CN110057798A - A kind of streaming sample multi-wavelength fluorescence detection method - Google Patents
A kind of streaming sample multi-wavelength fluorescence detection method Download PDFInfo
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- CN110057798A CN110057798A CN201910343195.2A CN201910343195A CN110057798A CN 110057798 A CN110057798 A CN 110057798A CN 201910343195 A CN201910343195 A CN 201910343195A CN 110057798 A CN110057798 A CN 110057798A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N2021/6417—Spectrofluorimetric devices
- G01N2021/6419—Excitation at two or more wavelengths
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Abstract
A kind of streaming sample multi-wavelength fluorescence detection method, including, make laser after dichroscope enters objective lens on focusing illumination to sample cell；Fluorescent samples in sample cell emit multi-wavelength fluorescence when flowing through laser focal spot, after multi-wavelength fluorescence is by focusing objective len shots, sequentially pass through dichroscope, long pass filter, grating, and a zero level hot spot and several level-one stripeds are formed on optical imaging instrument；According to fluorescence zero level hot spot and level-one striped that optical imaging instrument records, each emission spectrum and concentration of survey calculation fluorescent samples.Method of the invention, it is only necessary to which the multi-wavelength fluorescence detection of streaming sample can be realized in a fluorescence detector, avoids and uses equipment instrument caused by multiple fluorescence detectors excessive in the past and the not high enough problem of sensitivity.
Description
Technical field
The present invention relates to biochemical analysis and field of sensing technologies, and in particular to a kind of streaming sample multi-wavelength fluorescence inspection
Survey method.
Background technique
For fluorescent material when being excited by light, ground state electron transits to excitation state, shines when ground state is returned in electron transition
Phenomenon is known as fluorescence.Fluorescence analysis is a kind of extremely important and common biochemical analysis and bio-sensing method.In numerous instrument
It is widely used in device, for example spectrophotometer, Fluorescence Spectrometer, fluorescence microscope, Laser Scanning Confocal Microscope, total internal reflection are micro-
Mirror, two-photon fluorescence spectrometer and such as flow cytometer.These instruments mostly can only divide single fluorescent material
Analysis.It is detected while streaming fluorescent samples for emitting multi-wavelength, it usually will be more using the optical filters of multiple and different wave bands
The fluorescence of different wave length, is introduced different sense channels by wavelength fluorescent light splitting, and each sense channel needs a detector.This
Cause multi-wavelength fluorescence detection to need multiple detectors, on the one hand increase instrument cost, on the other hand reduce fluorescence signal,
Reduce detection sensitivity.
Summary of the invention
The object of the present invention is to provide a kind of highly sensitive, low cost streaming sample multi-wavelength fluorescence detection methods.
For achieving the above object, technical solution of the present invention is specific as follows:
A kind of streaming sample multi-wavelength fluorescence detection method, comprising the following steps:
S1: make laser after dichroscope enters objective lens on focusing illumination to sample cell；
S2: the fluorescent samples in sample cell emit multi-wavelength fluorescence when flowing through laser focal spot, and multi-wavelength fluorescence is by conglomeration
After lens head is collected, dichroscope, long pass filter, grating are sequentially passed through, forms a zero level hot spot on optical imaging instrument
With several level-one stripeds；
S3: the fluorescence zero level hot spot and level-one striped recorded according to optical imaging instrument, each hair of survey calculation fluorescent samples
Penetrate spectrum and concentration.
Further, the sample is flowing sample, and the sample cell is sample flow pond, and sample flow pond is capillary
Pipe or microchannel, sample flow direction are vertical with laser irradiation direction.
Further, the cross-sectional shape in the sample flow pond is round, rectangular or trapezoidal, the sample flow pond
Material be glass, quartz or high polymer.
Further, cross-section lengths, width or the diameter in the sample flow pond are between 1 micron~3 millimeters.
Further, multi-wavelength fluorescent samples are that simple sample label multi-wavelength fluorescence probe or mixing sample mark respectively
The fluorescence probe of different wave length.
Further, also there is reflecting mirror and slit between the dichroscope and long pass filter
Further, the grating is transmission grating or reflecting grating, and grating constant is 30~1000 lines per millimeters.
Further, the laser excitation is single beam laser excitation or multiple laser while exciting.
Further, the optical imaging instrument is charge coupled device or complementary metal oxide semiconductor sensor.
Further, the step S3 is specifically included: the fluorescence zero level hot spot and level-one item recorded according to optical imaging instrument
Line, by measure respectively zero level hot spot at a distance from each level-one striped and the intensity distribution of each level-one striped measure fluorescent samples
Multiple emission spectrum；By the strength detection fluorescent material concentration for measuring corresponding level-one striped.
Compared with prior art, beneficial effects of the present invention:
Method of the invention excites streaming multi-wavelength fluorescent samples using laser as excitation light source in laser focal spot, by
The multi-wavelength that excitation is penetrated is zero level hot spot and multiple level-one stripeds, the record by imaging on optical imaging instrument by grating beam splitting；With one
Grade fringe position determines wavelength of fluorescence and type, does curve with level-one striped spectral intensity and fluorescent material concentration relationship, so
The fluorescent material in unknown sample is quantified afterwards；The multi-wavelength for only needing a fluorescence detector that streaming sample can be realized is glimmering
Light detection avoids and uses equipment instrument caused by multiple fluorescence detectors excessive in the past and the not high enough problem of sensitivity.
Detailed description of the invention
Fig. 1 is the detection method light path schematic diagram of the embodiment of the present invention 1；
Fig. 2 is 625 imaging results of quantum dot (QD) in the embodiment of the present invention 2；
Fig. 3 is QD 625 and the strength interaction curve of corresponding level-one striped of the various concentration in the embodiment of the present invention 2,
In, interior illustration is linear correlation part；
Fig. 4 is the imaging results of QD 525, QD 585 and QD 655 the mixing sample in the embodiment of the present invention 3；
In Fig. 1,1 is sample flow pond, and 2 be object lens, and 3 be laser, and 4 be dichroscope, and 5 be reflecting mirror, and 6 be slit, 7
It is transmission grating for long pass filter, 8,9 be optical imaging instrument.
Specific embodiment:
Embodiment 1
A kind of streaming sample multi-wavelength fluorescence detection method, comprising the following steps:
(1) optical path is built according to Fig. 1；
(2) dimethyl silicone polymer and coverslip is used to produce microchannel (100 microns × 60 microns) as sample flow
Pond 1；
(3) microchannel is placed in 2 top of object lens, is mixed using three kinds of quantum dots that syringe pump pushes launch wavelength different molten
Liquid flows through microchannel with certain speed；
(4) with wavelength 488nm, the argon ion laser 3 that beam diameter is about 100 microns of m vertically shines after the focusing of object lens 2
Penetrate microchannel；Fluorescent samples in sample flow pond 1 emit multi-wavelength fluorescence when flowing through laser focal spot, and multi-wavelength fluorescence is by focusing
After 2 shots of object lens, after sequentially passing through dichroscope 4, reflecting mirror 5, slit 6, long pass filter 7, grating 8, optics at
As forming a zero level hot spot and several level-one stripeds on instrument 9 (CCD)；
(5) with the zero level and first-order spectrum image of CCD record sample；With the qualitative quantum dot emission wavelengths in first-order spectrum position
That is quantum dot type, using first-order spectrum Gaussian peak most strength fluorescence intensity as concentration foundation.
Detection of the embodiment 2 to QD625 solution concentration
A. 8 μM of QD625 is taken to be diluted to 0.05,0.1,0.2,0.5,1,2 respectively with 8.3 borate buffer solution of 10mM pH,
4,8nM.
B. microchannel is placed on 100 times of object lens, the QD625 of above-mentioned concentration is pushed by syringe pump with certain speed and is led to
In road.
C. it is imaged using the Laser Scanning Confocal Microscope for the laser that light source is 488nm, CCD shoots the zero order light by grating image
Spot and first-order spectrum (Fig. 2).
D. quantitative experiment: experimental procedure b~c is repeated, the first-order spectrum image of various concentration QD625 is obtained, uses image
The intensity of processing software acquisition various concentration QD625 level-one striped.Using QD625 concentration as abscissa, level-one fringe intensity is vertical
Coordinate mapping, obtains dependent linearity curve, and the range of linearity is 0.01~1nM, dependent linearity coefficients R2=0.997 (Fig. 3).
Detection of the embodiment 3 to multi-wavelength fluorescent samples
A. a certain amount of 8 μM of QD525, QD585, QD655 mixing, with the molten dilution of 10mM pH8.3 borate buffer, mixed liquor are taken
Middle QD525, QD585, QD655 ultimate density are respectively 2nM, 1nM, 0.5nM.
B. microfluidic channel is placed on 100 times of object lens, so that laser is just perpendicular to channel, three kinds of quantum azeotropic mixtures
It is pushed into channel by syringe pump with certain speed.
C. it is imaged using the Laser Scanning Confocal Microscope for the laser that light source is 488nm, CCD shooting passes through one zero of grating image
Grade hot spot and three first-order spectrums (Fig. 4).According to the position of first-order spectrum and intensity distribution, three kinds of fluorescent materials can be accurate
It recognizes, from left to right respectively QD525, QD585, QD655.
It should be noted that being intended merely to further illustrate the contents of the present invention above, but should not be construed as to the present invention
Limitation.Without departing from the spirit and substance of the case in the present invention, it modifies or replaces to made by the method for the present invention, step or condition
It changes, all belongs to the scope of the present invention.
Claims (10)
1. a kind of streaming sample multi-wavelength fluorescence detection method, which comprises the following steps:
S1: make laser after dichroscope enters objective lens on focusing illumination to sample cell；
S2: the fluorescent samples in sample cell emit multi-wavelength fluorescence when flowing through laser focal spot, and multi-wavelength fluorescence is by focusing objective len mirror
After head is collected, sequentially pass through dichroscope, long pass filter, grating, if formed on optical imaging instrument zero level hot spot and
Dry level-one striped；
S3: the fluorescence zero level hot spot and level-one striped recorded according to optical imaging instrument, each transmitting light of survey calculation fluorescent samples
Spectrum and concentration.
2. the sample cell is sample the method according to claim 1, wherein the sample is flowing sample
Product flow cell, sample flow pond are capillary or microchannel, and sample flow direction is vertical with laser irradiation direction.
3. according to the method described in claim 2, it is characterized in that, the cross-sectional shape in the sample flow pond is round, side
Shape is trapezoidal, and the material in the sample flow pond is glass, quartz or high polymer.
4. according to the method described in claim 3, it is characterized in that, the cross-section lengths in the sample flow pond, width or straight
Diameter is between 1 micron~3 millimeters.
5. the method according to claim 1, wherein multi-wavelength fluorescent samples are that simple sample label multi-wavelength is glimmering
Light probe or mixing sample mark the fluorescence probe of different wave length respectively.
6. the method according to claim 1, wherein also having between the dichroscope and long pass filter anti-
Penetrate mirror and slit.
7. grating is normal the method according to claim 1, wherein the grating is transmission grating or reflecting grating
Number is 30~1000 lines per millimeters.
8. the method according to claim 1, wherein the laser excitation is single beam laser excitation or multiple laser
It excites simultaneously.
9. the method according to claim 1, wherein the optical imaging instrument is charge coupled device or complementary gold
Belong to oxide-semiconductor sensor.
10. the method according to claim 1, wherein the step S3 is specifically included: being remembered according to optical imaging instrument
The fluorescence zero level hot spot and level-one striped of record, by measuring zero level hot spot respectively at a distance from each level-one striped and each level-one item
Multiple emission spectrum of the intensity distribution measurement fluorescent samples of line；By the strength detection fluorescent material for measuring corresponding level-one striped
Concentration.
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| CN102538683A (en) * | 2012-01-11 | 2012-07-04 | 徐州师范大学 | Optical imaging method for measuring distance smaller than diffraction limit distance |
| CN103575712A (en) * | 2013-10-15 | 2014-02-12 | 中国科学院苏州生物医学工程技术研究所 | Particle fluorescence detection wavelength instant configuration beam splitting system |
| CN104502255A (en) * | 2014-12-29 | 2015-04-08 | 中国科学院长春光学精密机械与物理研究所 | Three-dimensional imaging flow cytometer device |
| CN204269552U (en) * | 2014-12-16 | 2015-04-15 | 南京融智生物科技有限公司 | Multicolor fluorescence pick-up unit |
| 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 |
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2019
- 2019-04-26 CN CN201910343195.2A patent/CN110057798A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH02147840A (en) * | 1988-11-29 | 1990-06-06 | Res Dev Corp Of Japan | Method and apparatus for multiwavelength fluorescent and phosphorescent analysis |
| CN102538683A (en) * | 2012-01-11 | 2012-07-04 | 徐州师范大学 | Optical imaging method for measuring distance smaller than diffraction limit distance |
| CN103575712A (en) * | 2013-10-15 | 2014-02-12 | 中国科学院苏州生物医学工程技术研究所 | Particle fluorescence detection wavelength instant configuration beam splitting system |
| CN204269552U (en) * | 2014-12-16 | 2015-04-15 | 南京融智生物科技有限公司 | Multicolor fluorescence pick-up unit |
| CN104502255A (en) * | 2014-12-29 | 2015-04-08 | 中国科学院长春光学精密机械与物理研究所 | Three-dimensional imaging flow cytometer device |
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