CN108844936A - A kind of three-dimensional drop detection system and detection method - Google Patents
A kind of three-dimensional drop detection system and detection method Download PDFInfo
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- 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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- 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
- G01N21/6402—Atomic fluorescence; Laser induced fluorescence
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- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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- 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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Abstract
The invention discloses a kind of three-dimensional drop detection systems, micro-fluidic chip including the interior drop for thering is three-dimensional state to be distributed, light beam is focused the point light source projected on the drop through confocal detection system, the imaging unit of Formation cross-section image is acquired to the fluorescence signal of drop reflection, scanning lens and each layer cross section image for handling the drop form the three-dimensional drop imaging unit of the imaging of three-dimensional drop, the scanning lens includes the object lens of galvanometer or mechanical system movement, focusing launching position of the mobile point light source to drop in the micro-fluidic chip；Detection method passes through point light source, burnt being detected and being imaged to distributed in three dimensions drop of copolymerization, quantitative detection fast high-sensitive degree, error is low, the detection of opposite conventional monolayers drop, the order of magnitude of processing sample in the unit time is improved, imaging clearly, precision are high；The biopsy of detection and analysis and tumour suitable for albumen and nucleic acid and early clinical diagnosis.
Description
Technical field
The present invention is to be related to a kind of three-dimensional drop detection system and detection method, belongs to biological chip testing technology field,
The biopsy of quantitative detection and tumour especially suitable for protein and nucleic acid and early clinical diagnosis.
Background technique
High throughput screening system originates from the research of drug screening, mainly uses 96 or 384 orifice plates for array reactor,
Liquid is carried out by automatic machinery people and distributes with sample mixed the screening flux, it can be achieved that daily at least 10000 samples, due to it
Powerful screening and analytic function, the application range of High Throughput Screening Assay expand to biology, medicine, change from drug screening
In etc. multiple scientific domains, and research is concentrated in the micromation of high throughput screening system.
How the generation of microlayer model is mainly implemented to deposit between continuous phase and dispersed phase with sufficiently large active force to disturb
Interfacial tension make up to unstability, in general, when separate phase somewhere apply power be greater than its interfacial tension when, micro liquid at this
Cognition breakthrough interfacial tension, which enters, forms drop in continuous phase.Micro-fluidic chip (microfluidicchip) refers to will be chemical
The core of one piece of very little is integrated into basic operation units such as sample preparation, reaction, separation, detections involved in the fields such as biology
On piece forms network by microchannel, runs through whole system with controlled fluid, to realize each of conventional chemical or biology laboratory
Kind function；It, which has, generates droplet particle labyrinth, for generating and storing the carrier of microlayer model particle.In micro-fluidic core
Injection oil-phase component solution is first had in piece, is reinjected water phase reagent and is somewhere applied more than the power of its interfacial tension, then water
Phase reagent breaks through interfacial tension and enters oil-phase component solution, and here it is water phase reagents in oil-phase solution, due to surface tension
One independent Water-In-Oil particle of effect and formation.Oil-phase component solution be for generating droplet particle necessary condition, and
The heat transfer for needing to provide temperature for microlayer model particle when providing environment for the existence of microlayer model particle, while also being expanded is situated between
Matter.
Sample quickly and accurately can be divided into several independent units by micro-fluidic chip, and carry out multistep parallel reaction,
It is at low cost, small in size, high-throughput, it is current ideal digital pcr platform.PCR reaction solution is divided into using micro-fluidic chip
Large number of and equal volume reaction member, the quantity for the reaction member being positive by fluorescence signal after detection amplification is to core
Acid template is quantified, and microfluidic chip liquid drop technology is used for digital pcr, flux is will test and further increases.In conjunction with
In the laser scanning co-focusing microscope (Laser Scanning Confocal Microscopy, LSCM) of research fine structure
Albumen or nucleic acid are detected and analyzed, there is very high imaging resolution and signal-to-noise ratio.
To improve detection accuracy, those skilled in the art are concentrated using generating the several of single layer tiling or single discrete state
The form of microlayer model carries out, and makes great efforts to improve drop formation precision, reduces droplet size, sample in high flux screening is effectively reduced
Product/actual consumption, save experimental cost with this, as a kind of with picoliters grade in disclosed in Chinese patent 20121589055.1
The application method of the automation microlayer model array screening system of precision；Single layer or single drop are made using existing micro-fluidic chip
Carry out quantitative detection and analysis, separation process is complicated, low separation efficiency, can detect that item is single, the unit time when for detecting
Interior amount detection is limited；Single part of volume of separation is smaller, sample number is more, and the number for detecting operation is also more, is improving
Detection workload is also increased while certain detection accuracy.
Obviously, existing microflow control technique is used in the form of the several microlayer models for generating single layer tiling or single discrete state
In quantitative detection and analysis, it is not able to satisfy people to while processing sample size improves at least one order of magnitude in the unit time
Realize fast high-sensitive, therefore how the application demand for the drop detection technology for improving absolute quantitation is realized to distributed in three dimensions
Drop is precisely detected, and is improved detection efficiency while improving detection accuracy, is the R&D direction with applications well prospect,
It is also those skilled in the art's technical problem urgently to be resolved.
Summary of the invention
In view of the above-mentioned problems existing in the prior art and demand, the object of the present invention is to provide a kind of three-dimensional drop detection systems
System and detection method improve detection efficiency while improving detection accuracy, and it is absolute to realization fast high-sensitive, raising to meet people
The application demand of quantitative drop detection technology.
To achieve the above object, the technical solution adopted by the present invention is as follows：
A kind of three-dimensional drop detection system, it is characterised in that：Including：Micro-fluidic chip has three in the micro-fluidic chip
The drop of dimension state distribution；Point light source, the light beam of the point light source is focused through confocal detection system projects the liquid
In drop；Imaging unit, the imaging unit are acquired Formation cross-section image to the fluorescence signal that the drop reflects；Scanning mirror
Head, the scanning lens include the object lens of galvanometer or mechanical system movement, and the mobile point light source is in the micro-fluidic chip
The focusing launching position of drop；Three-dimensional drop imaging unit, each layer that the three-dimensional drop imaging unit handles the drop are cut
The imaging of face image formation three-dimensional drop.
Preferably, the point light source forms point light source using laser, LED or halogen lamp and projects drop.
Preferably, the point light source is projected light beams upon on the drop by filter plate.
As further preferred scheme, it for the first laser device of 488nm and wavelength is 561nm that the point light source, which uses wavelength,
The laser that issues of second laser spread that mirror, convergence is closed beam and projected on the drop after optical filter through laser respectively.
As further preferred scheme, the angle for controlling the laser that the first laser device issues using dichroscope is inclined
Turn, the angular deflection for the laser that the second laser issues is controlled using reflecting mirror, later, is controlled and converged using dichroscope
It closes the angular deflection of the first laser device laser issued of beam and the laser of second laser sending and focuses projection
Onto the drop.
As further preferred scheme, optical filter or/and lens phase are passed through using the point light source and the fluorescence signal
The wrong 10nm wavelength that at least misplaces, is staggered to form 1~20 sense channel of detection in 300nm~1100nm wave-length coverage.
Preferably, the spot size is adjusted by beam expanding lens using point light source, the beam expanding lens adjusts multiplying power
It is 1~20 times.
Preferably, the imaging unit is PMT detector, APD detector or EMCCD photon detector.
As further preferred scheme, collected using the imaging unit, detection is by four-way fluorescence filter and thoroughly
Second polarization state light of mirror processing.
As further preferred scheme, the four-way fluorescence filter is controlled using switch motor.
Preferably, the drop is water phase or aqueous mixture, and the drop is stored in oily phase, the oil phase
Including one of mineral oil, vegetable oil, fluorinated oil or a variety of mixtures or/and compound.
As further preferred scheme, the oil mutually further includes that mass fraction ratio is 0.1%~10% activating agent.
As further preferred scheme, the water phase is that pure water or pure water and other contact angles are hydrophilic more than or equal to 90 °
The mixture of substance, the substance to be detected being coated in the water phase can be the reaction system of PCR amplification and nucleic acid-templated mixed
Close one of object, albumen and antibody mixture.
Preferably, the micro-fluidic chip is provided with transparency window, and the point light source passes through the transparency window for light
Beam projects on the drop in the micro-fluidic chip.
Preferably, the drop memory block height of the micro-fluidic chip is 5-10 times of the liquid-drop diameter, from
And drop is enable to store in the chip in three-dimensional form.
Preferably, the drop is in the arrangement of layer-by-layer or staggered floor construction, orderly or nothing in the micro-fluidic chip
Sequence arrangement, close-packed arrays or loosely-packed.
A kind of detection method of above-mentioned three-dimensional drop detection system, it is characterised in that：Include the following steps：
(1) light beam that point light source emits is projected on the drop inside micro-fluidic chip；
(2) reflected light reflected from drop is acquired by photon detector；
(3) imaging unit acquires the reflected light signal of the drop reflection to form the cross-sectional image of the drop.
Preferably, further comprising the steps of：
(4) mobile point light source to project on drop light beam again；
(5) step (2) to step (4) are repeated, until completing the layer-by-layer Scanning Detction to drop in the micro-fluidic chip
And the imaging of three-dimensional drop is formed in a computer.
Compared with prior art, the present invention has the advantages that：
Three-dimensional drop detection system of the present invention by point light source, copolymerization it is burnt to distributed in three dimensions drop carry out detection and
The quantitative detection to the fast high-sensitive degree of drop to be detected is realized in imaging, reduces detection error, can only relative to traditional detection
The detection for carrying out single layer drop improves the order of magnitude of processing sample in the unit time, it is high to obtain imaging clearly, precision；Not only
The quantitative detection and analysis that can be used for albumen and nucleic acid apply also for biopsy and the early clinical diagnosis of tumour, pseudoreaction
Rate is low, detection process is pollution-free.
Detailed description of the invention
Fig. 1 is a kind of structural schematic diagram for three-dimensional drop detection system that the embodiment of the present invention 1 provides；
Fig. 2 is the knot of the three-dimensional drop detection system for the object lens mechanically moved that the embodiment of the present invention 1 provides
Structure schematic diagram.
Figure label is schematically as follows：1, micro-fluidic chip；2, point light source；3, imaging unit；4, scanning lens；41, three axis
Motion platform；5, dichroscope；6, four-way fluorescence filter；7, lens.
Specific embodiment
Technical solution of the present invention is described in further detail below in conjunction with drawings and examples.
Embodiment 1
As shown in connection with fig. 1, a kind of three-dimensional drop detection system provided in this embodiment, including micro-fluidic chip 1, point light source
2, imaging unit 3, scanning lens 4 and three-dimensional drop imaging unit, the liquid for thering is in the micro-fluidic chip 1 three-dimensional state to be distributed
Drop；The light beam of the point light source 2 is focused through confocal detection system to be projected on the drop；The imaging unit is to institute
The fluorescence signal for stating drop reflection is acquired Formation cross-section image；The scanning lens includes that galvanometer or mechanical system are mobile
Object lens move the point light source to the focusing launching position of drop in the micro-fluidic chip, for example, three-axis moving can be used
Platform 41 controls movement of the scanning lens in X-axis, Y-axis, Z-direction；Described in the three-dimensional drop imaging unit processing
Each layer cross section image of drop forms the imaging of three-dimensional drop.
In the present embodiment, the point light source 2 forms point light source using laser, LED or halogen lamp and projects the liquid
Drop.
In the present embodiment, the point light source 2 is projected light beams upon on the drop by filter plate, in order to obtain precisely
Spend higher cross-sectional image, it for the first laser device of 488nm and wavelength is the second of 561nm to swash that the point light source 2, which uses wavelength,
Convergence conjunction beam projects on the drop laser that light device issues after laser spreads mirror, optical filter respectively；In view of point light source 2
The control of opticpath controls the angular deflection for the laser that the first laser device issues using dichroscope, using reflecting mirror
The angular deflection for the laser that the second laser issues is controlled, later, described that beam is closed in convergence is controlled using dichroscope 5
The angular deflection and focusing for the laser that the laser and the second laser that one laser issues issue project on the drop.
In order to faster more accurately capture object section image, in the present embodiment, using the point light source 1 with it is described
Fluorescence signal by optical filter or/and lens be staggered at least misplace 10nm wavelength, be staggered in 300nm~1100nm wave-length coverage
1~20 sense channel of detection is formed, making the point light source 1 and the fluorescence signal, physical light wavelength zone is staggered, passes through tune each other
The whole point light source or/and the fluorescence signal can significantly improve described cut to change it in the light focusing degree of the drop
The precision of face image.
In the present embodiment, the spot size is adjusted by beam expanding lens using point light source 1, the beam expanding lens adjusts multiplying power
It is 1~20 times.
In the present embodiment, the imaging unit 3 is PMT detector, APD detector or EMCCD photon detector, is used for
Acquisition converges fluorescence optical signals to form the cross-sectional image of the drop；In order to realize while multi-channel detection, using it is described at
As the fluorescence signal that unit 3 is collected, detection is handled by four-way fluorescence filter 6 and lens 7, using different fluorescence
Channel synthesis drop independent or combined includes that the character numerical value of fluorescence signal forms the imaging of droplet profile and fluorescence intensity；In order to
The convenience that the four-way fluorescence filter 6 controls is improved, switch motor progress can be used in the four-way fluorescence filter 6
Control.
In the present embodiment, the drop is water phase or aqueous mixture, and the drop is stored in oily phase, the oil phase
Including one of mineral oil, vegetable oil, fluorinated oil or a variety of mixtures or/and compound, preferably, the oil mutually also wraps
Including mass fraction ratio is 0.1%~10% activating agent；The water phase is pure water or pure water and other contact angles more than or equal to 90 °
The mixture of hydrophilic substance, the substance to be detected being coated in the water phase can be the reaction system and nucleic acid mould of PCR amplification
One of plate mixture, albumen and antibody mixture.
In the present embodiment, the micro-fluidic chip is provided with transparency window, and the point light source passes through the transparency window for light
Beam projects on the drop in the micro-fluidic chip.
In the present embodiment, the drop can arrange, orderly in layer-by-layer or staggered floor construction in the micro-fluidic chip 1
Or disorderly arranged, close-packed arrays or loosely-packed, the present embodiment even droplet distribution is required it is low, improve detection precision and
While detection efficiency, operation difficulty is reduced.
Embodiment 2
The detection method of three-dimensional drop detection system described in a kind of Application Example 1 provided in this embodiment, including with
Lower step：
(1) light beam that point light source 2 emits is projected on the drop inside micro-fluidic chip 1；
(2) reflected light reflected from drop is acquired by photon detector；
(3) imaging unit 3 acquires the reflected light signal of the drop reflection to form the cross-sectional image of the drop；
(4) mobile point light source 2 to project on drop light beam again；
(5) step (2) to step (4) are repeated, until completing the layer-by-layer Scanning Detction to drop in the micro-fluidic chip 1
And the imaging of three-dimensional drop is formed in a computer.
In the present embodiment, it is coated with nucleic acid to be detected in the drop, is prepared by following steps：
S1：With liquefaction phase；
S2：Prepare water phase, i.e. PCR reaction system；
Template is the plasmid containing T790M wild type and saltant type independently constructed
Primer sequence is：
F：5'-GCCTGCTGGGCATCTG-3'(SEQ ID NO:1)；
R：5'-TCTTTGTGTTCCCGGACATAGAC-3'(SEQ ID NO:2)；
Probe sequence is：
5'-FAM-ATGAGCTGCATGATGAG-MGB-NFQ-3'(SEQ ID NO:3)；
5'-VIC-ATGAGCTGCGTGATGAG-MGB-NFQ-3'(SEQ ID NO:4)；
5'-ROX-ATGAGCTGCATGATGAG-MGB-NFQ-3'(SEQ ID NO:5)；
5'-CY5-ATGAGCTGCATGATGAG-MGB-NFQ-3'(SEQ ID NO:6), wherein FAM/VIC/ROX/CY5
For fluorescent reporter group, NFQ is fluorescent quenching group.
Water phase is prepared according to system in below table：
| 2 × PCR reaction buffer (including Taq enzyme, dNTP, magnesium ion) | 7.5uL |
| BSA (1%) | 1.5uL |
| Upstream primer F (10 μM) | 0.3uL |
| Downstream primer R (10 μM) | 0.3uL |
| Probe (5 μM, each) | 0.1uL |
| Template | 1.0uL |
| Deionized water | 4.0uL |
| Total volume | 15uL |
S3：Oily phase, water phase are injected separately into micro-fluidic chip 1 and form the drop with three-dimensional superposed state distribution；
S4：Above-mentioned micro-fluidic chip 1 is put into PCR instrument, carries out situ PCR by following procedure：First in 96 DEG C of conditions
Lower progress initial denaturation is handled 10 minutes, then is carried out denaturation treatment 30 seconds under the conditions of 98 DEG C, later, is moved back under the conditions of 62 DEG C
After fire extends processing 1 minute, above-mentioned processing cycle 39 times, annealing is carried out under the conditions of 62 DEG C and extends processing 1 minute, finally 25
Isothermal holding is carried out under the conditions of DEG C, completes the PCR amplification to three-dimensional drop, and the three-dimensional drop for storing and completing PCR amplification is made
Micro-fluidic chip, wait to be detected.
It is visible in summary：Three-dimensional drop detection system of the present invention is by point light source 2, copolymerization coke to distributed in three dimensions liquid
Drop is detected and is imaged, and realizes the quantitative detection to the fast high-sensitive degree of drop to be detected, reduces detection error, relatively
The detection of single layer drop can only be carried out in traditional detection, the order of magnitude of processing sample in the unit time is improved, it is clear to obtain imaging
It is clear, precision is high；The quantitative detection and analysis that cannot be only used for albumen and nucleic acid apply also for biopsy and the early clinic of tumour
Diagnosis, pseudoreaction rate is low, detection process is pollution-free.
It is last it is necessarily pointed out that：The foregoing is merely the preferable specific embodiment of the present invention, but the present invention
Protection scope be not limited thereto, anyone skilled in the art in the technical scope disclosed by the present invention,
Any changes or substitutions that can be easily thought of, should be covered by the protection scope of the present invention.
Claims (10)
1. a kind of three-dimensional drop detection system, it is characterised in that：Including：
Micro-fluidic chip, the drop for thering is in the micro-fluidic chip three-dimensional state to be distributed；
Point light source, the light beam of the point light source is focused through confocal detection system to be projected on the drop；
Imaging unit, the imaging unit are acquired Formation cross-section image to the fluorescence signal that the drop reflects；
Scanning lens, the scanning lens include the object lens of galvanometer or mechanical system movement, and the mobile point light source is to described micro-
The focusing launching position of drop in fluidic chip；
Three-dimensional drop imaging unit, each layer cross section image that the three-dimensional drop imaging unit handles the drop form three-dimensional liquid
The imaging of drop.
2. three-dimensional drop detection system according to claim 1, it is characterised in that：The point light source uses laser, LED
Or halogen lamp forms point light source and projects drop.
3. three-dimensional drop detection system according to claim 1, it is characterised in that：The point light source passes through filter plate for light
Beam projects on the drop.
4. three-dimensional drop detection system according to claim 3, it is characterised in that：Using the point light source and the fluorescence
Signal by optical filter or/and lens be staggered at least misplace 10nm wavelength, be staggered to be formed in 300nm~1100nm wave-length coverage
Detect 1~20 sense channel.
5. three-dimensional drop detection system according to claim 1, it is characterised in that：It is adjusted using point light source by beam expanding lens
The spot size, the beam expanding lens adjustment multiplying power is 1~20 times.
6. three-dimensional drop detection system according to claim 1, it is characterised in that：The imaging unit be PMT detector,
APD detector or EMCCD photon detector.
7. three-dimensional drop detection system according to claim 1, it is characterised in that：The drop is that water phase or water phase mix
Object, the drop are stored in oily phase, and the oil mutually includes one of mineral oil, vegetable oil, fluorinated oil or a variety of mixtures
Or/and compound.
8. three-dimensional drop detection system according to claim 1, it is characterised in that：The micro-fluidic chip is provided with transparent
Window, the point light source are projected light beams upon by the transparency window on the drop in the micro-fluidic chip.
9. a kind of detection method of three-dimensional drop detection system according to claim 1, it is characterised in that：Including following step
Suddenly：
(1) light beam that point light source emits is projected on the drop inside micro-fluidic chip；
(2) reflected light reflected from drop is acquired by photon detector；
(3) imaging unit acquires the reflected light signal of the drop reflection to form the cross-sectional image of the drop.
10. detection method according to claim 9, it is characterised in that：It is further comprising the steps of：
(4) mobile point light source to project on drop light beam again；
(5) step (2) to step (4) are repeated, until complete to the layer-by-layer Scanning Detction of drop in the micro-fluidic chip and
The imaging of three-dimensional drop is formed in computer.
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| CN110195016A (en) * | 2019-07-17 | 2019-09-03 | 中国人民解放军军事科学院军事医学研究院 | Temperature control objective table and detection device |
| CN111307693A (en) * | 2020-02-24 | 2020-06-19 | 东南大学 | Passive wireless multi-stage droplet micro-fluidic detection device |
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