CN107435022A - Gene sequencing reaction cell and gene sequencing reaction equipment - Google Patents
Gene sequencing reaction cell and gene sequencing reaction equipment Download PDFInfo
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- CN107435022A CN107435022A CN201610363327.4A CN201610363327A CN107435022A CN 107435022 A CN107435022 A CN 107435022A CN 201610363327 A CN201610363327 A CN 201610363327A CN 107435022 A CN107435022 A CN 107435022A
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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 gene sequencing reaction cell and gene sequencing reaction equipment；Described gene sequencing reaction cell 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 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 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 gene sequencing reaction cell and gene sequencing reaction equipment.
Background technology
Prior art gene sequencing reaction device generally includes gene sequencing reaction cell, temperature control equipment and temperature-detecting device, the temperature control equipment has generally comprised cooling piece, cooling piece contacts with gene sequencing reaction cell, enters the control of trip temperature to gene sequencing reaction cell by cooling piece；Temperature-detecting device has generally comprised temperature sensor, and temperature sensor also contacts with gene sequencing reaction cell, by detecting the temperature of gene sequencing reaction cell, obtains the temperature of gene sequencing reaction.
This deficiency that following two aspects for gene sequencing reaction progress temperature-detecting device be present：On the one hand, because the temperature measured by temperature sensor is not the temperature for the reaction reagent for carrying out gene sequencing reaction, therefore there is error for detection of the prior art gene sequencing reaction device for the temperature of gene sequencing reaction；The temperature for detecting the reaction reagent of gene sequencing reaction is always the problem in gene sequencing field, generally, because gene sequencing reaction cell then needs to change after a gene sequencing reaction is carried out, but the problem of considering cost, temperature-detecting device need not typically be changed, therefore temperature measuring equipment not be fitted into gene sequencing reaction cell typically；Even more important is a bit, if the temperature-detecting device for carrying out thermometric is loaded to the inside of gene sequencing reaction cell, complete sealing is difficult to realize between temperature measuring point and gene sequencing reaction cell, the problem of leakage easily occurs, so that gene sequencing reaction can not be carried out normally.On the other hand, the temperature sensor of temperature-detecting device contacts with gene sequencing reaction cell, when carrying out thermometric, due to the cross-ventilation of gene sequencing reaction 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 gene sequencing reaction cell is needed, the cross-ventilation of gene sequencing reaction chamber surface can be avoided to the influence of temperature survey, 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.
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 first groove is provided with the lower surface of first substrate, after the first substrate is bonded with the second substrate, first groove and the second substrate, which enclose, forms described reaction channel, or
The first groove is provided with the upper surface of second substrate, after the first substrate is bonded with the second substrate, first groove and the first substrate, which enclose, forms described reaction channel.
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.
Further, 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.
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.
Further, the raised structure being formed in one with described second substrate.
In addition, present invention further teaches a kind of gene sequencing reaction equipment, it is theed improvement is that：Including above-mentioned gene sequencing reaction cell 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 gene sequencing reaction cell is arranged in the cell mounting seat, and cell compressing member is used to the gene sequencing reaction cell being pressed on cell mounting seat.
Further, it is provided with the lower surface of the cell compressing member for withstanding on the bulb plunger on the gene sequencing reaction cell.
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 gene sequencing reaction cell.
Further, first screwed hole corresponding with reagent exit is provided with the cell mounting seat or cell compressing member, second screwed hole corresponding with reagent exit is provided with the cell mounting seat or cell compressing member.
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 second substrate and raised dimensional structure diagram in another example of the invention.
Fig. 4 is the principle schematic of temperature survey mode of the prior art.
Fig. 5 is the partial enlarged drawing at A in Fig. 1 of the present invention.
Fig. 6 is the second glass substrate of gene sequencing reaction cell and raised dimensional structure diagram 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 is the cross-sectional view of gene sequencing reaction cell in another example of the invention.
Figure 10 be Fig. 9 described in example in gene sequencing reaction cell the second substrate dimensional structure diagram.
Figure 11 is the cross-sectional view of gene sequencing reaction cell in another example of the invention.
Figure 12, Figure 13 are the structural representation of the reagent buffering area of the gene sequencing reaction cell of the present invention.
Figure 14 is the cross-sectional view of gene sequencing reaction cell in another example of the invention.
Figure 15 is the dimensional structure diagram of PDMS interlayers and the second glass substrate in the example described in Figure 14.
Figure 16 is the cross-sectional view of gene sequencing reaction cell in another example of the invention.
Figure 17 is the dimensional structure diagram of the PDMS interlayers of gene sequencing reaction cell in Figure 16.
Figure 18 is the cross-sectional view of gene sequencing reaction equipment in one embodiment of the invention.
Figure 19 is the cross-sectional view of gene 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 gene sequencing reaction cell, by the way that multiple DNA fragmentations are fixed in gene sequencing reaction cell, reaction reagent is flowed through DNA fragmentation, realize reaction reagent and DNA fragmentation producer sequencing reaction.As Figure 1 and Figure 4, described gene sequencing reaction cell 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 zone of heating 30 for being heated to the reaction reagent in reaction channel is provided 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 zone of heating 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 addition, the position being arranged on for described raised 40 on second substrate 20, the invention also provides an example, as shown in Figure 3, described reaction channel is projected in figure on the second substrate 20 as shown in dash area 400 in Fig. 3, in the heating region such as Fig. 3 that zone of heating 30 is formed on the second substrate 20 shown in dash area 800, when described projection 40 is arranged on dash area 800（That is heating region）When interior, measured temperature value is more or less the same in thermometer hole 401, and therefore, in this example, the projection is arranged on one side of dash area 400, and is located at dash area 800（That is heating region）It is interior；The design of described raised 40 this position, the temperature probe of thermometer hole 401 can be avoided inserting, with connecting the interference of the pipeline on reagent inlet 101 or reagent exit 102 when mounted, while temperature measurement accuracy is ensured, make the easier for installation of sequencing reaction small chamber.
In the present embodiment realized by the heating of zone of heating 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 gene sequencing reaction cell 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, gene sequencing reaction cell 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 gene sequencing reaction cell, 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 gene sequencing reaction cell, 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 of gene sequencing reaction 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 in gene sequencing reaction chamber surface, the cross-ventilation of gene sequencing reaction 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 gene sequencing reaction cell using temperature sensor, as shown in figure 4, temperature sensor 50 is attached on gene sequencing reaction cell 100, gene sequencing reaction cell 100 is heated by zone of heating 30, inside gene sequencing reaction cell 100, thermoisopleth 1001 as shown in Figure 4 is formed, the surface temperature of gene sequencing reaction cell 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 gene sequencing reaction cell 100, the cross-ventilation on the surface of gene sequencing reaction cell 100 will impact to the measurement of temperature sensor 50.As shown in Figure 5, the present embodiment sets projection 40 on the second substrate 20 of gene sequencing reaction cell, 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 5, 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 gene sequencing reaction cell 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 gene sequencing reaction cell, 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 gene sequencing reaction cell 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 zone of heating 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 zone of heating 30 heated is arranged to the upper surface of gene sequencing reaction cell, the thermometer hole 401 for carrying out thermometric is arranged to the lower surface of gene sequencing reaction cell, or the zone of heating 30 heated is arranged to the lower surface of gene sequencing reaction cell, the thermometer hole 401 for carrying out thermometric is arranged to the upper surface of gene sequencing reaction cell, thermometer hole 401 is located away from zone of heating 30, influence during so as to avoid zone of heating 30 from heating for temperature survey, improve the accuracy of gene sequencing reaction temperature survey.
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 6, 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 11, 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 7, 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. 7, 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 gene sequencing reaction cell, and reaction reagent flows out at reagent exit.
For described gene sequencing reaction cell, present invention also offers an example, it is in this example with Fig. 1 embodiment differences, the reaction channel both ends of the gene sequencing reaction cell 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 8, 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.
Gene sequencing reaction cell 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 gene sequencing reaction cell of this structure it is very cumbersome with it is complicated, when being cleaned to gene sequencing reaction cell, need to be dismantled in more pipelines, it is very inconvenient, 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 gene sequencing reaction cell 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 gene sequencing reaction cell；When needing to carry out disassembly, cleaning to gene sequencing reaction cell, 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 gene sequencing reaction cell, 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 gene sequencing reaction cell 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 9, in this example, the gene sequencing reaction cell includes the first substrate 10 and the second substrate 20, and gene sequencing reaction cell 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 9, the reaction channel both ends of gene sequencing reaction cell 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, 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 10,The three-dimensional structure diagram of a specific embodiment of the second substrate 20 in as Fig. 9,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 gene sequencing reaction cell 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 gene sequencing reaction cell of multichannel, when needing to carry out disassembly, cleaning to gene sequencing reaction cell, 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 gene sequencing reaction cell, 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.Gene sequencing reaction cell 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 11, the invention also provides an example, and the structure and Fig. 9 of the second substrate 20 described in this example, structure in example described in Figure 11 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 gene sequencing reaction cell 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 12, 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 gene sequencing reaction cell is vertically arranged on sequencing device, as shown in figure 12, 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 12 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 12 is only a preferable example.As shown in figure 13, 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. 8, Fig. 9 and Figure 11, 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 gene sequencing reaction cell, the invention also provides an example, as shown in figure 14, gene sequencing reaction cell 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；Zone of heating 30 is arranged on the upper surface of the first substrate 10, and reagent exit 101 and reagent exit 102 are provided with the second glass substrate 202, and reagent exit 101 is connected with reagent exit 102 with reaction channel.Further,As shown in figure 14,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 15,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 zone of heating 30 heated and progress thermometric is respectively arranged on upper surface and the PDMS interlayers 201 of the first substrate 10 of gene sequencing reaction cell, thermometer hole 401 is located away from zone of heating 30, influence for temperature survey during so as to avoid zone of heating 30 from heating；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 of gene sequencing reaction 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 gene sequencing reaction cell when carrying out gene sequencing reaction, need to by adopt map device seizure gene sequencing reaction when fluorescence signal.When gene sequencing reaction cell is two-piece type structure, in example as shown in Figure 7, 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, gene sequencing reaction cell 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 gene sequencing reaction cell, on the basis of example described in Fig. 9, the invention also provides an example, as shown in figure 16, in this example, gene sequencing reaction cell 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. 9, is then no longer described in detail in this example.For described PDMS interlayers 201, as shown in Figure 16, Figure 17, 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 16, Figure 17, 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 of gene sequencing reaction 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 gene sequencing reaction equipment based on said gene sequencing reaction small chamber, the gene sequencing reaction equipment includes above-mentioned any gene sequencing reaction cell 100, therefore then no longer gene sequencing reaction cell 100 is described in detail herein；In addition, gene sequencing reaction equipment also includes cell fixing device 200, for described cell fixing device 200, the present invention proposes second embodiment, as shown in figure 18, the cell fixing device 200 includes cell mounting seat 2001 and cell compressing member 2002, the cell compressing member 2002 is fixed in cell mounting seat 2001, cell compressing member 2002 is used to the gene sequencing reaction cell being pressed on cell mounting seat 2001, by the effect of cell compressing member 2002, the fixation to gene sequencing reaction cell is realized.In this example, gene sequencing reaction cell 100 needs to be changed in use for some time, i.e. gene sequencing reaction cell 100 is consumptive material, therefore gene sequencing reaction cell 100 is fixed in cell mounting seat 2001 by the way of detachable.It is provided with lower surface on the cell compressing member 2002 for withstanding on the bulb plunger 2003 on the gene sequencing reaction cell, after gene sequencing reaction cell loads in cell mounting seat 2001, bulb plunger 2003 plays fixed effect to gene sequencing reaction cell, in addition, in the upper surface of gene sequencing reaction cell, the position of bulb plunger 2003 is provided with locating slot, after gene sequencing reaction cell is fixed in cell mounting seat 2001, bulb plunger 2003 is withstood on described locating slot, realizes the positioning of gene sequencing reaction cell 100.In this example, by the effect of cell compressing member 2002, bulb plunger 2003 and locating slot, the dismounting and fixation of gene sequencing reaction cell 100 are realized, facilitates the replacing of gene sequencing reaction cell 100, improves the efficiency of gene sequencing reaction.
For described gene sequencing reaction equipment, the invention also provides an example, same, gene sequencing reaction equipment includes above-mentioned any gene sequencing reaction cell, also the cell mounting seat 2001 and cell compressing member 2002 in example as shown in figure 18 are included, therefore, this example is that another example is proposed on the basis of the example described in Figure 18.As shown in figure 19, the structural representation of gene sequencing reaction equipment as in this example, gene sequencing reaction equipment described in this example also includes temperature measuring equipment, the temperature measuring equipment includes temperature probe 300 and data processing unit, the temperature probe 300 is used to insert in the thermometer hole 401 of the gene sequencing reaction cell, the temperature probe 300 is connected with the data processing unit, temperature probe 300 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 300, zone of heating is realized and controlled, such as, the temperature of gene sequencing reaction is set as T1, temperature measured by temperature probe 300 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, then control zone of heating, improve the temperature of heating；In general, the zone of heating is the coating being coated on gene sequencing reaction cell 100, such as ITO coatings, and ITO coatings realize the heating to gene sequencing reaction cell after being powered, data processing unit realizes the lifting to the small room temperature of gene sequencing reaction by improving the heating powers of ITO coatings.
In the example described in Figure 19,First screwed hole 2004 corresponding with reagent exit 101 is provided with the cell compressing member 2002,Second screwed hole 2005 corresponding with reagent exit 102 is additionally provided with cell compressing member 2002,It should be noted that,First screwed hole 2004,The position of second screwed hole 2005 can be set according to the position of reagent exit 101 and reagent exit 102,Such as,When the reagent exit 101 and reagent exit 102 of gene sequencing reaction cell are all disposed within the second substrate 20,First screwed hole 2004 and the second screwed hole 2005 can be then arranged in cell mounting seat 2001,For the position of the first screwed hole 2004 and the second screwed hole 2005,A kind of scheme that can implement is provide only in this example.After gene sequencing reaction cell is fixed in cell mounting seat 2001, need to be screwed into fastening bolt 2006 in the screwed hole 2005 of the first screwed hole 2004 and second, and the through hole being respectively communicated with reagent exit 101 and reagent exit 102 is respectively arranged with two fastening bolts 2006, the through hole for the fastening bolt 2006 being arranged on reagent exit 101 is connected with the pipeline for being passed through reaction reagent, and the through hole for the fastening bolt 2006 being arranged on reagent exit 102 is connected with the pipeline for flowing out reaction reagent.In this example, the material of the fastening bolt 2006 is flexible material, such as rubber etc., when fastening bolt 2006 is screwed into the first screwed hole 2004 and the second screwed hole 2005, because the quality of fastening bolt 2006 is softer, it can realize with gene sequencing reaction cell and closely be bonded, so as to prevent reagent exit 101 and reagent exit 102 from the situation of leakage occur, there is good sealing property.
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 (12)
- A kind of 1. gene sequencing reaction cell, 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 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.
- 2. gene sequencing reaction cell according to claim 1, it is characterised in that：The first groove is provided with the lower surface of first substrate, after the first substrate is bonded with the second substrate, first groove and the second substrate, which enclose, forms described reaction channel, orThe first groove is provided with the upper surface of second substrate, after the first substrate is bonded with the second substrate, first groove and the first substrate, which enclose, forms described reaction channel.
- 3. gene sequencing reaction cell 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.
- 4. gene sequencing reaction cell 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.
- 5. gene sequencing reaction cell according to claim 4, it is characterised in that：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.
- 6. gene sequencing reaction cell according to claim 5, 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.
- 7. the gene sequencing reaction cell according to any one of claim 1 to 6, 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.
- 8. gene sequencing reaction cell according to claim 7, it is characterised in that：The raised structure being formed in one with described second substrate.
- A kind of 9. gene sequencing reaction equipment, it is characterised in that：Including the gene sequencing reaction cell and cell fixing device described in claim 1；The cell fixing device includes cell mounting seat and cell compressing member, the cell compressing member is fixed in cell mounting seat, the gene sequencing reaction cell is arranged in the cell mounting seat, and cell compressing member is used to the gene sequencing reaction cell being pressed on cell mounting seat.
- 10. gene sequencing reaction equipment according to claim 9, it is characterised in that：The cell compressing member is provided with for withstanding on the bulb plunger on the gene sequencing reaction cell.
- 11. gene sequencing reaction equipment according to claim 9, 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 gene sequencing reaction cell.
- 12. gene sequencing reaction equipment according to claim 9, it is characterised in that：First screwed hole corresponding with reagent exit is provided with the cell mounting seat or cell compressing member, second screwed hole corresponding with reagent exit is provided with the cell mounting seat or cell compressing member.
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