CN107400628A - Sequencing reaction small chamber, sequencing reaction fixture and sequencing reaction equipment - Google Patents
Sequencing reaction small chamber, sequencing reaction fixture and sequencing reaction equipment Download PDFInfo
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- CN107400628A CN107400628A CN201610338059.0A CN201610338059A CN107400628A CN 107400628 A CN107400628 A CN 107400628A CN 201610338059 A CN201610338059 A CN 201610338059A CN 107400628 A CN107400628 A CN 107400628A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
Abstract
The present invention relates to gene sequencing field, there is provided a kind of sequencing reaction small chamber, sequencing reaction fixture and sequencing reaction equipment；Described sequencing reaction small chamber includes the first substrate and the second substrate, and the lower surface of the first substrate is bonded with the upper surface of the second substrate, and at least one hollow reaction channel is formed between first substrate and the second substrate；Reagent exit is provided with first substrate or the second substrate, reagent exit is additionally provided with the first substrate or the second substrate；The heating slide for being heated to the reagent in reaction channel is fitted with the upper surface of first substrate；It is corresponding on second substrate that a projection is provided with the position of the reaction channel, a thermometer hole is provided with projection, temperature probe is accommodated in thermometer hole；The beneficial effects of the invention are as follows：Avoid the cross-ventilation of gene sequencing reaction chamber surface influences to caused by thermometric, improves the accuracy of gene sequencing reaction temperature survey, and the temperature for making to measure is closer to the temperature of the reaction reagent of gene sequencing reaction.
Description
Technical field
The present invention relates to gene sequencing field, it is more particularly related to a kind of sequencing reaction small chamber, sequencing reaction fixture and sequencing reaction equipment.
Background technology
In the prior art, a kind of sequencing reaction device, including sequencing reaction small chamber, temperature control equipment and temperature-detecting device, the temperature control equipment have generally comprised cooling piece, cooling piece contacts with sequencing reaction small chamber, enters the control of trip temperature to sequencing reaction small chamber by cooling piece；Temperature-detecting device has generally comprised temperature sensor, and temperature sensor also contacts with sequencing reaction small chamber, by detecting the temperature of sequencing reaction small chamber, obtains the temperature of sequencing reaction.
There is the deficiencies of following two aspects in this device for carrying out temperature detection for sequencing reaction, on the one hand, because the temperature measured by temperature sensor is not the temperature for the reagent for carrying out sequencing reaction, therefore there is error for the detection of temperature for sequencing reaction；The temperature for detecting the reagent of sequencing reaction is always the problem in gene sequencing field, generally, because sequencing reaction small chamber then needs to change after once sequencing reaction is carried out, and the device for carrying out temperature survey need not be changed, the problem of in view of cost, temperature measuring equipment can not be changed when being changed to sequencing reaction small chamber, therefore temperature measuring equipment is not fitted into sequencing reaction small chamber typically；Even more important is a bit, if the temperature measuring equipment for carrying out thermometric to be loaded to the inside of sequencing reaction small chamber, complete sealing is difficult to realize between temperature measuring point and sequencing reaction small chamber, the problem of leakage easily occurs, so that sequencing reaction can not be carried out normally.On the other hand, the temperature sensor of temperature-detecting device contacts with sequencing reaction small chamber, when carrying out thermometric, due to the cross-ventilation on sequencing reaction small chamber surface, easily the measurement to temperature impacts, and causes the measurement of temperature deviation easily occur, the precision of temperature survey is relatively low.
Therefore a kind of new sequencing reaction small chamber is needed, the cross-ventilation on sequencing reaction small chamber surface can be avoided to the influence of temperature survey, improve the accuracy of sequencing reaction temperature survey, the temperature for making to measure is closer to the temperature of the reagent of sequencing reaction.
The content of the invention
It is an object of the invention to provide a kind of gene sequencing reaction cell and gene sequencing reaction equipment, aim to solve the problem that measurement of the cross-ventilation of gene sequencing reaction chamber surface in the prior art easily to temperature impacts, the measurement of temperature is caused the problem of deviation, the precision of temperature survey is relatively low easily occur.
In order to realize goal of the invention, a kind of gene sequencing reaction cell, it is theed improvement is that：Including the first substrate and the second substrate, the lower surface of first substrate is bonded with the upper surface of the second substrate, and at least one hollow reaction channel is formed between first substrate and the second substrate；
The reagent exit connected with reaction channel is provided with first substrate or the second substrate, the reagent exit connected with reaction channel is additionally provided with first substrate or the second substrate；
The zone of heating for being heated to the reaction reagent in reaction channel is provided with the upper surface of first substrate；It is corresponding on second substrate that a projection is provided with the position of the reaction channel, a thermometer hole is provided with the projection, and the thermometer hole is used to accommodate the temperature probe for carrying out the reaction reagent in reaction channel temperature detection.
In such a configuration, the both ends of the reaction channel are respectively provided with the reagent buffering area for accommodating reaction reagent, the reagent exit is connected with reagent buffering area therein, the reagent exit is connected with another reagent buffering area, the reagent buffering area be arranged on the first substrate lower surface and positioned at the first groove both ends the second groove or the reagent buffering area be arranged on the second substrate upper surface and positioned at the first groove both ends the second groove.
In such a configuration, first substrate includes the first glass substrate, and second substrate includes PDMS interlayers and the second glass substrate, and the PDMS interlayers are between the first glass substrate and the second glass substrate；The through hole of bar shaped is provided with the PDMS interlayers, the reaction channel is enclosed and formed by the first glass substrate, the through hole of bar shaped and the second glass substrate；The PDMS interlayers are slightly longer than the second glass substrate of described, after first glass substrate, PDMS interlayers and the fitting of the second glass substrate, the PDMS interlayers have the lower surface do not blocked by second glass substrate, and the projection is arranged on the lower surface not being blocked of PDMS interlayers.
Further, the both ends of the reaction channel are respectively provided with the reagent buffering area for accommodating reaction reagent, and the reagent buffering area is the through hole being arranged on PDMS interlayers；The reagent exit is connected with a wherein reagent buffering area, and the reagent exit is connected with another reagent buffering area.
Further, the bearing of trend of the raised upper thermometer hole and the second described substrate are perpendicular；Or the bearing of trend of the raised upper thermometer hole is parallel with the second described substrate.
In addition, present invention further teaches a kind of sequencing reaction fixture, including sequencing reaction small chamber as described above, it is theed improvement is that：Sequencing reaction fixture stationary fixture under stationary fixture and cell also on cell, the sequencing reaction small chamber are arranged on the cell under stationary fixture and cell between stationary fixture.
Further, it is provided with the 3rd groove for housing the sequencing reaction small chamber on the cell under stationary fixture or cell on stationary fixture, or
It is provided with the 4th groove on the cell on stationary fixture, is provided with the 5th groove on stationary fixture under the cell, the sequencing reaction small chamber is arranged in the 4th groove and the 5th groove.
Further, on the cell first screwed hole corresponding with reagent inlet is provided with stationary fixture or cell on stationary fixture, the screw with through hole is locked in first screwed hole, the top of through hole on the screw is connected with being passed through the pipeline of reagent, and the bottom of the through hole on the screw is connected with described reagent inlet；
On the cell second screwed hole corresponding with reagent exit is provided with stationary fixture or cell on stationary fixture, the screw with through hole is locked in second screwed hole, the top of through hole on the screw is connected with discharging the pipeline of reagent, and the bottom of the through hole on the screw is connected with described reagent exit.
Further, when being provided with three groove for housing the sequencing reaction small chamber on stationary fixture under stationary fixture or cell on the cell, the 6th groove of the heating slide for accommodating the sequencing reaction small chamber is additionally provided with the 3rd groove；Or
It is provided with the 4th groove on the cell on stationary fixture, when being provided with five grooves on stationary fixture under the cell, the 6th groove of the heating slide for accommodating the sequencing reaction small chamber is additionally provided with the 4th groove or the 5th groove.
Further, it is provided with the window for being taken pictures to sequencing reaction on the cell under stationary fixture and cell on stationary fixture, the recessed groove for being easy to the conducting probe contact heating slide is provided with the side wall of the window.
In addition, present invention further teaches a kind of sequencing reaction equipment, it is characterised in that：Including sequencing reaction fixture and cell fixing device；The cell fixing device includes cell mounting seat and cell compressing member, the cell compressing member is fixed in cell mounting seat, the sequencing reaction small chamber is arranged in the cell mounting seat, and cell compressing member is used to the sequencing reaction small chamber being pressed on cell mounting seat.
Further, it is provided with the cell compressing member for withstanding on the bulb plunger on the sequencing reaction small chamber.
Further, in addition to temperature measuring equipment, the temperature measuring equipment include temperature probe, and the temperature probe is used to insert in the thermometer hole of the sequencing reaction small chamber.
Further, cell end cap is rotatablely equipped with the cell mounting seat, the window on stationary fixture under stationary fixture on cell and cell is provided with the second through hole on the cell end cap, the cell end cap is provided with third through-hole corresponding to the recessed groove on stationary fixture under stationary fixture on the cell and cell.
As from the foregoing, the setting that the present invention passes through thermometer hole, temperature inside thermometer hole is more close to the temperature of reaction reagent, and due to the presence of thermometer hole, temperature probe or temperature sensor are placed in thermometer hole, it can be very good to avoid the cross-ventilation of gene sequencing reaction chamber surface to be influenceed to caused by thermometric, improve the accuracy of gene sequencing reaction temperature survey, the temperature for making to measure is closer to the temperature of the reaction reagent of gene sequencing reaction.
Brief description of the drawings
Fig. 1 is the cross-sectional view of gene sequencing reaction cell in one embodiment of the invention.
Fig. 2 is the dimensional structure diagram of gene sequencing reaction cell in one embodiment of the invention.
Fig. 3 is the principle schematic of temperature survey mode of the prior art.
Fig. 4 is the partial enlarged drawing at A in Fig. 1 of the present invention.
Fig. 5 is the second glass substrate of gene sequencing reaction cell and raised dimensional structure diagram in another example of the invention.
Fig. 6 is the cross-sectional view of gene sequencing reaction cell in another example of the invention.
Fig. 7 is the cross-sectional view of gene sequencing reaction cell in another example of the invention.
Fig. 8 is the cross-sectional view of gene sequencing reaction cell in another example of the invention.
Fig. 9 be Fig. 8 described in example in gene sequencing reaction cell the second substrate dimensional structure diagram.
Figure 10 is the cross-sectional view of gene sequencing reaction cell in another example of the invention.
Figure 11, Figure 12 are the structural representation of the reagent buffering area of the gene sequencing reaction cell of the present invention.
Figure 13 is the cross-sectional view of gene sequencing reaction cell in another example of the invention.
Figure 14 is the dimensional structure diagram of PDMS interlayers and the second glass substrate in the example described in Figure 13.
Figure 15 is the cross-sectional view of gene sequencing reaction cell in another example of the invention.
Figure 16 is the dimensional structure diagram of the PDMS interlayers of gene sequencing reaction cell in Figure 15.
Figure 17 is the dimensional structure diagram of one embodiment of the invention sequencing reaction fixture.
Figure 18 is the dimensional structure diagram of sequencing reaction fixture in another example of the invention.
Figure 19 is the dimensional structure diagram of stationary fixture on cell in Figure 18.
Figure 20 is the cross-sectional view of sequencing reaction equipment in one embodiment of the invention.
Figure 21 is the cross-sectional view of sequencing reaction equipment in another example of the invention.
Embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, below in conjunction with drawings and Examples, the present invention will be described in further detail.
The present invention proposes first embodiment, and the present embodiment proposes a kind of sequencing reaction small chamber, by the way that multiple DNA fragmentations are fixed in sequencing reaction small chamber, reaction reagent is flowed through DNA fragmentation, realize reaction reagent and DNA fragmentation producer sequencing reaction.As Figure 1 and Figure 4, described sequencing reaction small chamber includes the first substrate 10 and the second substrate 20, and the lower surface of first substrate 10 and the upper surface of the second substrate 20 fit, and realize the sealing between the first substrate 10 and the second substrate 20；At least one hollow reaction channel is formed between the substrate 20 of the first substrate 10 and second, when an only reaction channel, realize single pass gene sequencing reaction, when with a plurality of reaction channel, the parallel sequencing of multiple passages can then be realized, for described reaction channel, it will be described in detail below, here is omitted.The reagent exit 101 connected with reaction channel is provided with second substrate 20, the pipeline communication of reagent exit 101 and outside supply reaction reagent, is passed through the reaction reagent needed for gene sequencing reaction into reaction channel by the reagent exit 101；The reagent exit 102 connected with reaction channel is additionally provided with second substrate 20, the pipeline communication of reagent exit 102 and discharge reaction reagent, reaction reagent after gene sequencing reaction occurs is flowed out by the reagent exit 102, in the present embodiment, as shown in figure 1, described reagent exit 101 and reagent exit 102 is arranged on the second substrate 20, but for described reagent exit 101 and reagent exit 102, the different examples having, will be described in detail below.
Further, the heating slide 30 for being heated to the reagent in reaction channel is fitted with the upper surface of first substrate 10, when being passed through reaction reagent from reagent exit 101, when reaction reagent flows through from reaction channel, then by heating slide 30 to realizing that reaction reagent heats, hereafter reaction reagent flows out at reagent exit 102.Position on second substrate 20 corresponding to the reaction channel is provided with a projection 40, and a thermometer hole 401 is provided with described raised 40, and the temperature probe for carrying out temperature detection to the reaction reagent in reaction channel is accommodated in the thermometer hole 401.As shown in Figure 2, when an only reaction channel, after first substrate 10 is bonded with the second substrate 20, described reaction channel is projected in figure on the second substrate 20 as shown in dash area 400 in Fig. 2, described projection 40 is projected on the position of the second substrate 20 across the reaction channel, the fluorescence signal in the reaction channel is blocked in order to avoid projection 40 simultaneously, collection to fluorescence signal impacts, described raised 40 are positioned close in the position of reagent exit 101, but it should be noted that, described raised 40 can also be positioned close on the position of reagent exit 102.In the present embodiment realized by heating the heating of slide 30 to meet the needs of gene sequencing reaction, it is necessary to enter the regulation of trip temperature to the reaction reagent in reaction channel during gene sequencing reaction is carried out；Meanwhile during gene sequencing reaction is carried out, it is also necessary to the temperature of the reaction reagent in reaction channel is monitored, the temperature of the reaction reagent is maintained within the scope of required temperature.Because reaction channel needs to be passed through reaction reagent, the situation of leakage can not occur, the temperature sensor or temperature probe of detection temperature can not load in reaction channel, its reason is after temperature sensor or temperature probe load in reaction channel, the sealing of sequencing reaction small chamber can not be realized, the situation of leakage easily occurs；Because the first substrate 10 and the second substrate 20 are general all relatively thin, in temperature probe or all more difficult loading reaction channel of temperature sensor；In addition, sequencing reaction small chamber is needing to be changed after gene sequencing reaction, even if temperature probe or temperature sensor can load in reaction channel, when being changed to sequencing reaction small chamber, the assembly and disassembly of temperature sensor or temperature probe are all more troublesome.In view of the reason for above-mentioned, in the prior art in order to detect the temperature of the reaction reagent in reaction channel, more typical technical scheme is that temperature sensor or temperature probe are arranged on the surface of sequencing reaction small chamber, and the temperature of the reaction reagent in reaction channel is measured；But the mode of this temperature survey, it is a kind of indirect metering system, the temperature of temperature sensor measurement is the temperature on sequencing reaction small chamber surface, and when the temperature is not fully equal with the temperature of the reaction reagent in reaction channel, the result of temperature survey occurs as soon as larger error；Also, when temperature sensor is arranged on sequencing reaction small chamber surface, the cross-ventilation on sequencing reaction small chamber surface can then impact to the measurement of temperature, the measurement result of temperature sensor deviation is occurred.
Specifically, in the prior art, when directly carrying out temperature survey on the surface of sequencing reaction small chamber using temperature sensor, as shown in figure 3, temperature sensor 50 is attached on sequencing reaction small chamber 100, sequencing reaction small chamber 100 is heated by heating slide 30, inside sequencing reaction small chamber 100, thermoisopleth 1001 as shown in Figure 3 is formed, the surface temperature of sequencing reaction small chamber 100, then there is certain difference, causes error during temperature survey with internal temperature；Meanwhile temperature sensor 50 is directly arranged on the surface of sequencing reaction small chamber 100, the cross-ventilation on the surface of sequencing reaction small chamber 100 will impact to the measurement of temperature sensor 50.As shown in Figure 4, the present embodiment sets projection 40 on the second substrate 20 of sequencing reaction small chamber, and when being provided with thermometer hole 401 in projection 40, the measurement of realizing temperature is stretched into thermometer hole 401 by temperature probe, because projection 40 is arranged on the second substrate 20, the structure that the substrate 20 of preferred embodiment protrusions 40 and second is formed in one, in the second substrate 20, then form thermoisopleth 1001 as shown in Figure 4, therefore the temperature inside thermometer hole 401, more close to the temperature inside the second substrate 20, temperature probe stretches into thermometer hole 401 temperature measured, compared than the metering system in Fig. 2, then it is closer to the temperature of the reaction reagent of gene sequencing reaction；And, due to the presence of thermometer hole 401, temperature probe or temperature sensor are placed in thermometer hole 401, it then can be very good to avoid the cross-ventilation on the surface of sequencing reaction small chamber 100 from influenceing to caused by thermometric, the accuracy of gene sequencing reaction temperature survey is improved, the temperature for making to measure is closer to the temperature of the reaction reagent of gene sequencing reaction.
It should be further stated that, in the prior art, by setting a stable heat radiation surface to heat the reaction reagent in reaction channel on sequencing reaction small chamber, due to the limitation of sequencing device structure, temperature measuring point is generally located on one side of heat radiation surface, when heat radiation surface persistently heats to the reaction reagent in reaction channel, after a period of time, the temperature of heat radiation surface, the temperature of reaction reagent, the temperature of the temperature of sequencing reaction small chamber and temperature measuring point tend to consistent, and the measurement of temperature is realized by the temperature sensor of temperature measuring point；Although this mode can realize the measurement of reaction reagent temperature, because temperature measuring point and heat radiation surface are on the same face, the temperature of heat radiation surface can impact to the temperature survey of temperature measuring point, reduce the degree of accuracy of thermometric.And in the present embodiment, the heating slide 30 is arranged on the upper surface of the first substrate 10, projection 40 is arranged on the position for corresponding to reaction channel on the second substrate 20, described in the present embodiment raised 40 are projected in across the reaction channel on the position of the second substrate 20, the position of thermometer hole 401 is closer to reaction channel.Pass through the design of this structure, the heating slide 30 heated is arranged to the upper surface of sequencing reaction small chamber, the thermometer hole 401 for carrying out thermometric is arranged to the lower surface of sequencing reaction small chamber, or the heating slide 30 heated is arranged to the lower surface of sequencing reaction small chamber, the thermometer hole 401 for carrying out thermometric is arranged to the upper surface of sequencing reaction small chamber, thermometer hole 401 is located away from heating slide 30, so as to avoid heating influence when slide 30 heats for temperature survey, the accuracy of gene sequencing reaction temperature survey is improved.
For the thermometer hole 401 on described raised 40, in the example described in Fig. 1, Fig. 2, the bearing of trend of thermometer hole 401 and the second glass substrate 20 are perpendicular.As shown in Figure 5, the present invention also proposed an example for the thermometer hole 401 of projection 40, the thermometer hole 401 is arranged in raised 40 side wall, the bearing of trend of thermometer hole 401 is parallel with the second substrate 20, this structure of thermometer hole, it is located at the situation of sequencing reaction small chamber side suitable for temperature probe, and, in this example, because the bearing of trend of thermometer hole 401 is parallel with the second substrate 20, in the described thermometer hole 401 of temperature probe insertion, the surface of second substrate 20 can play a part of guiding to the insertion of temperature probe, it is easy in the described thermometer hole of temperature probe insertion.Further, in the opening of the thermometer hole 401, the chamfering playing the guiding role can also be set, be more convenient in the described thermometer hole of temperature probe insertion.
For described projection 40; in the example described in Fig. 1, Fig. 2; described raised 40 be square structure; but; raised 40 structure is not formed on protection scope of the present invention to be influenceed, for example, described raised 40 can be designed as cylinder; or in order to increase the substrate contact area of projection 40 and second, projection 40 can be designed as circular platform type；Other shapes can also be designed as according to demand.In addition, in the example described in Fig. 1, Fig. 2, the thermometer hole 401 is circular blind hole, but the shape of the thermometer hole 401 can be set according to demand, preferably, the shape of the thermometer hole 401 is identical with the profile of temperature probe, after temperature probe is stretched into described thermometer hole 401, the outer surface of temperature probe and the inwall of thermometer hole 401 formed it is good be bonded, gap is larger so as to avoiding between the inwall of the outer surface of temperature probe and thermometer hole 401, influences the result of temperature survey；Further, in order to lift the heat transfer efficiency between the inwall of thermometer hole and temperature probe, heat conductive silica gel is filled with the thermometer hole, by the effect of heat conductive silica gel, by the heat transfer in projection 40 to the temperature probe.
On the basis of above-described embodiment, for described reaction channel, the present invention proposes an example, in this example, as shown in Figure 1 and Figure 2, the first groove 103 is provided with the lower surface of first substrate 10, first groove 103 can be one, can also be parallel a plurality of, after the first substrate 10 and the second substrate 20 fit, the first groove 103 encloses the reaction channel described in composition with the second substrate 20；In addition, in this example, the reagent exit 101 and reagent exit 102 being connected with reaction channel are provided with second substrate 20, the quantity of the reagent exit 101 and reagent exit 102 need to be depending on the quantity of the first groove 103, when the first groove 103 is one, a reagent exit 101 and a reagent exit 102 are then provided with, is corresponded to respectively on the two ends of the first groove 103；When the first groove 103 is a plurality of, then reagent exit 101 and reagent exit 102 are provided with corresponding to each first groove 103.As shown in Figure 10, alternate embodiment as above-mentioned reaction channel, also the upper surface of second substrate 20 can be provided with the first groove 103, after the first substrate 10 is bonded with the second substrate 20, the first groove 103 and the first substrate 10 enclose and form described reaction channel；Likewise, the reagent exit 101 and reagent exit 102 being connected with reaction channel are provided with the second substrate 20.In addition, as shown in Figure 6, another alternate embodiment as above-mentioned reaction channel, the lower surface of first substrate 10 is provided with the first groove 103, the upper surface of second substrate 20 is also equipped with the first groove 103, and first the first groove 103 on substrate 10 is identical with the shape of the first groove 103 on the second substrate 20, after the first substrate 10 and the second substrate 20 fit, the first groove 103 on the first groove 103 and the second substrate 20 on the first substrate 10 is to constitute described reaction channel.
And for the position of reagent exit in above-mentioned example and reagent exit, there is the combination of a variety of different schemes, in the example described in Fig. 1 or Fig. 6, reagent exit and reagent exit are arranged on the second substrate 20.(not shown) in alternate embodiment, the reagent exit can also be arranged on the first substrate, and the reagent exit can also be arranged on the first substrate, in addition, also reagent exit is arranged on the first substrate, while reagent exit is arranged on the second substrate；Or reagent exit is arranged on the second substrate, reagent exit is arranged on the first substrate, can be toward being passed through reaction reagent in the reaction channel of sequencing reaction small chamber, and reaction reagent flows out at reagent exit.
For described sequencing reaction small chamber, present invention also offers an example, it is in this example with Fig. 1 embodiment differences, the reaction channel both ends of the sequencing reaction small chamber are separately provided for accommodating the reagent buffering area of reaction reagent, reagent exit 101 is connected with a wherein reagent buffering area, reagent exit 102 is connected with another reagent buffering area, in this example, as shown in Figure 7, the lower surface of first substrate 10 is provided with the first groove 103, first groove 103 and the second substrate 20, which enclose, forms described reaction channel, above-mentioned reagent buffering area is to be arranged on the lower surface of the first substrate 10 and positioned at second groove 104 at the both ends of the first groove 103.The second described groove 104 is compared with the first groove 103, second groove 104 can accommodate more reaction reagents, when the reaction reagent needed for gene sequencing reaction is passed through into reaction channel by reagent exit 101, before reaction reagent enters reaction channel, reaction reagent can enter in the reagent buffering area of reaction channel one end first, after reagent buffering area is filled, reaction reagent is entered back into hollow reaction channel, with testing sample producer sequencing reaction in reaction channel, when with multiple reaction channels, then reagent enters in each reaction channel simultaneously, with testing sample in multiple reaction channels while producer sequencing reaction；Hereafter reaction reagent is then flowed out into the reagent buffering area of the reaction channel other end, and reaction reagent is discharged after the reagent buffering area collects from the pipeline of discharge reaction reagent.Due to the presence of reagent buffering area, reagent buffering area can store a certain amount of reaction reagent, it can be flowed into reagent buffering area before reaction reagent is passed through reaction channel, reagent buffering area is advantageous to the air in uniform discharge reaction channel, internal pressure is consistent when reaction reagent enters reaction channel, reaction reagent can be uniformly by each reaction channel, therefore ensure that the uniformity of each reaction channel flow velocity and diffusible uniformity.
Sequencing reaction small chamber of the prior art with multiple passages, because reagent exit 101 and reagent exit 102 with multiple reaction channels, each reaction channel are required to access conduit, for flowing into and out for reaction reagent；The structure of the sequencing reaction small chamber of this structure it is very cumbersome with it is complicated, it is very inconvenient when being cleaned to sequencing reaction small chamber, it is necessary to which more pipelines are dismantled, dismantling with being also easy to poor sealing occur in assembling process, causing the situation of leakage occur during gene sequencing reaction.And the multichannel sequencing reaction small chamber in the present embodiment, due to the setting of reagent buffering area, it is only necessary to which a pipeline accesses reagent buffering area, it is possible to complete the gene sequencing reaction of multiple passages, simplify the structure of multichannel sequencing reaction small chamber；When needing to carry out disassembly, cleaning to sequencing reaction small chamber, only need the pipeline and a pipeline for going out liquid connected with reagent exit 102 of a piece feed liquor connected with reagent exit 101 of dismounting, can is cleaned to the inside of sequencing reaction small chamber, it is very convenient, reduce and cause the situation of leakage occur during gene sequencing reaction in the poor sealing dismantled with occurring in assembling process.Therefore such a sequencing reaction small chamber in this example, can be repeatedly used in actual use, and relative to reaction small chamber disposable in the prior art, the present invention greatly reduces the cost of equipment.
For described reagent buffering area, the invention also provides an example, as shown in figure 8, in this example, the sequencing reaction small chamber includes the first substrate 10 and the second substrate 20, and sequencing reaction small chamber and the difference of example described in Fig. 1 are in this example：First groove 103 is arranged at the upper surface of the second substrate 20.In this example, as shown in Figure 8, the reaction channel both ends of sequencing reaction small chamber are separately provided for accommodating the reagent buffering area of reaction reagent, reagent exit 101 is connected with a wherein reagent buffering area, reagent exit 102 is connected with another reagent buffering area, and in this example, the upper surface of second substrate 20 is provided with the first groove 103, first substrate 10 and the first groove 103, which enclose, forms described reaction channel, and reagent buffering area is the second groove 104 for being arranged on reaction channel both ends.
As shown in Figure 9,The three-dimensional structure diagram of a specific embodiment of the second substrate 20 in as Fig. 8,In this example,The upper surface of second substrate 20 is provided with six the first parallel grooves 103,First substrate 10 is bonded with the second substrate 20, constitutes the sequencing reaction small chamber of six passages,When carrying out gene sequencing reaction,The reagent injected by reagent exit 101 initially enters the reagent buffering area at reagent exit 101,After persistently injecting reaction reagent,After reaction reagent is filled with the reagent buffering area at reagent exit 101,It can just enter simultaneously in six reaction channels,Respectively gene sequencing reaction is carried out with the testing sample being fixed in reaction channel,It is then out and enters in the reagent buffering area at reagent exit 102,Reaction reagent is after the reagent buffering area collects,Discharged from the pipeline of discharge reaction reagent,Persistently inject reaction reagent,Reaction reagent then constantly flows through from reaction channel,With testing sample producer sequencing reaction.Due to the presence of reagent buffering area, before reaction reagent is passed through reaction channel, or after reaction channel outflow, reagent buffering area can store a certain amount of reaction reagent, ensure that the uniformity of each reaction channel flow velocity and diffusible uniformity.This invention simplifies the structure of the sequencing reaction small chamber of multichannel, when needing to carry out disassembly, cleaning to sequencing reaction small chamber, only need the pipeline and a pipeline for going out liquid connected with reagent exit 102 of a piece feed liquor connected with reagent exit 101 of dismounting, after first substrate 10 is separated with the second substrate 20, reaction channel of the can to sequencing reaction small chamber, reagent buffering area, reagent exit 101 and reagent exit 102 are cleaned, it is very convenient, reduce simultaneously and cause the situation of leakage occur during gene sequencing reaction in the poor sealing dismantled with occurring in assembling process.When carrying out the installation of the first substrate 10 and the second substrate 20, because reagent exit 101, reagent exit 102, reaction channel and reagent buffering area are arranged on the second substrate 20, therefore very facility is installed.Sequencing reaction small chamber in this example is placed vertically, and reagent exit 101 is when being located at lower position, after being passed through reaction reagent, the reaction reagent full reagent buffering area at reagent exit first, hereafter enter simultaneously in six reaction channels, so as to be advantageous to the air in uniform discharge reaction channel.
For the position of described reagent buffering area, as shown in Figure 10, the invention also provides an example, and the structure and Fig. 8 of the second substrate 20 described in this example, structure in example described in Figure 10 is identical, the structure of the second substrate 20 is then not described in detail in this example, its difference is, the lower surface of first substrate 10 is also equipped with the second groove 104, and first the second groove 104 set on substrate 10 shape it is identical with the shape of the second groove 104 on the second substrate 20, after first substrate 10 is bonded with the second substrate 20, the second groove 104 set on first substrate 10 then matches with the second groove 104 on the second substrate 20, to form described reagent buffering area.In this example, when the sequencing reaction small chamber is kept flat, that is the first substrate 10 is in the lower section of the second substrate 20, and it is horizontal, the reaction reagent being passed through by reagent exit 101, it can enter first in reagent buffering area, fill after the second groove on the first substrate 10, just enter in described reaction channel, the reagent buffering area serves the effect of buffering to reagent.
For the shape of reagent buffering area, the present invention proposes an example, as shown in figure 11, the semicircular in shape of reagent buffering area 500, reagent buffering area 500 has arc-shaped edges and straight line, and the straight line of the reagent buffering area 500 is located at close to the side of the reaction channel.Pass through the design of such a structure, after sequencing reaction small chamber is vertically arranged on sequencing device, as shown in figure 11, during reagent is injected toward reagent buffering area 500, because reagent Buffer Design is semicircle, the flow velocity of A points, B points and C points shown in Figure 11 is more uniformly distributed so that the flow velocity of optional position is more uniformly distributed in reagent buffering area, so as to ensure that the uniformity of each reaction channel flow velocity and diffusible uniformity；Make the sequencing reaction in multiple reaction channels while carry out.It should be noted that, above-mentioned scheme does not form any restrictions to the structure of reagent buffering area, the size and shape of reagent buffering area and it is designed according to being actually needed, such as reagent buffering area can use square or rectangular, the scheme shown in Figure 11 is only a preferable example.As shown in figure 12, for another example of reagent buffering area 500, in the example, the reagent buffering area 500 is triangular in shape, during injecting reagent toward reagent buffering area 500, by the design of triangular structure, ensure the uniformity of each reaction channel flow velocity and diffusible uniformity, make the sequencing reaction in multiple reaction channels while carry out.
In addition, in example described in Fig. 7, Fig. 8 and Figure 10, reaction channel both ends are separately provided for accommodating the reagent buffering area of reaction reagent, described raised 40 are arranged at the lower surface of the second glass substrate 20, described raised 40 are arranged at close to the position of reagent exit 101, due to being provided with reagent buffering area at reagent exit 101, thermometer hole 401 in projection 40 is then closer to reagent buffering area, the reaction reagent of reagent buffering area is more, its temperature, the accuracy of the temperature survey of raising can be detected by thermometer hole 401；It should be noted that described raised 40 can also be positioned close to the position of reagent exit 102, same temperature survey effect can also be realized.
For described sequencing reaction small chamber, the invention also provides an example, as shown in figure 13, sequencing reaction small chamber includes the first substrate 10 and the second substrate 20, in this example, first substrate 10 is the first glass substrate, second substrate 20 includes the glass substrate 202 of PDMS interlayers 201 and second, and the PDMS interlayers 201 are between the first glass substrate and the second glass substrate 202, the through hole 2010 of bar shaped is provided with PDMS interlayers 201, described reaction channel is then by the first glass substrate, the glass substrate 202 of through hole 2010 and second of bar shaped, which encloses, to be formed；Heating slide 30 is arranged on the upper surface of the first substrate 10, reagent exit 101 and reagent exit 102 is provided with the second glass substrate 202, reagent exit 101 is connected with reagent exit 102 with reaction channel.Further,As shown in figure 13,The equal length of the glass substrate of PDMS interlayers 201 and first,And PDMS interlayers 201 are slightly longer than the second described glass substrate 202,When the first glass substrate,After the fitting of the glass substrate 202 of PDMS interlayers 201 and second,Do not blocked by second glass substrate 202 one end of the lower surface of the PDMS interlayers 201,Described raised 40 are arranged on the lower surface do not blocked by the second glass substrate 202 of PDMS interlayers 201,As shown in figure 14,The as dimensional structure diagram of the glass substrate 202 of PDMS interlayers 201 and second,In this example,Described raised 40 across the bar shaped through hole 2010 on the extended line of PDMS interlayers 201,And,After the second glass substrate 202 is fitted on PDMS interlayers 201,Described raised 40 then lean against in the side wall 1020 of the second glass substrate 202,The design of this structure of projection 40,Thermometer hole 401 in projection 40 is set to be closely located to reaction channel,The temperature in thermometer hole 401 is set to be closer to the temperature of the reaction reagent of gene sequencing reaction,Improve the accuracy of gene sequencing reaction temperature survey.
In this example, described raised 40 structures being formed in one with PDMS interlayers 201, because the quality of described PDMS interlayers 201 is softer, after temperature probe is stretched into described thermometer hole 401, temperature probe can be closely bonded with the inwall of thermometer hole 401, temperature on PDMS interlayers 201 can be then transferred on temperature probe well by the effect of thermometer hole 401, realize the measurement to reaction reagent temperature in gene sequencing reaction.In addition, in this example, the thermometer hole 401 of the heating slide 30 heated and progress thermometric is respectively arranged on upper surface and the PDMS interlayers 201 of the first substrate 10 of sequencing reaction small chamber, thermometer hole 401 is located away from heating slide 30, so as to avoid heating influence when slide 30 heats for temperature survey；And due to the presence of thermometer hole 401, temperature probe or temperature sensor are placed in thermometer hole 401, it then can be very good to avoid the cross-ventilation on sequencing reaction small chamber surface to be influenceed to caused by thermometric, the temperature for making to measure is closer to the temperature of the reaction reagent of gene sequencing reaction.Improve the accuracy of gene sequencing reaction temperature survey.
In addition, the present invention sequencing reaction small chamber when carrying out gene sequencing reaction, need to by adopt map device seizure gene sequencing reaction when fluorescence signal.When sequencing reaction small chamber is two-piece type structure, in example as shown in Figure 6, adopt map device then needs, by the second substrate 20, to be adopted map device and captured in collection fluorescence signal, fluorescence signal；When the material of the second substrate 20 is PDMS, PDMS meeting autofluorescences, interfered to adopting the collection of fluorescence signal of map device.And in this example, sequencing reaction small chamber has three-decker, wherein PDMS interlayers 201 are arranged between the first glass substrate and the second glass substrate 202, and due to being provided with the through hole 2010 of bar shaped on PDMS interlayers 201, when adopting the collection of map device progress fluorescence signal, the autofluorescence on PDMS interlayers 201 will not be then collected, therefore the autofluorescence on PDMS interlayers 201 will not interfere to the collection for the fluorescence signal for adopting map device.
For described sequencing reaction small chamber, on the basis of example described in Fig. 8, the invention also provides an example, as shown in figure 15, in this example, sequencing reaction small chamber is three-decker, including the first glass substrate, the glass substrate 202 of PDMS interlayers 201 and second, PDMS interlayers 201 are arranged between the first glass substrate and the second glass substrate 202, wherein, the structure of first glass substrate and the second glass substrate 202 is identical with the structure in the example described in Fig. 8, is then no longer described in detail in this example.For described PDMS interlayers 201, as shown in Figure 15, Figure 16, the through hole 2010 of bar shaped is provided with the PDMS interlayers 201, after the first glass substrate, the fitting of the glass substrate 202 of PDMS interlayers 201 and second, the through hole 2010 of the first glass substrate, the second glass substrate 202 then with bar shaped constitutes reaction channel；In this example,The through hole 2010 of the bar shaped is provided at both ends with reagent buffering area 2011,The reagent buffering area 2011 is the through hole being arranged on PDMS interlayers 201,And,Reagent exit 101 is connected with a wherein reagent buffering area 2011,Reagent exit 102 is connected with another reagent buffering area 2011,The reaction reagent that reagent exit 101 is passed through,Before reaction reagent enters reaction channel,Reaction reagent can enter in the reagent buffering area 2011 of reaction channel one end first,After reagent buffering area 2011 is filled,Reaction reagent is entered back into reaction channel,With testing sample in reaction channel while producer sequencing reaction,Hereafter reaction reagent is then flowed out into the reagent buffering area 2011 of the reaction channel other end,Reaction reagent is after the reagent buffering area 2011 collects,Discharged from the pipeline of discharge reaction reagent.Due to the presence of reagent buffering area 2011, reagent buffering area 2011 can store a certain amount of reaction reagent, it can be flowed into reagent buffering area 2011 before reaction reagent is passed through reaction channel, reagent buffering area 2011 is advantageous to the air in uniform discharge reaction channel, when reaction reagent enters reaction channel internal pressure one to, reaction reagent can be uniformly by each reaction channel, therefore ensure that the uniformity of each reaction channel flow velocity and diffusible uniformity.Same, in this example, as shown in Figure 15, Figure 16, have and PDMS interlayers 201 integrally formed raised 40 on the PDMS interlayers 201, thermometer hole 401 is provided with the projection 40, the temperature probe of temperature measuring equipment is stretched into thermometer hole 401, and the temperature probe can be temperature sensor, or thermocouple.Due to the presence of thermometer hole 401, temperature probe or temperature sensor are placed in thermometer hole 401, it then can be very good to avoid the cross-ventilation on sequencing reaction small chamber surface to be influenceed to caused by thermometric, the temperature for making to measure is closer to the temperature of the reaction reagent of gene sequencing reaction, improves the accuracy of gene sequencing reaction temperature survey.
Present invention further teaches a kind of sequencing reaction fixture including above-mentioned sequencing reaction small chamber, the sequencing reaction fixture includes sequencing reaction small chamber 200, stationary fixture 400 under stationary fixture 300 and cell on cell, and the sequencing reaction small chamber 200 is arranged on the cell under stationary fixture 300 and cell between stationary fixture 400；As shown in figure 17, for described sequencing reaction fixture, the present invention proposes an embodiment, the 3rd groove 4001 for housing the sequencing reaction small chamber 200 is provided with the cell on stationary fixture 400, sequencing reaction small chamber 200 is attached in the 3rd groove 4001 of stationary fixture 400 under cell, realize the fixation of sequencing reaction small chamber 200, in the present embodiment, the depth of the 3rd groove 4001 is slightly larger than the thickness of the sequencing reaction small chamber 200；Meanwhile corresponding screw hole is provided with stationary fixture 400 under stationary fixture 300 and cell on the cell, by locking screw, fastening is realized to stationary fixture 400 under stationary fixture on cell 300 and cell.
For stationary fixture under stationary fixture on cell and cell, the invention also provides another example, the 4th groove is provided with the cell on stationary fixture, it is corresponding, the 5th groove is provided with stationary fixture under the cell, the shape of 4th groove and the 5th groove, size is identical, shape with the sequencing reaction small chamber simultaneously, size is identical, in this example, 4th groove and the 5th groove depth sum, slightly larger than the thickness of the sequencing reaction small chamber, after stationary fixture is fixed under cell on stationary fixture on cell, the sequencing reaction small chamber is then just fixed under the 4th groove and cell of stationary fixture on cell in the 5th groove of stationary fixture.
In the above-mentioned example of sequencing reaction fixture, it should be further stated that, for stationary fixture fixed structure under stationary fixture on the cell and cell, with a variety of enforceable modes, such as, on cell under stationary fixture and cell stationary fixture side installation hinge, the rotation connection of stationary fixture under stationary fixture and cell on cell is realized by hinge, the buckle that the opposite side installation of stationary fixture matches under stationary fixture and cell on cell simultaneously, by the effect of buckle, stationary fixture on cell is fixed under cell on stationary fixture；When needing to open sequencing reaction fixture, then buckle is untied, rotate on cell after stationary fixture, then sequencing reaction fixture can be opened, so as to be operated to sequencing reaction small chamber.
On the basis of example described in Figure 17, for described sequencing reaction fixture, the invention also provides an example, as shown in Figure 18, Figure 19, it is that the lower surface of stationary fixture 300 on cell is provided with the 4th groove 3001 with the difference of the example described in Figure 17 in this example, the 5th groove 4002, the 4th groove 3001 and the 5th groove 4002 depth sum, slightly larger than the thickness of the sequencing reaction small chamber 200 are provided with cell corresponding to the upper surface of stationary fixture 400；Also, the 6th groove 3002 is provided with the bottom surface of the 4th groove 3001, the width of the 6th groove 3002 is equal with the width of the 4th groove 3001, but the length for being shorter in length than the 4th groove 3001 of the 6th groove 3002；When cell upper fixture 300 is fixed under cell on stationary fixture 400, then in the 5th groove 4002 of stationary fixture 400 under the 4th groove 3001 and cell of cell upper fixture 300, heating slide is located in the 6th described groove 3002 sequencing reaction small chamber 200.Further, first screwed hole 3003 corresponding with reagent inlet 101 is provided with the cell upper fixture 300, second screwed hole 3004 corresponding with reagent exit 102 is additionally provided with cell upper fixture 300, the design of 3004 this structure of first screwed hole 3003 and the second screwed hole, after the screw with through hole is locked, after the screw with through hole being locked into the first screwed hole 3003, the top of through hole on the screw is connected with being passed through the pipeline of reagent, the bottom of through hole on the screw is then pressed on described sequencing reaction small chamber and is connected with reagent inlet；Same, after the screw with through hole being locked into the second screwed hole 3004, the top of through hole on the screw is then connected with discharging the pipeline of reagent, and the bottom of the screw is then pressed on described sequencing reaction small chamber and is connected with reagent exit, so as to realize being passed through and flowing out for reaction reagent；Due to the 4th groove 3001 and the 5th groove 4002 depth sum, slightly larger than the thickness of the sequencing reaction small chamber 200, sequencing reaction small chamber 200 is active on the vertical direction under cell upper fixture 300 and cell between stationary fixture 400, and in this example, the screw locked in the first screwed hole 3003 and the second screwed hole 3004 is typically made of the softer material of quality, when locking screw, the lower end of screw is then pressed on sequencing reaction small chamber 200, screw at reagent inlet 101 and between the reagent inlet 101 of sequencing reaction small chamber 200, good sealing is respectively formed between screw and reagent exit 102 at reagent exit 102, the problem of so as to avoid the occurrence of leakage.
In addition, as shown in Figure 18, Figure 19, the window 3005 for being taken pictures to sequencing reaction is provided with the cell upper fixture 300 and cell on stationary fixture 400, and, the window 3005 set in cell upper fixture 300 is the through hole being arranged on the bottom surface of the 4th groove 3001, the width of window 3005 is less than the width of the 6th groove 3002, but the length of window 3005 is more than the length of the 6th groove.When carrying out sequencing reaction, by adopting the effect of map device, the sequencing reaction in the reaction channel of sequencing reaction small chamber 200 is taken pictures before window 3005, to obtain fluorescence signal.Further, the recessed groove 3006 for being easy to conducting probe to contact the heating slide after passing through is provided with the side wall of the window 3005, pass through the design of recessed groove 3006, on the one hand conducting probe is touched heating slide, on the other hand avoid acquisition of the conducting probe to fluorescence signal from impacting.
In the example described in Figure 17, and in the example described in Figure 18, Figure 19, it should be noted that the position of first screwed hole 3003 is corresponding with the position of reagent inlet 101, the position of the first screwed hole 3003 can be adjusted according to the position of reagent inlet 101；The position of second screwed hole 3004 is corresponding with the position of reagent exit 102, and the position of the second screwed hole 3004 can be adjusted according to the position of reagent exit 102.
Present invention further teaches a kind of sequencing reaction equipment for including above-mentioned sequencing reaction fixture, sequencing reaction equipment includes cell fixing device and above-mentioned any sequencing reaction fixture, above-mentioned any sequencing reaction small chamber is provided with sequencing reaction fixture, therefore no longer the structure of sequencing reaction small chamber and sequencing reaction fixture is described in detail for this example.As shown in figure 20, the cell fixing device 500 includes cell mounting seat 5001 and cell compressing member 5002, the cell compressing member 5002 is fixed in cell mounting seat 5001, cell compressing member 5002 is used to the sequencing reaction fixture 600 being pressed on cell mounting seat 5001, by the effect of cell compressing member 5002, the fixation to sequencing reaction fixture 600 is realized.It is provided with the cell compressing member 5002 for withstanding on the bulb plunger 5003 on the sequencing reaction fixture 600, after sequencing reaction fixture 600 loads in cell mounting seat 5001, bulb plunger 5003 plays fixed effect to sequencing reaction fixture 600, in addition, in the upper surface of sequencing reaction fixture 600, the position of bulb plunger 5003 is provided with locating slot, after sequencing reaction fixture 600 is fixed in cell mounting seat 5001, bulb plunger 5003 is withstood on described locating slot, realizes the positioning of sequencing reaction fixture 600.In this example, by the effect of cell compressing member 5002, bulb plunger 5003 and locating slot, the dismounting and fixation of sequencing reaction fixture 600 are realized, facilitates the replacing of sequencing reaction fixture 600, improves the efficiency of gene sequencing reaction.
For described sequencing reaction equipment, as shown in figure 21, the invention also provides an example, sequencing reaction equipment also includes temperature measuring equipment, described temperature measuring equipment includes temperature probe 700 and data processing unit, the temperature probe 700 is used to insert in the thermometer hole of the sequencing reaction small chamber, temperature probe 700 is connected with the data processing unit, temperature probe 700 is used for the temperature for detecting reaction reagent in reaction channel, the temperature of detection is transferred to data processing unit, the temperature that data processing unit detects according to temperature probe 700, control is realized to heating slide, such as, the temperature of gene sequencing reaction is set as T1, temperature measured by temperature probe 700 is T2, as temperature T2<During T1, illustrate that the temperature of the reaction reagent in gene sequencing reaction passage is less than the temperature needed for gene sequencing reaction, after data processing unit receives the temperature measurement signal of temperature probe 700, then slide is heated in control, improves the temperature of heating.Further, in this example, it is rotatablely equipped with cell end cap in the cell mounting seat（Not shown in figure）, the window 3005 under cell upper fixture 300 and cell on stationary fixture 400 is corresponded on the cell end cap and is provided with the second through hole, by the setting of the second through hole, avoids cell end cap from impacting the figure of adopting for adopting map device；The recessed groove 3006 that the cell end cap corresponds under the cell upper fixture 300 and cell on stationary fixture 400 is provided with third through-hole, pass through the setting of third through-hole, conducting probe is set to be passed through from third through-hole, contact heating slide, after conducting probe is powered, heating slide then generates heat, and realizes the heating to sequencing reaction small chamber 200.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, and all any modification, equivalent and improvement made within the spirit and principles of the invention etc., should be included in the scope of the protection.
Claims (14)
- A kind of 1. sequencing reaction small chamber, it is characterised in that：Including the first substrate and the second substrate, the lower surface of first substrate is bonded with the upper surface of the second substrate, and at least one hollow reaction channel is formed between first substrate and the second substrate；The reagent exit connected with reaction channel is provided with first substrate or the second substrate, the reagent exit connected with reaction channel is additionally provided with first substrate or the second substrate；The heating slide for being heated to the reagent in reaction channel is fitted with the upper surface of first substrate；It is corresponding on second substrate that a projection is provided with the position of the reaction channel, a thermometer hole is provided with the projection, and the thermometer hole is used to accommodate the temperature probe for carrying out the reaction reagent in reaction channel temperature detection.
- 2. sequencing reaction small chamber according to claim 1, it is characterised in that：The both ends of the reaction channel are respectively provided with the reagent buffering area for accommodating reaction reagent, the reagent exit is connected with reagent buffering area therein, the reagent exit is connected with another reagent buffering area, the reagent buffering area be arranged on the first substrate lower surface and positioned at the first groove both ends the second groove or the reagent buffering area be arranged on the second substrate upper surface and positioned at the first groove both ends the second groove.
- 3. sequencing reaction small chamber according to claim 1, it is characterised in that：First substrate includes the first glass substrate, and second substrate includes PDMS interlayers and the second glass substrate, and the PDMS interlayers are between the first glass substrate and the second glass substrate；The through hole of bar shaped is provided with the PDMS interlayers, the reaction channel is enclosed and formed by the first glass substrate, the through hole of bar shaped and the second glass substrate；The PDMS interlayers are slightly longer than the second described glass substrate, after first glass substrate, PDMS interlayers and the fitting of the second glass substrate, the PDMS interlayers have the lower surface do not blocked by second glass substrate, and the projection is arranged on the lower surface not being blocked of PDMS interlayers.
- 4. sequencing reaction small chamber according to claim 3, it is characterised in that：The both ends of the reaction channel are respectively provided with the reagent buffering area for accommodating reaction reagent, and the reagent buffering area is the through hole being arranged on PDMS interlayers；The reagent exit is connected with a wherein reagent buffering area, and the reagent exit is connected with another reagent buffering area.
- 5. according to the sequencing reaction small chamber described in any one of Claims 1-4, it is characterised in that：The bearing of trend and the second described substrate of the raised upper thermometer hole are perpendicular；OrThe bearing of trend of the raised upper thermometer hole is parallel with the second described substrate.
- 6. a kind of sequencing reaction fixture, including the sequencing reaction small chamber described in claim 1, it is characterised in that：The sequencing reaction fixture also includes on cell stationary fixture under stationary fixture and cell, and the sequencing reaction small chamber is arranged on the cell under stationary fixture and cell between stationary fixture.
- 7. sequencing reaction fixture according to claim 6, it is characterised in that：It is provided with the 3rd groove for housing the sequencing reaction small chamber on the cell under stationary fixture or cell on stationary fixture, orIt is provided with the 4th groove on the cell on stationary fixture, is provided with the 5th groove on stationary fixture under the cell, the sequencing reaction small chamber is arranged in the 4th groove and the 5th groove.
- 8. sequencing reaction fixture according to claim 7, it is characterised in that：On the cell first screwed hole corresponding with reagent inlet is provided with stationary fixture or cell on stationary fixture, the screw with through hole is locked in first screwed hole, the top of through hole on the screw is connected with being passed through the pipeline of reagent, and the bottom of the through hole on the screw is connected with described reagent inlet；On the cell second screwed hole corresponding with reagent exit is provided with stationary fixture or cell on stationary fixture, the screw with through hole is locked in second screwed hole, the top of through hole on the screw is connected with discharging the pipeline of reagent, and the bottom of the through hole on the screw is connected with described reagent exit.
- 9. sequencing reaction fixture according to claim 7, it is characterised in that：When being provided with three groove for housing the sequencing reaction small chamber on stationary fixture under stationary fixture or cell on the cell, the 6th groove of the heating slide for accommodating the sequencing reaction small chamber is additionally provided with the bottom surface of the 3rd groove；OrIt is provided with the 4th groove on the cell on stationary fixture, when being provided with five grooves on stationary fixture under the cell, the 6th groove of the heating slide for accommodating the sequencing reaction small chamber is additionally provided with the bottom surface of the 4th groove or the 5th groove.
- 10. sequencing reaction fixture according to claim 9, it is characterised in that：It is provided with the window for being taken pictures to sequencing reaction on the cell under stationary fixture and cell on stationary fixture, the recessed groove for being easy to the conducting probe contact heating slide is provided with the side wall of the window.
- 11. a kind of sequencing reaction equipment, including the sequencing reaction fixture described in claim 6, it is characterised in that：Also include cell fixing device；The cell fixing device includes cell mounting seat and cell compressing member, the cell compressing member is fixed in cell mounting seat, the sequencing reaction small chamber is arranged in the cell mounting seat, and cell compressing member is used to the sequencing reaction small chamber being pressed on cell mounting seat.
- 12. sequencing reaction equipment according to claim 11, it is characterised in that：It is provided with the cell compressing member for withstanding on the bulb plunger on the sequencing reaction small chamber.
- 13. sequencing reaction equipment according to claim 11, it is characterised in that：Also include temperature measuring equipment, the temperature measuring equipment includes temperature probe, and the temperature probe is used to insert in the thermometer hole of the sequencing reaction small chamber.
- 14. sequencing reaction equipment according to claim 11, it is characterised in that：Cell end cap is rotatablely equipped with the cell mounting seat, the window on stationary fixture under stationary fixture on cell and cell is provided with the second through hole on the cell end cap, the cell end cap is provided with third through-hole corresponding to the recessed groove on stationary fixture under stationary fixture on the cell and cell.
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