CN104614351A - Rapid and multi-channel real-time fluorescence quantification detection device - Google Patents
Rapid and multi-channel real-time fluorescence quantification detection device Download PDFInfo
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- CN104614351A CN104614351A CN201510031523.7A CN201510031523A CN104614351A CN 104614351 A CN104614351 A CN 104614351A CN 201510031523 A CN201510031523 A CN 201510031523A CN 104614351 A CN104614351 A CN 104614351A
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Abstract
The invention discloses a rapid and multi-channel real-time fluorescence quantification detection device which comprises an excitation light source, an excitation light filtering structure, light conduction optical fibers, a lens line array, a fluorescence light filtering structure, a spatial light modulator and a photomultiplier, wherein excited light emitted from the excitation light source is coupled into the light conduction optical fibers after passing through the excitation light filtering structure; the excited light beam emitted from the optical fibers passes through the lens line array and is converged to radiate to a target to be detected so as to excite fluorescence; the fluorescence is collected by the fluorescence lens array and the light conduction optical fibers, and is further filtered by the fluorescence light filtering structure so as to filter off background interference light; the filtered fluorescence is further filtered by the spatial light modulator; the further filtered fluorescence finally reaches the photomultiplier, so as to form signals which can be detected and calculated. By adopting the rapid and multi-channel real-time fluorescence quantification detection device, rapid detection on multiple targets through multiple channels can be achieved, the system wholeness is improved, the mechanical vibration is alleviated, and the detection precision is improved.
Description
Technical field
The present invention relates to biomolecule fluoroscopic examination field, particularly a kind of quick, hyperchannel real time fluorescent quantitative pick-up unit, the fluorescent quantitation being mainly used in 96 hole PCR reaction plates or ELISA Plate detects.
Background technology
Real time fluorescent quantitative detection system, effectively can realize Real_time quantitative detection PCR (PolymeraseChain Reaction, PCR), it has the advantages such as specificity is good, zero degree is high, linear relationship is good, the range of linearity is wide, simple to operate, safety, automaticity are high, pollution-free, detection speed fast, high flux, has a wide range of applications in fields such as disease prevention and cure, detection GMOs, medical test, prenatal and postnatal care.Excitation source is mainly irradiated on fluorescent material by real time fluorescent quantitative detection method conventional at present, irradiates fluorescent material, make fluorescent material electronics generation energy level transition by exciting light, and unstable transition electronics is got back to ground state emission and gone out fluorescence.Because different fluorescent materials has different excitation wavelengths and wavelength of fluorescence, the different excitation wavelength used just can excite different fluorescent materials to produce specific fluorescent, by the specificity that different fluorescent material combines from different PCR reaction product, the fluorescent quantitation realizing different target PCR reaction product detects.After PCR reaction each time terminates, the fluorescence intensity that detection fluorescent material emits obtains the concentration of fluorescent material indirectly, and fluorescent material concentration is directly proportional to PCR reaction product concentration, therefore can detect PCR reaction product concentration by fluorescent material concentration.
For realizing hyperchannel, fast and accurately real time fluorescent quantitative, the method of usual employing has Single wavelength to excite multi-wavelength detection and multi-wavelength excitation multi-wavelength detection, conventional light channel structure mainly confocal version, sensitive detection parts generally adopt the photomultiplier that gain is larger.Because Single wavelength excites spectral response narrow, some wavelength fluorescent launching efficiency is low, be subject to a lot of restriction, and multi-wavelength excitation can ensure the launching efficiency of different fluorescence in multi-wavelength detection.But current multi-wavelength excitation detection method needs to arrange different gear trains to realize the conversion of different wave length, simultaneously because photomultiplier can only detect a target at every turn, so also need increase drive mechanism or construction of switch to realize the selection of different passage, the great number of driving mechanism added or construction of switch, destroy the stability of system, its accuracy of detection and detection speed condition each other to a certain extent.Therefore, the structure simplifying multichannel detection system is the developing direction realizing quick and precisely quantitatively detecting.
Summary of the invention
The object of the invention is the deficiency overcoming prior art existence, provide one to realize multi-wavelength excitation, multi-wavelength detection easily, and the real time fluorescent quantitative pick-up unit that globality is more optimized.
For realizing above-mentioned technical purpose, the present invention adopts following technical scheme:
A kind of quick, hyperchannel real time fluorescent quantitative pick-up unit, comprises the excitation light emission system of multi-wavelength, splitting optical fiber, coaxial optical fiber, linear lens array row, fluorescent modulation system and photomultiplier; Wherein said excitation light emission system comprises laser array, exciting light collimation lens, exciting light optical filtering mirror wheel and exciting light coupled lens; Described coaxial optical fiber is wrapped up by two-layer optical fiber and forming, outer optical fiber annular arrangement wrapping inner layer optical fiber, and coaxial optical fiber is in order to the conduction of the conduction and fluorescence that realize exciting light; Described fluorescent modulation system comprises fluorescence optical fiber bundle, fluorescence collimation lens, fluorescence optical filtering mirror wheel, plus lens and spatial light modulator; Described spatial light modulator comprises hole array, the arrangement mode of described hole array is corresponding with the arrangement mode of described fluorescence optical fiber bundle, its function is gating and the filtering parasitic light that can realize the intrafascicular corresponding optical fiber of fluorescence optical fiber simultaneously, by being loaded into hole array image in spatial light modulator, realizing the logical light of different channel fiber and blocking light.
The continuous agitation light that described laser array produces to be filtered mirror wheel by exciting light after exciting light collimation lens collimation, is filtered form the monochromatic excitation light outgoing of narrow wave band by exciting light optical filtering mirror wheel; Outgoing exciting light is evenly divided in some splitting optical fibers through exciting light coupled lens, then imports in the inner layer optical fiber of respective numbers coaxial optical fiber respectively; Exciting light converges on corresponding object to be checked respectively through the lens unit of respective numbers in linear lens array row after some coaxial optical fiber outgoing, the fluorescence that object to be checked excites is collected in the outer optical fiber importing coaxial optical fiber more respectively through linear lens array row, described fluorescence conducts to fluorescence collimation lens through the fluorescence optical fiber bundle of respective numbers, outgoing fluorescence is irradiated in spatial light modulator by being converged by plus lens after fluorescence optical filtering mirror wheel, is entered photomultiplier detect by the fluorescence in the optical fiber of spatial light modulator gating.
Preferably, described laser array is by the laser constitution of some different wave lengths, and the wavelength coverage that exciting light exports is 270nm-750nm, and exciting light exit facet is positioned on the focal plane of exciting light collimation lens, and collimation obtains directional light.
Preferred further, described exciting light optical filtering mirror wheel is made up of monochromatic filter, and its optical filter quantity is identical with the number of lasers in described laser array.
Preferably, described splitting optical fiber coupling section is positioned on the focal plane of exciting light plus lens.
Preferably, the lens unit in described linear lens array row is the linear alignment, and its numerical aperture is not less than 0.2, and focal length is not less than 11mm.
Preferably, described fluorescence collimation lens is made up of single or multiple lens, and described fluorescence optical fiber bundle outgoing end face is positioned on fluorescence collimation lens focal plane, and emergent light collimation obtains directional light.
Preferably, described fluorescence optical filtering mirror wheel is made up of 1,2,4,6 or 8 monochromatic filter annular arrangement.
Preferably, described spatial light modulator is positioned on plus lens focal plane.
Preferably, described splitting optical fiber and coaxial optical fiber are 1 × N optical fiber structure; Described fluorescence optical fiber bundle is that N closes bundle optical fiber, can be formed through surrounding layer is coated by N root single fiber, the wherein arrangement in rule between optical fiber; Described linear lens array arranges and is made up of 1 × N lens unit; Wherein N >=2;
Preferred further, described N is 8.
The technical program principle is:
Multiple light source produces the light of different wave length, wavelength coverage contains 270nm-750nm, collimated light beam is formed through collimation lens, then form the monochromatic excitation light of narrow wave band through the exciting light optical filtering wheel be made up of high-quality optical filtering, be coupled into splitting optical fiber finally by exciting light coupled lens.Different Light can mate the optical filter of respective wavelength, and with optical filtering wheels all-in-one-piece conversion equipment, the exciting light that can be realized different wave length by the conversion of light source and optical filtering is coupled into splitting optical fiber.
Linear lens array arranges and is made up of 1 × N number of lens cells, can realize exciting and phosphor collection of N number of destination agent hole simultaneously, drives the translation of reagent orifice plate can complete the detection of whole plate reagent wells by one dimension displacement device.
Arrange corresponding 1 × N coaxial optical fiber array with linear lens array, it is wrapped up by inside and outside two-layer optical fiber and forming, and internal layer completes exciting light conduction, and skin completes the corresponding phosphor collection conduction detecting target separately.Exciting light is irradiated to detection target from the outgoing of optical fiber internal layer through the convergence of linear lens array row, and fluorescence enters fiber outer layer through the collection of linear lens array row.
The fluorescence of fluorescence optical fiber bundle outgoing collimates through collimation lens, then arrowband fluorescence is formed by fluorescence optical filtering, other background stray light of filtering simultaneously, be irradiated in spatial light modulator after plus lens converges, the some spot array that formation one is corresponding with fluorescence optical fiber bundle optical fiber arrangement in spatial light modulator.Spatial light modulator is positioned on plus lens focal plane, and spatial light modulator completes the dual-use function of pin-hole filter-ing and photoswitch selection simultaneously.Spatial light modulator, by Fast transforms modulation system, realizes the logical light of different luminous point and blocks light, and realization quick control different target fluorescence enters the object that photomultiplier carries out detecting.
Technical solution of the present invention has the following advantages:
1. the linear lens array row that the present invention uses can realize the detection of multiple 96 hole PCR reaction plates or ELISA Plate reagent wells simultaneously, decrease translation dimension, simplify system architecture, enhance systematic entirety, improve system accuracy, avoid optical fiber simultaneously and too much make the shortcoming that system is too numerous and diverse, be difficult to maintenance;
2. the present invention uses coaxial optical fiber, avoids light in air dielectric, conducts brought problem, makes system more stable compact, is different from traditional approach and needs to arrange dichroic mirror to be separated the method for exciting light and fluorescence, simplify system;
3. usage space photomodulator, achieve the function of pin-hole filter-ing and photoswitch gating simultaneously, by traditional structure independently two devices be merged into one, greatly simplifie system architecture, instead of traditional Mechanical course by optical modulation simultaneously, improve detection speed;
Accompanying drawing explanation
Fig. 1 is embodiment of the present invention real time fluorescent quantitative structure of the detecting device stereographic map.
Wherein:
1. laser array; 2. exciting light collimation lens; 3. exciting light optical filtering mirror wheel; 4. exciting light coupled lens; 5. splitting optical fiber; 6. coaxial optical fiber; 7. linear lens array row; 8. object to be checked; 9. fluorescence optical fiber bundle; 10. fluorescence collimation lens; 11. fluorescence optical filtering mirror wheels; 12. plus lens; 13. spatial light modulators; 14. photomultipliers.
Embodiment
Below in conjunction with accompanying drawing, by embodiment, the present invention will be further described, to understand the present invention better.
Composition graphs 1, the present embodiment real time fluorescent quantitative pick-up unit is made up of laser array 1, exciting light collimation lens 2, exciting light optical filtering mirror wheel 3, exciting light coupled lens 4, splitting optical fiber 5, coaxial optical fiber 6, linear lens array row 7, fluorescence optical fiber bundle 9, fluorescence collimation lens 10, fluorescence optical filtering mirror wheel 11, plus lens 12, spatial light modulator 13 and photomultiplier 14; Produced the light of different wave length in laser array 1 by 4 laser light sources, wavelength coverage is between 270nm-750nm, and exciting light bandwidth is less than 20nm, and light-emitting face is positioned on the focal plane of exciting light collimation lens 2; The present embodiment adopts eight channels designs, and splitting optical fiber 5, coaxial optical fiber 6 and linear lens array row 7 are 1 × 8; Exciting light collimation lens 2 is coaxial at a distance of 50mm with exciting light coupled lens 4, exciting light optical filtering mirror wheel 3 comprises 4 optical filterings, between exciting light collimation lens 2 and exciting light coupled lens 4, splitting optical fiber 5 section that is coupled is positioned on exciting light plus lens 4 focal plane, exciting light is through coaxial optical fiber 6 outgoing, and converge on object 8 to be checked through linear lens array row 7, object 8 to be checked is contained in 96 orifice plates, Width is 8 row, with linear lens array row 7 one_to_one corresponding.The fluorescence that object 8 to be checked ejects is collected through linear lens array row 7 and is coupled into coaxial optical fiber 6, fluorescence is from fluorescence optical fiber bundle 9 outgoing, fluorescence optical fiber bundle 9 outgoing end face is positioned on fluorescence collimation lens 10 focal plane, fluorescence optical filtering mirror wheel 11 is positioned at 25mm place, fluorescence collimation lens 10 rear, fluorescence converges through plus lens 12, plus lens 12 is coaxial at a distance of 50mm with collimation lens 11, spatial light modulator 13 is positioned on plus lens 12 focal plane, and photomultiplier 14 is positioned at 10mm place, spatial light modulator 13 rear.
The principle of device is as follows: the exciting light penetrated by laser array 1, collimate through exciting light collimation lens 2 and form directional light, directional light forms narrow wave band light beam through the optical filter optical filtering of exciting light optical filtering mirror wheel 3, filtered exciting light converges through exciting light coupled lens 4 and is coupled into splitting optical fiber 5, evenly be divided into 8 bundles and imported coaxial optical fiber 6 respectively, exciting light is from coaxial optical fiber 6 outgoing, converge through linear lens array row 7 and be irradiated to target surface to be checked, object to be checked is excited to launch fluorescence, fluorescence is collected through linear lens array row 7 and is coupled into coaxial optical fiber 6, ring-type fluorescence optical fiber bundle 9 is formed at exit end, outgoing fluorescence collimates through fluorescence collimation lens 10 and forms directional light, then filter via fluorescence optical filtering mirror wheel 11 upper filter, to eliminate the interference of parasitic light light, converge in spatial light modulator 13 by plus lens 12 again, spatial light modulator periodic transformation is loaded into aperture mode, optical fiber in the corresponding different fluorescence optical fiber bundle 9 of aperture arrangement, the periodicity realizing different fiber is led to light and blocks light, the fluorescence passed through enters photomultiplier 14 and is collected detection.
Later stage carries out Treatment Analysis by computing machine to the photosignal obtained, and can obtain the fluorescence intensity level of corresponding object to be checked 8 and target concentration information to be checked.
Should be understood that above-described embodiment only for technical conceive of the present invention and feature are described, its object is to understand content of the present invention for those skilled in the art and implement according to this, not embodiment is exhaustive, can not limit the scope of the invention with this.All technical schemes according to the present invention's invention are modified or equivalent replacement, and do not depart from aim and the scope of technical solution of the present invention, and it all should be encompassed in the middle of right of the present invention.
Claims (10)
1. quick, a hyperchannel real time fluorescent quantitative pick-up unit, comprises the excitation light emission system of multi-wavelength, it is characterized in that:
Also comprise splitting optical fiber (5), coaxial optical fiber (6), linear lens array row (7), fluorescent modulation system and photomultiplier (14); Wherein
Described excitation light emission system comprises laser array (1), exciting light collimation lens (2), exciting light optical filtering mirror wheel (3) and exciting light coupled lens (4);
Described coaxial optical fiber (6) is wrapped up by two-layer optical fiber and forming, outer optical fiber annular arrangement wrapping inner layer optical fiber;
Described fluorescent modulation system comprises fluorescence optical fiber bundle (9), fluorescence collimation lens (10), fluorescence optical filtering mirror wheel (11), plus lens (12) and spatial light modulator (13); Described spatial light modulator (13) comprises hole array, and the arrangement mode of described hole array is corresponding with the arrangement mode of described fluorescence optical fiber bundle (9);
The continuous agitation light that described laser array (1) produces to be filtered mirror wheel (3) by exciting light after exciting light collimation lens (2) collimation, outgoing exciting light is evenly divided in some splitting optical fibers (5) through exciting light coupled lens (4), then imports in the inner layer optical fiber of respective numbers coaxial optical fiber (6) respectively, exciting light converges on corresponding object to be checked (8) respectively through the lens unit of respective numbers in linear lens array row (7) after some coaxial optical fibers (6) outgoing, the fluorescence that object to be checked (8) excites is collected in the outer optical fiber importing coaxial optical fiber (6) more respectively through linear lens array row (7), described fluorescence conducts to fluorescence collimation lens (10) through the fluorescence optical fiber bundle (9) of respective numbers, outgoing fluorescence filters to be converged by plus lens (12) after mirror wheel (11) by fluorescence and is irradiated in spatial light modulator (13), entered photomultiplier (14) by the fluorescence in the optical fiber of spatial light modulator (13) gating to detect.
2. real time fluorescent quantitative pick-up unit according to claim 1, it is characterized in that: described laser array (1) is by the laser constitution of some different wave lengths, the wavelength coverage that exciting light exports is 270nm-750nm, and exciting light exit facet is positioned on the focal plane of exciting light collimation lens (2).
3. real time fluorescent quantitative pick-up unit according to claim 2, it is characterized in that: described exciting light optical filtering mirror wheel (3) is made up of monochromatic filter, and its optical filter quantity is identical with the number of lasers in described laser array (1).
4. real time fluorescent quantitative pick-up unit according to claim 1, is characterized in that: described splitting optical fiber (5) coupling section is positioned on the focal plane of exciting light coupled lens (4).
5. real time fluorescent quantitative pick-up unit according to claim 1, is characterized in that: the lens unit in described linear lens array row (7) is the linear alignment, and its numerical aperture is not less than 0.2, and focal length is not less than 11mm.
6. real time fluorescent quantitative pick-up unit according to claim 1, it is characterized in that: described fluorescence collimation lens (10) is made up of single or multiple lens, described fluorescence optical fiber bundle (9) outgoing end face is positioned on fluorescence collimation lens (10) focal plane.
7. real time fluorescent quantitative pick-up unit according to claim 1, is characterized in that: described fluorescence optical filtering mirror wheel (11) is made up of 1,2,4,6 or 8 monochromatic filter annular arrangement.
8. real time fluorescent quantitative pick-up unit according to claim 1, is characterized in that: described spatial light modulator (13) is positioned on plus lens (12) focal plane.
9. the real time fluorescent quantitative pick-up unit according to any one of claim 1 to 8, it is characterized in that: described splitting optical fiber (5) and coaxial optical fiber (6) are 1 × N optical fiber structure, described fluorescence optical fiber bundle (9) restraints optical fiber for N closes, and described linear lens array row (7) is made up of 1 × N lens unit; Wherein N >=2.
10. real time fluorescent quantitative pick-up unit according to claim 9, is characterized in that: described N is 8.
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| CN105136684A (en) * | 2015-08-14 | 2015-12-09 | 上海蓝怡科技股份有限公司 | Multi-sample detection device and method |
| CN105527263A (en) * | 2016-01-15 | 2016-04-27 | 公安部第一研究所 | Optical fiber beam splitting method and device for laser-induced fluorescent light path |
| CN106872432A (en) * | 2017-03-30 | 2017-06-20 | 杭州晶格科学仪器有限公司 | A kind of fluorescence detection device and temperature control method with temperature control mechanism |
| CN107024460A (en) * | 2017-03-30 | 2017-08-08 | 杭州晶格科学仪器有限公司 | A kind of fluorescence detection device and fluorescence detection method |
| CN107746806A (en) * | 2017-11-20 | 2018-03-02 | 鲲鹏基因（北京）科技有限责任公司 | A kind of real-time fluorescence quantitative PCR instrument |
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| CN105527263A (en) * | 2016-01-15 | 2016-04-27 | 公安部第一研究所 | Optical fiber beam splitting method and device for laser-induced fluorescent light path |
| CN107024460B (en) * | 2017-03-30 | 2020-01-24 | 杭州晶格科学仪器有限公司 | Fluorescence detection method |
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| CN107746806A (en) * | 2017-11-20 | 2018-03-02 | 鲲鹏基因（北京）科技有限责任公司 | A kind of real-time fluorescence quantitative PCR instrument |
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| CN107817235A (en) * | 2017-12-11 | 2018-03-20 | 苏州合惠生物科技有限公司 | A kind of real time fluorescent quantitative augmentation detection instrument |
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| CN110161007A (en) * | 2019-06-03 | 2019-08-23 | 杭州安誉科技有限公司 | A kind of optical detection apparatus for the synchronous detection of multi-wavelength fluorescence |
| WO2021169071A1 (en) * | 2020-02-25 | 2021-09-02 | 杭州博日科技有限公司 | Optical path position calibration method, calibration tool, and fluorescence quantitative detection system |
| CN111307770A (en) * | 2020-02-25 | 2020-06-19 | 杭州博日科技有限公司 | PCR detection device and method |
| CN111289434A (en) * | 2020-02-25 | 2020-06-16 | 杭州博日科技有限公司 | Light path position calibration method, calibration tool and fluorescent quantitative detection system |
| WO2021203538A1 (en) * | 2020-04-10 | 2021-10-14 | 杭州博日科技有限公司 | Optical fiber mounting base, pcr optical module, and pcr amplifier |
| CN112304915A (en) * | 2020-10-29 | 2021-02-02 | 苏州雅睿生物技术有限公司 | Real-time fluorescence detection optical system and real-time fluorescence quantitative PCR instrument |
| CN112304915B (en) * | 2020-10-29 | 2021-05-04 | 苏州雅睿生物技术有限公司 | Real-time fluorescence detection optical system and real-time fluorescence quantitative PCR instrument |
| CN112683252A (en) * | 2021-01-16 | 2021-04-20 | 九江中科神光科技有限公司 | Laser positioning detection device |
| WO2022160726A1 (en) * | 2021-01-29 | 2022-08-04 | 广东润鹏生物技术有限公司 | Multi-channel fluorescence quantitative detection device and molecular diagnosis platform |
| CN112945921A (en) * | 2021-02-01 | 2021-06-11 | 广州市鹏达智能科技有限公司 | Fluorescence quantitative analysis device and method |
| CN114675411A (en) * | 2022-02-22 | 2022-06-28 | 徕卡显微***科技(苏州)有限公司 | Filter element, light source module, multi-channel fluorescence lighting system and fluorescence microscope |
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Application publication date: 20150513 |