CN207457068U - A kind of large area digital pcr droplet fluorescence high pass amount detecting device - Google Patents
A kind of large area digital pcr droplet fluorescence high pass amount detecting device Download PDFInfo
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- CN207457068U CN207457068U CN201721120614.9U CN201721120614U CN207457068U CN 207457068 U CN207457068 U CN 207457068U CN 201721120614 U CN201721120614 U CN 201721120614U CN 207457068 U CN207457068 U CN 207457068U
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Abstract
The utility model discloses a kind of large area digital pcr droplet fluorescence high pass amount detecting device, including fluorescence excitation light path and fluorescence detection optical path；Along fluorescence excitation light path, line focus device and objective apparatus are incident on the digital pcr chip for being placed in objective table the exciting light of light supply apparatus transmitting successively；Along fluorescence detection optical path, the fluorescence that digital pcr chip stimulated luminescence generates is transmitted to image acquiring sensor after the imaging of objective apparatus, focusing arrangement and imaging device successively；Wherein, objective table is additionally provided with temperature control device, and temperature control device controls the circulating temperature that digital pcr chip reacts in real time.The utility model can realize high-throughput, highly sensitive, high specific real-time digital qPCR detections, be of great significance for improving digital pcr detecting instrument performance, precision and flux.
Description
Technical field
The utility model is related to biomedical inspection field field of nucleic acid detection, more particularly to one kind can realize large area
Digital pcr droplet fluorescence high pass amount detecting device.
Background technology
In fields such as the analysis of early diagnosis of tumor examination, blood disease parting and carrying capacity, noninvasive pre-natal diagnosis, it is required for pair
The nucleic acid of super low concentration carries out the detection of highly sensitive high specific at a high speed, and conventional PCR increasingly can not meet demand.For
Realize this function, people have developed a kind of digital pcr technology on the basis of PCR, and detection sensitivity is improved 1~2 quantity
Grade.
Droplet type digital pcr use " dividing and rule " (divide and conquer) inspection policies, by sample,
PCR reagent mixture, using oil as continuous phase, using microflow control technique, sample mixture is formed one by one as dispersed phase
Suspending drops are in continuous phase.And then a standard PCR amplification is distributed into a large amount of droplets (nanoliter to picoliters), Mei Gewei
The target molecule (DNA profiling) of 0 or 1 copy is only included in drop.Independent PCR amplification is carried out to each droplet, amplification terminates
Afterwards, detect the fluorescence of each droplet, the droplet of positive signal counted, so as to obtain the absolute copy number of sample template and
Nucleic acid concentration.The size and number of droplet determine the detection sensitivity and lower limit of digital pcr, existing digital pcr droplet number
Between 20,000~10,000,000.
Existing commercialization droplet type digital pcr technical operation is as follows：It is prepared in dedicated droplet and drop is prepared on chip；It is logical
It crosses pipettor droplet is transferred in centrifuge tube, temperature cycles amplification is carried out using conventional PCR instrument；After the completion of amplification, utilize
Streaming technology carries out fluorescence excitation and detection, result of calculation to each droplet successively.Such detection mode has the following problems：
(1) flow cytometer detection speed is slower, and detection speed is in 200~500/s, and detection process takes long, and flux is low；
(2) end-point method detection can only be carried out to single droplet, it is impossible to which real-time qPCR reduces specificity and sensitivity；
(3) light path hardware is complicated, it is difficult to realize multiple digital pcr detection (more than 4 weights)；
(4) transfer process of drop will cause the outside contamination that droplet loses and may attract twice；
(5) the heat block thermal inertia of standard PCR amplification instrument is big, thermal cycle times length when small (2 or so).
To solve the problems, such as above-mentioned five, applicant has been presented for integral type digital pcr chip, and has applied for that utility model is special
Sharp (application number 201510961967.0).Referring to attached drawing 1, integral type digital pcr chip includes body 1, and disposed thereon is continuous
Phase injection hole 11, sample injection hole 12, individual layer droplet tiling cavity 15, vacuum access aperture 16, "+font " for generating droplet
Micro-structure 13 and for connect continuous phase injection hole 11 and "+font " micro-structure 13 fluid path 14, for connect sample injection
First fluid path 17 of hole 12 and "+font " micro-structure 13, for connecting "+font " micro-structure 13 and individual layer droplet tiling cavity 15
The second fluid path 18.All micro-structures are made on a piece of polymeric substrates by MEMS technology, then with another piece
Polymer flat plate is mutually bonded and forms the micro sprue system of closing, in continuous phase injection hole 11, sample injection hole 12, vacuum access
Perforate at hole 16 3, for injecting sample and applying negative pressure.
With reference to Fig. 2, basic principle is：After continuous phase injection hole 11 injects oil, sample injection hole 12 injects sample,
16 applying vacuum negative pressure of vacuum access aperture, sample, continuous phase will reach "+word under the action of negative pressure by runner 14 and 17
Type " micro-structure 13.Here, sample then passes through runner by because forming nanoliter level droplet 20 one by one during the shearing of continuous phase
18, into individual layer droplet tiling cavity 15, are laid into droplet individual layer 19.
The technical method has the advantage that：Droplet individual layer 19 is suitable for carrying out image conversion fluorescence using CCD taking pictures detection,
With higher flux and speed；It is bent that the variation of droplet fluorescence with Xun Huan at this time can be obtained by continuous fluorescence image detection
Line carries out single drop qPCR detections, has higher sensitivity and specificity；It, can be easily by increasing excitation light source and optical filter
Realize multiple digital pcr；Whole process integration is completed, and there is no droplet loss and extraneous pollutions.Finally, individual layer droplet will
With larger temperature control area and smaller thermal inertia, PCR cycle proliferation time can be reduced within half an hour.
In order to carry out detection of nucleic acids using the digital pcr chip, on the one hand need to carry out the digital pcr micro-fluidic chip
Temperature cycles expand, and on the other hand need to carry out digital pcr drop individual layer real time laser excitation and fluorescence imaging detection.Per temperature
Circulation primary is spent, a drop fluoroscopic image detection is just carried out, to obtain the fluorescence consecutive variations curve of each drop.To improve
Flux on one chip, can integrate multiple integral type digital pcr micro-fluidic structures, form 32,48 even 96 samples
While detection (with reference to Fig. 3), to improve flux, so just need chip that can carry out one-dimensional or two-dimentional movement, so as to
Can picture of large image scale acquisition be carried out by scan mode.In addition, to improve detection efficiency, using a kind of new algorithm, first pass through
Small multiplying power carries out rough big visual field viewing, has found that it is likely that there are after drop phosphor dot, switch to the fine small field of view observation of high magnification
Mode, to improve efficiency.But existing detection device such as fluorescence microscope etc., can not meet above-mentioned testing requirements.
Utility model content
One purpose of the utility model is to solve at least the above and/or defect, and provides and at least will be described later
The advantages of.
The utility model is to provide a kind of large area digital pcr droplet fluorescence high pass amount detecting device there are one purpose,
It is by the design of system light path and splits the continuous scanning of detection zone, can realize digital pcr it is high-throughput, in real time and
Precisely quantitative detection, in addition, the quick positioning and detection of drop phosphor dot can be achieved by the switching of size surface sweeping visual field.
In order to realize these purposes and further advantage according to the present utility model, it is micro- to provide a kind of large area digital pcr
Fluorescence high pass amount detecting device is dripped, including fluorescence excitation light path and fluorescence detection optical path；
Along the fluorescence excitation light path, line focus device and objective apparatus are incident to the exciting light of light supply apparatus transmitting successively
It is placed on the digital pcr chip of objective table；
Along the fluorescence detection optical path, the fluorescence that the digital pcr chip is generated by the exciting light is successively through the object
Image acquiring sensor is transmitted to after lens device, the focusing arrangement and imaging device imaging；
Wherein, wherein the objective table is additionally provided with temperature control device, the temperature control device controls the digital pcr chip in real time
The circulating temperature of reaction.
Preferably, wherein, the objective table is fixed on optical circuit path or translation freedoms at least there are one tool,
To meet the needs of picture of large image scale acquisition.
Preferably, wherein, the objective apparatus includes：
One object lens, is fixed in light path；
Or the object lens of several different multiplyings, it is configured respectively into light path in the form of changeable, so as to fulfill not
With the digital pcr droplet fluorescence imaging of multiplying power.
Preferably, wherein, the multiplying power of the object lens is selected from 2X, 4X, 5X or 10X.
Preferably, wherein, the light supply apparatus includes：
Light source assembly provides parallel mixing light source；
Light source optical filter box, has：
One light source optical filter, be fixed in the fluorescence excitation light path or
Several light source optical filters are configured in the form of changeable respectively into the fluorescence excitation light path.
Light source optical filter can be used for carrying out optical filtering processing to mixing light source, can be used for excitation PCR droplets dyestuff institute to provide
The monochromatic source needed.Monochromatic source is generated using mixing light source and optical filter in this programme, can be provided with relatively low cost
The monochromatic excitation light source of multiple wavelength realizes multiple digital pcr function.
Preferably, wherein, the light supply apparatus includes several sets of monochromatic light sources；
Along light path direction of advance, the sets of monochromatic light sources includes successively：Monochromatic source, shutter and light lens；Wherein, institute
It states shutter break-make and controls the fluorescence excitation light path.
Preferably, wherein, the light supply apparatus includes：
At least two sets of monochromatic light sources, to provide stronger excitation light source；；
The transmitting light path of the sets of monochromatic light sources is closed beam and imports the fluorescent exciting by several first dichronic mirrors
Road.
Dichronic mirror is a branch of available for two-way excitation light source is merged into, can be by all exciting lights using multiple dichroic mirrors
Source is merged into a branch of, so as to provide the excitation light source of multiple and different wavelength, realizes multiple digital pcr function.
Preferably, wherein, focusing arrangement includes：
Second dichronic mirror, the exciting light of reflection source device transmitting are excited to generate through the digital pcr chip simultaneously
Fluorescence.
Preferably, wherein, focusing arrangement further includes：
First focus lens assembly is arranged between the light supply apparatus and second dichronic mirror, for shaping and expansion
Shu Suoshu laser, so as to obtain preferable exciting light.
Preferably, wherein, focusing arrangement further includes：
Second focus lens assembly is arranged between second dichronic mirror and the imaging device, for shaping and expansion
Shu Suoshu fluorescence, so as to obtain preferable image quality.
Preferably, wherein, along the fluorescence detection optical path, the imaging device filters out component and glimmering including fluorescence successively
Light imaging len；
Preferably, along the fluorescence detection optical path, the imaging device includes single fluorescence filtering assembly and glimmering successively
Light imaging len；
Wherein, the fluorescence filtering assembly is fixedly arranged on the fluorescence detection optical path, and the fluorescence filtering assembly has：
Light source light blocking piece filters out the exciting light of the light supply apparatus transmitting；And
Fluorescent optical filter filters out the stray light of the non-wavelength of fluorescence.
Preferably, wherein, along the fluorescence detection optical path, the imaging device includes several fluorescence optical filtering groups successively
Part and fluorescence imaging lens；
Wherein, several described fluorescence filtering assemblies include：
The light source light blocking piece of several different wave lengths is respectively configured in the form of changeable to the fluoroscopic examination light
Road, to filter out the exciting light of the light supply apparatus transmitting respectively；And
The fluorescent optical filter of several different wave lengths is respectively configured in the form of changeable to the fluoroscopic examination light
Road, to filter out the stray light of the non-wavelength of fluorescence respectively.
The technical program is filtered out by light supply apparatus exciting light and the non-wavelength of fluorescence stray light, can improve system
Signal-to-noise ratio is switched by the combination between the light source light blocking piece and fluorescent optical filter of several different wave lengths, can gather PCR droplets
The fluoroscopic image of the different wave length distributed realizes multiple digital pcr function.
The concrete operation step of the high-throughput detection of large area digital pcr droplet fluorescence is realized such as using above-mentioned detection device
Under：
Step 1：The digital pcr chip for having completed droplet processing is placed on objective table, the digital pcr chip and temperature control
Device connects；
Step 2：A PCR temperature cycles are carried out, process is：92~96 degree are warming up to, 25~40s is stopped, is cooled to 53
~57 degree, 25~35s is stopped, is warming up to 70~74 degree, stops 35~45s；
Step 3：Objective apparatus is switched into minimum magnification objective；
Step 4：Light supply apparatus, imaging device are switched into the first fluorescent dye or the corresponding light path of probe；
Step 5：Decile is carried out to two dimension of level of the digital pcr chip, obtains several rectangles or rectangular region,
Each region area is S, and carries out droplet fluoroscopic image acquisition to each region, wherein the S is 1mm2~16mm2；
Step 6：Light supply apparatus, imaging device are switched into the second fluorescent dye or the corresponding light path of probe, performed again
The step 5 completes the detection of the image of the second fluorescent dye；
Step 7：Repeating said steps 6, until all fluorescent dyes or probe have been detected and finished；
Step 8：Objective apparatus is switched into time low range object lens, repeating said steps 4, step 5, step 6 and step 7；
Step 9：Repeating said steps 8, until the switching of all multiplying powers of object lens finishes, all fluorescent dyes have detected
Finish；
Step 10：Step 2~step 9 is repeated, and is counted, is often repeated once, is counted once, is more than N until counting, wherein
N represents amplification cycles number, and the N is 30~40 times；
Step 11：Test is completed, the qPCR for carrying out subsequent image processing and each digital pcr drop is calculated, and statistics is negative
Positive droplet number, and calculate concentration.
Preferably, wherein, in the step 5,
Any dimension of the digital pcr chip in the horizontal direction is subjected to decile, obtains the strip that several width are W
Shape detection zone continuously scans the strip detection zone, the consecutive image of each detection zone is obtained, to digital pcr
Micro-fluidic chip image is continuously acquired, wherein, the width W is 1mm~3mm.
The utility model includes at least following advantageous effect：
The utility model can realize high-throughput, highly sensitive, high specific real-time digital qPCR detections, for improving
Digital pcr detecting instrument performance and precision, flux are of great significance.It, can be to low dense using technical solutions of the utility model
The sample of nucleic acid of degree carries out quick high accuracy detection, for cancer, infects disease, pre-natal diagnosis etc. with important meaning
Justice, for improving people's accuracy of detection and time limit, improving medical detection technique, improvement clinical testing procedure with critically important meaning
Justice.
Part is illustrated to embody by the further advantage, target and feature of the utility model by following, and part will also pass through
Research and practice to the utility model and be understood by the person skilled in the art.
Description of the drawings
Fig. 1 is the structure diagram of integrated digital pcr chip in an example；
Fig. 2 is the structure diagram of integrated digital pcr chip in another example；
Fig. 3 is high-throughput integrated digital pcr chip structure diagram (48 array) in another example；
Fig. 4 is large area digital pcr droplet fluorescence high throughput structure of the detecting device block diagram in another example；
Fig. 5 is large area digital pcr droplet fluorescence high throughput structure of the detecting device schematic diagram in another example；
Fig. 6 is the large area digital pcr droplet fluorescence high pass for having in another example motion in one dimension mechanism objective table device
Amount detecting device structure diagram；
Fig. 7 is the large area digital pcr droplet fluorescence high pass for having in another example two-dimensional motion mechanism objective table device
Amount detecting device structure diagram；
Fig. 8 is the large area digital pcr droplet fluorescence height for having in another example multiple and different magnification objective switching devices
Flux structure of the detecting device schematic diagram；
Fig. 9 is the large area digital pcr droplet fluorescence high throughput structure of the detecting device with light source optical filter switching mechanism
Schematic diagram；
The large area digital pcr droplet fluorescence high throughput structure of the detecting device that Figure 10 has multiple sets of monochromatic light sources is illustrated
Figure；
Figure 11 is the high-throughput inspection of large area digital pcr droplet fluorescence for having in another example exciting light focus lens assembly
Survey apparatus structure schematic diagram；
Figure 12 is the high-throughput detection of large area digital pcr droplet fluorescence for having in another example fluorescent foci lens subassembly
Apparatus structure schematic diagram；
Figure 13 is glimmering to have the large area digital pcr droplet of exciting light and fluorescent foci lens subassembly in another example simultaneously
Light high throughput structure of the detecting device schematic diagram；
Figure 14 is the large area digital pcr droplet fluorescence high throughput for having in another example fluorescence filtering assembly switching mechanism
Structure of the detecting device schematic diagram；
Figure 15 is the schematic diagram that digital pcr chip is divided into hough transform region by bidimensional in another example；
Figure 16 is digital pcr chip in another example by the one-dimensional schematic diagram for being divided into strip detection zone；
Figure 17 is the large area digital pcr droplet fluorescence high pass amount detecting device that double digital pcr is used in another example
Structure diagram；
Figure 18 is the large area digital pcr droplet fluorescence high pass amount detecting device that sixfold digital pcr is used in another example
Structure diagram；
Figure 19 is the structure diagram of light source optical filter fixing device in another example；
Figure 20 is the structure diagram of fluorescent optical filter fixing device in imaging device in another example.
Specific embodiment
The utility model is described in further detail below in conjunction with the accompanying drawings, to make those skilled in the art with reference to explanation
Book word can be implemented according to this.
It should be appreciated that such as " having ", "comprising" and " comprising " term used herein do not allot one or more
The presence or addition of a other elements or its combination.
With reference to Fig. 4 and Fig. 5, as a kind of way of realization of the utility model, large area digital pcr droplet fluorescence is high-throughput
Detection device includes：
Objective apparatus 200, be located at digital pcr chip directly over, for entire digital pcr fluoroscopic image acquisition most before
End；
Light supply apparatus 300, for the fluorescence excitation of digital pcr droplet；
Focusing arrangement 400, one side converge to the light that light supply apparatus 300 is sent on digital pcr chip 1, the opposing party
The phosphor collection that face generates digital pcr chip 1, and imaging optical path device 500 and image acquiring sensor 600 are transmitted to, so as to
For light source illumination and the coupling of fluorescence imaging；
Imaging optical path device 500 is used to fluoroscopic image carrying out shaping and optical filtering, and by imaging and Image Acquisition biography
600 on sensor, for the fluorescence imaging of digital pcr droplet；
Image acquiring sensor 600 is used for the acquisition to fluoroscopic image and is converted into digitized image, to digital pcr
The fluoroscopic image of droplet gathers in real time.
PCR temperature control devices 700 are used to carry out digital pcr chip 1 accurate temperature cycles control, realize droplet number
The amplification procedure of word PCR；And
Objective table device 800 is used to carry digital pcr droplet micro-fluidic chip and its temperature control device.
In this technical solution, including fluorescence excitation light path and fluorescence detection optical path；
Along the fluorescence excitation light path, line focus device and objective apparatus are incident to the exciting light of light supply apparatus transmitting successively
It is placed on the digital pcr chip of objective table；
Along the fluorescence detection optical path, the fluorescence that the digital pcr chip is generated by the exciting light is successively through the object
Image acquiring sensor is transmitted to after lens device, the focusing arrangement and imaging device imaging；
Wherein, wherein the objective table is additionally provided with temperature control device, the temperature control device controls the digital pcr chip in real time
The circulating temperature of reaction.
By adding a variety of fluorescent dyes or probe to digital pcr reaction system, coordinate different mix, primer, can realize
Multiple digital pcr.Wherein, PCR tuples are identical with the fluorescent dye or the species of probe that add, and for example, double PCR then needs
Two fluorescent dyes or probe.
In another example, the objective table is fixed on optical circuit path or translation freedoms at least there are one tool, with
Realize that one-dimensional or two-dimensional scan mode carries out Image Acquisition, so as to expand detection zone.
With reference to Fig. 6, in a kind of embodiment for realizing the one-dimensional degree of freedom of objective table, objective table device 800 includes servo
Motor 803, transmission device 802, position detecting device 806 transport one-dimensional precise on the horizontal X direction of objective table 801 with realizing
Dynamic control.By the motion in one dimension to objective table, to realize that imaging system detects the one-dimensional scanning of digital pcr chip, to carry
High detection scope realizes the high-throughput detection of large area.Wherein, servomotor 803 can be step-servo motor, DC servo electricity
Machine, AC servo motor, transmission device 802 can be ball leading screw driving device, and position sensor can be that linear grating is compiled
Code device, resistor type displacement sensor.
With reference to Fig. 7, in a kind of embodiment for realizing objective table two-dimensional freedom, objective table device 800 includes a pair
Servomotor 803 and 805, a pair of of transmission device 802 and 804, a pair of of position detecting device 806 and 807, to realize to objective table
X, Y bidimensional precise flange in 801 horizontal direction.By the two-dimensional motion to objective table, to realize imaging system logarithm
The bidimensional Scanning Detction of word pcr chip to improve detection range, realizes the high-throughput detection of large area.
In another example, the objective apparatus includes an object lens, is fixed in light path.
In another example, the objective apparatus includes the object lens of several different multiplyings, is divided in the form of changeable
It is not configured into light path.
With reference to Fig. 8, it can be achieved that a kind of embodiment that different multiplying object lens automatically switch is：Objective apparatus 200 is included and watched
Take the control being depicted without in motor 205, transmission device 204, turning joint 203,202, one groups of object lens 201 of object lens fixed plate and figure
Unit processed.Wherein, the isometrical distribution centered on turning joint 203 in object lens fixed plate 202 of object lens 201.Servomotor 205 is logical
Transmission device 204, turning joint 203 are crossed, object lens fixed plate 202 can be driven to be rotated, and then can be by required for
Enlargement ratio object lens switch to 400 front end of focusing arrangement in light path.Wherein, servomotor 205 can be step-servo motor,
DC servo motor, AC servo motor, transmission device 204 can be belt wheel transmission device, gear drive etc., rotating hinge
Chain 203 can be swivel bearing.The droplet fluorogram of different enlargement ratios can be realized by automating switching using this scheme
As obtaining.Also, this mode is a kind of explanation of preferred embodiments, but is not limited thereto.When implementing the utility model,
It can implement the different switching aspect of object lens according to user's demand.
In another example, the multiplying power of the object lens is selected from 2X, 4X, 5X or 10X.
In another example, with reference to Fig. 9, the light supply apparatus 300 includes：
Light source assembly by mixing light source emission element 301 and convex lens 302, provides parallel mixing light source；
Light source optical filter box, has：
One light source optical filter 303, be fixed in the fluorescence excitation light path or
Several light source optical filters 303 are respectively configured in the form of changeable into the fluorescence excitation light path.
In the technical scheme, realize that the embodiment that light sources with different wavelengths optical filter automatically switches is：Light supply apparatus 300
By halogen lamp 301, lens 302, a series of optical filters 303, optical filter fixed plate 305, turning joint 304, transmission device 306,
Servomotor 307 and control unit (being not drawn into figure) composition.Lens 302 are for the light that light source 301 is sent to be collimated, shape
Into collimated light beam.Servomotor 307 drives optical filter fixed plate 305 to do rotation fortune by transmission device 306 and turning joint 304
It is dynamic, the optical filter 303 of different wave length is switched into 302 front end of lens, realizes that the monochromatic light of 300 different wave length of light supply apparatus is defeated
Go out.Wherein, transmission device 306 can be belt wheel transmission, gear drive, and turning joint 304 can be that the dynamic hinge of rotation is held.Servo
Motor can be step-servo motor, DC servo motor or AC servo motor.Also, this mode is a kind of preferable reality
The explanation of example, but be not limited thereto.When implementing the utility model, can light source optical filter be implemented according to user's demand
Difference switching aspect.
In another example, with reference to Figure 10, the light supply apparatus 300 includes several sets of monochromatic light sources；
Along light path direction of advance, the sets of monochromatic light sources includes successively：Monochromatic source 301, shutter 308 and light lens
302；Wherein, 308 break-make of shutter controls the fluorescence excitation light path.
In another example, the light supply apparatus 300 includes：
Two and above sets of monochromatic light sources；
The transmitting light path of the sets of monochromatic light sources is closed beam and imports the fluorescence and swashed by several first dichronic mirrors 309
Luminous road.
In this embodiment, light supply apparatus 300 is by multiple monochromatic sources 301, lens 302, shutter 308, the first color separation
Mirror 309 forms.Wherein, each light source 301 corresponds to a shutter 308, for controlling the break-make of the light source.First dichronic mirror
309 are used to implement the conjunction beam of two-beam, and the quantity of the first dichronic mirror 309 subtracts 1 for 301 quantity of light source, realizes that all light sources are sent
Light conjunction beam.Which road monochrome light output is controlled by shutter 308 and introduces system.Light source can be lasing light emitter, LED etc., but
It is without being limited thereto.
In another example, with reference to Fig. 5, focusing arrangement includes：
Second dichronic mirror, the exciting light of reflection source device transmitting are excited to generate through the digital pcr chip simultaneously
Fluorescence.
In this embodiment, focusing arrangement 400 is only comprising second dichronic mirror 401, and the second dichronic mirror 401 is on the one hand
For the light reflection that light-source system 300 is sent to digital pcr chip 1, to excite digital pcr drop；On the other hand
For the fluorescence generated through drop on digital pcr chip, and feed them into imaging device 500.
In another example, referring to Figure 11, focusing arrangement 400 further includes：
First focus lens assembly, it includes lens 402 and lens 403, the first focus lens assembly is arranged on the light source
Between device 300 and second dichronic mirror 401, the light for light supply apparatus 300 to be sent is collimated and expanded, to change
Excite spot size and quality.
In another example, referring to Figure 12, focusing arrangement further includes：
Second focus lens assembly, it includes lens 404 and lens 405, the second focus lens assembly by object lens to filling
It puts 200 fluorescence sent to be collimated and expanded, coordinate with imaging len 501, improve image quality.
In another example, referring to Figure 13, focusing arrangement also simultaneously comprising lens 402, lens 403, lens 404 and 405, changes
Become excitation spot size, improve image quality.
In another example, along the fluorescence detection optical path, the imaging device 500 includes single fluorescence filtering assembly successively
With fluorescence imaging lens 501；
Wherein, the fluorescence filtering assembly is fixedly arranged on the fluorescence detection optical path, and the fluorescence filtering assembly has：
Light source light blocking piece filters out the exciting light of the light supply apparatus transmitting；And
Fluorescent optical filter filters out the stray light of the non-wavelength of fluorescence.
In another example, with reference to Figure 14, along the fluorescence detection optical path, the imaging device includes fluorescence optical filtering group successively
Part and fluorescence imaging lens 501；
Wherein, the fluorescence filtering assembly includes：
The light source light blocking piece of several different wave lengths is respectively configured in the form of changeable to the fluoroscopic examination light
Road, to filter out the exciting light of the light supply apparatus transmitting respectively；And
The fluorescent optical filter of several different wave lengths is respectively configured in the form of changeable to the fluoroscopic examination light
Road, to filter out the stray light of the non-wavelength of fluorescence respectively.
In this embodiment, comprising multigroup optical filter, (every group of optical filter includes a light source light blocking piece to imaging device 500
With a fluorescent optical filter) and for switch between each group optical filter optical filter fixed plate 505, turning joint 504, transmission
Device 506, servomotor 507 and control unit (being not drawn into figure).Servomotor 507 passes through transmission device 506 and rotating hinge
Chain 505 drives the optical filter 502 being located in optical filter fixed plate 505 and optical filter 503 to rotate, by required any group
Optical filter 502 and optical filter 503 switch to 700 front end of image acquiring sensor, realize filtering out and required fluorescence ripple for exciting light
The acquisition of section.Each group optical filter 502 and optical filter 503 mutually collect with each monochromatic light that excitation light source is sent in the imaging device
It closes, can realize digital pcr fluorescence excitation and the detection of different dyes, and then realize multiple digital pcr.
In another example, the high-throughput inspection of large area digital pcr droplet fluorescence is realized using the technical solution of the utility model
The method of survey, includes the following steps：
Step 1) places the micro-fluidic chip 1 for having completed the generation of digital pcr droplet to objective table 801, with PCR temperature controls
Device 700 connects.
Step 2), carries out a PCR temperature cycles, and process is：92~96 degree are warming up to, 25~40s is stopped, is cooled to 53
~57 degree, 25~35s is stopped, is warming up to 70~74 degree, stops 35~45s；
Step 3), objective apparatus 200 switch to minimum magnification objective；
Step 4), light supply apparatus, imaging device switch to the first dyestuff or the corresponding light path of probe.
Step 5), referring to Figure 15, by digital pcr micro-fluidic chip 1, X, Y carry out decile in two vertical direction of level, are
Several rectangles or rectangular region 101, each region has long L and width W, and carries out droplet fluoroscopic image to each region and obtain
It takes.The long L is 1mm~4mm, and the width W is 1mm~4mm；
Step 6), light supply apparatus 300, imaging device 500 switch to the corresponding light path of next dyestuff.Step is performed again
5) detection of the image of next fluorescent dye or probe, is completed；
Step 7) repeats step 6), until all fluorescent dyes or probe detection finish；
Step 8), objective apparatus 200 switch to time low range object lens；Repeat step 4), step 5), step 6), step 7)；
Step 9) repeats step 8), until the object lens switching of all multiplying powers finishes, all fluorescent dyes or probe have detected
Finish；
Step 10) repeats step 2)~step 9), and counts, and is often repeated once, and counts once, is more than N until counting；
The N represents amplification cycles number, is 30~40；
Step 11) completes all tests, and the qPCR for carrying out subsequent image processing and each digital pcr drop is calculated, statistics
Negative positive droplet number, and calculate concentration.
In another example, the high-throughput inspection of large area digital pcr droplet fluorescence is realized using the technical solution of the utility model
The method of survey, includes the following steps：
Step 1) places the micro-fluidic chip 1 for having completed the generation of digital pcr droplet to objective table 801, with PCR temperature controls
Device 700 connects.
Step 2), carries out a PCR temperature cycles, and process is：92~96 degree are warming up to, 25~40s is stopped, is cooled to 53
~57 degree, 25~35s is stopped, is warming up to 70~74 degree, stops 35~45s；
Step 3), objective apparatus 200 switch to minimum magnification objective；
Step 4), light supply apparatus, imaging device switch to the first dyestuff or the corresponding light path of probe.
The dimension of digital pcr chip in the horizontal direction referring to Figure 16, is carried out decile, is divided into one by step 5)
Serial width is the strip detection zone of W, by continuous scan mode, obtains the consecutive image of each detection zone, right
Digital pcr micro-fluidic chip image is continuously acquired, and the width W is 1mm~3mm；
Step 6), light supply apparatus 300, imaging device 500 switch to the corresponding light path of next dyestuff.Step is performed again
5) detection of the image of next fluorescent dye or probe, is completed；
Step 7) repeats step 6), until all fluorescent dyes or probe detection finish；
Step 8) objective apparatus 200 switches to time low range object lens；Repeat step 4), step 5), step 6), step 7)；
Step 9) repeats step 8), until the object lens switching of all multiplying powers finishes, all fluorescent dyes or probe have detected
Finish；
Step 10) repeats step 2)~step 9), and counts, and is often repeated once, and counts once, is more than N until counting；
The N represents amplification cycles number, is 30~40；
Step 11) completes all tests, and the qPCR for carrying out subsequent image processing and each digital pcr drop is calculated, statistics
Negative positive droplet number, and calculate concentration.
The utility model is described in further detail with reference to specific embodiment：
<Example 1>
The digital pcr micro-fluidic chip parameter of required detection is：It realizes double digital pcr, uses the first dyestuff and second
Dyestuff, excitation wavelength are respectively 488nm, 532nm, and wavelength of fluorescence is respectively 515nm and 560nm, the micro-fluidic core of droplet digital pcr
Piece is integrated with 48 monocyte sample detection structures, integral core length of a film 150mm, wide 100mm.
Referring to Figure 17, for this chip, propose that a kind of real-time fluorescence image for carvel-built digital pcr drop obtains
Device is taken, including：
Objective apparatus 200, two object lens 2011,2012 comprising 4X and 10X enlargement ratios；
Light supply apparatus 300, for the fluorescence excitation of digital pcr droplet, including two lasers 3011,3012, two groups fast
Door 3081,3082, two groups of lens 3021,3022 and a dichroic mirror 3091；Wherein, 3011 wavelength of laser be 488nm, laser
3012 wavelength of device is 532nm, and lens 3021,3022 have front end focal length 50mm, rear end focal length 47mm.
Focusing arrangement 400, for light source illumination and the coupling of fluorescence imaging, including 401, two groups of lens 402 of dichroic mirror,
403、404、405；The light that lens 402,403 send light source expands, and is allowed to be formed diameter 3mm circle hot spots and exposes to number
On PCR droplet micro-fluidic chips；The fluorescence shaping that lens 404,405 will be distributed from digital pcr droplet micro-fluidic chip, with into
As light path is combined, diameter 2mm image illuminations are made to image acquiring sensor 700.
Imaging optical path device 500, for the fluorescence imaging of digital pcr droplet, including two groups of optical filters 5021,5031,
5022、5032；With plus lens 501, optical filter fixed plate 505, turning joint 504, transmission device 506,507 groups of servomotor
Into.Wherein, filter out optical source wavelength optical filter 5031,5032 be respectively long wave pass filter, cutoff frequency be respectively 500nm,
540nm, fluorescent optical filter 5021,5022 respectively centre wavelength 515nm, 560nm, bandwidth 30nm optical filters, lens 501 have
Front end focal length 50mm, rear end focal length 47mm；
Image acquiring sensor 600, the fluoroscopic image for digital pcr droplet gather in real time, are sensed for Array CCD
Device；
PCR temperature control devices 700, for the temperature cycles control of digital pcr droplet, including temperature sensor 702, heating
Using Peltier as heating actuator 704 and cooling actuator 703, heat transfer fast 701；Wherein, 701 be to be formed by machining
Al-alloy parts, heating actuator 704 be resistive heater, cooling actuator 703 is fan, and temperature sensor 702 is thermoelectricity
Even sensor.
Objective table device for carrying digital pcr droplet micro-fluidic chip and its temperature control device, including objective table 801, is watched
Take motor 803,805, transmission device 802,804, position sensor 806,807.Wherein, servomotor 803,805 is two-phase four
Line step-servo motor, transmission device 802,804 are ball screw structure, and stroke is respectively 200mm and 150mm, position sensing
Device 806,807 is linear grating encoder, and range is 200mm and 150mm, precision 0.01mm.
Specific detection process is as follows：
Step 1) places the micro-fluidic chip for having completed the generation of digital pcr droplet to objective table, with PCR temperature control devices
Connect.
Step 2), carries out a PCR temperature cycles, and process is：92~96 degree are warming up to, 25~40s is stopped, is cooled to 53
~57 degree, 25~35s is stopped, is warming up to 70~74 degree, stops 35~45s；
Step 3), objective apparatus switch to minimum magnification objective；
Step 4), light supply apparatus, imaging device switch to the corresponding light path of the first dyestuff.
Digital pcr micro-fluidic chip is carried out decile by step 5) in two vertical direction of level, is several rectangles or rectangular
Shape region, each region area are S, and carry out droplet fluoroscopic image acquisition to each region.The rectangular area is width 2mm's
Square；
Step 6), light supply apparatus, imaging device switch to the corresponding light path of next dyestuff.Step 5) is performed again, is completed
The detection of the image of next fluorescent dye；
Step 7) repeats step 6), until all fluorescent dyes have been detected and finished；
Step 8), objective apparatus switch to time low range object lens；Repeat step 4), step 5), step 6), step 7)；
Step 9) repeats step 8), until the object lens switching of all multiplying powers finishes, all fluorescent dye detections finish；
Step 10) repeats step 2)~step 9), and counts, and is often repeated once, and counts once, is more than N until counting；
The N represents amplification cycles number, is 30~40；
Step 11) completes all tests, and the qPCR for carrying out subsequent image processing and each digital pcr drop is calculated, statistics
Negative positive droplet number, and calculate concentration.
<Example 2>
The digital pcr micro-fluidic chip parameter of required detection is：Sixfold digital pcr, dyestuff used are respectively：
Fluorescent dye 1：Excitation wavelength 390nm, wavelength of fluorescence 420nm；
Fluorescent dye 2：Excitation wavelength 475nm, wavelength of fluorescence 522nm；
Fluorescent dye 3：Excitation wavelength 525nm, wavelength of fluorescence 577nm；
Fluorescent dye 4：Excitation wavelength 572nm, wavelength of fluorescence 628nm；
Fluorescent dye 5：Excitation wavelength 630nm, wavelength of fluorescence 675nm；
Fluorescent dye 6：Excitation wavelength 650nm, wavelength of fluorescence 710nm；
Droplet digital pcr micro-fluidic chip is integrated with 48 monocyte sample detection structures, integral core length of a film 150mm, wide
100mm.For this chip, referring to Figure 18, a kind of real-time fluorescence image acquisition for carvel-built digital pcr drop is proposed
Device, including：
Objective apparatus 200, two object lens 2011,2012 comprising 4X and 10X enlargement ratios；
Light supply apparatus 300, for the fluorescence excitation of digital pcr droplet, including halogen tungsten lamp light source 301, lens 302 filter
Piece fixed plate 305, turning joint 3041, transmission device 306, servomotor 307 and the filter in optical filter fixed plate 305
Mating plate 3031,3032,3033,3034,3035,3036, is shown in Figure 19.Filter center wavelength be respectively 390nm, 475nm,
525nm, 572nm, 630nm, 650nm, bandwidth 20nm；Wherein, lens 302 have front end focal length 50mm, rear end focal length 47mm.It watches
Motor 307 is taken as four line step-servo motor of two-phase, transmission device 306 is belt wheel transmission mechanism, and turning joint is rolling bearing；
Focusing arrangement 400, for light source illumination and the coupling of fluorescence imaging, including 401, two groups of lens 402 of dichroic mirror,
403、404、405；The light that lens 402,403 send light source expands, and is allowed to be formed diameter 3mm circle hot spots and exposes to number
On PCR droplet micro-fluidic chips；The fluorescence shaping that lens 404,405 will be distributed from digital pcr droplet micro-fluidic chip, with into
As light path is combined, diameter 2mm image illuminations are made to image acquiring sensor 700.
Imaging optical path device 500 for the fluorescence imaging of digital pcr droplet, includes filtering out optical source wavelength referring to Figure 20
Band resistance optical filter 5021,5022,5053,5024,5025,5026 and through droplet fluorescence fluorescent optical filter 5031,5032,
5033rd, 5034,5035,5036, plus lens 501, optical filter fixed plate 505, turning joint 504, transmission device 506, servo
Motor 507 forms.Wherein, cardiac wave in the band resistance optical filter 5021,5022,5053,5024,5025,5026 of optical source wavelength is filtered out
Long is respectively 390nm, 475nm, 525nm, 572nm, 630nm, 650nm, bandwidth 20nm, fluorescent optical filter 5031,5032,
5033rd, 5034,5035,5036 centre wavelengths are respectively 420nm, 522nm, 577nm, 628nm, 675nm, 710nm, bandwidth 20nm
Optical filter, lens 501 have front end focal length 50mm, rear end focal length 47mm；
Image acquiring sensor 700, the fluoroscopic image for digital pcr droplet gather in real time, are sensed for Array CCD
Device；
PCR temperature control devices 700, for the temperature cycles control of digital pcr droplet, including temperature sensor 702, heating
Using Peltier as heating actuator 704 and cooling actuator 703, heat transfer fast 701；Wherein, 701 be to be formed by machining
Al-alloy parts, heating actuator 704 be resistive heater, cooling actuator 703 is fan, and temperature sensor 702 is thermoelectricity
Even sensor.
Objective table device for carrying digital pcr droplet micro-fluidic chip and its temperature control device, including objective table 801, is watched
Take motor 803,805, transmission device 802,804, position sensor 806,807.Wherein, servomotor 803,805 is two-phase four
Line step-servo motor, transmission device 802,804 are ball screw structure, and stroke is respectively 200mm and 150mm, position sensing
Device 806,807 is linear grating encoder, and range is 200mm and 150mm, precision 0.01mm.
Specific detection process is as follows：
Step 1) places the micro-fluidic chip for having completed the generation of digital pcr droplet to objective table, with PCR temperature control devices
Connect.
Step 2), carries out a PCR temperature cycles, and process is：92~96 degree are warming up to, 25~40s is stopped, is cooled to 53
~57 degree, 25~35s is stopped, is warming up to 70~74 degree, stops 35~45s；
Step 3), objective apparatus switch to minimum magnification objective；
Step 4), light supply apparatus, imaging device switch to the corresponding light path of the first dyestuff.
The dimension of digital pcr chip in the horizontal direction is carried out decile by step 5), and being divided into a series of width is
The strip detection zone of W passes through continuous scan mode, the consecutive image of acquisition each detection zone, to digital pcr miniflow
Control chip image is continuously acquired.The width W is 2mm；
Step 6), light supply apparatus, imaging device switch to the corresponding light path of next dyestuff.Step 5) is performed again, is completed
The detection of the image of next fluorescent dye；
Step 7) repeats step 6), until all fluorescent dyes have been detected and finished；
Step 8), objective apparatus switch to time low range object lens；Repeat step 4), step 5), step 6), step 7)；
Step 9) repeats step 8), until the object lens switching of all multiplying powers finishes, all fluorescent dye detections finish；
Step 10) repeats step 2)~step 9), and counts, and is often repeated once, and counts once, is more than N until counting；
The N represents amplification cycles number, is 30~40；
Step 11) completes all tests, and the qPCR for carrying out subsequent image processing and each digital pcr drop is calculated, statistics
Negative positive droplet number, and calculate concentration.
Number of devices and treatment scale described herein are the explanations for simplifying the utility model.To the utility model
Large area digital pcr droplet fluorescence high pass amount detecting device and the application of method, modifications and variations to those skilled in the art
It is obvious for member.
Although the embodiment of the utility model is disclosed as above, it is not restricted in specification and embodiment
Listed utilization.It can be applied to the field of various suitable the utility model completely.For those skilled in the art,
Other modification is easily achieved.Therefore without departing from the general concept defined in the claims and the equivalent scope, this reality
Specific details is not limited to new and shown here as the legend with description.
Claims (12)
1. a kind of large area digital pcr droplet fluorescence high pass amount detecting device, which is characterized in that including fluorescence excitation light path and glimmering
Light light path；
Along the fluorescence excitation light path, line focus device and objective apparatus are incident to and are placed in the exciting light of light supply apparatus transmitting successively
On the digital pcr chip of objective table；
Along the fluorescence detection optical path, the digital pcr chip is filled by the fluorescence that the exciting light generates through the object lens successively
It puts, be transmitted to image acquiring sensor after the imaging of the focusing arrangement and imaging device；
Wherein, wherein the objective table is additionally provided with temperature control device, the temperature control device controls the digital pcr chip to react in real time
Circulating temperature.
2. large area digital pcr droplet fluorescence high pass amount detecting device as described in claim 1, which is characterized in that the load
Object platform is fixed on optical circuit path or at least there are one translation freedoms for tool.
3. large area digital pcr droplet fluorescence high pass amount detecting device as described in claim 1, which is characterized in that the object
Lens device includes：
One object lens, is fixed in light path；
Or the object lens of several different multiplyings, it is configured respectively into light path in the form of changeable.
4. large area digital pcr droplet fluorescence high pass amount detecting device as claimed in claim 3, which is characterized in that the object
The multiplying power of mirror is selected from 2X, 4X, 5X or 10X.
5. large area digital pcr droplet fluorescence high pass amount detecting device as described in claim 1, which is characterized in that the light
Source device includes：
Light source assembly provides parallel mixing light source；
Light source optical filter box, has：
One light source optical filter, be fixed in the fluorescence excitation light path or
Several light source optical filters are configured in the form of changeable respectively into the fluorescence excitation light path.
6. large area digital pcr droplet fluorescence high pass amount detecting device as described in claim 1, which is characterized in that the light
Source device includes several sets of monochromatic light sources；
Along light path direction of advance, the sets of monochromatic light sources includes successively：Monochromatic source, shutter and light lens；Wherein, it is described fast
Door break-make controls the fluorescence excitation light path.
7. large area digital pcr droplet fluorescence high pass amount detecting device as claimed in claim 6, which is characterized in that the light
Source device includes：
At least two sets of monochromatic light sources；
The transmitting light path of the sets of monochromatic light sources is closed beam and imports the fluorescence excitation light path by several first dichronic mirrors
In.
8. large area digital pcr droplet fluorescence high pass amount detecting device as described in claim 1, which is characterized in that focus on dress
Put including：
Second dichronic mirror, the exciting light for reflecting the light supply apparatus transmitting are excited to generate through the digital pcr chip simultaneously
Fluorescence.
9. large area digital pcr droplet fluorescence high pass amount detecting device as claimed in claim 8, which is characterized in that focus on dress
It puts and further includes：
First focus lens assembly is arranged between the light supply apparatus and second dichronic mirror, for shaping and expands institute
State exciting light.
10. large area digital pcr droplet fluorescence high pass amount detecting device as claimed in claim 9, which is characterized in that focus on dress
It puts and further includes：
Second focus lens assembly is arranged between second dichronic mirror and the imaging device, for shaping and expands institute
State fluorescence.
11. large area digital pcr droplet fluorescence high pass amount detecting device as described in claim 1, which is characterized in that along described
Fluorescence detection optical path, the imaging device include single fluorescence filtering assembly and fluorescence imaging lens successively；
Wherein, the fluorescence filtering assembly is fixedly arranged on the fluorescence detection optical path, and the fluorescence filtering assembly has：
Light source light blocking piece filters out the exciting light of the light supply apparatus transmitting；And
Fluorescent optical filter filters out the stray light of the wavelength of the non-fluorescence.
12. large area digital pcr droplet fluorescence high pass amount detecting device as described in claim 1, which is characterized in that along described
Fluorescence detection optical path, the imaging device include several fluorescence filtering assemblies and fluorescence imaging lens successively；
Wherein, several described fluorescence filtering assemblies include：
The light source light blocking piece of several different wave lengths, is respectively configured in the form of changeable to the fluorescence detection optical path, with
The exciting light of the light supply apparatus transmitting is filtered out respectively；And
The fluorescent optical filter of several different wave lengths is respectively configured in the form of changeable to the fluorescence detection optical path, with
The stray light of the wavelength of the non-fluorescence is filtered out respectively.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107478629A (en) * | 2017-09-04 | 2017-12-15 | 中国科学院苏州生物医学工程技术研究所 | A kind of large area digital pcr droplet fluorescence high pass amount detecting device and method |
| CN108489957A (en) * | 2018-06-07 | 2018-09-04 | 江苏大学 | A kind of Portable fluorescence detection device and method |
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| CN115901702A (en) * | 2022-11-02 | 2023-04-04 | 苏州中科医疗器械产业发展有限公司 | Digital microdroplet quantitative detection system, detection method and medium |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107478629A (en) * | 2017-09-04 | 2017-12-15 | 中国科学院苏州生物医学工程技术研究所 | A kind of large area digital pcr droplet fluorescence high pass amount detecting device and method |
| CN108489957A (en) * | 2018-06-07 | 2018-09-04 | 江苏大学 | A kind of Portable fluorescence detection device and method |
| CN111560310A (en) * | 2020-05-29 | 2020-08-21 | 上海交通大学 | Random access type digital nucleic acid detection device and use method |
| CN111560310B (en) * | 2020-05-29 | 2023-01-03 | 上海交通大学 | Random access type digital nucleic acid detection device and use method |
| CN112414983A (en) * | 2020-11-05 | 2021-02-26 | 北京中科生仪科技有限公司 | Biological detection method based on excitation light source |
| CN115901702A (en) * | 2022-11-02 | 2023-04-04 | 苏州中科医疗器械产业发展有限公司 | Digital microdroplet quantitative detection system, detection method and medium |
| CN115901702B (en) * | 2022-11-02 | 2024-02-09 | 苏州中科医疗器械产业发展有限公司 | Digital microdroplet quantitative detection system, detection method and medium |
| CN115598103A (en) * | 2022-11-16 | 2023-01-13 | 上海芯像生物科技有限公司（Cn） | Optical assembly and fluorescence microscopic detection system |
| CN115598103B (en) * | 2022-11-16 | 2023-03-24 | 上海芯像生物科技有限公司 | Optical assembly and fluorescence microscopic detection system |
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