CN110069084A - Temperature control device - Google Patents
Temperature control device Download PDFInfo
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- CN110069084A CN110069084A CN201810070360.7A CN201810070360A CN110069084A CN 110069084 A CN110069084 A CN 110069084A CN 201810070360 A CN201810070360 A CN 201810070360A CN 110069084 A CN110069084 A CN 110069084A
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- microlayer model
- temperature control
- control device
- temperature
- circuit board
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Classifications
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- G—PHYSICS
- G05—CONTROLLING; REGULATING
- G05D—SYSTEMS FOR CONTROLLING OR REGULATING NON-ELECTRIC VARIABLES
- G05D23/00—Control of temperature
- G05D23/19—Control of temperature characterised by the use of electric means
- G05D23/20—Control of temperature characterised by the use of electric means with sensing elements having variation of electric or magnetic properties with change of temperature
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- Physics & Mathematics (AREA)
- General Physics & Mathematics (AREA)
- Engineering & Computer Science (AREA)
- Automation & Control Theory (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
The present invention provides a kind of temperature control device, the temperature control device include flexible circuit board, with the flexible circuit board is spaced heats the substrate and multiple semi-conductor electricity couples.It is described to heat the substrate the first surface and second surface including being oppositely arranged.The multiple semi-conductor electricity couple is set between the flexible circuit board and the first surface, and the multiple semi-conductor electricity couple is serially connected, in parallel or Hybrid connections.The flexible circuit board in heating and cooling process with the eliminating deformation thermal stress of itself, to extend the service life of the temperature control device.When the multiple microlayer model carries out nucleic acid amplification within the scope of different temperatures, by the flexible circuit board, heat the substrate and multiple semi-conductor electricity couples may be implemented to switch within several seconds time rapidly.The cooling that instantaneously heats up may be implemented in the temperature control device, and then the process for the cooling that heats up shortens, to realize the circulation of high/low temperature, the detection time of the digital pcr detector is shortened, detection efficiency is improved.
Description
Technical field
The present invention relates to digital pcr field of analytic instrument, more particularly to a kind of temperature control device.
Background technique
Polymerase chain reaction (Polymerase Chain Reaction, PCR) is a kind of specific for amplifying amplification
DNA segment Protocols in Molecular Biology.PCR detection technique is used to diagnosing hereditary illness, detects pathogen in clinical samples
Nucleic acid, make science of heredity identification to legal medical expert's sample, and catastrophe in analysis activated oncogene etc. obtains extensively
Application.PCR reaction condition is temperature, time and cycle-index.Three step of based on PCR principle and denaturation-is set and anneals-prolongs
Stretch three temperature spots.It the time of PCR extension, can be depending on the length of segment to be amplified.Also, extension of time is too long
It will lead to the appearance of non-specific amplification band.Amplification to low concentration template, extension of time want a little longer.
PCR amplification instrument in the prior art realizes that alternating temperature is completed by semiconductor chilling plate and heat conducting module, by
After temperature probe temperature collection in heat conducting module, by the direct temperature control of PID temperature control method, heat conducting module temperature reaches target
After temperature, remain unchanged.The heating of testing sample solution has hysteresis quality, as the temperature of testing sample solution is close to target temperature
It is smaller and smaller to spend internal-external temperature difference, heats up slower and slower.Existing temperature control device, temperature rate is slow, heats up or cools down each time
It requires tens seconds to several minutes, does tens PCR cycles and need or so 1~2 hour.Therefore, this temperature rate meeting
The time needed for completing nucleic acid amplification is caused to extend, nucleic acid amplification efficiency is low.
Summary of the invention
Based on this, it is necessary to it is slow for existing temperature control device temperature rate, the time required to causing to complete nucleic acid amplification
Long problem provides a kind of temperature control device that temperature rate is fast.
The present invention provide a kind of temperature control device include flexible circuit board, with the spaced heating base of the flexible circuit board
Plate and multiple semi-conductor electricity couples.It is described to heat the substrate the first surface and second surface including being oppositely arranged.It is the multiple
Semi-conductor electricity couple is set between the flexible circuit board and the first surface, and the multiple semi-conductor electricity couple is mutual
Series, parallel or Hybrid connections.
The semi-conductor electricity couple includes a p-type galvanic couple and one and the p-type galvanic couple in one of the embodiments,
Spaced N-type galvanic couple.
The first surface includes multiple spaced first electrode sheets in one of the embodiments, described in one
First electrode sheet is corresponding with a semi-conductor electricity couple.The p-type galvanic couple and the N of the semiconductor galvanic centering
Type galvanic couple passes through institute's first electrode sheet series connection.
The flexible circuit board includes the second electrode that multiple intervals are arranged and are serially connected in one of the embodiments,
Piece.The semi-conductor electricity couple of adjacent two passes through a second electrode sheet series connection.
The temperature control device further includes enhanced thermal conduction layer in one of the embodiments, is set to the second surface.
It in one of the embodiments, include graphene in the material of the enhanced thermal conduction layer.
The temperature control device further includes second controller in one of the embodiments, with the multiple semiconductor galvanic
To electrical connection, for controlling size of current.
The temperature control device further comprises temperature sensor in one of the embodiments, is set to second table
Face is simultaneously electrically connected with the second controller, for detecting the temperature of the second surface and the temperature being sent to described
Two controllers.
The second controller includes temperature control unit and control circuit in one of the embodiments,.The temperature
Control unit is connect with the temperature sensor, the temperature to second surface described in real-time detection.The control circuit and institute
Flexible circuit board connection is stated, to regulate and control the temperature change of the multiple semi-conductor electricity couple.
The flexible circuit board is provided with first electrode and second electrode in one of the embodiments,.It is the multiple
It connects after second electrode sheet series connection with the first electrode and the second electrode.The first electrode and the second electrode
It is connect respectively with the control circuit.
The temperature control device further includes radiator in one of the embodiments,.The radiator include substrate with
And the cooling fin being connect with the substrate.The flexible circuit board is set to the surface of the substrate.
The temperature control device further includes fan in one of the embodiments,.The fan is set to the cooling fin week
It encloses.
The present invention provides a kind of temperature control device, and the temperature control device includes between flexible circuit board and the flexible circuit board
Every setting heat the substrate and multiple semi-conductor electricity couples.The first surface heated the substrate including being oppositely arranged and
Two surfaces.The multiple semi-conductor electricity couple is set between the flexible circuit board and the first surface, and the multiple half
Conductor galvanic couple to be serially connected, in parallel or Hybrid connections.The flexible circuit board high, light-weight, thickness with Distribution density
Feature thin, bending is good.The flexible circuit board in heating and cooling process with the eliminating deformation thermal stress of itself, to prolong
The service life of the temperature control device is grown.When the multiple microlayer model carries out nucleic acid amplification within the scope of different temperatures, lead to
It crosses the flexible circuit board, heat the substrate and multiple semi-conductor electricity couples may be implemented to be cut within several seconds time rapidly
It changes.The cooling that instantaneously heats up may be implemented in the temperature control device, and then the process for the cooling that heats up shortens, to realize height
The circulation of temperature, the detection time of the digital pcr detector is shortened, detection efficiency is improved.
Detailed description of the invention
Fig. 1 is the overall structure diagram of digital pcr detector provided by the invention；
Fig. 2 is temperature control device structural schematic diagram of the present invention；
Fig. 3 is temperature control device structure of the present invention section structural schematic diagram；
Fig. 4 is the semi-conductor electricity couple electrode connecting structure schematic diagram of temperature control device of the present invention；
Fig. 5 is the mapping test schematic diagram of temperature control device of the present invention；
Fig. 6 is the steady-state performance test schematic diagram of temperature control device of the present invention；
Fig. 7 is microlayer model structure of container schematic diagram of the present invention；
Fig. 8 is microlayer model structure of container planar structure schematic diagram of the present invention；
Fig. 9 is the reaction member structural schematic diagram of microlayer model container of the present invention；
Figure 10 is a kind of section structural schematic diagram of microlayer model container of the present invention；
Figure 11 is a kind of section structural schematic diagram of microlayer model container of the present invention；
Figure 12 is a kind of section structural schematic diagram of microlayer model container of the present invention；
Figure 13 is the analysis method flow chart of digital pcr detector of the present invention；
Figure 14 is microlayer model Tiling methods flow chart of the present invention；
Figure 15 is that microlayer model container floor microlayer model of the present invention accumulates schematic diagram.
Wherein: 10- microlayer model generating means；20- temperature control device；30- fluorescence signal detection device；40- quantitative analysis dress
It sets；50- controller；210- second controller；212- temperature control unit；214- control circuit；220- flexible circuit board；
221- second electrode sheet；222- first electrode；223- second electrode；The more a semi-conductor electricity couples of 230-；231-P type galvanic couple；
232-N type galvanic couple；240- is heated the substrate；241- first surface；242- second surface；243- first electrode sheet；The thermally conductive increasing of 250-
Strong layer；260- temperature sensor；270- radiator；271- substrate；272- cooling fin；273- fan；Microlayer model container 60；
610- container floor；The bottom surface 611-；The first annular side plate of 620-；The first annular side 621-；630- storage space；631- is opened
Mouthful；640- annular slab；641- annular surface；The second annular side plate of 650-；660- third annular side plate；612- reaction member；613-
Multiple ring ribs；614- microlayer model accommodating groove；670- sealing cover.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, by the following examples, it and combines attached
Figure, the present invention will be described in further detail.It should be appreciated that specific embodiment described herein is only to explain this hair
It is bright, it is not intended to limit the present invention.
It should be noted that it can directly on the other element when element is referred to as " being fixed on " another element
Or there may also be elements placed in the middle.When an element is considered as " connection " another element, it, which can be, is directly connected to
To another element or it may be simultaneously present centering elements.On the contrary, when element is referred to as " directly existing " another element "upper"
When, intermediary element is not present.Term as used herein "vertical", "horizontal", "left" and "right" and similar statement
For illustrative purposes only.Various difference objects are in the ratio drafting convenient for enumerating explanation in embodiment attached drawing, rather than press real
The ratio of border component is drawn.
Referring to Figure 1, the present invention provides a kind of digital pcr detector 1, and the digital pcr detector 1 includes: microlayer model
Generating means 10, temperature control device 20, fluorescence signal detection device 30, quantitative analysis device 40 and controller 50.Micro- liquid
Generating means 10 are dripped to form multiple microlayer models for nucleic acid amplification reaction liquid droplet.The temperature control device 20 with it is described micro-
Drop formation device 10 is connected by track, the multiple microlayer model is transferred to the temperature control device 20, carries out temperature
Circulation realizes nucleic acid amplification.The fluorescence signal detection device 30 is oppositely arranged with the temperature control device 20, to nucleic acid
The multiple microlayer model after amplification carries out detection of taking pictures.The quantitative analysis device 40 and the fluorescence signal detection device
30 are connected by data line, to realize the transmission of the multiple microlayer model fluorescence information, carry out quantitative analysis.The controller
50 fill with the microlayer model generating means 10, the temperature control device 20, fluorescence signal detection device 30 and quantitative analysis respectively
Set 40 connections, to control the microlayer model generating means 10, the temperature control device 20, fluorescence signal detection device 30 and
Quantitative analysis device 40.
The digital pcr detector 1 can be by the microlayer model generating means 10, the temperature control device 20, the fluorescence
Signal supervisory instrument 30 and the quantitative analysis device 40 are integrated, so that automation behaviour may be implemented in operator
Make.The digital pcr detector 1 working efficiency with higher.
At work, the microlayer model generating means 10 can expand the determined nucleic acid digital pcr detector 1
Reaction solution carries out droplet, to form multiple microlayer models.The temperature control device 20 can carry out core to the multiple microlayer model
Acid amplification.The fluorescence signal detection device 30 claps the change in fluorescence picture for surveying the multiple microlayer model in real time.By described more
The change in fluorescence picture of a microlayer model, the change in fluorescence curve of available the multiple microlayer model.According to the change in fluorescence
Curve, the Ct value of available the multiple microlayer model, and by Ct value and the relationship of starting copy number to the dense of initial DNA
Degree carries out quantitative analysis.Wherein, Ct value refers to circulation experienced when the fluorescence signal of each microlayer model reaches the threshold value of setting
Number.
The temperature control device 20 carries out nucleic acid amplification reaction to the multiple microlayer model, and is detected by the fluorescence signal
Device 30 acquires the product signal of the multiple microlayer model after nucleic acid amplification reaction, such as fluorescence, UV absorption, turbidity letter
Number.Difference using the multiple amplification with non-amplification microlayer model in composition, to the amount of droplets for obtaining target sequence amplification
It is analyzed, the final quantitative analysis realized to nucleic acid molecules.Pass through the change in fluorescence figure of the multiple microlayer model of real-time monitoring
The problem of piece, testing result has substantivity, can solve the false positive and false negative in the multiple microlayer model.
The digital pcr detector 1 is by the microlayer model generating means 10, the temperature control device 20, the fluorescence signal
Detection device 30 and the quantitative analysis device 40 are integrated, so that automatic operation may be implemented in the operator, no
Into improving work efficiency, also have the advantages that rapid reaction, reproducible, high sensitivity, high specificity and result are clear.
In one embodiment, the microlayer model 199 is determined nucleic acid amplification reaction solution, is generated and is filled by the microlayer model
10 are set by the determined nucleic acid amplification reaction solution droplet, forms multiple microlayer models, to pass through the digital pcr detector 1
It is detected.The determined nucleic acid amplification reaction solution is passed through to the microlayer model generating means 10 of integral type digital pcr detector 1,
It is handled by dropletization and is converted into multiple microlayer models 199, so that the detection segment in sample to be tested is from a large amount of complex background
It separates, and is placed in microlayer model container 60, wait to be detected.It can be generated by microlayer model generating means 10 multiple big
Small uniform microlayer model 199.In the micron-scale, and each microlayer model 199 can regard each 199 size of the microlayer model
Make an independent reactor, is equivalent to common test tube in biochemical reaction.The multiple microlayer model 199 is placed in microlayer model
In container 60, convenient for detection observation.Meanwhile it is different by the microlayer model generating means 10 multiple volumes also to can be generated
Microlayer model, to carry out clinical medicine detection.The multiple microlayer model 199 is small in size, quantity is more, has many conventional test tubes
No advantage.A large amount of microlayer models 199 can be generated by the microlayer model generating means 10, so that the digital pcr detects
Instrument 1 has the advantages that flux is high, consumables cost is low low with ambient noise, has good industrial prospect.
In one embodiment, the temperature control device 20 includes between flexible circuit board 220 and the flexible circuit board 220
240 and multiple semi-conductor electricity couples 230 are heated the substrate every setting.It is described that heat the substrate 240 include the be oppositely arranged
One surface 241 and second surface 242.The multiple semi-conductor electricity couple 230 be set to the flexible circuit board 220 with it is described
Between first surface 241, the multiple semi-conductor electricity couple 230 is serially connected, in parallel or Hybrid connections.
The flexible circuit board 220 (Flexible Printed Circuit, FPC) is with polyimides or polyester film
To have height reliability, excellent flexible printed circuit made of substrate.The flexible circuit board 220 has wiring
The advantage that density is high, light-weight, thickness is thin, bending is good.The flexible circuit board 220 is light-weight, thickness is thin, can be effective
Save small product size.
When temperature changes, object prevents it from complete due to the mutual constraint between external restraint and internal each section
Free to contract and expand and the stress generated.Thermal stress is also known as temperature changing stress.It carries and balances each other outside thermal stress and zero, be by thermal deformation by about
Self balancing stress caused by beam is compressed in temperature eminence, and tensile deformation occurs for temperature lower.It controls under certain condition
Stress is allowed to reasonable layout, so that it may which the mechanical performance and service life for improving part are turned bane into boon.
A substrate in conventional semiconductors refrigerator is replaced by the flexible circuit board 220, so that the semiconductor
Refrigerator heating conduction is more preferable.The flexible circuit board 220 in heating and cooling process with the eliminating deformation thermal stress of itself, into
And extend the service life of the semiconductor cooler.
When the multiple microlayer model carries out nucleic acid amplification within the scope of different temperatures, may be implemented rapidly in several seconds time
Inside switch over.The cooling that instantaneously heats up may be implemented in the temperature control device 20, and then the process for the cooling that heats up shortens, thus
The detection time of the digital pcr detector 1 is shortened, improves detection efficiency by the circulation for realizing high/low temperature.
In one embodiment, the temperature control device 20 further includes second controller 210 and temperature sensor 260.It is described
Second controller 210 is electrically connected with the flexible circuit board 220, for controlling size of current.The temperature sensor 260 is set
240 surfaces are heated the substrate described in being placed in, the temperature sensor 260 is electrically connected with the second controller 210, for detecting
It is described heat the substrate 240 temperature and the temperature is sent to the second controller 210.
Based on this, it is necessary to it is slow for existing temperature control device temperature rate, the time required to causing to complete nucleic acid amplification
Long problem provides the temperature control device that a kind of temperature rate is fast, service life is high.
As Figure 2-3, the present invention also provides a kind of temperature control devices 20 comprising flexible circuit board 220 and the flexibility
Circuit board 220 is spaced to heat the substrate 240 and multiple semi-conductor electricity couples 230.It is described heat the substrate 240 include phase
To the first surface 241 and second surface 242 of setting.The multiple semi-conductor electricity couple 230 is set to the flexible circuit board
Between 220 and the first surface 241, the multiple semi-conductor electricity couple 230 is serially connected, in parallel or Hybrid connections.
The temperature control device 20 is usually applied in high/low temperature circulation environment, and temperature needs fast lifting, so to described
Temperature control device 20 requires high.In order to meet the application demand of the temperature control device 20, the temperature control device 20 is using described soft
Property circuit board 220.The flexible circuit board 220 has the characteristics that Distribution density is high, light-weight, thickness is thin, bending is good.Institute
Flexible circuit board 220 is stated in heating and cooling process with the eliminating deformation thermal stress of itself.It can by the flexible circuit board 220
To reduce thermal stress present in heating and cooling process, to extend the service life of the temperature control device 20.Meanwhile passing through
The flexible circuit board 220, solves the problems, such as that temperature distribution is non-uniform.When the multiple microlayer model is in different temperatures range
When interior progress nucleic acid amplification, by the flexible circuit board 220, heat the substrate 240 and multiple semi-conductor electricity couples 230 can
It was switched within several seconds time rapidly with realizing.The cooling that instantaneously heats up may be implemented in the temperature control device 20, and then the drop that heats up
The process of temperature shortens, to realize the circulation of high/low temperature, the detection time of the digital pcr detector 1 is shortened,
Improve detection efficiency.
The flexible circuit board 220 (Flexible Printed Circuit, FPC) can be with polyimides or polyester
Film is that one kind made of substrate has height reliability, excellent flexible printed circuit.The flexible circuit board has
The feature that Distribution density is high, light-weight, thickness is thin, bending is good.The flexible circuit board is light-weight, thickness is thin, can be effective
Save small product size.Wherein, the semiconductor cooler (Thermo Electric Cooler, TEC) is to utilize semiconductor material
Made of the Peltier effect of material.So-called Peltier effect refers to when DC current passes through the electricity that two kinds of semiconductor materials form
When even, one end heat absorption, the phenomenon that the heat release of one end.It is replaced in conventional semiconductors refrigerator by the flexible circuit board 220
One substrate, so that the semiconductor cooler heating conduction is more preferable.
When temperature changes, object prevents it from complete due to the mutual constraint between external restraint and internal each section
Full free to contract and expand and the stress generated.Thermal stress is also known as temperature changing stress.Thermal stress and zero outside carry balance each other, be by thermal deformation by
Self balancing stress caused by constraining, compresses in temperature eminence, and tensile deformation occurs for temperature lower.It controls under certain condition
Stress processed is allowed to reasonable layout, so that it may which the mechanical performance and service life for improving part are turned bane into boon.
It is in one embodiment, described that heat the substrate 240 can be superconduction aluminum substrate circuit.
Aluminum substrate is a kind of metal-based copper-clad plate with good heat radiating function, and general single sided board is by three-decker institute group
At being circuit layer (copper foil), insulating layer and metal-based layer respectively.The superconduction aluminum substrate circuit is that the material of wiring board is aluminium
Alloy, can be thermally conductive fast.Aluminum substrate can minimize thermal resistance, so that aluminum substrate is had fabulous heat-conductive characteristic, with thick film
Ceramic circuit is compared, its mechanical performance is again extremely excellent.
As described in Figure 4, in one embodiment, the semi-conductor electricity couple 230 includes a p-type galvanic couple 231 and one
With the spaced N-type galvanic couple 232 of the p-type galvanic couple 231.
The p-type galvanic couple 231 and the N-type galvanic couple 232 be welded on the flexible circuit board 220 and the substrate 240 it
Between.The semi-conductor electricity couple 230 includes some a pair of electricity formed by the p-type galvanic couple 231 and the N-type galvanic couple 232
It is even, it is connected together between the multipair semi-conductor electricity couple 230 by electrode, and be clipped in the flexible circuit board 220 and institute
It states between first surface 241.When a current flows through, " heat " side and " cold " side can be generated.It is refrigeration or heating, Yi Jizhi
Cold, heating rate, by being determined by its current direction and size.The heat that a pair of semi-conductor electricity couple 230 generates
Electrical effect very little, so be all cascaded in practice by up to a hundred to the semi-conductor electricity couple 230, the heat generated in this way
Electrical effect just will increase.
In one embodiment, the first surface 241 includes multiple spaced first electrode sheets 243, an institute
It is corresponding with a semi-conductor electricity couple 230 to state first electrode sheet 243, the p-type in the semi-conductor electricity couple 230
Galvanic couple 231 is connected with the N-type galvanic couple 232 by the first electrode sheet 243.
In one embodiment, the flexible circuit board 220 includes the second electrode that multiple intervals are arranged and are serially connected
Piece 221, the semi-conductor electricity couple 230 of adjacent two are connected by a second electrode sheet 221.
When there is electric current logical in the semi-conductor electricity couple 230 that the p-type galvanic couple 231 and the N-type galvanic couple 232 are coupled to
It is out-of-date, heat transfer will be generated between both ends, heat will be transferred to the other end from one end, thus generate the temperature difference formed it is cold
Hot end.But there are resistance when electric current is by the p-type galvanic couple 231 for the p-type galvanic couple 231 and the N-type galvanic couple 232 itself
Heat will be generated when with the N-type galvanic couple 232, to will affect hot transmitting.And the flexible circuit board 220 with it is described
Heat the substrate heat between 240 also can by air and the p-type galvanic couple 231 and 232 material of N-type galvanic couple itself into
The reverse heat transmitting of row.When hot and cold side reaches certain temperature difference, and the amount of both heat transmitting is equal, an equalization point will be reached,
Just reverse heat transmitting is cancelled out each other.The temperature of hot and cold side would not continue to change at this time.In order to reach lower temperature,
The modes such as heat dissipation can be taken to reduce the temperature in hot end to realize.
In one embodiment, the temperature control device 20 further includes enhanced thermal conduction layer 250, is set to the second surface
242。
The enhanced thermal conduction layer 250 has very good intensity, flexible, conductive, thermally conductive, optical characteristics.It is described thermally conductive
Enhancement layer 250 is directly contacted with the microlayer model container 60, and the multiple microlayer model can be made to be heated evenly, to pass through
Nucleic acid amplification is realized to the control of temperature.The enhanced thermal conduction layer 250 can be graphite thermal conductive layer or silicone grease heat-conducting layer, add
It is fast thermally conductive, the temperature uniformity of the second surface 242 for heating the substrate 240 is increased, and then guarantee close to described micro-
The surface temperature of drop receptacle 60 is uniform, so that the multiple microlayer model is heated evenly.And then nucleic acid amplification is completed, it mentions
High detection efficiency, saves the time.
It in one embodiment, include graphene in the material of the enhanced thermal conduction layer 250.The graphene is a kind of flat
Face film has extraordinary heat-conductive characteristic, and uniform heat conduction laterally may be implemented.
In one embodiment, the temperature control device 20 further includes second controller 210, with the flexible circuit board 220
Electrical connection, for controlling size of current.
In one embodiment, the temperature control device 20 further comprises temperature sensor 260, is set to second table
Face 242, and be electrically connected with the second controller 210, for detecting the temperature of the second surface 242 and sending out the temperature
Give the second controller 210.
The temperature sensor 260 is set to the second surface 242 of the enhanced thermal conduction layer 250, to detect
The real time temperature of second surface 242 is stated, and then temperature information is fed back into the second controller 210, to realize to described
The control of multiple microlayer model heating temperatures.The temperature sensor 260 is used for the resistance variations by detecting metal to measure
The temperature for stating microlayer model container 60 carries out the temperature change in amplification process to the multiple microlayer model of real-time detection,
To which temperature information is fed back to the second controller 210, and then the regulation that the control circuit carries out temperature is controlled, it is real
Existing temperature control, more preferably progress nucleic acid amplification.
In one embodiment, the second controller 210 includes temperature control unit 212 and control circuit 214.Institute
It states temperature control unit 212 to connect with the temperature sensor 260, the temperature to second surface 242 described in real-time detection.
The control circuit 214 is connect with the flexible circuit board 220, to regulate and control the temperature of the multiple semi-conductor electricity couple 230
Variation.
The temperature control unit 212 is set on one piece of circuit board with the control circuit 214.The temperature control is single
The relationship of member 212 and the control circuit 214 is the logical operation relationship of internal algorithm, can use Packet
Identifier closed loop control algorithm, that is, PID closed loop control algorithm.The temperature that the temperature control unit 212 detects is
The temperature feedback of nucleic acid amplification, as the input of internal algorithm, the result after the control circuit 214 calculates is calculated as internal
The output of method, to form closed loop relationship.The temperature feedback of circuit part is actually a sample circuit.Acquisition be exactly
Electric signal in platinum resistance is converted to the input terminal that temperature value passes to control circuit.The temperature sensor 260 with it is described
Temperature control unit 212 is connected by the platinum resistance three-wire system of standard.
In one embodiment, the flexible circuit board 220 is provided with first electrode 222 and second electrode 223.It is described
Multiple second electrode sheets 221 are connected after connecting with the first electrode 222 and the second electrode 223.The first electrode
222 connect with the control circuit respectively with the second electrode 223.
Connection between the control circuit 214 and the flexible circuit board 220 is both threads, is separately connected described first
Electrode 222 and the second electrode 223.
In one embodiment, the temperature control device 20 further includes radiator 270, and the radiator 270 includes base
Plate 271 and the cooling fin 272 being connect with the substrate 271.The flexible circuit board 220 is set to the table of the substrate 271
Face.
Since the cooling fin 272 is set to 271 surface of substrate, in no feelings for reducing 271 area of substrate
Under condition, heat exchange area is further increased, extends the time that cool breeze acts on 271 surface of substrate, the multiply radiation air of formation
Road is also beneficial to accelerate heat exchange, more heats is taken away from 271 surface of substrate, to reach more ideal
Heat dissipation effect.
In one embodiment, the temperature control device further includes that fan 273 is set to around the cooling fin 273.
The radiator 270 can be assisted to radiate by the fan 273.Wherein, the fan 273 is set to
Around the cooling fin 273, can be set it is multiple, so as to reach better heat dissipation effect, so that the temperature control
The heating cooling of device 20 is quicker.
In one embodiment, alternating current is passed to the temperature control device 20, and passes through 210 pairs of electricity of the second controller
Big minor adjustment is flowed to control the rate that the temperature control device 20 is refrigeration or heating and refrigeration, heating.Meanwhile passing through institute
Heated in real-time temperature of the temperature sensor 260 to detect the microlayer model container 60 is stated, and then temperature information is fed back into institute
State temperature control unit 212.Temperature variations are fed back to the control circuit 214 by the temperature control unit 212, from
And control the temperature of the multiple microlayer model.The multiple microlayer model can be made to carry out nucleic acid by the temperature control device 20
Amplification.Three step of based on PCR principle and denaturation-temperature spot of annealing-extension three is set.Using three temperature in standard reaction
Degree point method, double-stranded DNA is denaturalized at 90~95 DEG C, then is rapidly cooled to 40~60 DEG C, and primer annealing is simultaneously integrated on target sequence,
Then it is rapidly heated to 70~75 DEG C, under the action of Taq archaeal dna polymerase, extends primer strand along template, in suitable temperature
Nucleic acid is expanded in degree range.Meanwhile in amplification process, 60 bottom of microlayer model container and the temperature control
The close note of device 20 closes, and does not have gap between the two, improves the accuracy of the digital pcr detector 1.
In one embodiment, the operating method for calculate amplification by the temperature control device 20 is as follows:
Firstly, the microlayer model container 60 to be placed in the enhanced thermal conduction layer 250 of the temperature control device 20.
Then, the multiple microlayer model is subjected to heating heating, temperature is heated to 95 DEG C, and heat 10min.It will be described
Multiple micro- liquid are heated to 95 DEG C, and heat 10min, the enzyme in the multiple microlayer model is carried out thermal starting.
Secondly, carrying out denaturation 30s to the multiple microlayer model after the multiple microlayer model completes enzyme thermal starting；
Again, after the multiple micro- liquid denaturation, 55 DEG C are cooled to, and anneal and extend 45s, to the multiple pico- liquid into
Row is taken pictures, and carries out 45 circulations；
Finally, being cooled to 4 DEG C after circulation 45 times, long-time preservation is carried out to the multiple micro- liquid.
Fig. 5 is referred to, the temperature control device 20 tests temperature-control performance under normal circumstances, and mainly there are two indexs, respectively in wink
When state and stable state under observe the lifting temperature variations of the temperature control device 20.By to the multiple microlayer model
The monitoring of heating process, when the temperature control device 20 carries out gradient of temperature to the multiple microlayer model, temperature rate maximum can
To reach 13.34448 DEG C/s., control precision is 0.02722 DEG C.Also, the temperature control device 20 is warming up to stable state sometimes
When the rate that measures most fastly can be to 18.953894 DEG C/s.Therefore, the transient response of the temperature control device 20 is good, passes through institute
Stating temperature control device 20 may be implemented the cooling that instantaneously heats up, and saves the time, improves detection efficiency.
Fig. 6 is referred to, when the temperature control device 20 is in stable state, that is, the temperature fluctuations after reaching stable
Situation.When the temperature control device 20 is in stable state, temperature change is more steady, and temperature fluctuations are smaller.Therefore, described
Temperature control device 20 can achieve quickly heating down cycles, and warmly taken after stablizing float it is smaller, save digital pcr detection
It the time of solution sample, improves work efficiency.When can be shortened needed for completing nucleic acid amplification by this temperature rate
Between, nucleic acid amplification efficiency is improved, and improve the accuracy of digital pcr detection system.
The determined nucleic acid amplification reaction solution is carried out droplet by the microlayer model generating means 10, forms multiple micro- liquid
Drop.Then, during being heated by the temperature control device 20 to the multiple microlayer model, using the fluorescence signal
Detection device 30 claps the change in fluorescence image for surveying the multiple microlayer model in real time.By the quantitative analysis device 40 to described
The change in fluorescence image of multiple microlayer models is analyzed, and obtains the Ct value of the multiple microlayer model, and copy with starting by Ct value
The relationship of shellfish number carries out quantitative analysis to the concentration of original nucleic acid.
The determined nucleic acid amplification reaction solution is carried out droplet by the microlayer model generating means 10, forms multiple micro- liquid
Drop.Then, nucleic acid amplification is carried out to the multiple microlayer model by the temperature control device 20.Meanwhile using the fluorescence signal
Detection device 30 claps the change in fluorescence picture for surveying the multiple microlayer model in real time.Pass through the change in fluorescence of the multiple microlayer model
Picture obtains the change in fluorescence curve of the multiple microlayer model.It is available the multiple according to the change in fluorescence curve
The Ct value of microlayer model, and quantitative analysis is carried out to the concentration of initial DNA by Ct value and the relationship of starting copy number.Wherein, Ct
Value refers to recurring number experienced when the fluorescence signal of each microlayer model reaches the threshold value of setting.
The generation of microlayer model generating means 20 is uniform size microlayer model, by temperature control device 30 to the multiple micro-
Drop carries out nucleic acid amplification reaction, and acquires product signal, such as fluorescence, UV absorption, turbidity signal.Using the multiple
Difference of the amplification with non-amplification microlayer model in composition, analyzes the amount of droplets for obtaining target sequence amplification, final real
Now to the quantitative analysis of nucleic acid molecules.By the change in fluorescence picture of the multiple microlayer model of real-time monitoring, sequencing result has
Substantivity, the problem of can solve the false positive and false negative in the multiple microlayer model.
Drop formula PCR detection method is the reaction member that sample is dispersed into Water-In-Oil at present, single to each reaction later
Member carries out in real time or end point fluorescence is analyzed.Theoretically, the quantity of reaction chamber determine instrument dynamic range and to
Determine the detection accuracy under concentration.The reaction member structure of drop formula PCR detection method is usually microcavity or dimple structure at present,
Belong to expendable consumed product, carries out one-time detection and need to be abandoned, prevent cross contamination.But in actually detected process
In, from microlayer model occur card be transferred to PCR reaction plate, from PCR reaction plate to liquid drop analyzer during can all be damaged
It loses, limitation is still had to the number of the microlayer model.So if current microlayer model reaction member progress is a large amount of micro-
Drop detection still has limitation to the number of the microlayer model, and consumables cost is higher.
Fig. 7-12 is referred to, the embodiment of the present invention provides a kind of microlayer model container 60 comprising bottom surface 611, around described
The first annular side 621 and annular surface 641 that bottom surface 611 is arranged.The first annular side 621 and 611 phase of bottom surface
Even, it and surrounds to form a storage space 630 with opening 631, the first annular side 621 is perpendicular to the bottom surface
611.The annular surface 641 is connected around 631 setting of opening with the first annular side 621, the annular surface
641 is parallel with the bottom surface 611.
The annular surface 641 is parallel with the bottom surface 611, to ensure that the liquid surface in the microlayer model container 60 is
Horizontal plane.By being set to the annular surface 641, the liquid level of the microlayer model container 60 can be made to show flat state,
The whole liquid level for avoiding the microlayer model container 60 is arc.Therefore, it will not influence by the microlayer model container 60 described
Container floor carries out imaging of taking pictures convenient for the camera 331, improves described more close to the observation of the microlayer model at edge position
The detection efficiency of a microlayer model.
In one embodiment, the microlayer model container 60 further comprises container floor 610, around the container floor
The first annular side plate 620 and annular slab 640 of 610 settings.The surface of the container floor 610 is the bottom surface 611.Institute
The inner surface for stating first annular side plate 620 is the first annular side 621, the first annular side plate 620 and the container
Bottom plate 610 is fixedly connected, and is surrounded jointly with the container floor 610 and formed the storage space 630.The annular slab 640
Surface be the annular surface 641.The annular slab 640 is with the first annular side plate 620 far from the container floor 610
One end be fixedly connected, and the annular slab 640 is parallel with the container floor 610.
When preparing the multiple microlayer model by the microlayer model generating means 10, first by the second liquid (oily phase
Composition) it is placed in the microlayer model container 60.Liquid level and the 641 horizontal plane phase of annular surface when the second liquid
Meanwhile stopping that the second liquid is added.At this point, the surface of the liquid level of the second liquid and the annular surface 641 is same
On horizontal plane, it is ensured that the pasta of the second liquid in the microlayer model container 60 is plane, facilitates guarantee container bottom
The top surface of oil liquid above face is horizontal plane, convenient for imaging, the utilization rate of the microlayer model container 60 is improved, to accommodate
The microlayer model of even larger amounts.
In one embodiment, the inner circumferential of the annular slab 640 and the first annular side plate 620 are far from the container bottom
One end of plate 610 connects.The microlayer model container 60 further includes the second annular side plate 650.Second annular side plate, 650 ring
It is arranged around the annular slab 640 and is fixedly connected with the annular slab 640, the radius of second annular side plate 650 is greater than institute
State the internal diameter of annular slab 640.
Pass through the annular slab 640, the first annular side plate 620, the container floor 610 and second annular
Side plate 650 can cooperate with the annular surface 641, the first annular side 621 with the bottom surface 611 respectively,
Form the storage space 630.In the storage space 630, to accommodate the second liquid (oil phase composition).Institute
The liquid level of second liquid and the surface of the annular surface 641 are stated in same level, it is ensured that the microlayer model container 60
In the pasta of the second liquid be plane, facilitate and guarantee that the top surface of oil liquid above container bottoms is horizontal plane, avoid
The problem of whole liquid level of the microlayer model container is arc, and spill liquid level is presented in liquid level.To be examined by the fluorescence signal
When surveying device 30 to the progress fluorescence detection of the multiple microlayer model, more convenient for imaging, the microlayer model container 60 is improved
Utilization rate, to accommodate the microlayer model of even larger amounts.
In one embodiment, the periphery of the annular slab 640 and the first annular side plate 620 are far from the container bottom
One end of plate 610 connects.
In one embodiment, the periphery of the first annular side plate 620 is fixedly connected on the first annular side
621.By the way that the microlayer model container 60 can be made by the connection of the annular slab 640 and the first annular side plate 620
Form a horizontal platform.When the second liquid is added in the microlayer model container 60, the second liquid can be made
Liquid level and the annular surface 641 surface in same level, thereby may be ensured that the institute in the microlayer model container 60
The pasta for stating second liquid is plane, facilitates and guarantees that the top surface of the oil liquid above container bottoms is horizontal plane, avoids described micro-
The problem of whole liquid level of drop receptacle is arc, and spill liquid level is presented in liquid level.To pass through the fluorescence signal detection device
When 30 pairs of the multiple microlayer models carry out fluorescence detection, more convenient for imaging, the utilization of the microlayer model container 60 is improved
Rate, to accommodate the microlayer model of even larger amounts.
In one embodiment, the microlayer model container 60 further includes third annular side plate 660.The third annular side plate
660 one end is fixedly connected on the bottom surface 611.The other end of the third annular side plate 660 is fixedly connected on described first
The inner circumferential of annular side plate 620.The third annular side plate 660 surrounds jointly with the container floor 610 and forms the storage sky
Between 630.
In one embodiment, the third annular side plate 660 is vertical with the container floor 610.Pass through micro- liquid
The setting for dripping third annular side plate 660 described in container 60 can make the liquid level and the annular surface of the second liquid
641 surface is in same level, so that liquid level in the microlayer model container 60 is plane, the traditional feelings in surface
The generation of concave meniscus under condition improves the utilization rate of the microlayer model container 60 convenient for imaging.
In one embodiment, the microlayer model container 60 further includes multiple ring ribs 613, is arranged at intervals at the bottom
Face 611, each ring rib 613 is surrounded with the bottom surface 611 forms a microlayer model accommodating groove 614.The microlayer model
Accommodating groove 614 is used to store the multiple microlayer model of generation, and the multiple microlayer model and passes through microlayer model Tiling methods
It is laid in the bottom surface 611, forms single layer microlayer model, for observation of taking pictures.Meanwhile multiple microlayer model accommodating grooves 614 it
Between spacing can be configured according to the distance between row's needle of the microlayer model generating means 10 so that one
Secondary property forms multiple microlayer models in multiple microlayer model accommodating grooves 614, improves the receiving of the microlayer model container 60
Amount, can also be used to detect different types of nucleic acid.
In one embodiment, the height of multiple ring ribs 613 is 0.1mm-1mm.By to the ring
The setting of 613 height of shape raised line can be conducive to the shade caused by excluding when exciting light is irradiated from side, so that the phase
Machine can obtain the fluorescence information of all microlayer models, improve the sensitivity of the fluorescence detection device.
In one embodiment, 614 inner wall surface of microlayer model accommodating groove is provided with oleophobic layer.
By doing oleophobic processing on 610 surface of container floor, so that between the bottom plate 610 and the microlayer model
Viscous stickiness reduces, and surface tension reduces, and then frictional force reduces, and is easy to slide, the microlayer model can be spread automatically, it is therefore prevented that
The multiple microlayer model flocks together.Meanwhile the multiple microlayer model can be made quicker when being tiled, have
The microlayer model container floor 610 is laid in conducive to the multiple microlayer model.When the surface tension of the container floor is less than institute
When stating the surface tension of second liquid (oils) 699, the resistance of the microlayer model and the bottom plate becomes smaller, the microlayer model meeting
It is spread from microlayer model reaction member bottom described in trend, realizes tiling.
The oleophobic membrane is also oleophobic layer, is a kind of composite coating material, is a kind of functional material coating, often has
Oleophobic function.The oleophobic layer is generally using nano silica as raw material (SiO2), using spraying process, formed on surface
Coating has good translucency and hydro-oleophobicity.When the multiple microlayer model and the reaction member contact, contact
Angle can achieve 90 degree, it can be achieved that tumbling and leaving no trace automatically, so as to realize that it is described that the multiple microlayer model is laid in
Microlayer model container bottom 610.
In one embodiment, it includes that the microlayer model mentioned in above embodiments holds that a kind of microlayer model, which generates kit,
Device 60, sealing cover 670 and oil phase composition, the oil phase composition are placed in the storage space 630, the sealing
Lid 670 is set to the opening 631, to seal the storage space 630.
In one embodiment, each reaction member 612 includes each ring rib 613 and each ring
Shape raised line 613 and the bottom surface 611, which surround, forms a microlayer model accommodating groove 614.
The container floor 610 is provided with multiple reaction members 612, and each reaction member 612 can place multiple micro- liquid
Drop, so that the microlayer model container 60 can accommodate large batch of microlayer model, so that the number of drops really detected
Mesh can be considerably beyond 20000, and there is no the limitations of the number to the microlayer model.Meanwhile if carrying out a large amount of microlayer model
Detection, it may be desirable to expend more times.
In one embodiment, multiple 612 shapes of the reaction member are rectangle.The microlayer model container 60 be it is rectangular or
Rectangle.Since most of film and number photosensitive element CCD/CMOS are rectangular so far, so by institute
It states 60 shape of microlayer model container and is designed as rectangular, the space utilization rate of the microlayer model container, and the side of being conducive to can be improved
Just the splicing of the fluorescent image formed, to realize real-time tracing.
In one embodiment, multiple reaction members 612 are equidistantly arranged in the container floor 610.Multiple institutes
The spacing for stating reaction member 612 is identical as the distance between row's needle of the microlayer model generating means 10, in order to simultaneously more
A large amount of microlayer model is formed in a reaction member 612, the speed of microlayer model generation is improved, saves the time.Meanwhile
It is different multiple volumes can also to be generated in the multiple reaction member 612 by the microlayer model generating means 10
Microlayer model.
In one embodiment, the height of multiple ring ribs 613 is 0.1mm-1mm.By to the ring
The setting of 613 height of shape raised line can be conducive to the shade caused by excluding when exciting light is irradiated from side, so that the phase
Machine can obtain the fluorescence information of all microlayer models, improve the sensitivity of the fluorescence detection device.
Each independent reaction unit of traditional digital pcr detection system is generally placed with a microlayer model.Also, in reality
In the detection process of border, the drop number really detected is not up to 20000, still has to the number of the microlayer model
Limitation.So can solve problem above using the microlayer model container 60, limitation will not be generated to the microlayer model number.
Therefore, a large amount of microlayer model can be accommodated by multiple reaction members 612 on the container floor 610,
The storage amount of the microlayer model container 60 is increased, the detection greater than 20000 microlayer models may be implemented, and can be to difference
The nucleic acid of type is detected.It, can be to avoid making the multiple microlayer model be scattered to adjacent by the reaction member frame
Reaction member 612 in.
In one embodiment, the cross section of the microlayer model container 60 is rectangle.The microlayer model container 60 is rectangular
Or rectangle.60 shape of microlayer model container is consistent with the shape of camera lens, improves the space of the microlayer model container
Utilization rate, and be conducive to facilitate the splicing for the fluorescent image to be formed, to realize real-time tracing.
In one embodiment, the annular surface 641 is a square box.
By doing oleophobic processing on 610 surface of container floor, so that between the bottom plate 610 and the microlayer model
Viscous stickiness reduces, and surface tension reduces, and then frictional force reduces, and is easy to slide, the microlayer model can be spread automatically, it is therefore prevented that
The multiple microlayer model flocks together.Meanwhile the multiple microlayer model can be made quicker when being tiled, have
The microlayer model container floor 610 is laid in conducive to the multiple microlayer model.When the surface tension of the container floor is less than institute
When stating the surface tension of second liquid (oils), the resistance of the microlayer model and the bottom plate becomes smaller, and the microlayer model can be automatic
It is spread to microlayer model reaction member bottom, realizes tiling.
The oleophobic membrane is also oleophobic layer, is a kind of composite coating material, is a kind of functional material coating, often has
Oleophobic function.The oleophobic layer is generally using nano silica as raw material (SiO2), using spraying process, formed on surface
Coating has good translucency and hydro-oleophobicity.When the multiple microlayer model and the reaction member contact, contact
Angle can achieve 90 degree, it can be achieved that tumbling and leaving no trace automatically, so as to realize that it is described that the multiple microlayer model is laid in
Microlayer model container bottom 610.
In one embodiment, the height of the first annular side plate 620 or second annular side plate 650 is 5mm-
15mm.By the setting of the first annular side plate 620 or 650 height of the second annular side plate, can make described micro-
Drop formation device 10 avoids the multiple microlayer model from throwing away during preparing the multiple microlayer model.And can have
Shade caused by conducive to excluding when exciting light is irradiated from side, enables the camera 331 to obtain the glimmering of all microlayer models
Optical information improves the sensitivity of the fluorescence detection device 30.
In one embodiment, the material of the container floor 610 is glass, quartz or stainless steel etc..
In one embodiment, the material of the container floor 610 is glass, and cheap, consumables cost is low.
If carrying out a large amount of microlayer model detection, the microlayer model container 60 use glass material, it is cheap, consumptive material at
This is low, and carrying out one-time detection can be abandoned, it is therefore prevented that cross contamination saves detection time, improves the number
The detection efficiency of PCR detector 1.
In one embodiment, the ring rib 613 is identical as the material of the microlayer model container floor 610.It utilizes
Technology can make the microlayer model container floor 610 form multiple reaction members 612.Multiple reactions are single
Member 612 is set to the microlayer model container floor 610 in the form of an array, forms multiple nucleic acid amplification units.
In one embodiment, the geomery size of the container floor 610 and 24 orifice plates and 96 orifice plate outer dimensions
Unanimously, so that the convenient instrument for being applied to other models of the microlayer model container 60, has more practicability and compatibility.
In one embodiment, the material of the first annular side plate 620 or second annular side plate 650 is resistance to height
Temperature, oil resistant, non-blooming black silicon rubber.The black silicon rubber have it is tasteless it is nontoxic, be not afraid of high temperature and resist severe cold
Feature.Also, the black silicon rubber have good electrical insulating property, oxytolerant ageing resistance, fast light ageing resistance, mildew resistance with
And the advantages that chemical stability, receive the attention of Modern Medical Field.
Testing cost can be reduced using glass or stainless steel material by the microlayer model container 60.Meanwhile passing through
Multiple reaction members 612 on the container floor 610 can accommodate a large amount of microlayer model, increase the microlayer model
The storage amount of container may be implemented the detection greater than 20000 microlayer models, and can detect to different types of nucleic acid,
It is economical and practical.
340 oblique illumination of excitation light source is to the microlayer model container 60, to irradiate the multiple microlayer model.Pass through
Exciting light scattering background can be effectively reduced in the oblique fire optical path that the excitation light source 340 is formed.Meanwhile reducing the microlayer model
The first annular side plate 620 of container 60 or the height of second annular side plate 650 are conducive to exclude exciting light from side
Shade caused by when face is irradiated, enables the camera 331 to obtain the fluorescence information of all microlayer models, improves described
The sensitivity of fluorescence detection device 30.
In one embodiment, the determined nucleic acid amplification reaction solution is carried out droplet by the microlayer model generating means 10
Change, forms multiple microlayer models.Wherein, the generation of microlayer model generating means 10 is the microlayer model of uniform size.Then, lead to
It crosses the temperature control device 20 and nucleic acid amplification is carried out to the multiple microlayer model.Meanwhile using the fluorescence signal detection device 30
The change in fluorescence image for surveying the multiple microlayer model is clapped in real time.By the change in fluorescence image of the multiple microlayer model, institute is obtained
State the change in fluorescence curve of multiple microlayer models.According to the change in fluorescence curve, the Ct of available the multiple microlayer model
Value, and quantitative analysis is carried out to the concentration of original nucleic acid by Ct value and the relationship of starting copy number.
PCR cycle just enters real exponential amplification phase (logarithmic phase), at this time when reaching the recurring number where Ct value
Slight error is not yet amplified, therefore the reproducibility of Ct value is fabulous, i.e., same nucleic acid-templated different time amplification or same time are not
With expanding in microlayer model container, obtained Ct value is constant.When the corresponding fluorescence curve of the microlayer model is amplification curve
When, show to contain target gene ingredient in the microlayer model at this time.When the corresponding fluorescence curve of the microlayer model is straight line
When, show in the microlayer model at this time without target gene ingredient.From the real-time fluorescence curves of acquisition, Ct value can be obtained, often
The Ct value of a microlayer model carries out derivation when obtaining, to the real-time fluorescence curves, and the slope of the real-time fluorescence curves is fixed
Fluorescence curve initial cycles number be required for Ct value.
The generation of microlayer model generating means 10 is uniform size microlayer model, by temperature control device 20 to the multiple micro-
Drop carries out nucleic acid amplification reaction, and acquires product signal, such as fluorescence, UV absorption, turbidity signal.The digital pcr inspection
Instrument 1 is surveyed by the microlayer model generating means 10, the temperature control device 20, the fluorescence signal detection device 30 and described fixed
It is integrated to measure analytical equipment 40, the operator is allowed to realize automation behaviour by integral type digital pcr detection machine 1
Make, improves work efficiency, rapid reaction, reproducible, high sensitivity, high specificity, result are clear.
The detection process of the digital pcr detector 1 mainly includes 5 links: prepare determined nucleic acid amplification reaction solution, to
Survey the acquisition and quantitative analysis of nucleic acid amplification reaction liquid droplet, nucleic acid amplification, fluorescence information.
In one embodiment, a kind of analysis method of digital pcr detector, comprising the following steps:
S10 prepares determined nucleic acid amplification reaction solution；
The determined nucleic acid amplification reaction solution droplet is formed multiple microlayer models by S20；
The multiple microlayer model is carried out nucleic acid amplification, and obtains the fluorescence information of the multiple microlayer model in real time by S30；
S40 carries out quantitative analysis to the multiple microlayer model according to the fluorescence information of the multiple microlayer model.At one
Implement
In example, the step S10 includes:
Preparation needs the nucleic acid amplification reaction liquid detected.It include nucleic acid mould to be detected in the nucleic acid amplification reaction liquid
Plate, reaction buffered aqueous solution, deoxyribonucleoside triphosphate, primer, polymerase and Product Labeling substance etc..
Wherein, nucleic acid amplification reaction liquid can be the nucleic acid amplification reaction liquid with DNA (DNA) for template
(can be described as DNA amplification reaction liquid), being also possible to (can with the reverse transcription nucleic acid amplification reaction liquid that ribonucleic acid (RNA) is template
Referred to as RNA inverse transcription reaction liquid), it can also be other nucleic acid amplification reaction liquid, as ring mediated isothermal amplification (LAMP) reacts
Liquid.Wherein, the characteristics of DNA amplification reaction liquid be containing dNTP, buffer required for DNA cloning, inorganic ion,
Polymerase, primer, DNA profiling to be detected and fluorescent dye or fluorescence probe etc..Fluorescent dye or fluorescence in reaction solution
Probe can indicate nucleic acid amplification, can be the fluorescent dye in conjunction with DNA such as SYBR Green, be also possible to simultaneously containing glimmering
The oligosaccharides nucleotide probe of light group and quenching group, such as TaqMan fluorescence probe.
In one embodiment, prepare to specialize in the reagent set and solution that digital pcr uses, for external source is reduced or avoided
Potential pollution of the DNA to template DNA sample.Used all appts and consumptive material should carry out high-temperature sterilization, at high temperature drying
Reason.The nucleic acid amplification reaction liquid ingredient to be detected includes: template DNA to be amplified, the few core of specificity for expanding template
Thuja acid primer, hot resistant DNA polymerase, 4 kinds of triphosphate deoxyribose nucleotide substrates, divalent metal Mg2+、Taqman
Probe or fluorescent dye and PCR buffer etc..
In one embodiment, when preparing the determined nucleic acid amplification reaction solution, using Taqman probe to described to be measured
Nucleic acid amplification reaction liquid is marked.
In one embodiment, when preparing the determined nucleic acid amplification reaction solution, using SYBR fluorescent dye to it is described to
Nucleic acid amplification reaction liquid is surveyed to be marked.
In one embodiment, the step S20 is multiple to be formed by the determined nucleic acid amplification reaction solution droplet
Microlayer model includes two kinds of microlayer model generation methods: the microlayer model generation method and the microlayer model in speed change period instantaneously accelerated is raw
At method.
The determined nucleic acid amplification reaction solution is subjected to droplet processing by the microlayer model generating means 10, can be obtained
Large batch of microlayer model is taken, the detection for the digital pcr detector 1.Wherein, the driving liquid 1214 be it is a kind of with
The determined nucleic acid amplification reaction solution objectionable intermingling and the liquid being independent of each other.First liquid is determined nucleic acid expansion
Increase reaction solution, the second liquid is oil mixture.The nucleic acid amplification reaction liquid prepared is generated by the microlayer model
Device can prepare large batch of microlayer model.During preparing the multiple microlayer model, by the multiple microlayer model
It is placed in the microlayer model container, the multiple microlayer model is detected to convenient.
In one embodiment, a large amount of micro- liquid is generated in the second liquid by the microlayer model generating means 10
Drop, can keep not merging between the multiple microlayer model.
Digital pcr (Digital PCR, dPCR) is a kind of nucleic acid molecules absolute quantitation technology.By described in digital pcr
Microlayer model generating means generate multiple microlayer models, and the bottom that will drop down onto the microlayer model container in the microlayer model container,
It is irregular to be packed together.The multiple microlayer models prepared by microlayer model generating means are concentrated in downward infall process
It is integrated into the intermediate position of microlayer model container, is flocked together, is unfavorable for observing.Based on this, it is necessary to be gathered in for concentration
The problem of microlayer model container bottom, provides a kind of microlayer model Tiling methods.
In one embodiment, the step S30 includes:
S310: the multiple microlayer model is laid in the microlayer model container；S320: will be the multiple micro- after tiling
Drop carries out nucleic acid amplification；S330: the multiple microlayer model carry out nucleic acid amplification when, in real time to the multiple microlayer model into
Capable detection of taking pictures.Referring to Figure 14, in one embodiment, the step S310 includes a kind of microlayer model Tiling methods.It is described
Microlayer model Tiling methods include:
S311, provides a microlayer model container 60, and the microlayer model container 60 has opening 631, and the microlayer model container
Second liquid is filled in 60；
S312, provides the first liquid, the density of first liquid be greater than the second liquid and with the second liquid
It is immiscible, and first liquid is generated into multiple microlayer model stackings and is piled up in the microlayer model container floor 610；
S313 carries out high/low temperature circulation to the multiple microlayer model, until the multiple microlayer model is laid in the container
Bottom plate 610.
Multiple microlayer models, and the container that the microlayer model container 60 will be dropped down onto are generated in the microlayer model container 60
Bottom plate 610, it is irregular to be packed together.It, can be in the appearance when a large amount of microlayer model drop to the container floor 610
The microlayer model of the formation multilayer of device bottom plate 610.And the multiple microlayer models prepared by microlayer model generating means were settled downwards
Cheng Dangzhong is integrated into the intermediate position of microlayer model container, flocks together, is unfavorable for observing.
In one embodiment, the second liquid is oil phase composition.
In one embodiment, the component of the oil phase composition includes mineral oil and surfactant.The oil phase
The percent by volume of composition mineral oil in fluid is 88%-98.5%.The surfactant includes the silicon oxygen chain containing chain alkyl
Nonionic surface active agent, the body of the silicon oxygen chain nonionic surface active agent containing chain alkyl in the oil phase composition
Product percentage is 1.5%-12%.
In one embodiment, first liquid is determined nucleic acid amplification reaction solution.
In one embodiment, the step S312 includes: S3122, provides and spits liquid pipette tips with outlet end, described to spit
The first liquid 190 is stored in liquid pipette tips；The liquid level of the second liquid is inserted into the outlet end for spitting liquid pipette tips by S3124
Under, and the movement that velocity magnitude is in mechanical periodicity is done, it is described to spit in the first half cycle and later half period of velocity magnitude variation
The velocity magnitude of the outlet end of liquid pipette tips is monotonically changed；
S3126, according to the movement of the mechanical periodicity of the outlet end for spitting liquid pipette tips, first liquid spits liquid by described
The outlet end of pipette tips is discharged, and forms multiple microlayer models under the second liquid liquid level, and be piled up in the microlayer model container bottom
Plate 610.
In one embodiment, in the step S3124, liquid of the outlet end for spitting liquid pipette tips in the second liquid
Velocity magnitude under face changes in cosine curve.
In one embodiment, described in the step S3124 spits the outlet end of liquid pipette tips under second liquid liquid level
The motion profile of mechanical periodicity include one of a variety of tracks such as straightway, arc section, polygon or a variety of combinations.
In one embodiment, the step S312 includes:
S3121 is provided and is spat liquid pipette tips with outlet end, and described spit stores the first liquid in liquid pipette tips；
The outlet end for spitting liquid pipette tips is inserted under the liquid level of the second liquid by S3123, instantaneous to accelerate；
S3125, according to the instantaneous accelerated motion of the outlet end for spitting liquid pipette tips, first liquid spits liquid rifle by described
The outlet end discharge of head, forms multiple microlayer models, and be laminated and be piled up in the microlayer model container under the second liquid liquid level
Bottom plate 610.
In one embodiment, in the step S3123, the period fortune of the outlet end for spitting liquid pipette tips instantaneously accelerated
In dynamic first half cycle and later half period, the velocity magnitude of the outlet end for spitting liquid pipette tips is identical, contrary.
In one embodiment, the motion profile of the periodic motion instantaneously accelerated in the step S3123 includes straight line
One of a variety of tracks such as section, arc section, polygon or a variety of combinations.
In one embodiment, the step S313 includes: S3131: the multiple microlayer model is heated up；S3133: by institute
State multiple microlayer model coolings；S3135: the multiple microlayer model is subjected to high/low temperature circulation repeatedly, until the multiple microlayer model
It is laid in the microlayer model container floor.
In one embodiment, when preparing the multiple microlayer model by the microlayer model generating means 10, first by institute
Second liquid is stated to be placed in the microlayer model container 60.When the liquid level of the second liquid is identical as the annular surface 641
When, stop that the second liquid is added.At this point, the surface of the liquid level of the second liquid and the annular surface 641 is in same water
In plane, it is ensured that the pasta of 699 body of the second liquid in the microlayer model container 60 is plane, facilitates guarantee container
The top surface of oil liquid above bottom surface is horizontal plane, convenient for imaging.
By the microlayer model generating means 10 by determined nucleic acid amplification reaction solution droplet, shape in the second liquid
At a large amount of microlayer models.The multiple microlayer model drop to multiple reaction members 612 of the microlayer model container floor 610
It is interior.Container floor 610 described in the annular surface 641 is parallel, to ensure the second liquid in the microlayer model container for level
Face.Multiple microlayer models can be placed in each reaction member 612, so that the microlayer model container 60 can accommodate
Microlayer model more than 20000.
In one embodiment, the instantaneous acceleration fortune of the outlet end of liquid pipette tips is spat according in the step S3125
Dynamic motion profile, the multiple microlayer model are laid in the microlayer model container floor 610.Going out for liquid pipette tips is spat by described
The motion profile instantaneously accelerated at mouth end can make the multiple microlayer model in dropping to the microlayer model container 60
When, it can mutually stagger, so that the multiple microlayer model does not have each other when dropping in the microlayer model container 60
Mutually accumulation.So that the multiple microlayer model is laid in the microlayer model container 60, facilitates and carry out observation of taking pictures.
In one embodiment, the microlayer model container floor 610 is coated with oleophobic layer.The oleophobic layer also makes oleophobic apply
Layer, is a kind of composite coating material, is a kind of functional material coating, often has the function of oleophobic.The oleophobic layer generally with
Nano silica is raw material (SiO2), using spraying process, coating is formed in screen surface, has good translucency
And hydro-oleophobicity.
In one embodiment, for the microlayer model Tiling methods in above embodiments, the temperature control device 20 includes soft
Property circuit board 220, with the flexible circuit board 220 is spaced heats the substrate 240 and be set to the flexible circuit board
220 and the multiple semi-conductor electricity couples 230 heated the substrate between 240.
High/low temperature circulation is carried out by the temperature control device 20 to tile using the principle expanded with heat and contract with cold.When object temperature
When degree increases, the kinetic energy of molecule increases, and the mean free path of molecule increases, so showing as heat expansion.Similarly, when object temperature drop
When low, the kinetic energy of molecule reduces, and the mean free path of molecule is reduced, so showing as shrinkage.With the variation of temperature, work as temperature
When degree increases, the viscosity of sample droplets is lower, volume contraction.Meanwhile temperature more high viscosity is lower, when temperature is on 60 DEG C of left sides
When right, sample droplets shape is most soft, and shape is probably rendered as hexagon at this time, however under other temperature conditions, sample liquid
The changeability for dripping shape is poor, it is not easy to which realization is tiled in drop receptacle.
Referring to Figure 15, multiple microlayer models are dropped in microlayer model container 60, the multiple microlayer model is deposited in described
Microlayer model container floor 610, that is, the multiple microlayer model forms multilayer microlayer model on the microlayer model container floor 610.
In fluorescence signal detection process, when taking pictures to the multiple microlayer model, influencing each other between multilayer is caused, is influenced
The detection of taking pictures of the multiple microlayer model.Therefore, the microlayer model container 60 for accommodating the multiple microlayer model is subjected to height
Low-temperature circulating.The multiple microlayer model is subjected to high/low temperature circulation repeatedly, until the multiple microlayer model is laid in micro- liquid
Container floor 610 is dripped, so that the large batch of microlayer model is laid in the reaction member 612, is convenient for the big rule of magnanimity drop
Mould parallel observation.
In one embodiment, the step of carrying out high/low temperature circulation by the temperature control device 20 is as follows:
Firstly, the multiple microlayer model, which is carried out heating, is heated to 90 DEG C~95 DEG C, and heat 5min~10min；Then,
The multiple microlayer model is cooled to 40 DEG C~60 DEG C, and anneals and extends 30s~60s；Finally, circuiting sequentially repeatedly, it is cooled to
0 DEG C~10 DEG C, the multiple microlayer model is saved.
In one embodiment, the step of high/low temperature circulation being carried out by the temperature control device 20 further include: firstly, by institute
It states multiple microlayer models and carries out heating heating, temperature is heated to 95 DEG C, and heat 10min；The multiple micro- liquid is heated to 95
DEG C, and 10min is heated, the enzyme in the multiple microlayer model is carried out thermal starting.Then, the multiple microlayer model is completed
After enzyme thermal starting, denaturation 30s is carried out to the multiple microlayer model；Secondly, being cooled to 55 after the multiple micro- liquid denaturation
DEG C, and anneal and extend 45s, it takes pictures to the multiple pico- liquid, and carry out 45 circulations；Finally, after circulation 45 times,
4 DEG C are cooled to, long-time preservation is carried out to the multiple micro- liquid.
The determined nucleic acid amplification reaction solution generates multiple microlayer models by the microlayer model generating means 10, to carry out
Detection.The multiple microlayer model prepared by the microlayer model generating means 10 concentrates set in downward infall process
It at the intermediate position of the microlayer model container 60, flocks together, is unfavorable for observing.So acquisition institute in order to be more accurate
The relevant information for stating the nucleic acid amplification reaction of multiple microlayer models needs for the multiple microlayer model to be laid in the microlayer model and holds
In device.By the way that the multiple microlayer model to be laid in the microlayer model container, one layer is formed, so as to avoid the micro- liquid of multilayer
Influencing each other between drop, the detection so that the fluorescence signal detection device 30 is taken pictures obtain more accurate ground fluorescence information,
In order to quantitative analysis.
PCR reaction condition is temperature, time and cycle-index.The setting of temperature and time: three step of based on PCR principle and
Denaturation-temperature spot of annealing-extension three is set.Three temperature spot methods are used in standard reaction, double-stranded DNA becomes at 90~95 DEG C
Property, then 40~60 DEG C are rapidly cooled to, and primer annealing is simultaneously integrated on target sequence, it is then rapidly heated to 70~75 DEG C,
Under the action of Taq archaeal dna polymerase, extend primer strand along template.It can for shorter target gene (when length is 100~300bp)
Using two temperature spot methods, in addition to denaturation temperature, annealing can be combined into one with elongating temperature, general using 94 DEG C of denaturation, 65 DEG C of left sides
Right annealing and extension (this temperature Taq DNA enzymatic still has higher catalytic activity).Denaturation temperature is with the time: denaturation temperature is low, solution
Chain is not exclusively the main reason for leading to PCR failure.Under normal circumstances, 93 DEG C~94 DEG C min are enough to be denaturalized template DNA,
It needs to extend the time if lower than 93 DEG C, but temperature cannot be excessively high, because hot environment has an impact to the activity of enzyme.If this step is not
Target gene template or PCR product can be made to be denaturalized completely, will lead to PCR failure.
Annealing (renaturation) temperature and time: annealing temperature is to influence the more important factor of PCR specificity.Temperature is fast after denaturation
Speed is cooled to 40 DEG C~60 DEG C, and primer and template can be made to combine.Since template DNA is more much more complex than primer, primer and mould
Collision bonding machine between plate can be significantly larger than the collision between template complementary chain.Annealing temperature and time, depending on primer
Length, base composition and its concentration, there are also the length of target motif column.For 20 nucleotide, the primer of G+C content about 50%,
55 DEG C are to select the starting point of most suitable annealing temperature ideal.
Within the allowable range, select higher renaturation temperature that can greatly reduce the non-specific binding between primer and template,
Improve the specificity of PCR reaction.The renaturation time is generally 30sec~60sec, it is sufficient to make to be completely combined between primer and template.
Elongating temperature and time: the biological activity of Taq archaeal dna polymerase: 70~80 DEG C of 150 nucleotide/S/ enzyme molecule；70 DEG C of 60 core
Thuja acid/S/ enzyme molecule；55 DEG C of 24 nucleotide/S/ enzyme molecule；When higher than 90 DEG C, DNA synthesis can hardly be carried out.
So the elongating temperature of PCR reaction is typically chosen between 70~75 DEG C, normal temperature is 72 DEG C, and excessively high prolongs
Stretch the combination that temperature is unfavorable for primer and template.The time of PCR extension, can depending on the length of segment to be amplified, one
As DNA fragmentation within 1Kb, extension of time 1min is enough.The target sequence of 3~4kb needs 3min~4min；Expand 10Kb
15min need to be extended to.The too long appearance that will lead to non-specific amplification band of extension of time.Amplification to low concentration template extends
Time wants a little longer.
In one embodiment, the step of the multiple microlayer model is carried out nucleic acid amplification by the step S320 is as follows: first
First, the microlayer model container 60 is placed in described in the temperature control device 20 and is heated the substrate on 240；It then, will be the multiple
Microlayer model carries out heating heating, temperature is heated to 95 DEG C, and heat 10min；The multiple micro- liquid is heated to 95 DEG C, and
10min is heated, the enzyme in the multiple microlayer model is carried out thermal starting.It is opened secondly, the multiple microlayer model completes enzyme heat
After dynamic, denaturation 30s is carried out to the multiple microlayer model；Again, after the multiple micro- liquid denaturation, 55 DEG C are cooled to, and move back
Fire extends 45s, and carries out 45 circulations；Finally, 4 DEG C are cooled to, when carrying out long to the multiple micro- liquid after circulation 45 times
Between save.
The temperature control device 20 uses the flexible circuit board 220 and the enhanced thermal conduction layer 250, holds the microlayer model
60 uniformity of temperature profile of device, accelerates the heating conduction of the semiconductor cooler.When the multiple microlayer model is in different temperatures
When carrying out nucleic acid amplification in range, it is set to the temperature sensor 260 and described second on 250 surface of enhanced thermal conduction layer
Controller 210 connects, and can detecte the real time temperature of the microlayer model container 60, and then temperature information is fed back to described the
Two controllers 210, to realize the control to the multiple microlayer model heating temperature.It may be implemented rapidly within several seconds time
It switches over.The cooling that instantaneously heats up may be implemented in the temperature control device 20, and then the process for the cooling that heats up shortens, thus real
The detection time of the digital pcr detector 1 is shortened, improves detection efficiency by the circulation for having showed high/low temperature.
The multiple microlayer model can be made to carry out nucleic acid amplification by the temperature control device 20.Three step of based on PCR principle
Suddenly denaturation-temperature spot of annealing-extension three is set.Use three temperature spot methods in standard reaction, double-stranded DNA 90~
95 DEG C of denaturation, then 40~60 DEG C are rapidly cooled to, primer annealing is simultaneously integrated on target sequence, is then rapidly heated to 70~75
DEG C, under the action of Taq archaeal dna polymerase, extend primer strand along template, nucleic acid is expanded within the scope of suitable temperature
Increase.Meanwhile in amplification process, the enhanced thermal conduction of the microlayer model container floor 610 and the temperature control device 20
The close note of layer 250 closes, and does not have gap between the two, homogeneous heating improves the accuracy of the digital pcr detector 1.
Each technical characteristic of embodiment described above can be combined arbitrarily, for simplicity of description, not to above-mentioned reality
It applies all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, all should be considered as described in this specification.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
Limitations on the scope of the patent of the present invention therefore cannot be interpreted as.It should be pointed out that for those of ordinary skill in the art
For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to of the invention
Protection scope.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Claims (12)
1. a kind of temperature control device characterized by comprising
Flexible circuit board (220)；
With the flexible circuit board (220) is spaced heats the substrate (240), described heat the substrate (240) include opposite set
The first surface (241) and second surface (242) set；
Multiple semi-conductor electricity couples (230), are set between the flexible circuit board (220) and the first surface (241), institute
State multiple semi-conductor electricity couples (230) be serially connected, in parallel or Hybrid connections.
2. temperature control device as described in claim 1, which is characterized in that the semi-conductor electricity couple (230) includes a p-type electricity
Even (231) and one and the spaced N-type galvanic couple (232) of the p-type galvanic couple (231).
3. temperature control device as claimed in claim 2, which is characterized in that the first surface (241) includes multiple interval settings
First electrode sheet (243), a first electrode sheet (243) is corresponding with one semi-conductor electricity couple (230), described
The p-type galvanic couple (231) and the N-type galvanic couple (232) in semi-conductor electricity couple (230) pass through institute's first electrode sheet
(243) it connects.
4. temperature control device as claimed in claim 2, which is characterized in that the flexible circuit board (220) includes that multiple intervals are set
The second electrode sheet (221) set and be serially connected, the semi-conductor electricity couple (230) of adjacent two pass through one described second
Electrode slice (221) series connection.
5. temperature control device as described in claim 1, which is characterized in that further include enhanced thermal conduction layer (250), be set to described
Two surfaces (242).
6. temperature control device as claimed in claim 4, which is characterized in that include stone in the material of the enhanced thermal conduction layer (250)
Black alkene.
7. such as temperature control device of any of claims 1-6, which is characterized in that it further include second controller (210), with
The multiple semi-conductor electricity couple (230) electrical connection, for controlling size of current.
8. temperature control device as claimed in claim 7, which is characterized in that further comprise temperature sensor (260), be set to institute
It states second surface (242) and is electrically connected with the second controller (210), for detecting the temperature of the second surface (242)
And the temperature is sent to the second controller (210).
9. temperature control device as claimed in claim 8, which is characterized in that the second controller (210) includes:
Temperature control unit (212) is connect with the temperature sensor (260), to second surface described in real-time detection (242)
Temperature；
Control circuit (214) is connect with the flexible circuit board 220, to regulate and control the multiple semi-conductor electricity couple (230)
Temperature change.
10. temperature control device as claimed in claim 7, which is characterized in that the flexible circuit board (220) is provided with first electrode
(222) and second electrode (223), after the multiple second electrode sheet (221) series connection with the first electrode (222) and described
Second electrode (223) series connection, the first electrode (222) connect with the control circuit respectively with the second electrode (223).
11. temperature control device as described in claim 1, which is characterized in that the temperature control device further includes radiator 270, institute
Stating radiator (270) includes substrate (271) and the cooling fin (272) connecting with the substrate (271), the flexible circuit
Plate (220) is set to the surface of the substrate (271).
12. temperature control device as claimed in claim 11, which is characterized in that the temperature control device further includes fan (273) setting
Around the cooling fin (273).
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| CN110702588A (en) * | 2019-09-25 | 2020-01-17 | 深圳先进技术研究院 | Digital PCR analysis device and PCR analysis method |
| CN114167912A (en) * | 2021-12-07 | 2022-03-11 | 苏州华星光电技术有限公司 | Temperature control device and service life testing method of display panel |
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