CN209039495U - Digital PCR system - Google Patents
Digital PCR system Download PDFInfo
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- CN209039495U CN209039495U CN201821298506.5U CN201821298506U CN209039495U CN 209039495 U CN209039495 U CN 209039495U CN 201821298506 U CN201821298506 U CN 201821298506U CN 209039495 U CN209039495 U CN 209039495U
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- digital pcr
- pcr system
- spray orifice
- drop
- droplet formation
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Abstract
The utility model provides a digital PCR system, this digital PCR system include that the liquid drop forms subassembly and liquid drop orifice subassembly, liquid drop orifice subassembly connect in liquid drop forms subassembly below, the liquid drop form the subassembly including the apron and connect in at least one annular step of apron lower surface, liquid drop orifice subassembly includes a plurality of liquid drop orifices, wherein, the upper surface of liquid drop orifice subassembly the lower surface of apron reaches annular step encloses into the liquid drop formation cavity jointly, be equipped with vaporization part in the liquid drop orifice, be used for making digital PCR solution liquid layer vaporization in the liquid drop orifice pushes fast in the liquid drop formation oil in the liquid drop formation cavity to form digital PCR liquid drop. The utility model discloses use the hot bubble technique to carry out high-speed digital PCR liquid drop formation, have efficient digital PCR oil utilization rate to can carry out normal position PCR control by temperature change and normal position digital PCR signal collection.
Description
Technical field
The utility model belongs to biomedicine field, especially disease detection field, is related to a kind of integration number in situ
PCR system.
Background technique
Polymerase chain reaction (polymerase chain reaction, PCR) proposes have 20 years, phase so far
Between PCR have evolved into a key technology and routine techniques of molecular biology field, greatly pushed life science each
The development in a field.The especially later period nineties, American AB I company release real-time fluorescence quantitative PCR (real time PCR,
QPCR) technology and Related product are even more by PCR by synthesizing in vitro and qualitative/half-quantitative detection technology develops into a kind of Gao Ling
The gene analysis technique of quick, high specific and accurate quantification.
Although qPCR technology has been used to own in addition to wound and deficiency disease by the rapid development of more than ten years
The diagnosis of disease still influences many because being known as of its amplification efficiency, it cannot be guaranteed that in reaction process during PCR amplification
Middle amplification efficiency remain unchanged the amplification efficiency between actual sample and standard sample and different samples be it is identical, thus
It is directed at the basis that its quantitative analysis is relied on --- cycle threshold (CT) is not constant between.Therefore the quantitative of qPCR is
Still it is impossible to meet the requirements of molecular biology quantitative analysis for " relative quantification ", accuracy and reproducibility.
At the end of the 20th century, Vogelstein etc. proposes the concept of digital pcr (digital PCR, dPCR), by by a sample
Originally it is divided into tens to tens of thousands of parts, is assigned to different reaction members, each unit includes the target molecule of one or more copies
(DNA profiling) carries out PCR amplification to target molecule respectively in each reaction member, to each reaction member after amplification
Fluorescence signal carry out statistical analysis.Unlike qPCR, digital pcr is independent of CT value, therefore not by amplification efficiency
It influences, by directly counting or Poisson distribution formula calculates the mean concentration (content) of each reaction member after amplification,
Can be by control errors within 5%, digital pcr can not need reference standard sample and standard curve to realize absolute quantitation
Analysis.
Digital pcr (can also claim single-molecule PCR) generally comprises two parts content, i.e. PCR amplification and fluorescence signal analysis.?
The PCR amplification stage, different from traditional technology, digital pcr, which is generally required, is diluted to single molecules level for sample, and is evenly distributed to
Tens are reacted into tens of thousands of a units.Real-time fluorescence method for measuring, digital pcr are carried out to each circulation different from qPCR
Technology is to be acquired after amplification to the fluorescence signal of each reaction member.Finally by directly counting or Poisson distribution
The original concentration or content of sample is calculated in formula.
Since digital pcr is that a kind of nucleic acid molecules absolute quantitation technology compared to qPCR can directly count DNA molecular
Number, be the absolute quantitation to initial sample, be therefore particularly suitable for the application field that cannot be differentiated very well by CT value, example
Such as copy number variation, abrupt climatic change, gene relative expression research (such as allele imbalance express), two generation sequencing results are tested
Card, miRNA expression analysis, unicellular gene expression analysis etc..
At present there are mainly three types of digital pcr technologies on the market.One is by using the oil of flowing in particular instrument
The PCR solution for cutting off water phase forms drop, and PCR and detection are then completed in two other instruments；One is by by PCR
Solution is distributed on the silicon wafer hollowed out, then carries out being detected in PCR and an other instrument in particular instrument；Finally
One is liquid is formed drop by narrow Channeling implantation cavity on a kind of instrument, and PCR is completed, then at another
Detection is completed in instrument.However, the droplet formation speed or flux of three kinds of current methods are each restricted.In addition, above-mentioned three
The more large-scale instruments of dependence of kind technology without exception.This not only increases the cost of instrument purchased, and limits digital pcr
It is widely used；And increase the complexity of experimental implementation.
Therefore, how digital pcr droplet formation technology, the liquid of a kind of high speed greater than 1000 drops of formation per second are provided
Drop forms the situ PCR integrated with PCR temperature control and detecting instrument, efficient digital pcr oil utilization rate method, becomes ability
A field technique personnel important technological problems urgently to be resolved.
Utility model content
In view of the foregoing deficiencies of prior art, the purpose of this utility model is to provide a kind of integration numbers in situ
PCR system and droplet formation method, for solving, droplet formation speed is slow in the prior art, flux is small, complicated for operation, PCR is oily
The low problem of utilization rate.
In order to achieve the above objects and other related objects, the utility model provides a kind of digital pcr system, comprising:
Droplet formation component including cover board and is connected at least one ring-shaped step of the cover board lower surface；
Drop spray orifice component is connected to below the droplet formation component, including several drop spray orifices, the drop spray orifice
It is open, and extends toward the lower surface direction of the drop spray orifice component, but do not run through from the upper surface of the drop spray orifice component
The lower surface of the drop spray orifice component, wherein the upper surface of the drop spray orifice component, the lower surface of the cover board and described
Ring-shaped step surrounds droplet formation chamber, the drop spray orifice and the droplet formation chamber jointly, and the drop sprays
Vaporization member is equipped in hole, for vaporizing the digital pcr aqueous liquids layer in the drop spray orifice and being quickly pushed into the liquid
Drop is formed in the oil of the droplet formation in chamber, to form digital pcr drop.
Optionally, the drop spray orifice component includes thermal printing chip.
Optionally, the height of the ring-shaped step is less than 2 times of the diameter for the digital pcr drop to be formed, so as to obtain
Digital pcr drop one layer of structure is laid into the droplet formation chamber.
Optionally, the droplet formation component further includes at least one droplet formation oil injection hole, the droplet formation oil
Cover board described in injection hole break-through, with the droplet formation chamber.
Optionally, the droplet formation component further includes at least one droplet formation chamber exhaust hole, the droplet formation
Cover board described in chamber exhaust hole break-through, with the droplet formation chamber.
Optionally, the vaporization member is set to bottom surface or the side wall of the drop spray orifice.
Optionally, the opening shape of the drop spray orifice includes round, ellipse, any one in polygon.
Optionally, the vaporization member includes heating element, makes its vapour by heating the digital pcr aqueous liquids layer
Change.
Optionally, the heating element includes at least one layer of metal layer.
Optionally, the PCR system further includes at least one PCR reagent chamber for being used to store digital pcr solution, described
Runner is equipped in drop spray orifice component, the drop spray orifice passes through the runner and the PCR reagent chamber.
Optionally, the runner includes at least one sprue and a plurality of branch flow passage that connect with the sprue, each
The drop spray orifice is connect with a branch flow passage respectively.
Optionally, the digital pcr system further includes pedestal, and the upper surface of PCR reagent chamber from the pedestal is opened
Mouthful, and extend toward the lower surface direction of the pedestal, but do not run through the lower surface of the pedestal, the drop spray orifice component connection
In the pedestal upper surface, and cover the opening of the PCR reagent chamber.
Optionally, the lower surface of the pedestal is equipped at least one digital pcr solution injection hole, the digital pcr solution
Injection hole and the PCR reagent chamber.
Optionally, the lower surface of the pedestal is equipped at least one PCR reagent chamber exhaust hole, the PCR reagent chamber
Gas vent and the PCR reagent chamber.
Optionally, the digital pcr system further includes flexible circuit board, and the flexible circuit board is connected on the pedestal
It is square, the through-hole for accommodating the drop spray orifice component is equipped in the flexible circuit board, and the flexible circuit board surface is set
There are several first connection weld pads and several second connection weld pads, the drop spray orifice component to connect weldering with described first by conducting wire
Pad connection.
Optionally, the flexible circuit board is connect by adhesive means with the pedestal.
Optionally, the cross-sectional area of the drop spray orifice component is greater than the opening area of the PCR reagent chamber, the base
Seating face is equipped at least one for preventing glue from flowing to the drop spray orifice by the region that the drop spray orifice component is covered
Channel on component.
Optionally, the base-plates surface is equipped with one for preventing glue from flowing to the annular ditch on the drop spray orifice component
Road, the annular channel surround the surrounding of the drop spray orifice component.
Optionally, in the flexible circuit board be equipped at least two positional punches, the base-plates surface be equipped with it is described fixed
The corresponding positioning protrusion of position punch position.
Optionally, the digital pcr system further includes a controller, and the controller includes controller housing and is located at institute
The controller circuit board in controller housing is stated, the controller housing has the supporting part for placing the pedestal, described
Supporting part surface is equipped with several circuit connection conductive pins connecting with the controller circuitry connecting plate, and the circuit connection is led
Acusector is corresponding with the second connection position of weld pad.
Optionally, one end of the pedestal is provided at least one limiting slot, and the controller housing is provided at least one
A locating part corresponding with the limiting slot.
Optionally, the pedestal is provided with a limiting through hole, and the limiting through hole runs through the front and the back side of the pedestal,
The controller housing is provided with locating part corresponding with the limiting through hole.
Optionally, the controller further includes a cover, and the cover is connect with the controller housing, for covering
State pedestal.
Optionally, the digital pcr system further includes the heating module for heating the droplet formation chamber.
Optionally, the heating module is integrated in the drop spray orifice component.
Optionally, the digital pcr system is also configured with the external heat radiation wind for cooling down for the droplet formation chamber
Fan.
Optionally, the digital pcr system is also configured with the external semiconductor for cooling down for the droplet formation chamber
Refrigerator.
Optionally, the digital pcr system further includes the temperature sensor for testing the droplet formation chamber temp.
Optionally, the temperature sensor is integrated in the drop spray orifice component.
Optionally, the digital pcr system is also configured with Systems for optical inspection, in the case where not shifting sample into
The detection of row PCR signal collection.
As described above, the digital pcr system of the utility model, has the advantages that
(1) the utility model carries out high-speed figure PCR droplet formation using thermal technology, and the quick formation of drop depends on
Vaporization member in drop spray orifice vaporizes the transient heating of nanometer grade thickness liquid level, thus by the number in drop spray orifice
PCR solution is quickly pushed into droplet formation oil to form digital pcr drop, compared to the shape of 100 drops each second on the market
At speed, the droplet formation technology in the utility model may be implemented to be greater than 1000 droplet formation speed per second.
(2) compared to it is oily mutually and the water phase associated movement method that generates drop, oil in the technical solution of the utility model
It is mutually static, therefore the consumption of oily phase is greatly reduced, and reduces 50% or so oily phase dosage.
(3) due to being integrated with accurate temperature control on the drop spray orifice component of silicon substrate or thermal printing chip, it is achieved that
Temperature control PCR in situ.And the optical system of integration can be detected in the case where not shifting sample.This had both reduced operation
Time improves the accuracy of detection also by reduction human error.
(4) digital pcr drop in situ can be laid into one layer of structure.
Detailed description of the invention
Fig. 1 is shown as the schematic perspective view of the digital pcr system of the utility model.
Fig. 2 is shown as the top view of the digital pcr system of the utility model.
Fig. 3 is shown as the bottom view of the digital pcr system of the utility model.
Fig. 4-Fig. 7 is shown as the side view of the digital pcr system of the utility model.
Fig. 8 is shown as the decomposition texture schematic diagram of the digital pcr system of the utility model.
Fig. 9 is shown as the front that drop spray orifice component is combined with flexible circuit board in the digital pcr system of the utility model
Schematic perspective view.
Figure 10 is shown as the back side that drop spray orifice component is combined with flexible circuit board in the digital pcr system of the utility model
Schematic perspective view.
Figure 11 is shown as the vertical view that drop spray orifice component is combined with flexible circuit board in the digital pcr system of the utility model
Figure.
What Figure 12 was shown as that drop spray orifice component in the digital pcr system of the utility model combines with flexible circuit board looks up
Figure.
Figure 13-Figure 16 is shown as drop spray orifice component in the digital pcr system of the utility model and combines with flexible circuit board
Side view.
Figure 17 is shown as the partial perspective view of drop spray orifice component in the digital pcr system of the utility model.
Figure 18 is shown as the partial cutaway view of drop spray orifice component in the digital pcr system of the utility model.
Figure 19 is shown as the schematic diagram of front three-dimensional structure of droplet formation component in the digital pcr system of the utility model.
Figure 20 is shown as the back side dimensional structure diagram of droplet formation component in the digital pcr system of the utility model.
Figure 21 is shown as the top view of droplet formation component in the digital pcr system of the utility model.
Figure 22 is shown as the bottom view of droplet formation component in the digital pcr system of the utility model.
Figure 23-Figure 26 is shown as the side view of droplet formation component in the digital pcr system of the utility model.
Figure 27 is shown as the schematic diagram of front three-dimensional structure of pedestal in the digital pcr system of the utility model.
Figure 28 is shown as the back side dimensional structure diagram of pedestal in the digital pcr system of the utility model.
Figure 29 is shown as the top view of pedestal in the digital pcr system of the utility model.
Figure 30 is shown as the bottom view of pedestal in the digital pcr system of the utility model.
Figure 31-Figure 34 is shown as the side view of pedestal in the digital pcr system of the utility model.
Figure 35 is shown as the schematic perspective view of controller in the digital pcr system of the utility model.
Figure 36 is shown as cover in the digital pcr system of the utility model and is taken away the controller top view presented afterwards.
Figure 37 is shown as controller housing bottom plate in the digital pcr system of the utility model and is taken away the control presented afterwards
Device bottom view.
Figure 38 is shown as external radiator fan in the digital pcr system of the utility model and is set to showing in controller housing
It is intended to.
Figure 39 is shown as the optical microscope of the digital pcr drop formed using the digital pcr system of the utility model.
Figure 40 is shown as the fluorogram of the digital pcr drop formed using the digital pcr system of the utility model.
Component label instructions
1 droplet formation component
2 cover boards
3 ring-shaped steps
4 drop spray orifice components
5 drop spray orifices
6 droplet formation oil injection holes
7 droplet formation chamber exhaust holes
8 PCR reagent chambers
9 sprues
10 branch flow passages
11 pedestals
12 digital pcr solution injection holes
13 PCR reagent chamber exhaust holes
14 flexible circuit board
15 through-holes
16 enclosure support structures
17 second connection weld pads
18 channels
19 annular channels
20 positional punches
21 positioning protrusion
22 controllers
23 controller housings
24 supporting parts
25 circuit connection conductive pins
26 limiting slots
27,29 locating part
28 limiting through hole
30 covers
31 external heat radiation fans
32 sink decks
33 protrusions
34 ventilation openings
35 circuit board tie points
Specific embodiment
Illustrate the embodiments of the present invention below by way of specific specific example, those skilled in the art can be by this theory
Content disclosed by bright book understands other advantages and effect of the utility model easily.The utility model can also be by addition
Different specific embodiments are embodied or practiced, and the various details in this specification can also be based on different viewpoints and answer
With carrying out various modifications or alterations under the spirit without departing from the utility model.
Please refer to Fig.1 to Fig.4 0.It should be noted that diagram provided in the present embodiment only illustrates this in a schematic way
The basic conception of utility model is only shown with related component in the utility model rather than when according to actual implementation in schema then
Component count, shape and size are drawn, when actual implementation kenel, quantity and the ratio of each component can arbitrarily change for one kind
Become, and its assembly layout kenel may also be increasingly complex.
Embodiment one
The utility model provides a kind of digital pcr system, please refers to Fig. 1 to Fig. 7, wherein Fig. 1 is shown as the number
The schematic perspective view of PCR system, Fig. 2 are shown as the top view of the digital pcr system, and Fig. 3 is shown as the digital pcr
The bottom view of system, Fig. 4, Fig. 5, Fig. 6 and Fig. 7 are shown as side view of the digital pcr system on four direction.
Referring to Fig. 8, being shown as the decomposition texture schematic diagram of the digital pcr system, it is seen then that the digital pcr system
Including droplet formation component 1 and drop spray orifice component 4, wherein the drop spray orifice component 4 is connected to the droplet formation component
1 lower section.
As an example, the drop spray orifice component 4 may include thermal printing chip.Thermal printing technique is printer field
A major technique, the basic principle is that ink droplet sprayed by heating.In the utility model, the drop spray orifice component 4
Existing thermal printing chip can be used.
In the present embodiment, the drop spray orifice component 4 is connected to a flexible circuit board 14.Fig. 9 to Figure 16 is please referred to,
In, Fig. 9 is shown as the drop spray orifice component 4 schematic diagram of front three-dimensional structure combined with the flexible circuit board 14, Figure 10
It is shown as the drop spray orifice component 4 the back side dimensional structure diagram combined with the flexible circuit board 14, Figure 11 is shown as
The drop spray orifice component 4 top view combined with the flexible circuit board 14, Figure 12 be shown as the drop spray orifice component 4 with
The combined bottom view of the flexible circuit board 14, Figure 13, Figure 14, Figure 15 and Figure 16 are shown as the drop spray orifice component 4 and institute
It states flexible circuit board 14 and combines the side view on four direction.
Specifically, being equipped with the through-hole 15 for accommodating the drop spray orifice component 4 in the flexible circuit board 14, and described
14 surface of flexible circuit board is equipped with several first connections weld pad (not shown) and several second connection weld pads 17, the drop spray orifice
Component 4 connect weld pad with described first by conducting wire and connects, with by the flexible circuit board 14 by the drop spray orifice component 4
It is connect with peripheral control unit.The drop spray orifice component 4 can pass through routing (Wire Bond) technique and described first of standard
Connect weld pad connection.
Specifically, as shown in Fig. 9 and Figure 11, the drop spray orifice component 4 includes several drop spray orifices 5, in the present embodiment,
The drop spray orifice 4 is arranged in two rows at one end close to the drop spray orifice component 4, and every row drop spray orifice uniformly divides
Cloth.In other embodiments, the drop spray orifice 4 can also use other arrangement modes, should not excessively limit herein practical
Novel protection scope.
Please refer to Figure 17 and Figure 18, wherein Figure 17 is shown as the partial perspective view of the drop spray orifice component 4, Tu18Xian
It is shown as the partial cutaway view of drop spray orifice component in the digital pcr system, it is seen then that the drop spray orifice 5 is sprayed from the drop
The upper surface of aperture member 4 is open, and extends toward the lower surface direction of the drop spray orifice component 4, but does not spray through the drop
The lower surface of aperture member 4.The opening shape of the drop spray orifice 5 includes but is not limited to round, oval, any in polygon
It is a kind of.
Figure 19 is please referred to Figure 26, wherein Figure 19 is shown as the positive stereochemical structure signal of the droplet formation component 1
Figure, Figure 20 are shown as the back side dimensional structure diagram of the droplet formation component 1, and Figure 21 is shown as the droplet formation component
1 top view, Figure 22 are shown as the bottom view of the droplet formation component 1, and Figure 23, Figure 24, Figure 25 and Figure 26 are shown as described
Side view of the droplet formation component 1 on four direction.
Specifically, the droplet formation component 1 includes cover board 2 and at least one ring for being connected to 2 lower surface of cover board
Shape step 3.It should be pointed out that annular herein not merely represents in annulus, such as the present embodiment, the ring-shaped step 3
Exterior contour rectangle is presented, round rectangle is presented in the in-profile of the ring-shaped step 3.In other embodiments, the ring
Shape step 3 or other shapes should not excessively limit the guarantor of the utility model as long as step is end to end herein
Protect range.
Specifically, the upper surface of the drop spray orifice component 4, the lower surface of the cover board 2 and the ring-shaped step 3 are common
Surround droplet formation chamber, the drop spray orifice 5 and the droplet formation chamber
As shown in figure 18, it is equipped with vaporization member 34 in the drop spray orifice 5, for making the number in the drop spray orifice 5
In the vaporization of PCR aqueous liquids layer and the droplet formation oil being quickly pushed into the droplet formation chamber, to form digital pcr liquid
Drop.Wherein, the volume of the drop spray orifice 5 determines the volume of digital pcr drop to be formed.
As an example, the vaporization member 34 is set to the bottom surface of the drop spray orifice 5, the vaporization member 34 can be adopted
With heating element, make its vaporization by heating the digital pcr aqueous liquids layer.In the present embodiment, the heating element includes
Heating sheet, the heating sheet can be single metal layer, be also possible to composite multi-layer metal layer.The shape of the vaporization member 34
Including but not limited to round or rectangular, area can be 0.5~2 times of 5 floor space of drop spray orifice.In other embodiments
In, the vaporization member 34 also can be set in the side wall of the drop spray orifice 5, should not excessively limit the utility model herein
Protection scope.
In the present embodiment, the height of the ring-shaped step 3 is less than 2 times of the diameter for the digital pcr drop to be formed, so that
Obtained digital pcr drop is laid into one layer of structure in the droplet formation chamber.
Specifically, the droplet formation component 1 further includes the injection of at least one droplet formation oil as shown in Figure 21 and Figure 22
Hole 6, cover board 2 described in 6 break-through of droplet formation oil injection hole, with the droplet formation chamber.The droplet formation group
Part 1 further includes at least one droplet formation chamber exhaust hole 7, cover board described in the same break-through of the droplet formation chamber exhaust hole 7
2, with the droplet formation chamber.
As shown in figure 8, the PCR system further includes at least one PCR reagent chamber 8 for being used to store digital pcr solution.
As shown in figure 17, runner is equipped in the drop spray orifice component 4, the drop spray orifice 5 passes through the runner and the PCR reagent
Chamber 8 is connected to.
As an example, the runner includes at least one sprue 9 and a plurality of branch flow passage that connect with the sprue 9
10, each drop spray orifice 5 is connect with a branch flow passage 10 respectively.Show to be the drop spray in Figure 10 and Figure 17
Aperture member 4 includes the situation of a sprue 9, and in other embodiments, the quantity of the sprue 9 can also be with the drop
The quantity for forming chamber matches, that is, matches with the quantity of the ring-shaped step 3.Figure 20 shows to be the drop shape
It include the situation of a ring-shaped step 3 at component 1, in other embodiments, the quantity of the ring-shaped step 3 may be more
It is a, to construct multiple droplet formation chambers.
As an example, the material for constructing the runner and the drop spray orifice 5 includes but is not limited to silicon, polymer, photoetching
Glue etc..
Specifically, as shown in Fig. 1 and Fig. 8, the digital pcr system further includes pedestal 11, and the PCR reagent chamber 8 is set
It is placed in the pedestal 11.As an example, the material of the pedestal 11 includes but is not limited to transparent or opaque plastics, glass
In any one, the pedestal 11 can also use metal material.
Figure 27 is please referred to Figure 34, wherein Figure 27 is shown as the schematic diagram of front three-dimensional structure of the pedestal, and Figure 28 is shown
For the back side dimensional structure diagram of the pedestal, Figure 29 is shown as the top view of the pedestal, and Figure 30 is shown as the pedestal
Bottom view, Figure 31, Figure 32, Figure 33 and Figure 34 are shown as side view of the pedestal on four direction.
Specifically, the PCR reagent chamber 8 is open from the upper surface of the pedestal 11, and the following table of the past pedestal 11
Face direction extends, but does not run through the lower surface of the pedestal 11, and the drop spray orifice component 4 is connected to table on the pedestal 11
Face, and cover the opening of the PCR reagent chamber 8.
Specifically, the lower surface of the pedestal 11 is equipped at least one digital pcr solution injection hole 12, the digital pcr
Solution injection hole 12 is connected to the PCR reagent chamber 8.The lower surface of the pedestal is additionally provided at least one PCR reagent chamber
Gas vent 13, the PCR reagent chamber exhaust hole 13 are connected to the PCR reagent chamber 8.
Specifically, the flexible circuit board 14 is connected to 11 top of pedestal.As an example, the flexible circuit board is logical
Adhesive means (for example, by using glue or double-sided adhesive) are crossed to connect with the pedestal 11.In the present embodiment, the drop spray orifice component 4
Cross-sectional area be greater than the PCR reagent chamber 8 opening area, as shown in Figure 27 and Figure 29,11 surface of pedestal is by institute
It states the region that drop spray orifice component 4 is covered and is equipped at least one for preventing glue from flowing to the ditch on the drop spray orifice component
Road 18.In the present embodiment, the channel 18 uses straight channels, and quantity is multiple.
As an example, it is described for preventing glue from flowing to that 11 surface of pedestal is additionally provided with one as shown in Figure 27 and Figure 29
Annular channel 19 on drop spray orifice component, the annular channel surround the surrounding of the drop spray orifice component.
In the present embodiment, 11 surface of pedestal has a sink deck 32 for being used to accommodate flexible circuit board, and institute
The quadrangle for stating sink deck 32 has arc-shaped wider space, and the protrusion 33 of 32 surrounding of sink deck is described soft
Property wiring board plays positioning action when pasting 32 surface of sink deck.
As shown in Figure 10, at least two positional punches 20, as shown in figure 29, the base are equipped in the flexible circuit board 14
11 surfaces of seat are equipped with positioning protrusion 21 corresponding with 20 position of positional punch.
Specifically, the digital pcr system further includes a controller, Figure 35 is please referred to, is shown as the controller 22
Schematic perspective view, the controller circuit board (not shown) including controller housing 23 and in the controller housing.
In the present embodiment, the controller 22 further includes a cover 30, and the cover 30 is connect with the controller housing 22, for hiding
The pedestal 11 is covered, provides a shading environment for PCR reaction.
Figure 36 is please referred to, the cover 30 is shown as and is taken away the controller top view presented afterwards, it is seen that the control
Device shell 23 has the supporting part 24 for placing the pedestal 11, and 24 surface of supporting part is equipped with several and controller
The circuit connection conductive pin 25 (also referred to as Pin) of circuit connecting plate connection, and the circuit connection conductive pin 25 and described second
The position for connecting weld pad 17 is corresponding.
Figure 37 is please referred to, controller housing bottom plate is shown as and is taken away the controller bottom view presented afterwards, wherein is described
24 back side of supporting part is equipped with several circuit board tie points 35 corresponding with the circuit connection conductive pin 25, controller circuit board
The circuit connection conductive pin 25 can be outputed signal to by the circuit board tie point 35.
Specifically, as shown in figure 28, one end of the pedestal 11 is provided at least one limiting slot 26, as shown in figure 36,
The controller housing 23 is provided at least one locating part 29 corresponding with the limiting slot 26.The locating part 29 can be adopted
Use spring-loaded plunger.
Specifically, as shown in figure 28, the pedestal 11 is provided with a limiting through hole 28, and the limiting through hole 28 is through described
The front and the back side of pedestal, as shown in figure 36, the controller housing 23 are provided with limit corresponding with the limiting through hole 28
Position part 29.
Specifically, the digital pcr system further includes the heating module for heating the droplet formation chamber, to provide
The reaction condition of specific temperature.As an example, the heating module is integrated in the drop spray orifice component 4.
Specifically, the digital pcr system further includes the temperature sensor for testing the droplet formation chamber temp,
To monitor the indoor temperature of droplet formation chamber.As an example, the temperature sensor is integrated in the drop spray orifice component 4.
Specifically, the digital pcr system is also configured with the external heat radiation wind for cooling down for the droplet formation chamber
Fan.As an example, the external heat radiation fan may be disposed in the controller housing 23.Figure 38 is please referred to, is shown as described
External heat radiation fan 31 is set to the schematic diagram in controller housing 23, is additionally provided with ventilation opening by the external heat radiation fan 31
34.Enclosure support structure 16 is also shown in Figure 38.
Specifically, the external heat radiation fan can also be replaced using external semiconductor cooler.Semiconductor cooler
(Thermo Electric Cooler, abbreviation TEC) is made of the Peltier effect using semiconductor material.So-called Peltier
Effect refers to when the galvanic couple that DC current is made up of two kinds of semiconductor materials, one end heat absorption, the phenomenon that the heat release of one end.
The N-type of heavy doping and the bismuth telluride of p-type are mainly used as the semiconductor material of TEC, and Bismuth Telluride elements are simultaneously using electrically coupled in series
Row fever.TEC includes some p-types and N-type to (group), they are connected together by electrode, and be clipped in two ceramic electrodes it
Between；When there is electric current to flow through from TEC, the heat that electric current generates can pass to the other side from the side of TEC, generate " heat " on TEC
Side and " cold " side, here it is the heating of TEC and refrigeration principles.
Further, the digital pcr system is also configured with Systems for optical inspection, in the case where not shifting sample
Carry out the detection of PCR signal collection.The major part of optical system includes: that fluorescent light source, bright field light source, control circuit, optics are put
Big lens set, fluorescent optical filter, CCD camera, the slide unit system for moving lens, and the shell for being protected from light.Optics
The photograph shooting area of system is the entire area of cover board.This shooting can be Polaroid or multiple shooting and spliced map
Piece.
The digital pcr system of the utility model can be used for the formation of digital pcr drop, and the quick formation of drop depends on liquid
Instant vaporization of the vaporization member in spray orifice to nanometer grade thickness liquid level is dripped, thus by the digital pcr solution in drop spray orifice
Quickly to form digital pcr drop in push-in droplet formation oil, compared to the formation speed of 100 drops each second on the market,
Droplet formation technology in the utility model may be implemented to be greater than 1000 droplet formation speed per second.Compared to oily Xiang Yushui
The method that phase associated movement generates drop, the oil in the technical solution of the utility model is mutually static, therefore the consumption of oil phase
Amount is greatly reduced, and reduces 50% or so oily phase dosage, has efficient digital pcr oil utilization rate.Due to the liquid of silicon substrate
It is integrated with accurate temperature control on drop spray orifice component or thermal printing chip, it is achieved that temperature control PCR in situ.And the light of integration
System can be detected in the case where not shifting sample.This both reduces the operating time, also by reduction human error
Improve the accuracy of detection.Digital pcr drop in situ can be laid into one layer of structure.
Embodiment two
The present embodiment provides it is a kind of using digital pcr system described in embodiment one formed digital pcr drop method,
The following steps are included: being made in digital pcr solution boils and quickly push-in droplet formation oil using vaporization member, to form number
PCR drop.
As an example, carrying out high-speed figure PCR droplet formation using thermal technology, the vaporization member includes heating part
Part makes its vaporization by heating the digital pcr aqueous liquids layer.
Specifically, by the fever time of the control heating element, fever number and fever interval time to control
State the formation speed of digital pcr drop.It can achieve using the digital pcr droplet formation method of the utility model greater than 1000
The digital pcr droplet formation speed of a/second.
As an example, the digital pcr droplet formation method the following steps are included:
S1: injecting digital pcr solution into PCR reagent chamber, enters digital pcr solution and the PCR reagent chamber
The drop spray orifice of connection forms liquid level；
S2: droplet formation oil is added into droplet formation chamber；
S3: it vaporizes the liquid level using vaporization member and is quickly pushed into the drop in the droplet formation chamber
It is formed in oil, to form the digital pcr drop.
Specifically, the liquid level with a thickness of nanoscale, and be greater than 0.2nm, in the present embodiment, the thickness of the liquid level
Degree ranges preferably from 0.2nm~30000nm.
Specifically, the height of the droplet formation chamber is less than 2 times of the diameter for the digital pcr drop to be formed, so that
Obtained digital pcr drop is laid into one layer of structure in the droplet formation chamber.
It is formed specifically, being pushed completely into the droplet formation chamber to the indoor digital pcr solution of the PCR reagent chamber
After digital pcr drop, PCR reagent chamber droplet formation oil is filled, the PCR reagent chamber is made to be in filling shape
State is back to the PCR reagent chamber to avoid the drop of formation.Then droplet formation chamber can will be set to using sealing element
Droplet formation oil injection hole, droplet formation chamber exhaust hole on locular wall, the digital pcr being set on PCR reagent chamber wall are molten
Liquid injection hole and the sealing of PCR reagent chamber exhaust hole.The sealing element includes but is not limited to rubber plug, sealed membrane, cushion rubber ring, sealing
With cushion etc..The sealing element can be using flexible plastics materials such as rubber, PDMS.
Specifically, utilizing what is integrated on drop spray orifice component (can be thermal printing chip) after carrying out above-mentioned sealing
Heating and cooling needed for heating module and temperature sensor and external fan carry out PCR control, and realize temperature control PCR in situ.Root
It is needed according to certain specific temperature, fan can also be replaced to cool down with semiconductor refrigerating film.
Specifically, PCR signal receipts can also be carried out in the case where not shifting sample using the optical system of integration
Collection detection.
Figure 39 is please referred to, the optics for being shown as the digital pcr drop formed using the digital pcr system of the utility model is aobvious
Micro mirror figure, it is seen then that the digital pcr droplet morphology of formation is symmetrical, uniformly.
After forming drop by using the digital pcr of standard, is reacted by Standard PCR temperature control in situ, followed by 40
After ring, it is seen that have the positive drop of fluorescence signal.Figure 40 is please referred to, the digital pcr system shape using the utility model is shown as
At digital pcr drop fluorogram.
The digital pcr system of the utility model can satisfy the use of all digital pcr biochemical reagents.Due to many lifes
The concentration of the significant molecule of object in blood is very low (such as Circulating tumor DNA only has 3 DNA moleculars in every 2ml blood), and
The digital pcr system of the utility model has the characteristics that droplet formation quantity not by using oil mass to be limited and the characteristics of high speed,
So that this kind of detection being applied in order to possible in digital pcr.
In conclusion the digital pcr system of the utility model carries out high-speed figure PCR droplet formation using thermal technology,
Quick formed of drop vaporizes the transient heating of nanometer grade thickness liquid level dependent on the vaporization member in drop spray orifice, thus
Digital pcr solution in drop spray orifice is quickly pushed into droplet formation oil to form digital pcr drop, it is every compared on the market
The formation speed of second 100 drops, the droplet formation technology in the utility model may be implemented to be greater than 1000 liquid per second
Drop forms speed.Compared to it is oily mutually and the water phase associated movement method that generates drop, oil in the technical solution of the utility model
It is mutually static, therefore the consumption of oily phase is greatly reduced, and reduces 50% or so oily phase dosage.Due to the drop of silicon substrate
It is integrated with accurate temperature control on spray orifice component or thermal printing chip, it is achieved that temperature control PCR in situ.And the optics of integration
System can be detected in the case where not shifting sample.This both reduces the operating time, mentions also by human error is reduced
The high accuracy of detection.Digital pcr drop in situ can be laid into one layer of structure.So the utility model effectively overcomes
Various shortcoming in the prior art and have high industrial utilization value.
The above embodiments are only illustrative of the principle and efficacy of the utility model, and not for limitation, this is practical new
Type.Any person skilled in the art can all carry out above-described embodiment under the spirit and scope without prejudice to the utility model
Modifications and changes.Therefore, such as those of ordinary skill in the art without departing from the revealed essence of the utility model
All equivalent modifications or change completed under mind and technical idea, should be covered by the claim of the utility model.
Claims (30)
1. a kind of digital pcr system characterized by comprising
Droplet formation component including cover board and is connected at least one ring-shaped step of the cover board lower surface；
Drop spray orifice component is connected to below the droplet formation component, including several drop spray orifices, and the drop spray orifice is from institute
The upper surface opening of drop spray orifice component is stated, and is extended toward the lower surface direction of the drop spray orifice component, but not through described
The lower surface of drop spray orifice component, wherein the upper surface of the drop spray orifice component, the lower surface of the cover board and the annular
Step surrounds droplet formation chamber, the drop spray orifice and the droplet formation chamber jointly, and in the drop spray orifice
Equipped with vaporization member, for vaporizing the digital pcr aqueous liquids layer in the drop spray orifice and being quickly pushed into the drop shape
At in chamber droplet formation oil in, to form digital pcr drop.
2. digital pcr system according to claim 1, it is characterised in that: the drop spray orifice component includes thermal printing
Chip.
3. digital pcr system according to claim 1, it is characterised in that: the height of the ring-shaped step, which is less than, to be intended to be formed
2 times of diameter of digital pcr drop so that obtained digital pcr drop is laid into one layer in the droplet formation chamber
Structure.
4. digital pcr system according to claim 1, it is characterised in that: the droplet formation component further includes at least one
A droplet formation oil injection hole, cover board described in the droplet formation oil injection hole break-through, with the droplet formation chamber.
5. digital pcr system according to claim 1, it is characterised in that: the droplet formation component further includes at least one
A droplet formation chamber exhaust hole, cover board described in the droplet formation chamber exhaust hole break-through connect with the droplet formation chamber
It is logical.
6. digital pcr system according to claim 1, it is characterised in that: the vaporization member is set to the drop spray
The bottom surface in hole or side wall.
7. digital pcr system according to claim 1, it is characterised in that: the opening shape of the drop spray orifice includes circle
Shape, ellipse, any one in polygon.
8. digital pcr system according to claim 1, it is characterised in that: the vaporization member includes heating element, is passed through
Heating the digital pcr aqueous liquids layer makes its vaporization.
9. digital pcr system according to claim 8, it is characterised in that: the heating element includes at least one layer of metal
Layer.
10. digital pcr system according to claim 1, it is characterised in that: the PCR system further includes at least one use
Runner is equipped in the PCR reagent chamber of storage digital pcr solution, the drop spray orifice component, the drop spray orifice passes through institute
State runner and the PCR reagent chamber.
11. digital pcr system according to claim 10, it is characterised in that: the runner includes at least one sprue
And a plurality of branch flow passage connecting with the sprue, each drop spray orifice are connect with a branch flow passage respectively.
12. digital pcr system according to claim 10, it is characterised in that: the digital pcr system further includes pedestal,
The PCR reagent chamber is open from the upper surface of the pedestal, and extends toward the lower surface direction of the pedestal, but does not run through institute
The lower surface of pedestal is stated, the drop spray orifice component is connected to the pedestal upper surface, and covers opening for the PCR reagent chamber
Mouthful.
13. digital pcr system according to claim 12, it is characterised in that: the lower surface of the pedestal is equipped at least one
A digital pcr solution injection hole, the digital pcr solution injection hole and the PCR reagent chamber.
14. digital pcr system according to claim 12, it is characterised in that: the lower surface of the pedestal is equipped at least one
A PCR reagent chamber exhaust hole, the PCR reagent chamber exhaust hole and the PCR reagent chamber.
15. digital pcr system according to claim 12, it is characterised in that: the digital pcr system further includes flexible wire
Road plate, the flexible circuit board are connected to above the pedestal, are equipped in the flexible circuit board for accommodating the drop spray
The through-hole of aperture member, and the flexible circuit board surface is equipped with several first connection weld pads and several second connection weld pads, it is described
Drop spray orifice component connect weld pad connection with described first by conducting wire.
16. digital pcr system according to claim 15, it is characterised in that: the flexible circuit board passes through adhesive means
It is connect with the pedestal.
17. digital pcr system according to claim 16, it is characterised in that: the cross-sectional area of the drop spray orifice component
Greater than the opening area of the PCR reagent chamber, the base-plates surface is equipped with by the region that the drop spray orifice component is covered
At least one for preventing glue from flowing to the channel on the drop spray orifice component.
18. digital pcr system according to claim 16, it is characterised in that: the base-plates surface is equipped with one for preventing
Glue flows to the annular channel on the drop spray orifice component, and the annular channel surrounds the surrounding of the drop spray orifice component.
19. digital pcr system according to claim 15, it is characterised in that: be equipped at least two in the flexible circuit board
A positional punch, the base-plates surface are equipped with positioning protrusion corresponding with the positional punch position.
20. digital pcr system according to claim 15, it is characterised in that: the digital pcr system further includes a control
Device, the controller include controller housing and the controller circuit board in the controller housing, outside the controller
Shell has the supporting part for placing the pedestal, and the supporting part surface is equipped with several and controller circuitry connecting plate and connects
The circuit connection conductive pin connect, and the circuit connection conductive pin is corresponding with the second connection position of weld pad.
21. digital pcr system according to claim 20, it is characterised in that: one end of the pedestal is provided at least one
A limiting slot, the controller housing are provided at least one locating part corresponding with the limiting slot.
22. digital pcr system according to claim 20, it is characterised in that: the pedestal is provided with a limiting through hole, institute
Front and the back side that limiting through hole runs through the pedestal are stated, the controller housing is provided with corresponding with the limiting through hole
Locating part.
23. digital pcr system according to claim 20, it is characterised in that: the controller further includes a cover, described
Cover is connect with the controller housing, for covering the pedestal.
24. digital pcr system according to claim 1, it is characterised in that: the digital pcr system further includes for adding
The heating module of the heat droplet formation chamber.
25. digital pcr system according to claim 24, it is characterised in that: the heating module is integrated in the drop
In spray orifice component.
26. digital pcr system according to claim 24, it is characterised in that: the digital pcr system, which is also configured with, to be used for
For the external heat radiation fan of droplet formation chamber cooling.
27. digital pcr system according to claim 24, it is characterised in that: the digital pcr system, which is also configured with, to be used for
For the external semiconductor cooler of droplet formation chamber cooling.
28. digital pcr system according to claim 1, it is characterised in that: the digital pcr system further includes for surveying
Try the temperature sensor of the droplet formation chamber temp.
29. digital pcr system according to claim 28, it is characterised in that: the temperature sensor is integrated in the liquid
It drips in spray orifice component.
30. digital pcr system according to claim 1, it is characterised in that: the digital pcr system is also configured with optics
Detection system, for carrying out the detection of PCR signal collection in the case where not shifting sample.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201821298506.5U CN209039495U (en) | 2018-08-13 | 2018-08-13 | Digital PCR system |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201821298506.5U CN209039495U (en) | 2018-08-13 | 2018-08-13 | Digital PCR system |
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| Publication Number | Publication Date |
|---|---|
| CN209039495U true CN209039495U (en) | 2019-06-28 |
Family
ID=67030883
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| CN201821298506.5U Active CN209039495U (en) | 2018-08-13 | 2018-08-13 | Digital PCR system |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110628879A (en) * | 2019-11-04 | 2019-12-31 | 上海新微技术研发中心有限公司 | Digital PCR (polymerase chain reaction) liquid drop forming method |
| CN110628610A (en) * | 2019-11-04 | 2019-12-31 | 上海新微技术研发中心有限公司 | Digital PCR system |
| CN110628609A (en) * | 2019-11-04 | 2019-12-31 | 上海新微技术研发中心有限公司 | Digital PCR system |
| CN110669661A (en) * | 2019-11-04 | 2020-01-10 | 上海新微技术研发中心有限公司 | Digital PCR (polymerase chain reaction) liquid drop forming method |
| CN110699250A (en) * | 2019-11-04 | 2020-01-17 | 上海新微技术研发中心有限公司 | Digital PCR system |
-
2018
- 2018-08-13 CN CN201821298506.5U patent/CN209039495U/en active Active
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110628879A (en) * | 2019-11-04 | 2019-12-31 | 上海新微技术研发中心有限公司 | Digital PCR (polymerase chain reaction) liquid drop forming method |
| CN110628610A (en) * | 2019-11-04 | 2019-12-31 | 上海新微技术研发中心有限公司 | Digital PCR system |
| CN110628609A (en) * | 2019-11-04 | 2019-12-31 | 上海新微技术研发中心有限公司 | Digital PCR system |
| CN110669661A (en) * | 2019-11-04 | 2020-01-10 | 上海新微技术研发中心有限公司 | Digital PCR (polymerase chain reaction) liquid drop forming method |
| CN110699250A (en) * | 2019-11-04 | 2020-01-17 | 上海新微技术研发中心有限公司 | Digital PCR system |
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