CN1339610A - Time-resolved fluorescnet detection method and detector for gene chip - Google Patents
Time-resolved fluorescnet detection method and detector for gene chip Download PDFInfo
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- CN1339610A CN1339610A CN 01134009 CN01134009A CN1339610A CN 1339610 A CN1339610 A CN 1339610A CN 01134009 CN01134009 CN 01134009 CN 01134009 A CN01134009 A CN 01134009A CN 1339610 A CN1339610 A CN 1339610A
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Abstract
The present invention relates to the field of biological information testing technology. By using rare earth element ions with unique fluorescent characteristic as marker tracer and through time resolving fluorescent spectroscopic analysis to eliminate the effect of background fluorescence, the sensitivity of chip analysis is raised greatly. The detector of the present invention consists of frame, support platform, platform transmission mechanism, optical detection unit and photoelectronic signal processing unit. The present invention is superior to available technology obviously. The multifunctional instrument combining the temporal fluorescent resolving technology, gene chip detection technology and biological information technology has the advantages of high sensitivity, high stability, wide linearity range and long marker maintaining period.
Description
Technical field
The present invention relates to a kind of detection method, especially a kind of detection method of gene chip.The invention still further relates to the special detection device of this method of realization simultaneously, belong to bioinformation detection technique field.
Background technology
Human genome (order-checking) is planned the enforcement of stepping into of being near completion of (Human genome project) and post genome project, make that increasing animals and plants, microbial genome sequence are measured, the just former speed that does not have of gene order data increases rapidly.Therefore, setting up novel sequencing by hybridization method carries out efficiently, detects fast, analyzes and just seem especially important a large amount of genetic information.Gene chip has obtained development energetically as a kind of high-throughout detection method.Although the prospect of biochip technology is very bright, should see, still exist many insoluble problems in its road that advances, especially in the context of detection of gene chip.
Gene chip detects existing method: at first according to the designing probe that requires that detects, then probe is adopted the whole bag of tricks to be fixed on the carrier substrate, to detect liquid and substrate again and carry out hybridization, clean the back and adopt the gene chip scanning device to scan, the line data of going forward side by side is handled biology and the relevant information of obtaining.Existing gene chip scanning device mainly is made up of substrate carrier platform, optical detection unit, data acquisition board, main control system etc.Optical detection unit generally includes light source, colour filter, beam splitter, collimating mirror, optoelectronic receiver.According to the difference of moving parts, two kinds of structures of platform scanner and camera lens scanning can be arranged; And, can have CCD to detect (the pale pinkish purple device of electric charge) and photomultiplier detection dual mode according to the difference that optoelectronic receiver is selected.
Because above-mentioned existing gene chip hybridization detection method is the target molecule of the common fluorochrome label of employing and chip solution hybridization in hybridizing box of point sample, then airing be placed on detect on the platform of detector or directly in hybridizing box the reaction back clean and detect, therefore ground unrest is difficult to eliminate, make existing detection method have problems such as sensitivity is lower, hybridization efficiency is low, analyst coverage is narrower, limited the improvement with the gene chip quality applied of biochip technology.Though existing detector has been taked certain elimination ground unrest measure, for example adopt the method for cofocus scanning to reduce on-chip fluorescent impurity noise; Adopt photomultiplier to replace CCD and improve the extraction of fluorescent signal etc., but the impurity noise still exists, background noise still can not thoroughly be eliminated.Therefore be necessary to take new detection thinking to improve the sensitivity of chip detector, and proofing unit is improved accordingly, thoroughly to address the above problem.
Summary of the invention
The objective of the invention is problem at the existence of existing hybridization detection technique, a kind of interference that can abate the noise, highly sensitive gene chip time resolved fluorescence detection method are proposed, the present invention simultaneously also will provide special detection device---the gene chip time resolved fluorescence proofing unit of this method of realization, detect obstacle thereby put down the biochip technology progressive.
In order to reach above purpose, gene chip time resolved fluorescence detection method of the present invention may further comprise the steps: A. is fixed on the dna probe array on the chip basal body, carry out hybridization with target molecule by rare earth chelate compound bonded dyestuff (for example inner complex such as europium, ruthenium ion) mark, clean chip afterwards, and the chip after will cleaning is placed on the detection platform; B. detection light source is modulated, make incident beam impinge upon on-chip hybridization region, excite dyestuff to send fluorescence by the fluorescence camera lens; C. allow fluorescence and gene chip that the reflected light of exciting light is entered the detection light path through camera lens simultaneously, by two-phase look mirror, the short reflected light that excites of wavelength is reflected, and the long fluorescence of wavelength see through two-phase look mirror, enters collimated light path again after the colour filter of specific wavelength filters; D. placement space filtering pin hole before the focal plane of collimated light path, the noise signal of out of focus is removed, and the fluorescence that arrives the object lens focal plane is radiated at optoelectronic receiver by pin hole and carries out opto-electronic conversion, photosignal after the conversion is sent into A/D (mould/number) collection plates, gather postponing the signal of 10 μ s-1000 μ s after the time, as effective processing data; E. the valid data that the A/D collection plates is received are transported to main control system.
After this, carry out digitized processing,, determine chip hybridization result's bioinformation, and output shows according to the original design of chip by main control system.
The gene chip time resolved fluorescence proofing unit of realizing aforesaid method adopts following proposal to realize: this installs mainly by frame (1), support platform (2), platform transmission rig (3,4), optical detection unit, the Photoelectric Signal Processing unit is formed, wherein support platform (2) is by platform transmission rig (3,4) be connected with frame (1), optical detection unit is by detection light source (7), pass optical device (9), and be installed in collimating mirror (12) on the detection head (10), fluorescence camera lens (14) and at least one group of dichroscope (13), pin hole (15), colour filter (17), optoelectronic receiver (16) constitutes, described Photoelectric Signal Processing unit is made of mould/number (A/D) collection plates (19) and main control system, its annexation or pass, position each other is: pass the collimating mirror (12) that optical device (9) coupling is fixed on the detection light source (7) on the frame (1) and is fixed on detection head (10) one sides, fluorescence camera lens (14) is fixed in the lower end of detection head (10), facing to support platform (2), meet at right angles with the light path of collimating mirror (12), two-phase look mirror (13) is positioned at fluorescence camera lens (14) and vertical with it light path intersection, pin hole (15) is positioned on the light path between fluorescence camera lens (14) and the optoelectronic receiver (16), colour filter (17) is equipped with in its front, and optoelectronic receiver (16) is by cable (18) and mould/number (A/D) collection plates (19), and then be connected with main control system.
The tracer because above method employing rare earth element ion serves as a mark for example adopts to form Eu
3+(NTA)
3(TOPO)
3The fluorescent composition of type, wherein NTA is a 2-naphthoyl trifluoroacetone, TOPO is a trioctylphosphine oxide, Eu
3+The probe of-sequestrant mark and target DNA post-hybridization washing, under low PH condition, Eu
3+Can discharge with strengthen liquid in DNA combine with TOPO and form final fluorescent composition.The result, characteristics such as can ingeniously utilize that its unique emission peak is narrow, fluorescence lifetime long (10-1000us), Stokes shift are big, the delay measurements time, after treating that background short life fluorescence disappears, again the long lifetime fluorescence of above-mentioned inner complex is sampled and data processing, therefore can get rid of because short-life background fluorescence such as substrate impurity disturbs, thus the sensitivity that improves chip analysis widely, and its high efficiency, linearity range are wide.The branch light action of dichroscope is blocked the optical excitation signal of reflection simultaneously, and the pin hole behind the color filter removes the noise signal of out of focus, thereby improves signal to noise ratio, further makes the sensitivity of detection and stability obviously be better than prior art.
In order to improve the comparability of detection signal, the further improvement of the inventive method is: with beam splitter the light beam of light source beam splitting is formed reference beam, accept the reference beam signal that obtained by light beam of light source and input main control system with optoelectronic receiver.Like this, main control system can relatively detect the relative value of fluorescent signal to light signal, thereby the principle of utilizing signal to compare is eliminated the fixed influence of flashing, improves the verity of detected result.
The improvement of corresponding preceding method, the further improvement of said apparatus is: also contain beam splitter and supporting with it optoelectronic receiver, described beam splitter is positioned between detection light source and two-phase look mirror, described optoelectronic receiver is positioned on the reference path that beam splitter tells, and is connected with the A/D collection plates and then with main control system by cable.
In a word, the present invention organically combines time fluorescence resolution techniques, gene chip detecting technique, biology information technology, has plurality of advantages such as highly sensitive, good stability, linearity range is wide, the marker retention period is long.
Description of drawings
Below in conjunction with accompanying drawing the present invention is described in further detail.
Fig. 1 is the structural representation of embodiment one gene chip time resolved fluorescence proofing unit.
Fig. 2 is the structure principle chart of embodiment two gene chip time resolved fluorescence proofing unit.
Fig. 3 is the detection system schema of embodiment two.
Fig. 4 is the automatic focusing schema of embodiment two.
Fig. 5 obtains schema for the data of embodiment two.
Concrete enforcement mode
Embodiment one
The step that the present embodiment genetic chip time is differentiated the fluorescence detection method realization is: A. the dna probe array is fixed on the chip basal body, with the dyestuff (example of being combined by the rare earth element chelate Such as chelates such as europium, ruthenium ions) target molecule of mark hybridizes reaction in hybridizing box, cleans afterwards Chip, and the chip after will cleaning is placed on the detection platform; B. adopt optical source wavelength 337nm nitrogen molecular laser as detection light source, after modulation, make the light pulse width be 10ns, light pulse repetition rate 15-20Hz, incident light impinges upon the assorted of genetic chip by the fluorescence camera lens Hand over the district, the dyestuff through exciting sends 613nm fluorescence, and the camera lens by 0.8 millimeter high-NA enters Detect light path, genetic chip also enters the detection light path to the utilizing emitted light (337nm) that excites light simultaneously; C. make fluorescence and reverberation all enter the detection light path through camera lens, under 340/613 two-phase look mirror effect, 337nm The short wavelength excite reverberation to be reflected, and long wavelength's fluorescence of 613 ± 5nm sees through two-phase look mirror, through not The colour filter of co-wavelength filters, and enters respectively collimated light path separately; D. in face of Jiao of each collimated light path, place sky and ask the filtering pin hole, the noise signal of out of focus is removed, and arrive The fluorescence of the burnt face of object lens is radiated at the response photomultiplier by pin hole and carries out opto-electronic conversion, with after the conversion The photoelectricity signal is sent into A/D (mould/number) collection plate, the signal after postponing about 500 μ s times gathered, As effective deal with data; The valid data of several wavelength fluorescent signals that E. the A/D collection plate received are transported to main control system.
After this, carry out digitized processing by main control system, with the fluorescence intensity sign gene chip sample applying of pixel point The fluorescence intensity of place's hybridization point, namely gray scale or the pseudo-coloured picture with the pixel point after the conversion resembles to characterize, and utilizes mobile The gray feature of trellis algorithm pair array point extracts, and obtains the fluorescence gray value of difference, according to chip Original design is determined chip hybridization result's biological information and output display.
Realize the special-purpose instrument of said process as shown in Figure 1, comprise that frame 1, X-Y support platform 2, platform Transmission mechanism, optical detection unit, Photoelectric Signal Processing unit form. Wherein X-Y support platform 2 passes through The platform transmission mechanism is connected with frame 1. The platform transmission mechanism is by rolling that clasp nut 3, stepper motor 6 drive Ballscrew 4 consists of. Optical detection unit is by laser instrument 7, optical fiber 9 and be installed in a standard that detects on 10 Straight mirror 12, fluorescence camera lens 14 and three groups of dichroscopes 13, pin hole 15, colour filter 17, photomultiplier 16 Consist of. (essence is a calculating by A/D collection plate 19 and external main control system in the Photoelectric Signal Processing unit Machine) consists of. Wherein, optical fiber 9 is fixed on frame 1 by optical fiber entrance 8 and optical fiber outlet 11 couplings respectively The laser instrument 7 of one end and the collimation mirror 12 that is fixed on detection 10 1 side. Fluorescence camera lens 14 as object lens Be fixed in and detect a lower end of 10, support the chip 21 in the chip cartridges 20 on the platform 2 facing to X-Y, with standard The light path of straight mirror 12 meets at right angles. Three two-phase look mirrors 13 lay respectively at: fluorescence camera lens 14 is with it vertical Collimation mirror 12 light path intersections, and fluorescence camera lens 14 and with it vertical in addition two detection light path intersections. Three pin holes 15 lay respectively near the burnt face of light path between fluorescence camera lens 14 and three photomultipliers 16, Different colour filter 17 is equipped with respectively in its front. Three photomultipliers 16 are respectively by cable 18 and A/D Collection plate 19 and then be connected with the main control system of outside.
During real work, the laser that the laser instrument 7 of present embodiment detection device sends is passed by optical fiber 9 and enters detection 10, this incident light focuses on X-Y through collimation mirror 12, dichroscope 13 and fluorescence camera lens 14 and supports Chip 21 surfaces on the platform 2. Chip surface hybridization point is excited and produces fluorescence signal, this glimmering optical signals Fluorescence camera lens 14 is collected. For the fluorescence to different wave length is collected, the fluorescence signal is respectively through dichroscope, Afterwards can be with the noise signal of out of focus by the three groups of different filter 17 in upper, middle and lower and each filter The pin hole 15 that removes is collected by photomultiplier 16, imports mould/number collection plates 19 by cable 18 separately again, The external main control system of final input is processed rear demonstration by software.
Experimental results show that, owing to adopt rare earth element ion as the mark tracer, after treating that background short life fluorescence disappears, again the long-life fluorescence of above-mentioned chelate is sampled and process with data, therefore can get rid of because short-life background fluorescence such as substrate impurity disturbs, the branch light action of dichroscope is with the optical excitation signal blocking-up of reflection in addition, and the pin hole behind the filter removes the noise signal of out of focus, the result, in DNA analysis, detection sensitivity can reach 10-18-10
-16The dna probe of mol, sensitivity and detection accuracy obviously improve. This enforcement One big advantage of example is to detect simultaneously multi-wavelength's fluorescence.
Embodiment two
The present embodiment genetic chip time is differentiated the step of fluorescence detection method and the difference master of embodiment one 3 points are arranged: the hybridization reaction is carried out at detector; Adopt and divide beam optical path; Time delay is short. Its concrete steps As follows: A. with mechanical deposition, specking or in-situ synthesis the dna probe array is fixed on the transparent chip basal body of spectrum On, the LB film of the material employing polymer of chip (also can be glass, the quartz of functionalization, Si, Ge, GaAs, GaP, SiO2、Si
3N
4, Si gel, the polymer modified, or such as polytetrafluoro, poly-The gel of alum, Merlon and so on, polymer, or their mixture); Chip be shaped as circle Dish type (also can be the various shapes such as square, circle); Various microfabrication are adopted on the surface of chip Method is made the surface characteristics such as required muscle, V-type groove, micropore; Gathering of the zone of hybridization and incident light Focus connects closely that (surface of substrate obtains the spy of Si-OH usually for quartzy, glass surface Levy); B. chip is placed on the support platform that detects device, fixes with vacuum suction or mechanical compaction, and with core Sheet is connected with the microfluidic pond that is used as the hybridization pond, with rubber seal or sealing ring fluid is sealed, and jointly is placed on Detect on the support platform of device; C. (for example the press ion forms Eu with the target molecule of lanthanide ion mark3+(NTA)
3(TOPO)
3The fluorescent composition of type, wherein NTA is 2 naphthoyl trifluoropropyl ketone, TOPO is three octyl group phosphorous oxide, Eu3+Washing after the probe of-chelating agent mark and the target DNA hybridization, under the low PH condition, Eu3+Can discharge and strengthen in the liquid DNA is combined with TOPO and is formed final fluorescent composition. ), the hybridization such as fluorescence enhancement solution, cleaning fluid are anti-Answer reagent to inject the microfluidic pond by Micropump from fluid input mouth; That D. controls little pond solution temperature slowly is warming up to hybridization temperature, and temperature control precision is ± 0.5 ℃; After hybridization is finished, clean chip, make the target that is combined with the rare earth element dyestuff that mates fully with probe molecule Molecule is combined on the chip, and unmatched target molecule is cleaned; E. detection light source is carried out impulse modulation, adopts optical source wavelength 337nm nitrogen molecular laser as detection light source, Light pulse width 10ns, light pulse repetition rate 15-20Hz, incident light impinges upon base by the fluorescence camera lens Because of the hybridization region of chip, the dyestuff through exciting sends 613nm fluorescence, by 0.8 millimeter high-NA Camera lens enter the detection light path, simultaneously genetic chip also enters inspection to the utilizing emitted light (337nm) that excites light The photometry road; F. after making fluorescence enter the detection light path from the hemisphere solid angle direction, through 340/613 two-phase look mirror, short wavelength 337nm excite light by anti-not, and the 613 ± 5nm long wavelength fluorescence that sees through two-phase look mirror is by specific wavelength Colour filter filter, enter collimated light path; G. placement space filtering pin hole in face of Jiao of collimated light path makes the fluorescence of the burnt face of object lens be radiated at by pin hole Photomultiplier carries out opto-electronic conversion, and the photoelectricity signal after the conversion is sent into the A/D collection plate, to postponing approximately Signal after 50 μ s times is gathered, as effective deal with data; H. with beam splitter the light beam of light source beam splitting is formed reference beam, accept by light with the photomultiplier of wanting with it to join The reference beam signal that source beam obtains is also sent into the A/D collection plate; I. all valid data that the A/D collection plate received are transported to main control system.
At last, main control system carries out digitized processing on the one hand, with the fluorescence intensity sign genetic chip of pixel point The fluorescence intensity of point sample place hybridization point utilizes the gray feature of mobile grid algorithm pair array point to extract, and obtains Get the fluorescence gray value of difference, according to the original design of chip, determine chip hybridization result's biological information, And output display. Meanwhile, main control system also relatively detects the fluorescence signal to the relative value of light signal, from And the principle of utilizing signal to compare is eliminated the unsettled impact of light source, improves the authenticity of testing result.
The detection device of present embodiment is referring to the principle figure of Fig. 2. (this figure considers the effect of polychromatophilia material, label Not general with Fig. 1). Wherein the laser that sends of laser instrument 1 is through colour filter 2, camera lens 3 and 5, confocal pinhole 4, beam splitter 6, dichroscope 7, camera lens 8, object lens 9 incide substrate 28 surfaces, excite substrate surface assorted Intersection point produces the fluorescence signal. This glimmering optical signals camera lens 9 is collected, by camera lens 8, dichroscope 7 and Thereafter camera lens, pin hole, colour filter arrive the Photoelectric Detection device. For to different phosphor collections, the fluorescence signal Carry out the different wave length phosphor collection by dichroscope 14: on the one hand, fluorescence by camera lens 15, pin hole 16, Colour filter 17 is collected by Photoelectric Detection device 18, on the other hand then by camera lens 19, pin hole 20, colour filter 21 Collected by Photoelectric Detection device 22. In order to control the parameters such as intensity of incident laser, the laser that laser instrument 1 sends Also by beam splitter 6 beam splitting, enter detector 13 through camera lens 10, pin hole 11, colour filter 12 and collect. Control Main frame is according to the operation parameter of the signal feedback control laser instrument 1 of detector 13. The letter of above-mentioned all detectors Number all finally enter and to detect circuit 23, by the processing sequential of counter controls to signal, the result is by the main control system place Reason output. The characteristics of this embodiment are that substrate 20 is placed on the fluid pool 32, fluid pool 32 placements In circulating water cavity 31, by the wriggling pump with reactant liquor 30 from input mouthfuls 33, output mouthfuls 34 at fluid pool 32 Interior circulation, substrate 20 can be passed through the vacuum suction of vavuum pump 29 generations on fluid pool 32. Fluid pool 32 Be placed on the 3D motion platform. In testing process, at first carry out coarse scanning, four angles of substrate 20 are swept Retouch, and record relative position, to substrate scanning, utilize interpolation method that substrate is carried out automatic focusing scanning then, The outcome record that detects is corresponding file and is presented on the main control system demonstration screen.
The relevant data of above-described embodiment are obtained flow process can be referring to Fig. 3, Fig. 4, Fig. 5.
Fig. 3 is the detection system flow chart. At first import detected parameters, initialize then micromotion platform, start certainly Move and seek the focusing that Jiao finishes light path. Can carry out coarse scanning and meticulous scanning to chip. Scanning is finished and is carried out next Wheel scan work.
Fig. 4 is the flow chart of automatically focusing. An angle of lens focus chip, electric machine rotation adjustable lens and chip Distance, the fluorescence signal that PMT collects positive point also is converted into output voltage. Along with the motion of camera lens, electricity Extreme value appears in the pressure value. Thereby the lens location of definite this point is also recorded. Four of chip have been scanned when camera lens The angle, scanning software is determined the virtual focusing plane of chip, so finish fast automatic focusing when meticulous scanning.
Fig. 5 obtains flow chart automatically for data. At first import detected parameters, initialize micromotion platform, then true Surely test sweep parameter, start A/D, begin scanning, postpone to gather the time resoluting signal that obtains chip. Heavy Multiple scanning carries out data and processes until chip scanning finishes.
Embodiment three
Postpone 800 microseconds in the process that is to gather with embodiment two differences of present embodiment, earlier with DNA The probe array is fixed on the transparent chip basal body of spectrum, again with the target molecule of rare earth ion chelate complex dye marker Hybridize reaction at hybridizing box; After the cleaning chip is placed on the support platform that detects device; According to reality Executing example one F step afterwards detects. The device that adopts is identical with embodiment one. Experiment showed, the result Feasible.
In addition to the implementation, the present invention can also have many variations. For example, during Practical manufacturing, photoelectricity is accepted Device can be photodiode, avalanche optoelectronic pipe, photomultiplier, CCD (charge-coupled image sensor) etc.; Light The source can be LASER Light Source, arc light light source, heated filament light source, semiconductor luminotron etc.; The spectrum of light source can be Singlet or multiline or compose continuously line; Adopt integrated chip-detecting apparatus or the independence of Micrometer-Nanometer Processing Technology Chip, detect device etc. These are the desired protection domain of present patent application.
Claims (6)
1. gene chip time resolved fluorescence detection method, it is characterized in that may further comprise the steps: A. is fixed on the dna probe array on the chip basal body, carry out hybridization with target molecule by rare earth chelate compound bonded dye marker, clean chip afterwards, and the chip after will cleaning is placed on the detection platform; B. detection light source is modulated, and makes incident beam impinge upon on-chip hybridization region by the fluorescence camera lens, excites dyestuff to send fluorescence; C. fluorescence and gene chip enter the detection light path through camera lens simultaneously to the reflected light of exciting light, by two-phase look mirror, the short reflected light that excites of wavelength is reflected, and the long fluorescence of wavelength see through two-phase look mirror, enters collimated light path again after the colour filter of specific wavelength filters; D. placement space filtering pin hole before the focal plane of collimated light path, the noise signal of out of focus is removed, and the fluorescence that arrives the object lens focal plane is radiated at optoelectronic receiver by pin hole and carries out opto-electronic conversion, photosignal after the conversion is sent into the A/D collection plates, gather postponing the signal of 10 μ s-1000 μ s after the time, as effective processing data; E. the valid data that the A/D collection plates is received are transported to main control system.
2. gene chip time resolved fluorescence detection method according to claim 1, it is characterized in that: also comprise with beam splitter the light beam of light source beam splitting is formed reference beam, accept the reference beam signal that obtains by light beam of light source and input main control system with optoelectronic receiver.
3. according to the described gene chip time resolved fluorescence of claim 1 detection method, it is characterized in that may further comprise the steps: A. is fixed on the dna probe array on the transparent chip basal body of spectrum with mechanical deposition, specking or in-situ synthesis, and the material of chip adopts the LB film of polymkeric substance; The surface of chip adopts the method for various microfabrication to make surface characteristic such as required muscle, V-type groove, micropore; The zone of hybridization closely is connected with the focus point of incident beam; B. chip is placed on the support platform of proofing unit, fixes, and chip is connected with the microfluidic pond that is used as the hybridization pond, fluid is sealed, be placed on jointly on the support platform of proofing unit with glue envelope or sealing-ring with vacuum suck or mechanical compaction; C. hybridization reagent such as the target molecule of lanthanide ion mark, fluorescence enhancement solution, scavenging solution are injected the microfluidic pond by Micropump from the fluid input aperture; That D. controls little pond solution temperature slowly is warming up to hybridization temperature, and temperature control precision is ± 0.5 ℃; After hybridization is finished, clean chip, the target molecule that is combined with the rare earth element dyestuff that mates fully with probe molecule is combined on the chip, unmatched target molecule is cleaned; E. detection light source is carried out pulsed modulation, adopt optical source wavelength 337nm nitrogen molecular laser as detection light source, light impulse length 10ns, light pulse repetition rate 15-20Hz, incident beam impinges upon the hybridization region of gene chip by the fluorescence camera lens, dyestuff through exciting sends 613nm fluorescence, and the camera lens by 0.8 millimeter high-NA enters the detection light path, and gene chip also enters the detection light path to the emission light of exciting light simultaneously; I. after making fluorescence enter the detection light path from the hemisphere solid angle direction, through 340/613 two-phase look mirror, short wavelength's 337nm exciting light is reflected, and sees through the colour filter filtration of 613 ± 5nm long wavelength fluorescence of two-phase look mirror by specific wavelength, enters collimated light path; J. placement space filtering pin hole before the focal plane of collimated light path, make the fluorescence of object lens focal plane be radiated at photomultiplier and carry out opto-electronic conversion by pin hole, photosignal after the conversion is sent into the A/D collection plates, the signal after postponing about 50 μ s times is gathered, as effective processing data; K. with beam splitter the light beam of light source beam splitting is formed reference beam, by the reference beam signal that light beam of light source obtains, also send into the A/D collection plates with the photomultiplier acceptance of wanting with it to join; I. all valid data that the A/D collection plates is received are transported to main control system.
4. gene chip time resolved fluorescence proofing unit, mainly by frame (1), support platform (2), platform transmission rig (3,4), optical detection unit, the Photoelectric Signal Processing unit is formed, wherein support platform (2) is by platform transmission rig (3,4) be connected with frame (1), optical detection unit is by detection light source (7), pass optical device (9), and be installed in collimating mirror (12) on the detection head (10), fluorescence camera lens (14) and at least one group of dichroscope (13), pin hole (15), colour filter (17), optoelectronic receiver (16) constitutes, described Photoelectric Signal Processing unit is made of mould/number (A/D) collection plates (19) and main control system, its annexation or pass, position each other is: pass the collimating mirror (12) that optical device (9) coupling is fixed on the detection light source (7) on the frame (1) and is fixed on detection head (10) one sides, fluorescence camera lens (14) is fixed in the lower end of detection head (10), facing to support platform (2), meet at right angles with the light path of collimating mirror (12), two-phase look mirror (13) is positioned at fluorescence camera lens (14) and vertical with it light path intersection, pin hole (15) is positioned on the light path between fluorescence camera lens (14) and the optoelectronic receiver (16), colour filter (17) is equipped with in its front, and optoelectronic receiver (16) is by cable (18) and A/D collection plates (19), and then be connected with main control system.
5. gene chip time resolved fluorescence proofing unit according to claim 4, it is characterized in that: also contain beam splitter and supporting with it optoelectronic receiver, described beam splitter is positioned between detection light source and two-phase look mirror, described optoelectronic receiver is positioned on the reference path that beam splitter tells, and is connected with the A/D collection plates and then with main control system by cable.
6. according to the described gene chip time resolved fluorescence of claim 1 detection method, it is characterized in that: contain three groups of dichroscopes (13), pin hole (15), colour filter (17) and photomultiplier (16), three two-phase look mirrors (13) lay respectively at: fluorescence camera lens (14) and vertical with it collimating mirror (12) light path intersection, and fluorescence camera lens (14) and vertical with it two detection light path intersections in addition, three pin holes (15) lay respectively near the focal plane of light path between fluorescence camera lens (14) and three photomultiplier (16), its front is equipped with different colour filter (17) respectively, and three photomultiplier (16) are respectively by cable (18) and A/D collection plates (19), and then be connected with the main control system of outside.
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| WO2005083432A1 (en) * | 2003-12-19 | 2005-09-09 | Chengdu Kuachang Medical Industrial Limited | The detecting method of chip and the detecting device |
| US9533879B2 (en) | 2008-06-02 | 2017-01-03 | Bionano Genomics, Inc. | Integrated analysis devices and related fabrication methods and analysis techniques |
| CN110530565A (en) * | 2019-09-23 | 2019-12-03 | 中国工程物理研究院流体物理研究所 | A kind of multi-channel Time measuring device and method based on optical fiber probe |
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| WO2005083432A1 (en) * | 2003-12-19 | 2005-09-09 | Chengdu Kuachang Medical Industrial Limited | The detecting method of chip and the detecting device |
| US9533879B2 (en) | 2008-06-02 | 2017-01-03 | Bionano Genomics, Inc. | Integrated analysis devices and related fabrication methods and analysis techniques |
| US10654715B2 (en) | 2008-06-06 | 2020-05-19 | Bionano Genomics, Inc. | Integrated analysis devices and related fabrication methods and analysis techniques |
| US11292713B2 (en) | 2008-06-06 | 2022-04-05 | Bionano Genomics, Inc. | Integrated analysis device analysis techniques |
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