CN1137990C

CN1137990C - Microstructure polyase chain reaction cloning device

Info

Publication number: CN1137990C
Application number: CNB991273281A
Authority: CN
Inventors: 崔大付
Original assignee: Institute of Electronics of CAS
Current assignee: Institute of Electronics of CAS
Priority date: 1999-12-29
Filing date: 1999-12-29
Publication date: 2004-02-11
Anticipated expiration: 2019-12-29
Also published as: CN1301810A

Abstract

The present invention relates to a novel microstructure polyase chain reaction (PCR) cloning device which belongs to a novel applied device in the biologic field and the medical field. The present invention is suitable for research of biology and medicine. The present invention is also suitable for DNA gene amplification without pollution during medical clinic treatment. The device is manufactured according to the technology of a microelectron mechanical system (MEMS). The device has the advantages of miniaturization, disposable use and no pollution. In the structure of the device, a Peltier effect semiconductor refrigerating effect component is adopted. In the process of DNA gene amplification heat circulation, controllable, motive and quick refrigeration cooling operation can be realized. The amplification process is quickened, and the efficiency is increased.

Description

Microstructure polyase chain reaction cloning device

The invention belongs to information technology and the biomedical crossing domain that combines, the miniature gene amplification device that adopts microelectron-mechanical processing technology in the information technology and conductor refrigeration principle to make, and be suitable for a kind of quick, the free of contamination DNA gene amplification device used in the multiple field such as biomedicine.

Polymerase chain reaction (Polymeraze Chain Reaction-PCR) amplification technique proposes during the last ten years, development rapidly, the very important instruments of research field such as molecular biology and medical science have now been become, it can be fast, amplification in vitro goes out any desirable dna fragmentation, even single copy gene.And be widely used in molecular biology, medical science, genetics every field, many-sided research such as be used for the detecting of gene clone, order-checking, mutant and recombinant construction, gene expression regulation, Polymorphism Analysis, inherited disease and transmissible disease diagnosis, tumour mechanism, legal medical expert identifies.

Polymerase chain reaction (PCR) augmentor that uses both at home and abroad all belongs to large-scale plant at present, rather than the disposable using appts of microstructure type.Exist to pollute that consumption sample amount is big, speed waits problem, particularly domestic device slowly, exist serious pollution problem, use and produced by strict control.

And the domestic not development still of microstructure type pcr amplification device is abroad being developed, and does not still have commodity to come into the market.External microstructure polymerase chain reaction (PCR) augmentor of developing is determine active cooling, heat dissipation problem in the heating cycle process of opinion not.If solve the active heat removal cooling, can further accelerate thermal cycling speed, raise the efficiency.If make the precise temperature measurement transmitter on augmentor, the temperature of Monitoring and Controlling amplification procedure is studied the influence of temperature variation to amplification procedure effectively, and bigger improvement will be arranged the performance of augmentor, and this all is very important, and this invention will address the above problem.

It is big to the objective of the invention is to overcome common augmentor equipment volume, length consuming time, and 30-40 circulation needs a few hours, and consumption sample amount is big, is easy to generate problems such as pollution; The little well type that adopts the microelectron-mechanical processing technology on the thick silicon chip of about 0.5mm, to produce the about μ l of the volume level chamber of increasing, place sample liquid to be amplified, microcavity is produced the polysilicon heating resistor on one side, and the other side adds glass cover-plate, and the last liquid entrance of making forms.The polysilicon heating resistor is used for circulating and heats during work, cooling naturally.Because polymerase chain reaction (PCR) is a kind of nucleic acid amplification technologies in in-vitro simulated nature dna replication dna process, its working process is a repeatedly thermal cycling process.Usually dna molecular sex change under 95 ℃ of temperature is unwind, thereafter temperature is reduced under 55 ℃, anneal, combination, be warmed up to 75 ℃ then once again, the bonded dna molecular extends shaping, finishes a reproduction process, and so forth, the single DNA gene through 35 recursive copying amplifications, can reach 34,000,000,000 more than.Realize complete gene amplification process.

Technical scheme of the present invention as shown in Figure 1, a kind of novel microstructure polymerase chain reaction (PCR) augmentor is proposed, it has adopted Po Er to paste semiconductor cooler refrigeration, the integral part of semiconductor cooler as augmentor, and two kinds of typical structures are proposed and be provided with that high-precision platinum resistor heats, thermometric and temperature control.

Fig. 2 and Fig. 3 are two kinds of typical structural representations of the present invention.

1 is the miniature cavity of Si among Fig. 2, the 2nd, and sample inlet, the 3, the 5th, refrigerator inducer, the 4,8P.N N-type semiconductorN refrigerating sheet, the 6th, hot coupling layer, the 7th, radiator element, the 9th, sample delivery port, 10 insulating films, the 11st, heating RTD, the 12nd, the accurate RTD of thermometric.Its structure belongs to monolateral heat temperature raising, monolateral refrigeration cooling structure;

1 is Si microstructure cavity among Fig. 3,2, the 9th, sample is imported and exported, 3, the 5,10, the 12nd, the refrigerator inducer, 4, the 8,11, the 15th, N.P N-type semiconductorN refrigerating sheet, 6, the 13rd, hot coupling layer, the 7, the 14th, radiator element, the 16th, insulation layer, the 17th, the accurate RTD of thermometric, it will be with electrical isolation between the inducer during manufacturing.

The structure of two kinds of augmentors is as follows:

A. the substrate of augmentor is the miniature cavity of Si, the substrate upper edge has sample inlet and sample export, above outlet, one deck refrigerator inducer is housed, the inducer top is equipped with P-type semiconductor refrigerating sheet and N-type semiconductor refrigerating sheet respectively, above refrigerating sheet, also be equipped with and cause the device inducer, hot coupling layer is housed on this inducer, on the hot coupling layer radiator element is arranged; Under the substrate bottom, add one deck insulating film, the accurate RTD of heating RTD and thermometric is housed under the insulating film;

B. the substrate of augmentor is a Si microstructure cavity, the substrate upper edge has sample inlet and the outlet of sample mouth, above outlet, one deck refrigerator inducer is housed, the inducer top is equipped with P-type semiconductor refrigerating sheet and N-type semiconductor refrigerating sheet respectively, the refrigerator inducer also is housed above refrigerating sheet, hot coupling layer is housed on this inducer, on the hot coupling layer radiator element is arranged; Under the bottom of Si microstructure cavity, add one deck insulating film, the accurate RTD of a thermometric is housed on insulating film; The refrigerator inducer is housed under the insulating film, is respectively N-type semiconductor refrigerating sheet and P-type semiconductor refrigerating sheet under the inducer, the refrigerator inducer is housed under the refrigerating sheet, inducer is hot coupling layer down.

Technical essential of the present invention is that also the semiconducter device in the assembling of the both sides of augmentor microcavity body is that Po Er pastes conductor refrigeration spare.The structure that Po Er pastes semiconductor cooler is: on N-type semiconductor cooling piece and P-type semiconductor cooling piece the metal inducer is housed, the below of refrigerating sheet is equipped with inducer respectively, and these two inducers are connected with direct supply.

The present invention has following advantage: the one, and the miniature pcr amplification device that this employing conductor refrigeration principle is carried out initiatively cooling at home and abroad is to propose first, can raise the efficiency greatly, accelerates amplification rate; Having adopted RTD heating and thermometric among the present invention is convenient feasible in the technology not only, and resistance is adjustable, platinum material temperature factor good linearity, be convenient to accurate thermometric, thereby temperature in the time of accurately controlling amplification, very crucial to the rule that improves amplification efficiency, research and grasp amplification procedure, be that common augmentor is irrealizable; Apparatus of the present invention can disposablely be used, and amplification does not have a pollution problem in the sealing microcavity, during to the amplification of the disease gene that is infectious with necessary especially; This invention also is one of novel hi-tech DNA chip of integrated, microminiaturized, multifunction, and superior performance is the direction and goal of future development.

Description of drawings:

Fig. 1 is common microstructure type polymerase chain reaction (PCR) augmentor synoptic diagram

1-Si substrate, 2-sample inlet, 3, cover plate, 4-sample export, 5-electrode.

Fig. 2 is monolateral refrigeration type polymerase chain reaction (PCR) augmentor synoptic diagram for novel single-side heats.

The miniature cavity of 1-Si, 2-sample inlet, 3-refrigerator inducer, 4-P N-type semiconductorN refrigerating sheet, 5-refrigerator inducer, the hot coupling sheet of 6-, 7-radiator element, 8-N N-type semiconductorN refrigerating sheet, 9-sample delivery port, 10-insulating film, 11-heating RTD, the accurate RTD of 12-thermometric.

Fig. 3 is novel heating and refrigeration integral structure polymerase chain reaction (PCR) augmentor synoptic diagram.

1-Si microstructure cavity, 2-sample inlet, 3-refrigerator inducer, 4-N N-type semiconductorN refrigerating sheet, 5-refrigerator inducer, the hot coupling layer of 6-, 7-radiator element, 8-P N-type semiconductorN refrigerating sheet, 9-sample export, 10-refrigerator inducer, 11-P N-type semiconductorN refrigerating sheet, 12-refrigerator inducer, the hot coupling layer of 13-, 14-backing, 15-N N-type semiconductorN refrigerating sheet, 16-insulation layer, the accurate RTD of 17-temperature

Fig. 4 pastes the semiconductor cooler schematic diagram for Po Er.

1-N N-type semiconductorN cooling piece, 2-P N-type semiconductorN cooling piece, 3-metal inducer, 4-inducer, 5-inducer, 6-power supply.

Embodiment

The present invention proposes directly to introduce Po Er first and pastes the refrigeration of conductor refrigeration effect device in the device of microstructure polymerase chain reaction (PCR) augmentor, thereby has accelerated the amplification cycles cycle, has improved efficient.

The pcr amplification device of a kind of employing microelectromechanical systems (MEMS) technology processing and manufacturing is also proposed, its amplification region is the microlaser cavity body structure, volume can design on request, usually be designed and manufactured into heating zone and cooling space respectively on the both sides of μ l level amplification region, temperature probe is all arranged in two districts, adopted high-precision platinum resistor temperature measuring, constitute a kind of new micro pcr amplification device, after sample injects, can realize amplification automatically, need not other operations.

And the microstructure type pcr amplification device of another peltier effect type proposed, this device is that the both sides of pcr amplification device microcavity body amplification region have manufactured and designed two Po Er subsides semiconductor coolers, form circulation heating and refrigeration integral structure, as long as change the working current polarity that Po Er pastes semiconducter device, realize the conversion of refrigeration and heating, can realize quick, the pollution-free amplification of gene.

After designed microcavity body pcr amplification device both sides adopt Po Er to paste device, form circulation heating and refrigeration integral structure.When changing the polarity chron that Po Er pastes the working current of semiconductor cooler, refrigeration and pyrogenicity will change, and speed is faster, and efficient is higher.

The invention reside in and adopt multinomial hi-tech to realize integrated, the microminiaturization and the multifunction of heating, refrigeration cooling and the precise temperature measurement system of miniature polyase chain reaction cloning device; Also adopt miniature electronic planar technology and microelectron-mechanical three-dimensional process technology on the silicon chip of twin polishing, to produce technically and have RTD heating and the three-dimensional structure microcavity body of accurate platinum resistor temperature measuring and the small semiconductor frigorific unit that is complementary, with their bondings or fit together, become complete novel microstructure pcr amplification device then.Frigorific unit in the packaging assembly is also reusable

Among Fig. 4: N, P-type

semiconductor cooling piece

1,2 usefulness metal inducers 3 connect, when the forward dc electricity by 1-N N-type semiconductorN cooling piece by the hole of inducer in the 2-P N-type semiconductorN cooling piece, inducer joint motion dorsad, be to produce electronics, hole respectively on inducer 3 tab N types and the P type, the energy that electronics, hole produce derives from the heat energy of lattice, so on inducer 3, produce the heat absorption phenomenon, and on the other end inducer 4.5 of N, P type, produce exothermic phenomenon, thereby produce the temperature difference.When the heat of the high temperature side of heat release can effectively diffuse, the low temperature of heat absorption then can constantly absorb heat, and makes it to play the refrigerative effect.The 6th, power supply.

That the thermoelectric cooling module of making according to peltier effect has is in light weight, volume is little with mutually To the high advantages such as refrigerating capacity, be specially adapted to the refrigeration of the confined space. Because refrigerating assembly is A kind of shaped solid state heat pumps, thereby its Maintenance free, noiselessness can be in the work of any position, anti-impact Hit with the anti-vibration ability strong. In addition, change assembly operating current polarity chron, it again can pyrogenicity, Change current strength capable of regulating cooling power.

Claims

1. microstructure polyase chain reaction cloning device that is made of conductor refrigeration sheet, refrigerator inducer, hot coupling layer, radiator element, insulating film, resistance is characterized in that augmentor has following two kinds:

A. the substrate of augmentor is the miniature cavity of Si, the substrate upper edge has sample inlet and sample export, above outlet, one deck refrigerator inducer is housed, the inducer top is equipped with P-type semiconductor refrigerating sheet and N-type semiconductor refrigerating sheet respectively, the refrigerator inducer also is housed above refrigerating sheet, hot coupling layer is housed on this inducer, on the hot coupling layer radiator element is arranged; Under the substrate bottom, add one deck insulating film, the accurate RTD of heating RTD and thermometric is housed under the insulating film;

B. the substrate of augmentor is a Si microstructure cavity, the substrate upper edge has sample inlet and the outlet of sample mouth, above outlet, one deck refrigerator inducer is housed, the inducer top is equipped with P-type semiconductor refrigerating sheet and N-type semiconductor refrigerating sheet respectively, the refrigerator inducer also is housed above refrigerating sheet, hot coupling layer is housed on this inducer, on the hot coupling layer radiator element is arranged; Under the bottom of Si microstructure cavity, add one deck insulating film, the accurate RTD of one thermometric is housed at insulating film; The refrigerator inducer is housed under the insulating film, is respectively N-type semiconductor refrigerating sheet and P-type semiconductor refrigerating sheet under the inducer, the refrigerator inducer is housed under the refrigerating sheet, inducer is hot coupling layer down.

2. microstructure polyase chain reaction cloning device according to claim 1 is characterized in that the semiconducter device in the assembling of the both sides of augmentor microcavity body is that Po Er pastes conductor refrigeration spare.

3. microstructure polyase chain reaction cloning device according to claim 2, the structure that it is characterized in that Po Er subsides semiconductor cooler is: on N-type semiconductor cooling piece and P-type semiconductor cooling piece the metal inducer is housed, the below of refrigerating sheet is equipped with inducer respectively, and these two inducers are connected with direct supply.