CN106226278A - A kind of multiplexing flow-through assay device for microlayer model fluoroscopic image and spectral scan - Google Patents
A kind of multiplexing flow-through assay device for microlayer model fluoroscopic image and spectral scan Download PDFInfo
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- CN106226278A CN106226278A CN201610641325.7A CN201610641325A CN106226278A CN 106226278 A CN106226278 A CN 106226278A CN 201610641325 A CN201610641325 A CN 201610641325A CN 106226278 A CN106226278 A CN 106226278A
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- microlayer model
- dichroscope
- fluoroscopic image
- spectral scan
- fluorescence
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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
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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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
Abstract
The present invention relates to a kind of multiplexing flow-through assay device for microlayer model fluoroscopic image and spectral scan.Flow focusing principle is utilized to generate microlayer model in microchip, the focusing of microlayer model is realized by contracted channel, after the laser that laser instrument is launched is expanded by beam expanding lens, reflect through dichroscope, focused on the single microlayer model in contracted channel by object lens, the fluorescent material in microlayer model is fluoresced after exciting and is collected by object lens and pass through dichroscope, is divided into two bundles through spectroscope, a branch of EMCCD that images in after optical filter, records fluoroscopic image；The most a branch of after transmissive diffraction grating, the optical diffraction of different wave length, to different directions forming frequency bands of a spectrum, is realized spectral scan by spectrofluorophotometer collection.The present invention is capable of the multiplexing flow cytometer detection of microlayer model fluoroscopic image and spectral scan in microchip runner, obtains the complete optical information of microlayer model, meets fluorescence multiplexing coding techniques application demand in microlayer model.
Description
Technical field
The invention belongs to technical field of optical detection, particularly to a kind of for microlayer model fluoroscopic image and spectral scan
Multiplexing flow-through assay device.
Background technology
Drop is micro-fluidic is the micro-total analysis technology risen 21 century.Calendar year 2001, Quake et al. uses T-shaped passage first
Micro-fluidic chip forms single dispersing drop, and the drop formation method of this Bottom-up formula significantly improves the operable of drop
Property, promote the micro-fluidic fast development of drop (Thorsen T, Roberts R W, Arnold F H, et al.Dynamic
pattern formation in a vesicle-generating microfluidic device[J].Physical
review letters,2001,86(18):4163.).Microlayer model has as a kind of microreactor and does not has cross-contamination, generation
The advantages such as frequency is high, response speed is fast, coupling single cell analysis.At present, the chip on-line analysis technology for microlayer model has
Analysis speed is fast, flux advantages of higher, and wherein optical detection is the important means of microlayer model on-line analysis.
2007, Schmidt et al. devised the fluorescence spectrophotometer of a kind of sandwich structure, every in cmos image sensor
One corresponding a kind of independent color of pixel, when molecules detected flows through fluid channel, can obtain complete spectrogram, but should
Method is difficult to spectral detection (Schmidt O, Bassler M, Kiesel P, the et al.Fluorescence of microlayer model
spectrometer-on-a-fluidic-chip[J].Lab on a Chip,2007,7(5):626-629.).2010 beautiful
Harvard University of state Jeremy et al. utilizes microlayer model parcel to transfect the yeast cells of horseradish peroxidase (HRP) gene, and
The fluorescence intensity building optical system detection HRP Enzymic catalyzed fluorescent reaction eliminates not as benchmark, the method for use dielectrophoresis
Active mutant clone, but this system can only realize the detection of fluorescence intensity at single wavelength, it is thus achieved that quantity of information relatively low
(Agresti J J,Antipov E,Abate A R,et al.Ultrahigh-throughput screening in
drop-based microfluidics for directed evolution[J].Proceedings of the
National Academy of Sciences,2010,107(9):4004-4009.).Harvard University Alexander in 2014
Et al. achieve drop formation, droplet array on the same chip, and identify the antibacterial quoting in water.Pass through antigen-antibody
It is connected on magnetic bead in conjunction with by antibacterial, and uses fluorochrome label antibacterial, utilize CCD to gather fluoroscopic image differentiation and quote in water thin
The quantity of bacterium.But this system cannot obtain the spectral information launching light, it is difficult to is applied to towards multiobject fluorescence-encoded inspection
Survey method (Golberg A, Linshiz G, Kravets I, et al.Cloud-enabled microscopy and
droplet microfluidic platform for specific detection of Escherichia coli in
water[J].PloS one,2014,9(1):e86341.)。
Generation and the manipulation technology of microlayer model are the most ripe, but component in microlayer model are carried out high-sensitivity detection and divides
Analysis technology is the weakest, and the most of drop detection system reported depend on the analysis hands of the Poor informations such as optical imagery
Section, limits the further development of drop microflow control technique.Such as, for wavelength and the multiplexing fluorescence of two dimensions of fluorescence intensity
Coding techniques, only obtains the fluorescence intensity in Single wavelength or a certain narrow-band and cannot realize fluorescence decoding, in addition it is also necessary to fluorescence
Signal carries out broadband spectral scan detection, but existing drop detection system is difficult to obtain fluorescence spectrum and fluorescence simultaneously
Image information, limits the application in the micro-fluidic field of drop of the fluorescence multiplexing coding techniques.
To sum up, it is achieved a kind of multiplexing streaming that can be used for microlayer model fluoroscopic image and spectral scan in micro-fluidic chip runner
Detection device is necessary, it may have be widely applied prospect.
Summary of the invention
For overcoming the fluorescence spectrum and the problem of fluoroscopic image information that can not detect microlayer model at present simultaneously, meet fluorescence multiple
By coding techniques application demand in microlayer model, the invention provides a kind of for microlayer model fluoroscopic image and spectral scan
Multiplexing flow-through assay device.
This detection device, including microfluidic system, for generation and the focusing of microlayer model；
Object lens 3 are positioned at the top of contracted channel 2 to be detected, are sequentially provided with dichroscope 4 and spectroscope 7 above object lens 3,
Wherein, dichroscope 4 and spectroscope 7 are respectively from the horizontal by certain angle；Laser instrument 6 and beam expanding lens 5 are positioned at the one of dichroscope 4
On side, and the same straight line that three is in same level；EMCCD 9 and optical filter 8 are positioned at the side of spectroscope 7, and three
On person's same straight line in same level；It is arranged over transmissive diffraction grating 10, fluorescence spectrophotometer light at spectroscope 7
Degree meter 11 is positioned at above transmissive diffraction grating 10；EMCCD 9 and spectrofluorophotometer 11 are respectively connecting to computer.
The detection method of this detection device is:
Utilize flow focusing principle to generate microlayer model in microchip, realized the focusing of microlayer model by contracted channel 2, swash
After the laser that light device 6 is launched is expanded by beam expanding lens 5, reflect through dichroscope 4, by object lens 3 focus in contracted channel 2 single
Microlayer model 1, the fluorescent material in microlayer model is fluoresced after exciting and is collected and pass through dichroscope 4, through spectroscope by object lens 3
7 are divided into two bundles, a branch of EMCCD 9 that images in after optical filter 8, record fluoroscopic image, and send data to computer；Additionally
A branch of after transmissive diffraction grating 10, the optical diffraction of different wave length is to different directions forming frequency bands of a spectrum, by fluorescence spectrophotometer
Photometer 11 gathers and realizes spectral scan, and sends data to computer.
Dichroscope 4 and horizontal direction are 45° angle；The light that exciter 6 sends expands as directional light through beam expanding lens 5 refraction,
In light path straight down after dichroscope 4 reflects, the battery of lens through object lens 3 focuses on microlayer model, the fluorescence in microlayer model
The material fluorescence produced that is excited is propagated straight up through dichroscope 4.
Spectroscope 7 and horizontal direction are 45° angle, and the transmitting fluorescence passed through by dichroscope 4 is divided into horizontally and vertically on direction
Two-way light, be respectively used to fluoroscopic image and fluorescence spectrum Scanning Detction.
The invention have the benefit that
The present invention is capable of the multiplexing flow cytometer detection of microlayer model fluoroscopic image and spectral scan in micro-fluidic chip runner,
Obtain the complete optical information of microlayer model, meet fluorescence multiplexing coding techniques application demand in microlayer model.Meanwhile, by soft
Part design realizes the scanning of EMCCD digital independent and fluorescence spectrum and synchronization carries out, and drastically increases picking rate and detects flux.
Accompanying drawing explanation
Fig. 1 is that microlayer model generates and focuses on schematic diagram.
A kind of multiplexing flow-through assay device schematic diagram for microlayer model fluoroscopic image and spectral scan of Fig. 2.
Label declaration: 1-microlayer model, 2-contracted channel, 3-object lens, 4-dichroscope, 5-beam expanding lens, 6-laser instrument, 7-light splitting
Mirror, 8-optical filter, 9-EMCCD, 10-transmissive diffraction grating, 11-spectrofluorophotometer.
Detailed description of the invention
The present invention will be further described with detailed description of the invention below in conjunction with the accompanying drawings.It is emphasized that the description below
That be merely exemplary rather than in order to limit the scope of the present invention and application thereof.
Microlayer model, as microreactor, has been successfully applied to crystallization of protein, materials synthesis, molecule and cytobiology
With fields such as analytical chemistry.The specific embodiment party of the present invention is further illustrated below in conjunction with microlayer model inner laser induced fluorescence detection
Formula.
A kind of embodiment is that microfluidic system comprises aqueous phase input equipment, oil phase input equipment and microchip, for micro-
The generation of drop 1 and focusing.
Step 1) processing of microchip:
The processing of microchip between cleaning technology in complete.MEMS lithography process is utilized to go out photoresist mould, first to silicon
Sheet substrate is carried out, dry after, spin coating SU-8 negative photoresist, by mask plate, photoresist is exposed, then passes through developing agent
Process obtains photoresist mould.Then overmolded obtains PDMS chip, is processed the key realizing PDMS and glass by oxygen plasma
Close, construct the fluid channel that microlayer model generates and focuses on.Microchip contains many sample introductions entrance, meets the sample introduction need of two phase liquid
Ask.By improving microchip designs, it is achieved microlayer model 1 quick, stably generate.
Step 2) microlayer model generate and focus on:
Wherein oil phase is silicone oil, and aqueous phase is the aqueous solution comprising the reactants such as fluorescent material.Use pulsometer or syringe pump
For silicone oil and the input of reactant aqueous solution, water phase and an oil phase liquid stream forms flow focusing in runner intersection and produces microlayer model 1,
And realize the focusing of microlayer model 1 by contracted channel 2, as shown in Figure 1.Microlayer model 1 is the Water-In-Oil two generated in microchip
Phase drop, in order to produce stable Water-In-Oil drop, can be passed through water-repelling agent (such as Aquapel) before microchip uses so that
Fluid channel wall is easier to be infiltrated by oil phase；In oil phase, add surfactant span to ensure the stability of microlayer model 1 simultaneously.
And investigate the size of contracted channel 2 for microlayer model 1 focusing effect, the impact of flowing velocity, in adaptive detector detection speed
On the premise of improve the microlayer model 1 flowing velocity in contracted channel 2 as far as possible, improve microlayer model 1 focusing effect.
A kind of embodiment is, according to Fig. 2 build Systems for optical inspection carrying out debug, parameters optimization design.
After the laser that laser instrument 6 is launched is expanded by beam expanding lens 5, reflect through dichroscope 4, object lens 3 focus on contractive set
Single microlayer model 1 in road 2, in microlayer model 1, fluorescent material is fluoresced after exciting and is collected and pass through dichroscope 4 by object lens 3,
It is divided into two bundles through spectroscope 7, a branch of images in EMCCD 9 through optical filter 8, adjust the parameter such as gain of EMCCD 9, gather
The fluoroscopic image of microlayer model 1.The most a branch of after transmissive diffraction grating 10, the optical diffraction of different wave length is to different directions shape
Become frequency bands, spectrofluorophotometer 11 gather fluorescence signal, it is achieved fluorescence spectrum scans.
Equipped with controlling software on computer, control fluoroscopic image data reading and synchronization carry out, and right with what fluorescence spectrum scanned
The data of gained fluoroscopic image and fluorescence spectrum scanning process.Improved further by software design acquisition speed and
Detection flux.
Claims (4)
1., for microlayer model fluoroscopic image and a multiplexing flow-through assay device for spectral scan, including microfluidic system, it is used for
The generation of microlayer model and focusing, it is characterised in that
Object lens (3) are positioned at the top of contracted channel to be detected (2), are sequentially provided with dichroscope (4) and light splitting in the top of object lens (3)
Mirror (7), wherein, dichroscope (4) and spectroscope (7) are respectively from the horizontal by certain angle；Laser instrument (6) and beam expanding lens (5)
It is positioned on the side of dichroscope (4), and the same straight line that three is in same level；EMCCD (9) and optical filter (8) position
In the side of spectroscope (7), and on the same straight line that three is in same level；Being arranged over of spectroscope (7)
Emitting diffraction grating (10), spectrofluorophotometer (11) is positioned at transmissive diffraction grating (10) top；EMCCD (9) and fluorescence
Spectrophotometer (11) is respectively connecting to computer.
2. the detection side of a kind of multiplexing flow-through assay device for microlayer model fluoroscopic image and spectral scan described in claim 1
Method, it is characterised in that
Utilize flow focusing principle to generate microlayer model in microchip, realized the focusing of microlayer model, laser by contracted channel (2)
After the laser that device (6) is launched is expanded by beam expanding lens (5), reflect through dichroscope (4), object lens (3) focus on contracted channel (2)
In single microlayer model (1), the fluorescent material in microlayer model is fluoresced after exciting and is collected by object lens (3) and passed through dichroscope
(4), it is divided into two bundles, a branch of EMCCD (9) that images in after optical filter (8) through spectroscope (7), records fluoroscopic image, and will
Data send to computer；The most a branch of after transmissive diffraction grating (10), the optical diffraction of different wave length is to different directions shape
Become frequency bands, spectrofluorophotometer (11) gather and realize spectral scan, and send data to computer.
Detection method the most according to claim 2, it is characterised in that dichroscope (4) and horizontal direction are 45° angle；Excite
The light that device (6) sends expands as directional light through beam expanding lens (5) refraction, in light path straight down after dichroscope (4) reflects,
Battery of lens through object lens (3) focuses on microlayer model, and the fluorescent material in microlayer model is excited the fluorescence produced through dichroscope
(4) propagate straight up.
4. according to the detection method described in Claims 2 or 3, it is characterised in that spectroscope (7) and horizontal direction are 45° angle, will
What dichroscope (4) passed through launches the two-way light that fluorescence is divided on horizontally and vertically direction, is respectively used to fluoroscopic image and fluorescence
Spectral scan detects.
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| CN107576639A (en) * | 2017-08-28 | 2018-01-12 | 博奥生物集团有限公司 | Portable fully integrated DNA spot examines micro-full analytical system light path |
| CN107831589A (en) * | 2017-12-04 | 2018-03-23 | 中国计量大学 | A kind of controllable super-resolution microscope equipment of focusing based on spherical micro-nano liquid lens |
| CN108020490A (en) * | 2017-06-23 | 2018-05-11 | 中国科学院天津工业生物技术研究所 | A kind of high flux screening equipment using drop micro-fluidic chip |
| CN108828206A (en) * | 2018-05-24 | 2018-11-16 | 广州天宝颂原生物科技开发有限公司 | A kind of dry type fluorescence immunity analyzer and its test method |
| CN108844936A (en) * | 2018-07-09 | 2018-11-20 | 领航基因科技（杭州）有限公司 | A kind of three-dimensional drop detection system and detection method |
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| CN110057798A (en) * | 2019-04-26 | 2019-07-26 | 江苏师范大学 | A kind of streaming sample multi-wavelength fluorescence detection method |
| CN110057794A (en) * | 2019-03-19 | 2019-07-26 | 天津大学 | A method of methyl measurement and imaging are realized based on laser photolysis induced fluorescence |
| CN110296963A (en) * | 2019-06-13 | 2019-10-01 | 深圳先进技术研究院 | A kind of fluorescence detection device and fluorescence detection method |
| WO2020011193A1 (en) * | 2018-07-11 | 2020-01-16 | The University Of Hong Kong | Automatic microfluidic system for continuous and quantitive collection of droplets |
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| CN111913293A (en) * | 2019-12-12 | 2020-11-10 | 南开大学 | Super-resolution microscopy system and method based on fluorescence spectrum |
| CN112295619A (en) * | 2020-10-06 | 2021-02-02 | 新羿制造科技（北京）有限公司 | Micro-droplet chip channel positioning device and method |
| CN114216402A (en) * | 2021-12-14 | 2022-03-22 | 西南科技大学 | Method and device for measuring soft substrate micro-deformation caused by surface tension |
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