CN208580336U - Temperature control device - Google Patents

Temperature control device Download PDF

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Publication number
CN208580336U
CN208580336U CN201820125231.9U CN201820125231U CN208580336U CN 208580336 U CN208580336 U CN 208580336U CN 201820125231 U CN201820125231 U CN 201820125231U CN 208580336 U CN208580336 U CN 208580336U
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China
Prior art keywords
microlayer model
control device
temperature control
temperature
circuit board
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CN201820125231.9U
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Chinese (zh)
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盛广济
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Suzhou sinafu Medical Technology Co., Ltd
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Beijing Optical Trap Management Consulting Partnership (limited Partnership)
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Abstract

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

Temperature control device
Technical field
The utility model 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, the too long meeting of extension of time 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 temperature After degree, remain unchanged.The heating of testing sample solution has hysteresis quality, as the temperature of testing sample solution is close in target temperature The outer temperature difference is smaller and smaller, heats up slower and slower.Existing temperature control device, temperature rate is slow, and heating up or cooling down each time all needs It wants tens seconds to several minutes, does tens PCR cycles and need or so 1~2 hour.Therefore, this temperature rate will lead to Time needed for completing nucleic acid amplification extends, and nucleic acid amplification efficiency is low.
Utility model content
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 utility model provide a kind of temperature control device include flexible circuit board, with the flexible circuit board it is spaced plus Hot substrate 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 described Multiple semi-conductor electricity couples are set between the flexible circuit board and the first surface, the multiple semi-conductor electricity couple phase 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 of the semiconductor galvanic centering and the N-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 second Controller.
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 point It is not connect 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 user of temperature control device application method temperature control device as described above in one of the embodiments, Method.
The temperature control device include flexible circuit board, with the flexible circuit board is spaced heats the substrate and multiple Semi-conductor electricity couple.It is described to heat the substrate the first surface and second surface including being oppositely arranged.The multiple semiconductor galvanic To being set between the flexible circuit board and the first surface, the multiple semi-conductor electricity couple is serially connected, it is in parallel or Person's Hybrid connections.The flexible circuit board has the characteristics that Distribution density is high, light-weight, thickness is thin, bending is good.The flexibility 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 with And multiple semi-conductor electricity couples may be implemented to switch within several seconds time rapidly.The temperature control device may be implemented instantaneously to rise Temperature drop temperature, and then the process for the cooling that heats up shortens, so that the circulation of high/low temperature is realized, by the digital pcr detector Detection time shortens, and improves detection efficiency.
Detailed description of the invention
Fig. 1 is the overall structure diagram of digital pcr detector provided by the utility model；
Fig. 2 is the utility model temperature control device structural schematic diagram；
Fig. 3 is the utility model temperature control device structure section structural schematic diagram；
Fig. 4 is the semi-conductor electricity couple electrode connecting structure schematic diagram of the utility model temperature control device；
Fig. 5 is the mapping test schematic diagram of the utility model temperature control device；
Fig. 6 is the steady-state performance test schematic diagram of the utility model temperature control device；
Fig. 7 is the utility model microlayer model structure of container schematic diagram；
Fig. 8 is the utility model microlayer model structure of container planar structure schematic diagram；
Fig. 9 is the reaction member structural schematic diagram of the utility model microlayer model container；
Figure 10 is a kind of section structural schematic diagram of the utility model microlayer model container；
Figure 11 is a kind of section structural schematic diagram of the utility model microlayer model container；
Figure 12 is a kind of section structural schematic diagram of the utility model microlayer model container；
Figure 13 is the analysis method flow chart of the utility model digital pcr detector；
Figure 14 is the utility model microlayer model Tiling methods flow chart；
Figure 15 is that the utility model microlayer model container floor microlayer model 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 purpose of the utility model, technical solutions and advantages more clearly understood, by the following examples, it and ties Attached drawing is closed, the present invention will be further described in detail.It should be appreciated that specific embodiment described herein is only to solve The utility model is released, is not used to limit the utility model.
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", There is no intermediary elements.Term as used herein "vertical", "horizontal", "left" and "right" and similar statement are For illustrative purposes.Various difference objects are in the ratio drafting convenient for enumerating explanation in embodiment attached drawing, rather than press practical group The ratio of part is drawn.
Referring to Figure 1, the utility model provides a kind of digital pcr detector 1, and the digital pcr detector 1 includes: micro- Drop formation device 10, temperature control device 20, fluorescence signal detection device 30, quantitative analysis device 40 and controller 50.It is described Microlayer model generating means 10 are to form multiple microlayer models for nucleic acid amplification reaction liquid droplet.The temperature control device 20 and institute It states microlayer model generating means 10 to connect by track, the multiple microlayer model is transferred to the temperature control device 20, carry out Temperature cycles realize nucleic acid amplification.The fluorescence signal detection device 30 is oppositely arranged with the temperature control device 20, to core The multiple microlayer model after acid 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 40 connections are set, to control the microlayer model generating means 10, the temperature control device 20, fluorescence signal detection device 30 and determine Measure analytical equipment 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 the relationship of Ct value and starting copy number to the concentration of initial DNA Carry out quantitative analysis.Wherein, Ct value refers to recurring number experienced when the fluorescence signal of each microlayer model reaches the threshold value of setting.
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.Multiple sizes can be generated by microlayer model generating means 10 Uniform microlayer model 199.In the micron-scale, and each microlayer model 199 can be regarded as one to each 199 size of the microlayer model A independent reactor is equivalent to common test tube in biochemical reaction.The multiple microlayer model 199 is placed in microlayer model container 60 In, convenient for detection observation.Meanwhile the different microlayer model of multiple volumes also can be generated by the microlayer model generating means 10, To carry out clinical medicine detection.The multiple microlayer model 199 is small in size, quantity is more, with no excellent of many conventional test tubes Gesture.A large amount of microlayer models 199 can be generated by the microlayer model generating means 10, lead to so that the digital pcr detector 1 has Amount height, the low advantage low with ambient noise of consumables cost, have 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 heat the substrate 240 include be oppositely arranged first Surface 241 and second surface 242.The multiple semi-conductor electricity couple 230 is set to the flexible circuit board 220 and described the Between one 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 close Spend the advantage high, light-weight, thickness is thin, bending is good.The flexible circuit board 220 is light-weight, thickness is thin, can effectively 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 constrained by thermal deformation Caused self balancing stress is compressed in temperature eminence, and tensile deformation occurs for temperature lower.Proof stress under certain condition It 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, in turn 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 real The detection time of the digital pcr detector 1 is shortened, improves detection efficiency by the circulation for having showed 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 arranged 240 surfaces are heated the substrate in described, the temperature sensor 260 is electrically connected with the second controller 210, described for detecting It heats 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 utility model also provides a kind of temperature control device 20 comprising flexible circuit board 220, with it is described Flexible circuit board 220 is spaced to heat the substrate 240 and multiple semi-conductor electricity couples 230.It is described to heat the substrate 240 and include The first surface 241 and second surface 242 being oppositely arranged.The multiple semi-conductor electricity couple 230 is set to the flexible circuit Between plate 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.It is described Flexible circuit board 220 is in heating and cooling process with the eliminating deformation thermal stress of itself.It can be dropped by the flexible circuit board 220 Thermal stress present in low heating and cooling process, to extend the service life of the temperature control device 20.Meanwhile by described soft Property circuit board 220, solves the problems, such as that temperature distribution is non-uniform.When the multiple microlayer model carries out core within the scope of different temperatures Acid amplification when, by the flexible circuit board 220, heat the substrate 240 and multiple semi-conductor electricity couples 230 may be implemented rapidly It was switched within several seconds time.The cooling that instantaneously heats up may be implemented in the temperature control device 20, and then the process contracting for the cooling that heats up It is short, to realize the circulation of high/low temperature, the detection time of the digital pcr detector 1 is shortened, improves detection effect Rate.
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.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.Control is answered under certain condition Power 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 that aluminium closes Gold, can be thermally conductive fast.Aluminum substrate can minimize thermal resistance, and aluminum substrate is made to have fabulous heat-conductive characteristic, make pottery with thick film Porcelain 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 of galvanic couples formed by the p-type galvanic couple 231 and the N-type galvanic couple 232, It is connected together between the multipair semi-conductor electricity couple 230 by electrode, and is clipped in the flexible circuit board 220 and described the Between one surface 241.When a current flows through, " heat " side and " cold " side can be generated.It is refrigeration or heating and refrigeration, adds The rate of heat, by being determined by its current direction and size.The pyroelectric effect that a pair of semi-conductor electricity couple 230 generates Very little, so being all cascaded in practice by up to a hundred to the semi-conductor electricity couple 230, the pyroelectric effect generated in this way is just It will increase.
In one embodiment, the first surface 241 includes multiple spaced first electrode sheets 243, an institute State that first electrode sheet 243 is corresponding with a semi-conductor electricity couple 230, the p-type electricity in the semi-conductor electricity couple 230 Even 231 are connected with the N-type galvanic couple 232 by institute's 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 and hot End.But there are resistance when electric current is by the p-type galvanic couple 231 and institute for the p-type galvanic couple 231 and the N-type galvanic couple 232 itself Heat will be generated when stating N-type galvanic couple 232, to will affect hot transmitting.And the flexible circuit board 220 and the heating base Heat between plate 240 also can carry out reverse heat by air and the p-type galvanic couple 231 and 232 material of N-type galvanic couple itself Transmitting.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, can take scattered The modes such as heat 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, thus by pair Nucleic acid amplification is realized in the control of temperature.The enhanced thermal conduction layer 250 can be graphite thermal conductive layer or silicone grease heat-conducting layer, accelerate to lead Heat, increases the temperature uniformity of the second surface 242 for heating the substrate 240, and then guarantees close to the microlayer model The surface temperature of container 60 is uniform, so that the multiple microlayer model is heated evenly.And then nucleic acid amplification is completed, it improves 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 the temperature To 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, realized 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.Institute It states control circuit 214 to connect with the flexible circuit board 220, the temperature to regulate and control the multiple semi-conductor electricity couple 230 becomes Change.
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 calculating of control circuit 214 is as internal algorithm Output, to form closed loop relationship.The temperature feedback of circuit part is actually a sample circuit.Acquisition is exactly platinum electricity Electric signal in resistance is converted to the input terminal that temperature value passes to control circuit.The temperature sensor 260 and the temperature control Unit 212 processed 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 It is connect respectively with the control circuit 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, thus Control the temperature of the multiple microlayer model.The multiple microlayer model can be made to carry out nucleic acid expansion by the temperature control device 20 Increase.Three step of based on PCR principle and denaturation-temperature spot of annealing-extension three is set.Three temperature are used in standard reaction 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, so After be rapidly heated to 70~75 DEG C, under the action of Taq archaeal dna polymerase, extend primer strand along template, in suitable temperature Nucleic acid is expanded in range.Meanwhile in amplification process, 60 bottom of microlayer model container and the temperature control device 20 close notes close, and do not have gap between the two, improve 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 adding to the multiple microlayer model The monitoring of thermal process, when the temperature control device 20 carries out gradient of temperature to the multiple microlayer model, temperature rate maximum can be with Reach 13.34448 DEG C/s, control precision is 0.02722 DEG C.Also, when the temperature control device 20 is warming up to stable state sometimes The rate of measurement most fastly can be to 18.953894 DEG C/s.Therefore, the transient response of the temperature control device 20 is good, passes through the temperature The cooling that instantaneously heats up may be implemented in control device 20, 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, the temperature Control device 20 can achieve quickly heating down cycles, and warmly taken after stablizing float it is smaller, save digital pcr detection solution It the time of sample, improves work efficiency.The time needed for completing nucleic acid amplification can be shortened by this temperature rate, improved Nucleic acid amplification efficiency, 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, it during being heated by the temperature control device 20 to the multiple microlayer model, is examined using the fluorescence signal It surveys device 30 and claps the change in fluorescence image for surveying the multiple microlayer model in real time.By the quantitative analysis device 40 to described more The change in fluorescence image of a microlayer model is analyzed, and obtains the Ct value of the multiple microlayer model, and pass through Ct value and starting copies Several relationships 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 micro- according to the change in fluorescence curve The Ct value of drop, 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 Refer 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.Utilize the multiple expansion Increase the difference in composition with non-amplification microlayer model, the amount of droplets for obtaining target sequence amplification is analyzed, it is final to realize Quantitative analysis to nucleic acid molecules.By the change in fluorescence picture of the multiple microlayer model of real-time monitoring, sequencing result has straight Connecing property, 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, is belonged to In expendable consumed product, carries out one-time detection and need to be abandoned, prevent cross contamination.But during atual detection, from Microlayer model occur card is transferred to PCR reaction plate, from PCR reaction plate to liquid drop analyzer during can all lose, to institute The number for stating microlayer model still has limitation.So if current microlayer model reaction member carries out a large amount of microlayer model detection, Limitation is still had to the number of the microlayer model, and consumables cost is higher.
Fig. 7-12 is referred to, the utility model embodiment provides a kind of microlayer model container 60 comprising bottom surface 611 surrounds The first annular side 621 and annular surface 641 that the bottom surface 611 is arranged.The first annular side 621 and the bottom surface 611 are connected, and surround and to form a storage space 630 with opening 631, and the first annular side 621 is perpendicular to described Bottom surface 611.The annular surface 641 is connected around 631 setting of opening with the first annular side 621, the annular Face 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 the multiple close to the observation of the microlayer model at edge position The detection efficiency of 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 is 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 more The microlayer model of how much amount.
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).It is described The surface of the liquid level of second liquid and the annular surface 641 is in same level, it is ensured that in the microlayer model container 60 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 institute The problem of whole liquid level for stating microlayer model container is arc, and spill liquid level is presented in liquid level.To be detected by the fluorescence signal When device 30 carries out fluorescence detection to 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 60 shape of microlayer model container can be made by the connection of the annular slab 640 and the first annular side plate 620 At a horizontal platform.When the second liquid is added in the microlayer model container 60, the second liquid can be made The surface of liquid level and the annular surface 641 thereby may be ensured that described in the microlayer model container 60 in same level The pasta of second liquid is plane, facilitates and guarantees that the top surface of the oil liquid above container bottoms is horizontal plane, avoids micro- liquid The problem of whole liquid level for dripping container is arc, and spill liquid level is presented in liquid level.To pass through the fluorescence signal detection device 30 When carrying out fluorescence detection to the multiple microlayer model, more 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 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 641 of the second liquid Surface in same level so that liquid level in the microlayer model container 60 is plane, under the conventional situation of surface Concave meniscus generation, convenient for imaging, improve the utilization rate of the microlayer model container 60.
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 once Property forms multiple microlayer models in multiple microlayer model accommodating grooves 614, improves the saturation of the microlayer model container 60, It can 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 camera The fluorescence information that all microlayer models can be obtained improves 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 institute Multiple microlayer models are stated to flock together.Meanwhile the multiple microlayer model can be made quicker when being tiled, be conducive to The multiple microlayer model is laid in the microlayer model container floor 610.When the surface tension of the container floor is less than described the When the surface tension of two liquid (oils) 699, the resistance of the microlayer model and the bottom plate becomes smaller, and the microlayer model can be from trend The diffusion of 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 using spraying process, is formed on surface and is applied generally using nano silica as raw material (SiO2) Layer, has good translucency and hydro-oleophobicity.When the multiple microlayer model and the reaction member contact, contact angle can To reach 90 degree, it can be achieved that tumbling and leaving no trace automatically, so as to realize that the multiple microlayer model is laid in micro- liquid Drip 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 cover 670 are 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 will be described 60 shape of microlayer model container is designed as rectangular, and the space utilization rate of the microlayer model container can be improved, and is conducive to conveniently The splicing of the fluorescent image of formation, 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 The different micro- liquid of multiple volumes can be generated in the multiple reaction member 612 by the microlayer model generating means 10 Drop.
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 camera The fluorescence information that all microlayer models can be obtained improves 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 limit to the number of the microlayer model System.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 inhomogeneity 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 In reaction member 612.
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 institute Multiple microlayer models are stated to flock together.Meanwhile the multiple microlayer model can be made quicker when being tiled, be conducive to The multiple microlayer model is laid in the microlayer model container floor 610.When the surface tension of the container floor is less than described the When the surface tension of two liquid (oils), the resistance of the microlayer model and the bottom plate becomes smaller, and the microlayer model can be from trend institute The diffusion of microlayer model reaction member bottom is stated, 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, is formed and applied on surface Layer, has good translucency and hydro-oleophobicity.When the multiple microlayer model and the reaction member contact, contact angle can To reach 90 degree, it can be achieved that tumbling and leaving no trace automatically, so as to realize that the multiple microlayer model is laid in micro- liquid Drip 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 digital pcr The detection efficiency of 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 reaction members 612 are set to the microlayer model container floor 610 in the form of an array, form 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 and The advantages that chemical stability, receives 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 and hold The storage amount of device may be implemented the detection greater than 20000 microlayer models, and can detect to different types of nucleic acid, pass through Ji material benefit.
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 it reducing the microlayer model and holding The first annular side plate 620 of device 60 or the height of second annular side plate 650 are conducive to exclude exciting light from side Shade caused by when irradiation enables the camera 331 to obtain the fluorescence information of all microlayer models, improves the fluorescence The sensitivity of 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, pass through The temperature control device 20 carries out nucleic acid amplification to the multiple microlayer model.Meanwhile it is real using the fluorescence signal detection device 30 When clap the change in fluorescence image for surveying the multiple microlayer model.By the change in fluorescence image of the multiple microlayer model, described in acquisition The change in fluorescence curve of multiple microlayer models.According to the change in fluorescence curve, the Ct value of available the multiple microlayer model, 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, 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, Show in the microlayer model at this time without target gene ingredient.From the real-time fluorescence curves of acquisition, Ct value, Mei Gewei can be obtained The Ct value of drop carries out derivation when obtaining, to the real-time fluorescence curves, and the slope of the real-time fluorescence curves is fixed glimmering The initial cycles number of light curve is required 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 quantitative Analytical equipment 40 is integrated, and the operator is allowed to realize automatic operation by integral type digital pcr detection machine 1, It 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.
Referring to Figure 13, 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, such as ring mediated isothermal amplification (LAMP) reaction solution. Wherein, the characteristics of DNA amplification reaction liquid is containing dNTP, buffer required for DNA cloning, inorganic ion, polymerization Enzyme, primer, DNA profiling to be detected and fluorescent dye or fluorescence probe etc..Fluorescent dye or fluorescence probe in reaction solution It can indicate nucleic acid amplification, can be the fluorescent dye in conjunction with DNA such as SYBR Green, be also possible to contain fluorophor simultaneously With the oligosaccharides nucleotide probe of 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 generates 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 is a kind of and institute The liquid stating determined nucleic acid amplification reaction solution objectionable intermingling and being independent of each other.First liquid is that determined nucleic acid amplification is anti- Liquid is answered, the second liquid is oil mixture.By the nucleic acid amplification reaction liquid prepared by the microlayer model generating means, Large batch of microlayer model can be prepared.During preparing the multiple microlayer model, the multiple microlayer model is placed in 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: when the multiple microlayer model carries out nucleic acid amplification, the multiple microlayer model is carried out in real time It takes pictures detection.Referring to Figure 14, in one embodiment, the step S310 includes a kind of microlayer model Tiling methods.It is described micro- Drop 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 volume of the silicon oxygen chain nonionic surface active agent containing chain alkyl in the oil phase composition 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 liquid in the first half cycle and later half period of velocity magnitude variation The velocity magnitude of the outlet end of 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, 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 level On, it is ensured that the pasta of 699 body of the second liquid in the microlayer model container 60 is plane, facilitates and guarantees on container bottoms The top surface of the oil liquid of side 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 it is super Cross 20000 microlayer models.
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.Pass through the outlet for spitting liquid pipette tips The motion profile instantaneously accelerated at end can make the multiple microlayer model when dropping in the microlayer model container 60, It can mutually stagger, so that the multiple microlayer model is when dropping in the microlayer model container 60, it is no mutual each other 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, forms coating in screen surface, have 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 hypothermia When, the kinetic energy of molecule reduces, and the mean free path of molecule is reduced, so showing as shrinkage.With the variation of temperature, when temperature liter Gao Shi, the viscosity of sample droplets is lower, volume contraction.Meanwhile temperature more high viscosity is lower, when temperature is at 60 DEG C or so, Sample droplets shape is most soft, and shape is probably rendered as hexagon at this time, however under other temperature conditions, sample droplets shape Changeability it is poor, it is not easy to realization tile 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, influences institute State the detection of taking pictures of multiple microlayer models.Therefore, the microlayer model container 60 for accommodating the multiple microlayer model is subjected to height Temperature circulation.The multiple microlayer model is subjected to high/low temperature circulation repeatedly, until the multiple microlayer model is laid in the microlayer model Container floor 610, so that the large batch of microlayer model is laid in the reaction member 612, it is extensive convenient for magnanimity drop 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 completes enzyme After thermal starting, denaturation 30s is carried out to the multiple microlayer model；Secondly, 55 DEG C are cooled to after the multiple micro- liquid denaturation, And anneal and extend 45s, it takes pictures to the multiple pico- liquid, and carry out 45 circulations；Finally, after circulation 45 times, cooling To 4 DEG C, 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 container In.By the way that the multiple microlayer model to be laid in the microlayer model container, form one layer, so as to avoid multilayer microlayer model it Between influence each other 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, primer annealing is simultaneously integrated on target sequence, is then rapidly heated to 70~75 DEG C, in Taq Under the action of archaeal dna polymerase, extend primer strand along template.Shorter target gene (when length is 100~300bp) can be used Two temperature spot methods, in addition to denaturation temperature, annealing can be combined into one with elongating temperature, and general using 94 DEG C of denaturation, 65 DEG C or so are moved back Fire and extension (this temperature Taq DNA enzymatic still has higher catalytic activity).Denaturation temperature is with the time: denaturation temperature is low, and unwinding is not It is entirely 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, if low It then needs to extend the time in 93 DEG C, but temperature cannot be excessively high, because hot environment has an impact to the activity of enzyme.If this step cannot make target Gene template or PCR product are 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 is added Hot 10min, the enzyme in the multiple microlayer model is carried out thermal starting.Secondly, the multiple microlayer model completes enzyme thermal starting Later, denaturation 30s is carried out to the multiple microlayer model；Again, after the multiple micro- liquid denaturation, 55 DEG C are cooled to, and anneal Extend 45s, and carries out 45 circulations；Finally, being cooled to 4 DEG C after circulation 45 times, long-time guarantor is carried out to the multiple micro- liquid It deposits.
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, the temperature sensor 260 and described second for being set to 250 surface of enhanced thermal conduction layer are controlled Device 210 processed connects, and can detecte the real time temperature of the microlayer model container 60, and then temperature information is fed back to described second Controller 210, to realize the control to the multiple microlayer model heating temperature.It may be implemented to carry out within several seconds time rapidly Switching.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, to realize height The detection time of the digital pcr detector 1 is shortened, improves detection efficiency by the circulation of 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.Three temperature spot methods are used in standard reaction, double-stranded DNA is 90~95 DEG C denaturation, then is rapidly cooled to 40~60 DEG C, and 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. Meanwhile in amplification process, the enhanced thermal conduction layer of the microlayer model container floor 610 and the temperature control device 20 250 close notes close, and do 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.
Above-described embodiments merely represent several embodiments of the utility model, the description thereof is more specific and detailed, But it should not be understood as limiting the scope of the patent of the utility model.It should be pointed out that for the common of this field For technical staff, without departing from the concept of the premise utility, various modifications and improvements can be made, these all belong to In the protection scope of the utility model.Therefore, the scope of protection shall be subject to the appended claims for the utility model patent.

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 3, 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 5, which is characterized in that include stone in the material of the enhanced thermal conduction layer (250) Black alkene.
7. temperature control device as described in any of claims 4, which is characterized in that further include second controller (210), with institute Multiple semi-conductor electricity couple (230) electrical connections are stated, 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 9, 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) and the second electrode (223) respectively with the control circuit (214) Connection.
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 (272).
CN201820125231.9U 2018-01-24 2018-01-24 Temperature control device Active CN208580336U (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110069084A (en) * 2018-01-24 2019-07-30 思纳福(北京)医疗科技有限公司 Temperature control device
CN110752171A (en) * 2019-11-01 2020-02-04 长江存储科技有限责任公司 Wafer curvature adjusting device and method

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110069084A (en) * 2018-01-24 2019-07-30 思纳福(北京)医疗科技有限公司 Temperature control device
CN110752171A (en) * 2019-11-01 2020-02-04 长江存储科技有限责任公司 Wafer curvature adjusting device and method
CN110752171B (en) * 2019-11-01 2022-07-29 长江存储科技有限责任公司 Device and method for adjusting wafer curvature

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