0.1%; The KD200 syringe pump is realized the communication that is connected with workstation through this machine RS232C and RS232/USB converter.The RS232/USB converter is that the logical Industrial Co., Ltd. of the emerging fine gold in Shenzhen side produces, and links to each other with computing machine USB mouth.Syringe is that Shanghai high dove industry and trade company limited produces, and pump line is Φ 1 emulsion tube.
The fluorescence excitation light source is 405nm semiconductor laser LD, and parameter is: laser output center wavelength 405nm ± 5nm; Optical fiber output power 0-160mW; Fiber lengths 1m, oversheath 3mmPVC, 600um inner core; Connector SMA905.
Optical imaging device comprises objective table, lens barrel, object lens, laser semi-transparent semi-reflecting lens, fluorescence semi-transparent semi-reflecting lens, optical fiber, filter plate etc.; The liquid-phase chip below is followed successively by objective table and lens barrel; Set gradually laser semi-transparent semi-reflecting lens, fluorescence semi-transparent semi-reflecting lens, filter plate and optical fiber in the lens barrel, main effect is the amplification imaging to the microballoon that is inspired fluorescence.Its concrete parameter or manufacturer are: objective table and lens barrel are the MJ100 of Chongqing special optics difficult to understand; Object lens 40X; Y-type optical fiber 600um inner core, the 3mmPVC oversheath; Filter plate is 430nm high-pass filtering sheet and 440nm-460nm band pass filter, 0.01% bias light transmitance.
The fluorescence signal pick-up unit comprises coding microball decoding device and antibody labeling thing low-light pick-up unit.The coding microball decoding device is the AvaSpec-2048-USB2 type fiber spectrometer that Dutch Avantes B.V. company produces; Scope 400nm-950nm, resolution is 0.7nm, parasitic light＜0.1%; Sample rate is that 1.1ms/ scans at every turn, and data rate 1.8ms/ scans at every turn; Antibody labeling thing low-light pick-up unit is the R105UH type side window photomultiplier (PMT) that Beijing Hamamatsu Technology Co., Ltd. produces; Spectral response range 300nm-650nm; Peak response wavelength 400nm, negative electrode smallest effective size 24mm * 8mm, anodic pulse rise time 2.2ns; Electron-transit time 22ns, conversion characteristic when 1000Vdc (25 ℃) is 30uA/Lm.
Coding microball framing and conjuncted fault-tolerant pick-up unit are the RZ300CCD camera that Tianjin Xi Ge Electro-optical Technology, INC. (US) 62 Martin Road, Concord, Massachusetts 017 produces; 1,300,000 cmos pixels, Pixel Dimensions 5.2um * 5.2um, spectral response 400nm～1000nm; Resolution 1280 * 1024; Transfer rate 12f/s1280 * 1024, sensitive 1.8V/lux-sec550nm, image output USB2.0,480Mb/s.
Wherein, fiber spectrometer, photomultiplier PMT, CCD camera, semiconductor laser LD all are connected with the analytical work station through the USB line.The KD200 syringe pump is connected with the RS232/USB converter through this machine RS232C, and the RS232/USB converter is connected with workstation USB mouth.Fiber spectrometer, PMT receive fluorescence through y-type optical fiber, and y-type optical fiber is through SMA905 interface and lens barrel, and LD is through optical fiber output 405nm laser.Syringe pump passes through emulsion tube to liquid-phase chip injection buffer and sample liquid.
When using this device; The start-up system workstation; Get into and analyze and control program; Program flow diagram is shown in 3, and concrete steps are following: the propulsion plant of the syringe that is connected with two buffer pools on program start CCD camera and the syringe pump, inject damping fluid with pushing syringe the Buffer Pool of liquid-phase chip.Treat damping fluid in liquid-phase chip, form stablize sheath stream after, start the propulsion plant of the syringe that is connected with sample cell on the syringe pump, the sample liquid that is loaded with coding microball is slowly injected the sample cell of liquid-phase chip as core liquid.At this moment, start semiconductor laser, fiber spectrometer and PMT.The 405nm laser of semiconductor laser emission behind the laser semi-transparent semi-reflecting lens, by the core liquid to be checked zone of object lens focusing to liquid-phase chip, is used for exciting the fluorescence of coding microball.When having microballoon to pass through zone to be checked in the core liquid; Thereby the quantum dot in the microballoon is produced the fluorescence that has coded message by laser excitation, and these fluorescence focus on the end face of y-type optical fiber through object lens and fluorescence semi-transparent semi-reflecting lens; Pass through Y Optical Fiber Transmission and distribution again; Get into fiber spectrometer and PMT respectively, fiber spectrometer detects the fluorescence spectrum of coding, the fluorescence intensity of PMT certification mark thing.In this process, the CCD camera is monitored the core liquid zone to be checked of liquid-phase chip in real time, if during to be two disjunctors or other the invalid monitoring of microballoon image, it is invalid that this moment fiber spectrometer and PMT Information Monitoring is set to.If Information Monitoring is effective, control analysis software extracts peak position and peak height from the fluorescence data of gathering, judge coded message, gathers the light intensity data of mark quantum dot according to PMT and whether crosses threshold value, judges the negative positive of diagnosis.
The invention is mainly used in fields such as biomedical check and clinical examination; Be suitable for the biomacromolecule and the cell of quantum point coding microball sign are detected; Be specially adapted to the detection of the tumor marker of quantum point coding microball sign, be applied to the early diagnosis of cancer, the detection of virus etc.
Claims (1)
1. quantum dot coding fluorescence immunity analyzer; It is characterized in that said analyser comprises analytical work station, liquid-phase chip, micro-fluidic stream control device, fluorescence excitation light source, optical imaging device, fluorescence signal pick-up unit and coding microball framing and conjuncted fault-tolerant pick-up unit;
Said analytical work station is to contain the computing machine of analyzing with control program, and the analytical work station connects micro-fluidic stream control device, fluorescence excitation light source, fluorescence signal pick-up unit and coding microball framing and conjuncted fault-tolerant pick-up unit respectively;
Said liquid-phase chip is made up of substrate and cover plate, and substrate surface is provided with two Buffer Pools, sample cell and waste liquid pools, and the liquid bath that connects two Buffer Pools is crosswise with the liquid bath that is connected sample cell and waste liquid pool and intersects;
Said micro-fluidic stream control device comprises syringe pump, damping fluid syringe, sample liquid syringe, pump line and communication interface; Syringe pump is connected with the sample liquid syringe with the damping fluid syringe respectively through pump line; The damping fluid syringe connects two Buffer Pools respectively, and the sample liquid syringe connects sample cell; Syringe pump is connected with the communication of analytical work station through communication interface;
Said fluorescence excitation light source is a semiconductor laser;
Said fluorescence signal pick-up unit comprises coding microball decoding device and antibody labeling thing low-light pick-up unit, and the coding microball decoding device is a fiber spectrometer, and antibody labeling thing low-light pick-up unit is the side window photomultiplier;
Said optical imaging device comprises objective table, lens barrel, object lens, laser semi-transparent semi-reflecting lens, fluorescence semi-transparent semi-reflecting lens, filter plate and y-type optical fiber, and the liquid-phase chip below is followed successively by objective table and lens barrel; Set gradually laser semi-transparent semi-reflecting lens, fluorescence semi-transparent semi-reflecting lens, filter plate and y-type optical fiber in the lens barrel; Object lens are between liquid-phase chip and laser semi-transparent semi-reflecting lens; Lens barrel is connected with the input end of y-type optical fiber, and two output terminals of y-type optical fiber connect fiber spectrometer and side window photomultiplier respectively;
Said coding microball framing and conjuncted fault-tolerant pick-up unit are the CCD camera, the adjacent setting of CCD camera and liquid-phase chip.
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| CN102793533A (en) * | 2012-08-09 | 2012-11-28 | 中国科学院苏州纳米技术与纳米仿生研究所 | Short wave near infrared quantum dot imaging system |
| CN102841196B (en) * | 2012-09-11 | 2014-11-05 | 济南格致生物技术有限公司 | Micro immune detector |
| CN106226278A (en) * | 2016-08-05 | 2016-12-14 | 清华大学 | A kind of multiplexing flow-through assay device for microlayer model fluoroscopic image and spectral scan |
| CN107677803B (en) * | 2017-08-25 | 2019-12-17 | 中国科学院苏州生物医学工程技术研究所 | coding and decoding system and method for liquid phase chip analyzer |
| US10753851B2 (en) * | 2017-11-28 | 2020-08-25 | Alentic Microscience Inc. | Classifying microbeads in near-field imaging |
| CN108414446A (en) * | 2018-03-30 | 2018-08-17 | 广东顺德墨赛生物科技有限公司 | Micro-fluidic chip fluorescence detection device, method and device |
| CN110514575B (en) * | 2019-08-16 | 2022-03-18 | 迈克医疗电子有限公司 | Decoding device and method for quantum dot coding microspheres |
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| US6781690B2 (en) * | 1999-05-17 | 2004-08-24 | New Mexico State University Technology Transfer Corporation | Sensors employing nanoparticles and microcavities |
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