CN101105455A - Laser induction fluorescence detector - Google Patents
Laser induction fluorescence detector Download PDFInfo
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- CN101105455A CN101105455A CNA2007101389248A CN200710138924A CN101105455A CN 101105455 A CN101105455 A CN 101105455A CN A2007101389248 A CNA2007101389248 A CN A2007101389248A CN 200710138924 A CN200710138924 A CN 200710138924A CN 101105455 A CN101105455 A CN 101105455A
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Abstract
The invention discloses a laser induction fluorescence detecting machine, which is mainly used for the micro sample inspecting in the field of clinical medicine and life science. The invention comprises an excitation light path (10), a separating micro column (20) and a fluorescence light collecting path (30). The axis shaft line of the excitation light path (10) is vertical with the axis shaft line of the fluorescence light collecting path (30), and the excitation light path (10) has the same axis with the separating micro column (20), an optical fiber (40) is equipped between the excitation light path (10) and the separating micro column (20); the front end of the optical fiber (40) is coupled with the excitation light path (10), and the rear end extends to the inspecting window (21) of the separating micro column (20). The invention has the advantage that the invention has high fluorescence stimulation efficiency, The optical structure is simple, the volume is small, the fluorescence stimulation of the sample can be directly realized, the sensitivity and accuracy can be enhanced; and the invention can be used together with the micro column separating systems like capillary tube electrophoresis, capillary tube liquid chromatography and flowing injection, etc.
Description
Technical field
The present invention relates to the check and analysis instrument, particularly a kind of post inner laser induced fluorescent tester that is applicable to that mainly the micro-example in the fields such as clinical drug and life science detects.
Background technology
In recent years, for adapting to the development trend of analytical instrument microminiaturization, various Micro-Column Separation methods are arisen at the historic moment.Highly sensitive detection method is the important content of analytical chemistry research always, and the sensitivity that therefore improves detecting device has become the key issue of Micro-Column Separation technical applicationization.Because the internal diameter very little (being generally tens to the hundreds of micron) of microtrabeculae, the volume of detection window is only received liters for several to hundreds of on the post, and the ultraviolet photometric detection method of routine can not satisfy the high sensitivity requirement of detection.Laser-induced fluorescence (LIF) (laser induced fluorescence, LIF) detection method has obtained fast-developing in recent years as a kind of novel high-sensitivity detection mode and has used widely, it is the highest optical detecting method of sensitivity up to now, its detectability to fluorescent material can reach the zmol order of magnitude, under suitable condition even can realize Single Molecule Detection [Lee Y H, Maus R G, Smith B W, et al.AnalChem, 1994,66 (23): 4142-4149].Therefore, laser-Induced Fluorescence Detection can be used as detection method on a kind of highly sensitive post and with differential such as capillary liquid chromatography, Capillary Electrophoresis from the pattern logotype, realize the high efficiency separation analysis of micro substance.
Used LASER Light Source is generally used Argon ion laser (488nm), he-Ne laser (594nm) in the laser induced fluorescence detector, and He-Cd laser device (325nm) uses less because of the life-span is short.Because gas laser, have that the light source volume is big, shortcomings such as energy consumption and cost height, limited applying of LIF.Semiconductor laser (or claim laser diode) is the perfect light source of the development in recent years laser-induced fluorescence (LIF) of getting up, has low price, volume is little, long service life, advantage such as reliable and stable, and its emission wavelength has developed into indigo plant, green glow and the ultraviolet light that adopts frequency tripling and quadruple from original far infrared region, range of application progressively enlarges.
Aspect light path design, traditional laser induced fluorescence detector mainly contains orthogonal type and conllinear type.What most detecting devices adopted is the orthogonal type design, and promptly incident light, detection window and fluoroscopic examination direction three are vertical mutually.In the orthogonal type design, because sterically hindered influence should not be adopted short focal length lens.Conllinear type design (incident light and detection light conllinear) then can be adopted the littler kapillary of short focal length lens and internal diameter, and easy operating.Yet; in these two kinds of optical textures; incident light all will come excited sample by microtrabeculae (kapillary) wall; certainly will produce the scattering of certain light reflection at capillary wall; make the light that sends by laser instrument can not fully be used for fluorescence excitation; and then cause higher background noise, and influence fluorescence excitation efficient and detection sensitivity, be difficult to guarantee or improve the detection efficiency of Micro-Column Separation system.
Summary of the invention
Technical matters to be solved by this invention provides a kind of laser induced fluorescence detector with higher fluorescence excitation efficient, can with Micro-Column Separation system couplings such as Capillary Electrophoresis, capillary liquid chromatography and flow injection, and mechanism is simple, volume is little.
The technical solution adopted for the present invention to solve the technical problems is: laser induced fluorescence detector of the present invention, comprise excitation light path, separate microtrabeculae and phosphor collection light path, it is characterized in that: the axis of described excitation light path, phosphor collection light path meets at right angles and crosses, and excitation light path separation microtrabeculae is coaxial; Be provided with light transmitting fiber between described excitation light path, the separation microtrabeculae, this fibre-optic front end and excitation light path coupling, its rear end then extends the detection window that separates microtrabeculae.
As a preferred embodiment of the present invention, for improving the collection efficiency of fluorescence signal, to reach best detection effect, the vertical distance of end face and phosphor collection light path axis is 0.10～0.20mm within the described light transmitting fiber.Accurately this distance of control not only can obtain lower background noise, can obtain higher fluorescence signal simultaneously when sample test, and this helps improving detection signal-to-noise ratio, thereby improves the sensitivity of detecting device.
The invention has the beneficial effects as follows to have the advantages that optical texture is simple, volume is little, can avoid effectively because of laser penetration separates anaclasis and the scattering that post jamb causes, so can obtain higher fluorescence excitation efficient; After exciting light scioptics and a light transmitting fiber effectively were coupled, light transmitting fiber directly stretched into to separate in the microtrabeculae exciting light is imported the detection window position, directly realizes the fluorescence excitation of sample, helps improving detection sensitivity and accuracy; Can with Micro-Column Separation system couplings such as Capillary Electrophoresis, capillary liquid chromatography and flow injection.
Description of drawings
This instructions comprises following five width of cloth accompanying drawings:
Fig. 1 is the structural representation of laser induced fluorescence detector of the present invention;
Fig. 2 is the structural representation of laser induced fluorescence detector of the present invention and capillary electrophoresis system coupling;
Fig. 3 is the structural representation of calibration assistance platform in the laser induced fluorescence detector of the present invention;
Optical fiber end and collect the influence curve figure of the fluorescence intensity that the distance between the object lens axis produced the NDA mark penicillamine solution of blank borax buffer solution (pH9.2) and 0.20-μ M on Fig. 4.
Fig. 5 is the electrophoresis pattern of laser induced fluorescence detector of the present invention.
Parts, toponym and pairing mark among the figure: excitation light path 10, light source 11, lens 12, hole diaphragm 13, separation microtrabeculae 20, detection window position 21, phosphor collection light path 30, lens combination 31, hole diaphragm 32, optical filter 33, photoelectricity testing part 34, light transmitting fiber 40, calibration assistance platform 50, upper plate 51, lower plate 52, bolt 53, vertical apart from d.
Embodiment
The present invention is further described below in conjunction with drawings and Examples.
Laser induced fluorescence detector of the present invention comprises excitation light path 10, separates microtrabeculae 20 and phosphor collection light path 30.With reference to Fig. 1, the axis of described excitation light path 10, phosphor collection light path 30 meets at right angles and crosses, and excitation light path 10 is with to separate microtrabeculae 20 coaxial.For traditional orthogonal type and conllinear type laser-Induced Fluorescence Detection device, have the advantages that optical texture is simple, volume is little, simultaneously owing to this structure can effectively be avoided obtaining higher fluorescence excitation efficient because of laser penetration separates anaclasis and the scattering that post jamb causes.With reference to Fig. 1, be provided with light transmitting fiber 40 between described excitation light path 10, the separation microtrabeculae 20, the front end of this light transmitting fiber 40 and excitation light path 10 couplings, its rear end then extends the detection window 21 that separates microtrabeculae 20.Light transmitting fiber 40 directly stretches into to separate in the microtrabeculae exciting light is imported its detection window, directly realizes the fluorescence excitation of sample, helps improving detection sensitivity and accuracy, can be referred to as " post inner laser induced fluorescent tester ".
For improving the collection efficiency of fluorescence signal, to reach best detection effect, the vertical of end face and phosphor collection light path 30 axis is 0.10-0.20mm apart from d within the described light transmitting fiber 40.
With reference to Fig. 1, as a kind of typical collocation form, described excitation light path 10 comprises light source 11, lens 12 and the hole diaphragm 13 that sets gradually, and acts on light source 11 and the lens 12 and make exciting light focus on the colimated light system of light transmitting fiber 40 front ends.Excitation light path 10, separation microtrabeculae 20 phosphor collection light paths 30 are by 50 calibrations of calibration assistance platform.Described phosphor collection light path 30 comprises lens combination 31, hole diaphragm 32, optical filter 33 and the photoelectricity testing part 34 that from bottom to top sets gradually, and photoelectricity testing part 34 can adopt photomultiplier or photon counter usually.Detector arrangement compactness of the present invention only need be changed corresponding light source 11 and optical filter 33 at different analysis systems, and these two parts all can be realized changing at an easy rate.This detecting device is in conjunction with the relevant detection pond, can be easily and analytic system on-line couplings such as Capillary Electrophoresis, capillary liquid chromatography and flow injection.
Shown in Fig. 2 is detection system sketch with the capillary electrophoresis system coupling.With reference to this figure, separating microtrabeculae 20 is kapillary, and described light source 11 preferably adopts semiconductor laser.With reference to Fig. 3, calibration assistance platform 50 comprises and is held on therebetween upper plate 51, lower plate 52 with separating microtrabeculae 20, and spaced apart upper plate 51, lower plate 52 are connected and pass the bolt 53 of lower plate 52.Detection cell is arranged on the calibration assistance platform 50.
With reference to Fig. 2, exciting light is by behind the hole diaphragm 13, focuses on the front end of light transmitting fiber 40 through lens 12, by this light transmitting fiber 40 exciting light directly is introduced into the kapillary inside at detection cell place, realizes the fluorescence excitation of sample.The fluorescence signal that produces is collected with lens combination 31, by regulating calibration assistance platform 50, can guarantee the collection efficiency of fluorescence signal.The fluorescence signal collected respectively through via hole diaphragm 32 and optical filter 33, to eliminate the background interference that exciting light brings, is utilized photoelectricity testing part 34 to convert fluorescence signal to electric signal at last and realizes fluoroscopic examination.
Test with naphthalene-2 with above-mentioned laser induced fluorescence detector-Capillary Electrophoresis coupling, the penicillamine (Pen) after 3-two acetals (NDA) are derived obtains electrophoresis pattern as shown in Figure 6.
In testing process, accurately adjust the vertical of end face and phosphor collection light path axis within the light transmitting fiber apart from d, very big to the detection sensitivity influence.Measure by respectively the NDA mark penicillamine solution of blank borax (pH9.2) buffer solution and 0.20-μ M being carried out fluorescence signal, having investigated and write down light transmitting fiber is in diverse location (fluorescence signal intensity that blank buffer solution and sample solution are produced during d=0.10～0.45mm) the results are shown in Figure 4.According to this result, the light transmitting fiber inner face is controlled at b point position d=0.10～0.20mm (the wherein optimum 0.15mm of being), can improve the signal to noise ratio (S/N ratio) of detection, thereby obtain maximum detection sensitivity.
NDA is derived in the sample test behind the penicillamine, its deposition condition is: (1) column front derivation condition is got the standard solution of 10 μ L penicillamines in the plastic centrifuge tube of 500 μ L, the borax buffer solution (pH is 9.2) that adds 150 μ L 10mM then successively, the naphthalene-2 of 50 μ L 2.0mM, the KCN aqueous solution of 3-two acetal methanol solutions and 50 μ L 20mM, fully shake up, after placing 4h under the room temperature, introduce post inner fiber type laser induced fluorescence detector of the present invention-Capillary Electrophoresis coupling system and carry out separation determination.(2) Capillary Electrophoresis condition buffer solution is the borax buffer solution (pH 9.2) of 10mM; The fused quartz kapillary: 100 μ m i.d. * 50/48cm (length overall/effectively); Electrokinetic injection: 10kV, 10s; Separation voltage: 18kV.Its test result: getting signal to noise ratio (S/N ratio) is 3, and post inner fiber type laser induced fluorescence detector of the present invention is to naphthalene-2, and the concentration of the penicillamine of 3-two acetal marks detects and is limited to 0.8nM.
The electrophoresis spectrogram of post inner fiber type laser induced fluorescence detector of the present invention, as shown in Figure 4.A is the electrophoretogram of the Pen of 0.20 μ M NDA mark; B is the Pen of NDA mark and the electrophoretogram of ispol solution, and wherein, peak a is NDA, and peak b is 0.10 μ M NDA-Pen, and peak c is the NDA-valine of 0.22 μ M, and peak d is the NDA-alanine of 0.16 μ M, and peak e is the NDA-glycocoll of 0.25 μ M.
Claims (10)
1. laser induced fluorescence detector, comprise excitation light path (10), separate microtrabeculae (20) and phosphor collection light path (30), it is characterized in that: the axis of described excitation light path (10), phosphor collection light path (30) meets at right angles and crosses, and excitation light path (10) is with to separate microtrabeculae (20) coaxial; Be provided with light transmitting fiber (40) between described excitation light path (10), the separation microtrabeculae (20), front end of this light transmitting fiber (40) and excitation light path (10) coupling, its rear end then extends the detection window (21) that separates microtrabeculae (20).
2. laser induced fluorescence detector as claimed in claim 1 is characterized in that: the vertical distance (d) of end face and phosphor collection light path (30) axis is 0.1～0.2mm within the described light transmitting fiber (40).
3. laser induced fluorescence detector as claimed in claim 1 is characterized in that: the vertical distance (d) of end face and phosphor collection light path (30) axis is 0.15m within the described light transmitting fiber (40).
4. as claim 1,2 or 3 described laser induced fluorescence detectors, it is characterized in that: described excitation light path (10) comprises light source (11), lens (12) and the hole diaphragm (13) that sets gradually, and acts on light source (11) and the lens (12) and make exciting light focus on the colimated light system of light transmitting fiber (40) front end.
5. laser induced fluorescence detector as claimed in claim 4 is characterized in that: what described light source (11) adopted is semiconductor laser.
6. as claim 1,2 or 3 described laser induced fluorescence detectors, it is characterized in that: described phosphor collection light path (30) comprises lens combination (31), hole diaphragm (32), optical filter (33) and the photoelectricity testing part (34) that from bottom to top sets gradually.
7. laser induced fluorescence detector as claimed in claim 6 is characterized in that: what described photoelectricity testing part (34) adopted is photomultiplier.
8. laser induced fluorescence detector as claimed in claim 6 is characterized in that: what described photoelectricity testing part (34) adopted is photon counter.
9. as claim 1,2 or 3 described laser induced fluorescence detectors, it is characterized in that: described excitation light path (10), separation microtrabeculae (20) phosphor collection light path (30) are by calibration assistance platform (50) calibration.
10. laser induced fluorescence detector as claimed in claim 9, it is characterized in that: described calibration assistance platform (50) comprises and is held on therebetween upper plate (51), lower plate (52) and the bolt (53) that upper plate (51), lower plate (52) is connected and pass lower plate (52) spaced apart with separating microtrabeculae (20).
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| CNA2007101389248A CN101105455A (en) | 2007-07-04 | 2007-07-18 | Laser induction fluorescence detector |
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Cited By (17)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102033124A (en) * | 2009-12-25 | 2011-04-27 | 北京博晖创新光电技术股份有限公司 | Immunofluorescence detection device and detection method |
| CN102147365A (en) * | 2010-12-22 | 2011-08-10 | 中国科学院上海微系统与信息技术研究所 | Handheld bioluminescent detector and detection method |
| CN102297854A (en) * | 2011-05-23 | 2011-12-28 | 公安部第一研究所 | High-efficiency multi-mode laser-induced fluorescence optical path exciting system |
| CN102879365A (en) * | 2012-09-21 | 2013-01-16 | 常州大学 | Capillary electrophoresis fluorescence detection device |
| CN103063640A (en) * | 2012-12-28 | 2013-04-24 | 西北核技术研究所 | Laser-induced fluorescence combustion field parameter measuring device |
| CN103245651A (en) * | 2013-05-12 | 2013-08-14 | 浙江大学 | Detection method of eccentric focusing-type laser-induced fluorescence detection device suitable for detection on capillary column |
| CN103308503A (en) * | 2013-06-06 | 2013-09-18 | 四川大学 | Separated micro-column coupling light guide fiber exciting light induction fluorescence component |
| CN104280088A (en) * | 2013-07-11 | 2015-01-14 | 中国科学院大连化学物理研究所 | Method for calibrating effective uniform light emitting gas volume in container |
| CN105241856A (en) * | 2015-09-29 | 2016-01-13 | 窦晓鸣 | System and method for detecting miRNA through capillary polymer electrophoretic analysis |
| CN106124461A (en) * | 2016-06-08 | 2016-11-16 | 四川大学 | The method for quick of Aristolochic Acid |
| CN106164665A (en) * | 2014-03-07 | 2016-11-23 | 生命技术公司 | Optical system for capillary electrophoresis |
| US9518923B1 (en) | 2015-12-07 | 2016-12-13 | International Business Machines Corporation | System and methods for fluorescence detection |
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| CN108088809A (en) * | 2017-12-29 | 2018-05-29 | 四川大学 | Based on thin-layer chromatography-high pressure gas pulse liquid phase component separator associated with bis- spectrum |
| CN108627489A (en) * | 2018-05-24 | 2018-10-09 | 大连民族大学 | A kind of 128 channel array capillary electrophoresis |
| US10274460B2 (en) | 2014-03-07 | 2019-04-30 | Life Technologies Corporation | Capillary array cartridge for capillary electrophoresis systems |
| CN113063759A (en) * | 2021-03-15 | 2021-07-02 | 国科大杭州高等研究院 | Somatic cell laser-induced fluorescence detection method based on hemispherical space compound eye structure |
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2007
- 2007-07-18 CN CNA2007101389248A patent/CN101105455A/en active Pending
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| CN102033124A (en) * | 2009-12-25 | 2011-04-27 | 北京博晖创新光电技术股份有限公司 | Immunofluorescence detection device and detection method |
| CN102147365A (en) * | 2010-12-22 | 2011-08-10 | 中国科学院上海微系统与信息技术研究所 | Handheld bioluminescent detector and detection method |
| CN102297854A (en) * | 2011-05-23 | 2011-12-28 | 公安部第一研究所 | High-efficiency multi-mode laser-induced fluorescence optical path exciting system |
| CN102879365A (en) * | 2012-09-21 | 2013-01-16 | 常州大学 | Capillary electrophoresis fluorescence detection device |
| CN103063640B (en) * | 2012-12-28 | 2015-09-09 | 西北核技术研究所 | A kind of laser-induced fluorescence (LIF) combustion field parameter measuring apparatus |
| CN103063640A (en) * | 2012-12-28 | 2013-04-24 | 西北核技术研究所 | Laser-induced fluorescence combustion field parameter measuring device |
| CN103245651A (en) * | 2013-05-12 | 2013-08-14 | 浙江大学 | Detection method of eccentric focusing-type laser-induced fluorescence detection device suitable for detection on capillary column |
| CN103245651B (en) * | 2013-05-12 | 2015-11-11 | 浙江大学 | A kind of detection method being applicable to the eccentric focus type laser-Induced Fluorescence Detection device that capillary column detects |
| CN103308503B (en) * | 2013-06-06 | 2015-11-25 | 四川大学 | Be separated microtrabeculae coupling light transmitting fiber exciting light induced fluorescence assembly |
| CN103308503A (en) * | 2013-06-06 | 2013-09-18 | 四川大学 | Separated micro-column coupling light guide fiber exciting light induction fluorescence component |
| CN104280088B (en) * | 2013-07-11 | 2017-07-07 | 中国科学院大连化学物理研究所 | A kind of method of effective uniformly light-emitting gas volume in demarcation container |
| CN104280088A (en) * | 2013-07-11 | 2015-01-14 | 中国科学院大连化学物理研究所 | Method for calibrating effective uniform light emitting gas volume in container |
| US10274460B2 (en) | 2014-03-07 | 2019-04-30 | Life Technologies Corporation | Capillary array cartridge for capillary electrophoresis systems |
| CN106164665A (en) * | 2014-03-07 | 2016-11-23 | 生命技术公司 | Optical system for capillary electrophoresis |
| CN105241856A (en) * | 2015-09-29 | 2016-01-13 | 窦晓鸣 | System and method for detecting miRNA through capillary polymer electrophoretic analysis |
| US9518923B1 (en) | 2015-12-07 | 2016-12-13 | International Business Machines Corporation | System and methods for fluorescence detection |
| CN106124461B (en) * | 2016-06-08 | 2018-08-21 | 四川大学 | The rapid detection method of aristolochic acid |
| CN106124461A (en) * | 2016-06-08 | 2016-11-16 | 四川大学 | The method for quick of Aristolochic Acid |
| CN106885836A (en) * | 2017-04-19 | 2017-06-23 | 冯超 | Handwriting color marks detector |
| CN108088809A (en) * | 2017-12-29 | 2018-05-29 | 四川大学 | Based on thin-layer chromatography-high pressure gas pulse liquid phase component separator associated with bis- spectrum |
| CN108627489A (en) * | 2018-05-24 | 2018-10-09 | 大连民族大学 | A kind of 128 channel array capillary electrophoresis |
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