CN107400628B - Sequencing reaction cell, sequencing reaction clamp and sequencing reaction equipment - Google Patents
Sequencing reaction cell, sequencing reaction clamp and sequencing reaction equipment Download PDFInfo
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- CN107400628B CN107400628B CN201610338059.0A CN201610338059A CN107400628B CN 107400628 B CN107400628 B CN 107400628B CN 201610338059 A CN201610338059 A CN 201610338059A CN 107400628 B CN107400628 B CN 107400628B
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- 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 invention relates to the field of gene sequencing, and provides a sequencing reaction chamber, a sequencing reaction clamp and sequencing reaction equipment; the sequencing reaction chamber comprises a first substrate and a second substrate, wherein the lower surface of the first substrate is attached to the upper surface of the second substrate, and at least one hollow reaction channel is formed between the first substrate and the second substrate; a reagent outlet is arranged on the first substrate or the second substrate, and a reagent outlet is also arranged on the first substrate or the second substrate; a heating glass slide for heating the reagent in the reaction channel is attached to the upper surface of the first substrate; a bulge is arranged on the second substrate corresponding to the position of the reaction channel, a temperature measuring hole is arranged on the bulge, and a temperature measuring probe is accommodated in the temperature measuring hole; the invention has the beneficial effects that: the influence of air convection on temperature measurement on the surface of the gene sequencing reaction chamber is avoided, the accuracy of gene sequencing reaction temperature measurement is improved, and the measured temperature is closer to the temperature of a reaction reagent of the gene sequencing reaction.
Description
Technical Field
The invention relates to the field of gene sequencing, in particular to a sequencing reaction chamber, a sequencing reaction clamp and sequencing reaction equipment.
Background
In the prior art, a sequencing reaction device comprises a sequencing reaction chamber, a temperature control device and a temperature detection device, wherein the temperature control device generally comprises a refrigeration piece, the refrigeration piece is in contact with the sequencing reaction chamber, and the temperature of the sequencing reaction chamber is controlled by the refrigeration piece; the temperature detecting device generally comprises a temperature sensor, the temperature sensor is also contacted with the sequencing reaction chamber, and the temperature of the sequencing reaction is obtained by detecting the temperature of the sequencing reaction chamber.
Such an apparatus for detecting the temperature of a sequencing reaction has two disadvantages in that, on the one hand, the temperature detected by the temperature sensor is not the temperature of a reagent for performing a sequencing reaction, and thus, there is an error in detecting the temperature of a sequencing reaction; the temperature of a reagent for detecting a sequencing reaction is always a difficult problem in the field of gene sequencing, and generally, a sequencing reaction chamber needs to be replaced after a sequencing reaction is carried out once, a temperature measuring device does not need to be replaced, so that the temperature measuring device cannot be replaced when the sequencing reaction chamber is replaced in consideration of the cost problem, and a temperature measuring device is not generally installed in the sequencing reaction chamber; more importantly, if the temperature measuring device for measuring the temperature is arranged in the sequencing reaction chamber, the temperature measuring point and the sequencing reaction chamber are difficult to realize complete sealing, and the problem of liquid leakage is easy to occur, so that the sequencing reaction can not be normally carried out. On the other hand, the temperature sensor of the temperature detection device is in contact with the sequencing reaction chamber, and when temperature measurement is carried out, air convection on the surface of the sequencing reaction chamber easily influences the temperature measurement, so that the temperature measurement is prone to deviation, and the temperature measurement precision is low.
Therefore, a new sequencing reaction chamber is needed, which can avoid the influence of the air convection on the surface of the sequencing reaction chamber on the temperature measurement, improve the accuracy of the sequencing reaction temperature measurement, and enable the measured temperature to be closer to the temperature of the reagent of the sequencing reaction.
Disclosure of Invention
The invention aims to provide a gene sequencing reaction chamber and gene sequencing reaction equipment, and aims to solve the problems that air convection on the surface of the gene sequencing reaction chamber in the prior art is easy to influence the measurement of temperature, so that the measurement of the temperature is easy to deviate, and the measurement precision of the temperature is low.
In order to achieve the object of the invention, a gene sequencing reaction chamber is improved in that: the device comprises a first substrate and a second substrate, wherein the lower surface of the first substrate is attached to the upper surface of the second substrate, and at least one hollow reaction channel is formed between the first substrate and the second substrate;
the first substrate or the second substrate is provided with a reagent inlet communicated with the reaction channel, and the first substrate or the second substrate is also provided with a reagent outlet communicated with the reaction channel;
a heating layer for heating the reaction reagent in the reaction channel is arranged on the upper surface of the first substrate; and a bulge is arranged on the second substrate corresponding to the reaction channel, a temperature measuring hole is arranged on the bulge, and the temperature measuring hole is used for accommodating a temperature measuring probe for detecting the temperature of the reaction reagent in the reaction channel.
In the structure, two ends of the reaction channel are respectively provided with a reagent buffer zone for containing a reaction reagent, the reagent inlet is communicated with one reagent buffer zone, the reagent outlet is communicated with the other reagent buffer zone, and the reagent buffer zone is a second groove which is arranged on the lower surface of the first substrate and is positioned at two ends of the first groove or the reagent buffer zone is a second groove which is arranged on the upper surface of the second substrate and is positioned at two ends of the first groove.
In the above structure, the first substrate comprises a first glass substrate, the second substrate comprises a PDMS interlayer and a second glass substrate, and the PDMS interlayer is located between the first glass substrate and the second glass substrate; the PDMS interlayer is provided with a strip-shaped through hole, and the reaction channel is formed by enclosing a first glass substrate, the strip-shaped through hole and a second glass substrate; the PDMS interlayer is slightly longer than the second glass substrate, after the first glass substrate, the PDMS interlayer and the second glass substrate are jointed, the PDMS interlayer has a lower surface which is not shielded by the second glass substrate, and the protrusion is arranged on the lower surface of the PDMS interlayer which is not shielded.
Furthermore, two ends of the reaction channel are respectively provided with a reagent buffer zone for containing a reaction reagent, and the reagent buffer zones are through holes arranged on the PDMS interlayer; the reagent inlet is communicated with one reagent buffer zone, and the reagent outlet is communicated with the other reagent buffer zone.
Furthermore, the extending direction of the temperature measuring hole on the bulge is vertical to the second substrate; or the extending direction of the temperature measuring hole on the bulge is parallel to the second substrate.
In addition, the invention also discloses a sequencing reaction clamp, which comprises the sequencing reaction chamber, and is improved in that: the sequencing reaction clamp is characterized by further comprising a fixing clamp on the small chamber and a fixing clamp under the small chamber, and the sequencing reaction small chamber is arranged between the fixing clamp on the small chamber and the fixing clamp under the small chamber.
Further, a third groove for accommodating the sequencing reaction chamber is arranged on the chamber upper fixing clamp or the chamber lower fixing clamp, or
Be provided with the fourth recess on the mounting fixture on the cell, be provided with the fifth recess under the cell on the mounting fixture, the sequencing reaction cell sets up in fourth recess and fifth recess.
Furthermore, a first threaded hole corresponding to the reagent inlet is formed in the small chamber upper fixing clamp or the small chamber lower fixing clamp, a screw with a through hole is locked into the first threaded hole, the top end of the through hole in the screw is communicated with a pipeline for introducing the reagent, and the bottom end of the through hole in the screw is communicated with the reagent inlet;
and a second threaded hole corresponding to the reagent outlet is formed in the upper fixing clamp of the small chamber or the lower fixing clamp of the small chamber, a screw with a through hole is locked in the second threaded hole, the top end of the through hole in the screw is communicated with a pipeline for discharging the reagent, and the bottom end of the through hole in the screw is communicated with the reagent outlet.
Further, when a third groove for accommodating the sequencing reaction chamber is formed in the upper small chamber fixing clamp or the lower small chamber fixing clamp, a sixth groove for accommodating a heating slide of the sequencing reaction chamber is further formed in the third groove; or
And when the fixing clamp is provided with a fifth groove, a sixth groove for accommodating the heating slide of the sequencing reaction chamber is further arranged in the fourth groove or the fifth groove.
Furthermore, windows for photographing the sequencing reaction are arranged on the fixing clamp on the small chamber and the fixing clamp under the small chamber, and a concave groove for facilitating the contact of a conductive probe with the heating glass slide is arranged on the side wall of each window.
In addition, the invention also discloses sequencing reaction equipment, which is characterized in that: comprises a sequencing reaction clamp and a chamber fixing device; the small chamber fixing device comprises a small chamber mounting seat and a small chamber compressing piece, the small chamber compressing piece is fixed on the small chamber mounting seat, the sequencing reaction small chamber is mounted on the small chamber mounting seat, and the small chamber compressing piece is used for compressing the sequencing reaction small chamber on the small chamber mounting seat.
Furthermore, a ball plunger used for being pressed against the sequencing reaction chamber is arranged on the chamber pressing piece.
Furthermore, the sequencing reaction chamber also comprises a temperature measuring device, wherein the temperature measuring device comprises a temperature measuring probe, and the temperature measuring probe is inserted into a temperature measuring hole of the sequencing reaction chamber.
Furthermore, the cell mounting seat is rotatably provided with a cell end cover, a second through hole is formed in the cell end cover corresponding to a window on the cell upper fixing clamp and a window on the cell lower fixing clamp, and a third through hole is formed in the cell end cover corresponding to a concave groove in the cell upper fixing clamp and the cell lower fixing clamp.
According to the invention, the temperature inside the temperature measuring hole is closer to the temperature of the reaction reagent by the arrangement of the temperature measuring hole, and the temperature measuring probe or the temperature sensor is arranged in the temperature measuring hole due to the existence of the temperature measuring hole, so that the influence of air convection on temperature measurement on the surface of the gene sequencing reaction chamber can be well avoided, the accuracy of gene sequencing reaction temperature measurement is improved, and the measured temperature is closer to the temperature of the reaction reagent of the gene sequencing reaction.
Drawings
FIG. 1 is a schematic sectional view of a reaction chamber for gene sequencing according to an embodiment of the present invention.
FIG. 2 is a schematic perspective view of a gene sequencing reaction chamber according to an embodiment of the present invention.
Fig. 3 is a schematic diagram of a temperature measurement method in the prior art.
Fig. 4 is a partial enlarged view of the invention at a in fig. 1.
FIG. 5 is a schematic perspective view of a second glass substrate and projections of the gene sequencing reaction chamber according to another embodiment of the present invention.
FIG. 6 is a schematic sectional view of a gene sequencing reaction chamber according to another embodiment of the present invention.
FIG. 7 is a schematic sectional view of a gene sequencing reaction chamber according to another embodiment of the present invention.
FIG. 8 is a schematic sectional view of a gene sequencing reaction chamber according to another embodiment of the present invention.
FIG. 9 is a schematic perspective view of a second substrate of the gene sequencing reaction chamber in the example shown in FIG. 8.
FIG. 10 is a schematic sectional view of a reaction chamber for gene sequencing according to another embodiment of the present invention.
FIGS. 11 and 12 are schematic diagrams showing the structure of a reagent buffer of the gene sequencing reaction chamber of the present invention.
FIG. 13 is a schematic sectional view of a reaction chamber for gene sequencing according to another embodiment of the present invention.
Fig. 14 is a schematic perspective view of the PDMS interlayer and the second glass substrate in the example shown in fig. 13.
FIG. 15 is a schematic sectional view of a gene sequencing reaction chamber according to another embodiment of the present invention.
FIG. 16 is a schematic perspective view of the PDMS sandwich of the gene sequencing reaction chamber in FIG. 15.
FIG. 17 is a schematic perspective view of a sequencing reaction fixture according to an embodiment of the present invention.
FIG. 18 is a schematic perspective view of a sequencing reaction holder according to another embodiment of the present invention.
Figure 19 is the cell in figure 18 on the stationary fixture three-dimensional structure diagram.
FIG. 20 is a schematic cross-sectional view of a sequencing reaction apparatus according to an embodiment of the present invention.
FIG. 21 is a schematic sectional view showing a sequencing reaction apparatus according to another example of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments.
The present invention provides a first embodiment, which provides a sequencing reaction chamber, wherein a plurality of DNA fragments are fixed in the sequencing reaction chamber, and a reaction reagent flows through the DNA fragments, so that a gene sequencing reaction between the reaction reagent and the DNA fragments is realized. As shown in fig. 1 and 4, the sequencing reaction chamber comprises a first substrate 10 and a second substrate 20, wherein the lower surface of the first substrate 10 is attached to the upper surface of the second substrate 20, so that the first substrate 10 and the second substrate 20 are sealed; at least one hollow reaction channel is formed between the first substrate 10 and the second substrate 20, when only one reaction channel is provided, a single-channel gene sequencing reaction is realized, and when a plurality of reaction channels are provided, a plurality of channels can be sequenced in parallel. The second substrate 20 is provided with a reagent inlet 101 communicated with the reaction channel, the reagent inlet 101 is communicated with a pipeline for supplying a reaction reagent from the outside, and the reaction reagent required by the gene sequencing reaction is introduced into the reaction channel through the reagent inlet 101; the second substrate 20 is further provided with a reagent outlet 102 communicating with the reaction channel, the reagent outlet 102 is communicated with a conduit for discharging a reaction reagent, and the reaction reagent after the gene sequencing reaction occurs flows out through the reagent outlet 102. in the present embodiment, as shown in fig. 1, the reagent inlet 101 and the reagent outlet 102 are both provided on the second substrate 20, but different examples of the reagent inlet 101 and the reagent outlet 102 will be described in detail below.
Further, a heating slide 30 for heating a reagent in the reaction channel is attached to the upper surface of the first substrate 10, and when the reaction reagent is introduced from the reagent inlet 101 and flows through the reaction channel, the heating slide 30 heats the reaction reagent, and thereafter the reaction reagent flows out from the reagent outlet 102. A protrusion 40 is disposed on the second substrate 20 corresponding to the reaction channel, a temperature measuring hole 401 is disposed on the protrusion 40, and a temperature measuring probe for detecting the temperature of the reaction reagent in the reaction channel is accommodated in the temperature measuring hole 401. When only one reaction channel is provided, as shown in fig. 2, after the first substrate 10 is attached to the second substrate 20, the pattern of the reaction channel projected on the second substrate 20 is shown by the shaded portion 400 in fig. 2, and the protrusion 40 is projected on the second substrate 20 across the reaction channel, and at the same time, in order to avoid the influence of the protrusion 40 blocking the fluorescence signal in the reaction channel and the collection of the fluorescence signal, the protrusion 40 is disposed near the reagent inlet 101, but it should be noted that the protrusion 40 may also be disposed near the reagent outlet 102. In the process of the gene sequencing reaction, the temperature of the reaction reagent in the reaction channel needs to be regulated to meet the requirement of the gene sequencing reaction, and the heating of the heating slide 30 is adopted in the embodiment; meanwhile, in the process of the gene sequencing reaction, the temperature of the reaction reagent in the reaction channel needs to be monitored, so that the temperature of the reaction reagent is kept in a required temperature range. Because the reaction channel needs to be filled with a reaction reagent, the leakage can not occur, and the temperature sensor or the temperature probe for detecting the temperature can not be arranged in the reaction channel, the reason is that the sealing of the sequencing reaction chamber can not be realized after the temperature sensor or the temperature probe is arranged in the reaction channel, and the leakage is easy to occur; because the first substrate 10 and the second substrate 20 are generally thin, the temperature measuring probe or the temperature sensor is difficult to be arranged in the reaction channel; in addition, the sequencing reaction chamber needs to be replaced after the gene sequencing reaction, and even if the temperature probe or the temperature sensor can be installed in the reaction channel, the temperature sensor or the temperature probe is troublesome to mount and dismount when the sequencing reaction chamber is replaced. In view of the above, in the prior art, in order to detect the temperature of the reaction reagent in the reaction channel, a common technical solution is to mount a temperature sensor or a temperature probe on the surface of the sequencing reaction chamber, and measure the temperature of the reaction reagent in the reaction channel; however, this temperature measurement method is an indirect measurement method, the temperature measured by the temperature sensor is the temperature on the surface of the sequencing reaction chamber, and when the temperature is not completely equal to the temperature of the reaction reagent in the reaction channel, a large error occurs in the temperature measurement result; when the temperature sensor is mounted on the surface of the sequencing reaction chamber, the air convection on the surface of the sequencing reaction chamber affects the measurement of the temperature, and the measurement result of the temperature sensor is deviated.
Specifically, in the prior art, when a temperature sensor is used to directly measure the temperature on the surface of the sequencing reaction chamber, as shown in fig. 3, the temperature sensor 50 is attached to the sequencing reaction chamber 100, the sequencing reaction chamber 100 is heated by heating the slide 30, an isotherm 1001 as shown in fig. 3 is formed inside the sequencing reaction chamber 100, and a certain difference exists between the surface temperature and the internal temperature of the sequencing reaction chamber 100, which causes an error in temperature measurement; meanwhile, by directly disposing the temperature sensor 50 on the surface of the sequencing reaction chamber 100, the air convection on the surface of the sequencing reaction chamber 100 can affect the measurement of the temperature sensor 50. As shown in FIG. 4, in this embodiment, when the protrusion 40 is disposed on the second substrate 20 of the sequencing reaction chamber and the temperature measuring hole 401 is disposed in the protrusion 40, the temperature is measured by the temperature measuring probe extending into the temperature measuring hole 401, because the protrusion 40 is disposed on the second substrate 20, in the preferred embodiment, the protrusion 40 and the second substrate 20 are integrally formed, and the isotherm 1001 as shown in FIG. 4 is formed in the second substrate 20, the temperature inside the temperature measuring hole 401 is closer to the temperature inside the second substrate 20, and the temperature measured by the temperature measuring probe extending into the temperature measuring hole 401 is closer to the temperature of the reaction reagent of the gene sequencing reaction than the measurement mode in FIG. 2; moreover, due to the existence of the temperature measuring hole 401, the temperature measuring probe or the temperature sensor is arranged in the temperature measuring hole 401, so that the influence of air convection on temperature measurement on the surface of the sequencing reaction chamber 100 can be well avoided, the accuracy of the temperature measurement of the gene sequencing reaction is improved, and the measured temperature is closer to the temperature of a reaction reagent of the gene sequencing reaction.
It should be further noted that, in the prior art, a stable thermal radiation surface is arranged on the sequencing reaction chamber to heat the reaction reagent in the reaction channel, due to the structural limitation of the sequencing device, the temperature measurement point is generally arranged beside the thermal radiation surface, when the thermal radiation surface continuously heats the reaction reagent in the reaction channel, and after a period of time, the temperature of the thermal radiation surface, the temperature of the reaction reagent, the temperature of the sequencing reaction chamber and the temperature of the temperature measurement point all tend to be consistent, and the temperature measurement is realized by the temperature sensor of the temperature measurement point; although the temperature of the reaction reagent can be measured by the method, the temperature measuring point and the thermal radiation surface are positioned on the same surface, so that the temperature of the thermal radiation surface can influence the temperature measurement of the temperature measuring point, and the temperature measuring accuracy is reduced. In this embodiment, the heating slide 30 is disposed on the upper surface of the first substrate 10, the protrusion 40 is disposed on the second substrate 20 at a position corresponding to the reaction channel, and in this embodiment, the protrusion 40 is projected on the second substrate 20 across the reaction channel, so that the position of the temperature measuring hole 401 is closer to the reaction channel. Through the design of the structure, the heating slide 30 for heating is arranged on the upper surface of the sequencing reaction chamber, the temperature measuring hole 401 for measuring temperature is arranged on the lower surface of the sequencing reaction chamber, or the heating slide 30 for heating is arranged on the lower surface of the sequencing reaction chamber, the temperature measuring hole 401 for measuring temperature is arranged on the upper surface of the sequencing reaction chamber, and the position of the temperature measuring hole 401 is far away from the heating slide 30, so that the influence on temperature measurement when the heating slide 30 is heated is avoided, and the accuracy of gene sequencing reaction temperature measurement is improved.
For the temperature measuring hole 401 on the protrusion 40, in the example shown in fig. 1 and 2, the extending direction of the temperature measuring hole 401 is perpendicular to the second glass substrate 20. As shown in fig. 5, the present invention further provides an example of the temperature measuring hole 401 of the protrusion 40, the temperature measuring hole 401 is disposed on the sidewall of the protrusion 40, the extending direction of the temperature measuring hole 401 is parallel to the second substrate 20, and this structure of the temperature measuring hole is suitable for the case that the temperature measuring probe is located at one side of the sequencing reaction chamber, and in this example, because the extending direction of the temperature measuring hole 401 is parallel to the second substrate 20, when the temperature measuring probe is inserted into the temperature measuring hole 401, the surface of the second substrate 20 can play a role of guiding the insertion of the temperature measuring probe, so that the temperature measuring probe can be conveniently inserted into the temperature measuring hole. Furthermore, a chamfer angle with a guiding function can be arranged at the opening of the temperature measuring hole 401, so that the temperature measuring probe can be more conveniently inserted into the temperature measuring hole.
As for the protrusion 40, in the example shown in fig. 1 and 2, the protrusion 40 has a square structure, however, the structure of the protrusion 40 does not affect the protection scope of the present invention, for example, the protrusion 40 may be designed to be cylindrical, or the protrusion 40 may be designed to be truncated cone in order to increase the contact area of the protrusion 40 and the second substrate; and the shape can be designed into other shapes according to requirements. In addition, in the example shown in fig. 1 and fig. 2, the temperature measuring hole 401 is a circular blind hole, but the shape of the temperature measuring hole 401 can be set according to requirements, preferably, the shape of the temperature measuring hole 401 is the same as the shape of the temperature measuring probe, and after the temperature measuring probe extends into the temperature measuring hole 401, the outer surface of the temperature measuring probe forms a good fit with the inner wall of the temperature measuring hole 401, so that the influence on the temperature measurement result due to a large gap between the outer surface of the temperature measuring probe and the inner wall of the temperature measuring hole 401 is avoided; further, in order to improve the heat conduction efficiency between the inner wall of the temperature measurement hole and the temperature measurement probe, heat-conducting silica gel is filled in the temperature measurement hole, and the heat on the protrusion 40 is transferred to the temperature measurement probe under the action of the heat-conducting silica gel.
On the basis of the above embodiments, the present invention provides an example for the reaction channel, in this example, as shown in fig. 1 and fig. 2, a first groove 103 is provided on the lower surface of the first substrate 10, the first groove 103 may be a single groove or multiple parallel grooves, and after the first substrate 10 and the second substrate 20 are attached to each other, the first groove 103 and the second substrate 20 surround to form the reaction channel; in addition, in this example, the second substrate 20 is provided with a reagent inlet 101 and a reagent outlet 102 which are communicated with the reaction channel, the number of the reagent inlet 101 and the reagent outlet 102 is determined according to the number of the first grooves 103, when there is one first groove 103, one reagent inlet 101 and one reagent outlet 102 are provided, which are respectively corresponding to the head end and the tail end of the first groove 103; when the first groove 103 is a plurality of grooves, each first groove 103 is provided with a reagent inlet 101 and a reagent outlet 102. As shown in fig. 10, as an alternative embodiment of the reaction channel, a first groove 103 may be further disposed on the upper surface of the second substrate 20, and after the first substrate 10 is attached to the second substrate 20, the first groove 103 and the first substrate 10 enclose to form the reaction channel; similarly, the second substrate 20 is provided with a reagent inlet 101 and a reagent outlet 102 communicating with the reaction channel. In addition, as shown in fig. 6, as another alternative embodiment of the reaction channel, the lower surface of the first substrate 10 is provided with a first groove 103, the upper surface of the second substrate 20 is also provided with a first groove 103, the first groove 103 on the first substrate 10 and the first groove 103 on the second substrate 20 have the same shape, and after the first substrate 10 and the second substrate 20 are attached to each other, the first groove 103 on the first substrate 10 and the first groove 103 on the second substrate 20 form the reaction channel.
While there are many different combinations of solutions for the location of the reagent outlet and the reagent outlet in the above examples, both the reagent outlet and the reagent outlet are provided on the second substrate 20 in the examples described in fig. 1 or fig. 6. In an alternative embodiment (not shown), the reagent outlet may be disposed on a first substrate, and the reagent outlet may be disposed on the first substrate, and in addition, the reagent outlet may be disposed on the first substrate while the reagent outlet is disposed on a second substrate; or the reagent outlet is arranged on the second substrate, the reagent outlet is arranged on the first substrate, and the reagent outlet can be communicated with the reaction channel of the sequencing reaction chamber and the reaction reagent flows out from the reagent outlet.
The present invention also provides an example of the sequencing reaction chamber, which is different from the embodiment of fig. 1 in that reagent buffer regions for accommodating reaction reagents are respectively disposed at two ends of a reaction channel of the sequencing reaction chamber, a reagent inlet 101 is communicated with one of the reagent buffer regions, a reagent outlet 102 is communicated with the other reagent buffer region, in this example, as shown in fig. 7, a first groove 103 is disposed on a lower surface of the first substrate 10, the first groove 103 and the second substrate 20 enclose to form the reaction channel, and the reagent buffer regions are second grooves 104 disposed on the lower surface of the first substrate 10 and at two ends of the first groove 103. Compared with the first groove 103, the second groove 104 can contain more reaction reagents, when the reaction reagents required by the gene sequencing reaction are introduced into the reaction channel through the reagent inlet 101, before the reaction reagents enter the reaction channel, the reaction reagents firstly enter the reagent buffer area at one end of the reaction channel, after the reagent buffer area is filled, the reaction reagents enter the hollow reaction channel again to generate the gene sequencing reaction with the sample to be detected in the reaction channel, when a plurality of reaction channels are provided, the reagents simultaneously enter each reaction channel to generate the gene sequencing reaction with the sample to be detected in the plurality of reaction channels; then the reaction reagent flows out and enters a reagent buffer zone at the other end of the reaction channel, and the reaction reagent is collected in the reagent buffer zone and then is discharged from a pipeline for discharging the reaction reagent. Because the reagent buffer zone exists, a certain amount of reaction reagent can be stored in the reagent buffer zone, the reaction reagent can flow into the reagent buffer zone before flowing into the reaction channel, the reagent buffer zone is favorable for uniformly discharging air in each reaction channel, the internal pressure of the reaction reagent is consistent when the reaction reagent enters the reaction channels, and the reaction reagent can uniformly pass through each reaction channel, so that the uniformity of the flow speed and the uniformity of the diffusivity of each reaction channel are ensured.
In the sequencing reaction chamber with a plurality of channels in the prior art, due to the plurality of reaction channels, the reagent inlet 101 and the reagent outlet 102 of each reaction channel need to be connected with a pipeline for the inlet and the outlet of the reaction reagent; the sequencing reaction chamber with the structure has a very complicated and complicated structure, and when the sequencing reaction chamber is cleaned, a plurality of pipelines need to be disassembled, so that the sequencing reaction chamber is very inconvenient, and the poor sealing is easy to occur in the disassembling and assembling processes, thereby causing the liquid leakage in the gene sequencing reaction process. In the multi-channel sequencing reaction chamber in the embodiment, due to the arrangement of the reagent buffer area, gene sequencing reactions of a plurality of channels can be completed only by accessing one pipeline into the reagent buffer area, so that the structure of the multi-channel sequencing reaction chamber is simplified; when the sequencing reaction chamber needs to be disassembled and cleaned, the inside of the sequencing reaction chamber can be cleaned only by disassembling a liquid inlet pipeline communicated with the reagent inlet 101 and a liquid outlet pipeline communicated with the reagent outlet 102, so that the convenience is high, and the condition of liquid leakage in the gene sequencing reaction process caused by poor sealing in the disassembling and assembling processes is reduced. Therefore, the sequencing reaction chamber in the example can be reused for many times in the actual use process, and compared with the disposable reaction chamber in the prior art, the invention greatly reduces the cost of equipment.
For the reagent buffer, the present invention also provides an example, as shown in fig. 8, in which the sequencing reaction chamber includes a first substrate 10 and a second substrate 20, and in which the sequencing reaction chamber is different from the example shown in fig. 1 in that: the first groove 103 is disposed on the upper surface of the second substrate 20. In this example, as shown in fig. 8, two ends of the reaction channel of the sequencing reaction chamber are respectively provided with a reagent buffer area for containing a reaction reagent, the reagent inlet 101 is communicated with one of the reagent buffer areas, the reagent outlet 102 is communicated with the other reagent buffer area, in this example, the upper surface of the second substrate 20 is provided with a first groove 103, the first substrate 10 and the first groove 103 enclose to form the reaction channel, and the reagent buffer areas are second grooves 104 arranged at two ends of the reaction channel.
As shown in fig. 9, which is a three-dimensional structure diagram of an embodiment of the second substrate 20 in fig. 8, in this example, six parallel first grooves 103 are formed on the upper surface of the second substrate 20, the first substrate 10 and the second substrate 20 are attached to form a six-channel sequencing reaction chamber, when performing a gene sequencing reaction, a reagent injected through the reagent inlet 101 first enters a reagent buffer area located at the reagent inlet 101, after continuously injecting a reaction reagent, the reaction reagent fills the reagent buffer area located at the reagent inlet 101, then simultaneously enters the six reaction channels, performs a gene sequencing reaction with a sample to be tested fixed in the reaction channels respectively, then flows out and enters the reagent buffer area located at the reagent outlet 102, after the reaction reagent is collected in the reagent buffer area, is discharged from a conduit for discharging the reaction reagent, and continuously injects the reaction reagent, the reaction reagent continuously flows through the reaction channel to perform gene sequencing reaction with the sample to be detected. Due to the existence of the reagent buffer zone, before the reaction reagent is introduced into the reaction channel or after the reaction reagent flows out of the reaction channel, the reagent buffer zone can store a certain amount of reaction reagent, so that the uniformity of the flow speed and the uniformity of the diffusivity of each reaction channel are ensured. The structure of the multi-channel sequencing reaction chamber is simplified, when the sequencing reaction chamber needs to be disassembled and cleaned, only a liquid inlet pipeline communicated with the reagent inlet 101 and a liquid outlet pipeline communicated with the reagent outlet 102 need to be disassembled, and after the first substrate 10 is separated from the second substrate 20, the reaction channel, the reagent buffer area, the reagent inlet 101 and the reagent outlet 102 of the sequencing reaction chamber can be cleaned. When the first substrate 10 and the second substrate 20 are mounted, since the reagent inlet 101, the reagent outlet 102, the reaction channel, and the reagent buffer are all disposed on the second substrate 20, the mounting is very convenient. When the sequencing reaction chamber in this example is placed vertically and the reagent inlet 101 is located at the lower position, after the reaction reagent is introduced, the reaction reagent first fills the reagent buffer zone located at the reagent outlet and then simultaneously enters the six reaction channels, thereby facilitating the uniform discharge of air from each reaction channel.
As for the position of the reagent buffer area, as shown in fig. 10, the present invention further provides an example, and the structure of the second substrate 20 in this example is the same as the structure in the example shown in fig. 8 and 10, and the structure of the second substrate 20 is not described in detail in this example, except that the lower surface of the first substrate 10 is also provided with a second groove 104, and the shape of the second groove 104 provided on the first substrate 10 is the same as the shape of the second groove 104 on the second substrate 20, and after the first substrate 10 is attached to the second substrate 20, the second groove 104 provided on the first substrate 10 is matched with the second groove 104 on the second substrate 20 to form the reagent buffer area. In this example, when the sequencing reaction chamber is laid flat, i.e. the first substrate 10 is located below the second substrate 20 and in a horizontal position, the reagent introduced through the reagent inlet 101 first enters the reagent buffer zone, and enters the reaction channel after filling the second groove located on the first substrate 10, and the reagent buffer zone buffers the reagent.
Regarding the shape of the reagent buffer area, the present invention provides an example, as shown in fig. 11, the reagent buffer area 500 is in a semicircular shape, the reagent buffer area 500 has a circular arc edge and a straight edge, and the straight edge of the reagent buffer area 500 is located at a side close to the reaction channel. Through the design of the structure, after the sequencing reaction chamber is vertically arranged on the sequencing device, as shown in fig. 11, in the process of injecting the reagent into the reagent buffer zone 500, as the reagent buffer zone is designed to be semicircular, the flow rates of the points A, B and C shown in fig. 11 are more uniform, so that the flow rates of any position in the reagent buffer zone are more uniform, and the uniformity of the flow rate and the uniformity of the diffusivity of each reaction channel are ensured; so that the sequencing reactions in the multiple reaction channels are performed simultaneously. It should be noted that the above-mentioned scheme does not set any limit to the structure of the reagent buffer, the size and shape of the reagent buffer can be designed according to actual needs, for example, the reagent buffer can be square or rectangular, and the scheme shown in fig. 11 is only a preferred example. As shown in FIG. 12, another example of the reagent buffer 500 is shown, in which the reagent buffer 500 has a triangular shape, and the flow rate uniformity and the diffusion uniformity of each reaction channel are ensured by the design of the triangular structure during the injection of the reagent into the reagent buffer 500, so that the sequencing reactions in a plurality of reaction channels can be performed simultaneously.
In addition, in the examples shown in fig. 7, fig. 8 and fig. 10, both ends of the reaction channel are respectively provided with a reagent buffer area for containing the reaction reagent, the protrusions 40 are both arranged on the lower surface of the second glass substrate 20, the protrusions 40 are both arranged at positions close to the reagent inlet 101, because the reagent buffer area is arranged at the reagent inlet 101, the temperature measuring holes 401 on the protrusions 40 are closer to the reagent buffer area, the reaction reagent in the reagent buffer area is more, and the temperature of the reaction reagent can be detected through the temperature measuring holes 401, thereby improving the accuracy of temperature measurement; it should be noted that the protrusion 40 may be disposed near the reagent outlet 102, and the same temperature measurement effect may be achieved.
As for the sequencing reaction chamber, the present invention further provides an example, as shown in fig. 13, the sequencing reaction chamber includes a first substrate 10 and a second substrate 20, in this example, the first substrate 10 is a first glass substrate, the second substrate 20 includes a PDMS interlayer 201 and a second glass substrate 202, the PDMS interlayer 201 is located between the first glass substrate and the second glass substrate 202, a strip-shaped through hole 2010 is disposed on the PDMS interlayer 201, and the reaction channel is enclosed by the first glass substrate, the strip-shaped through hole 2010, and the second glass substrate 202; the heated slide 30 is disposed on the upper surface of the first substrate 10, and the second glass substrate 202 is provided with a reagent inlet 101 and a reagent outlet 102, the reagent inlet 101 and the reagent outlet 102 being communicated with the reaction channel. Further, as shown in fig. 13, the length of the PDMS interlayer 201 is equal to that of the first glass substrate, and the PDMS interlayer 201 is slightly longer than the second glass substrate 202, when the first glass substrate, the PDMS interlayer 201, and the second glass substrate 202 are bonded, one end of the lower surface of the PDMS interlayer 201 is not covered by the second glass substrate 202, the bump 40 is disposed on the lower surface of the PDMS interlayer 201 that is not covered by the second glass substrate 202, as shown in fig. 14, which is a schematic three-dimensional structure diagram of the PDMS interlayer 201 and the second glass substrate 202, in this example, the bump 40 spans the through hole 2010 in the shape of a bar on the extension line of the PDMS interlayer 201, and when the second glass substrate 202 is bonded on the PDMS interlayer 201, the bump 40 leans against the side wall 1020 of the second glass substrate 202, and the structure of the bump 40 is designed such that the position of the temperature measuring hole 401 on the bump 40 is close to the reaction channel, the temperature in the temperature measuring hole 401 is closer to the temperature of a reaction reagent of the gene sequencing reaction, and the accuracy of the temperature measurement of the gene sequencing reaction is improved.
In this example, the protrusion 40 and the PDMS interlayer 201 are integrally formed, and since the PDMS interlayer 201 has a soft texture, after the temperature measuring probe extends into the temperature measuring hole 401, the temperature measuring probe can be tightly attached to the inner wall of the temperature measuring hole 401, and the temperature on the PDMS interlayer 201 can be well transferred to the temperature measuring probe through the action of the temperature measuring hole 401, so as to measure the temperature of the reaction reagent in the gene sequencing reaction. In addition, in this example, the heating slide 30 for heating and the temperature measuring hole 401 for measuring temperature are respectively arranged on the upper surface of the first substrate 10 of the sequencing reaction chamber and the PDMS interlayer 201, and the position of the temperature measuring hole 401 is far away from the heating slide 30, so as to avoid the influence on temperature measurement when the heating slide 30 is heated; and because the temperature measuring hole 401 exists, the temperature measuring probe or the temperature sensor is arranged in the temperature measuring hole 401, so that the influence of air convection on the surface of the sequencing reaction chamber on temperature measurement can be well avoided, and the measured temperature is closer to the temperature of a reaction reagent of the gene sequencing reaction. The accuracy of the gene sequencing reaction temperature measurement is improved.
In addition, in the sequencing reaction chamber of the present invention, when performing a gene sequencing reaction, a fluorescent signal at the time of the gene sequencing reaction needs to be captured by a mapping device. When the sequencing reaction chamber has a two-piece structure, as shown in fig. 6, the image capture device collects the fluorescence signal, and the fluorescence signal can be captured by the image capture device only by passing through the second substrate 20; when the second substrate 20 is made of PDMS, the PDMS may autofluorescence, and interfere with the collection of the fluorescence signal of the image capturing device. In the present example, the sequencing reaction chamber has a three-layer structure, wherein the PDMS interlayer 201 is disposed between the first glass substrate and the second glass substrate 202, and since the PDMS interlayer 201 is provided with the strip-shaped through hole 2010, when the image capture device collects the fluorescence signal, the autofluorescence on the PDMS interlayer 201 is not collected, and therefore the autofluorescence on the PDMS interlayer 201 does not interfere with the collection of the fluorescence signal of the image capture device.
For the sequencing reaction chamber, on the basis of the example shown in fig. 8, the present invention further provides an example, as shown in fig. 15, in this example, the sequencing reaction chamber is a three-layer structure, and includes a first glass substrate, a PDMS interlayer 201, and a second glass substrate 202, the PDMS interlayer 201 is disposed between the first glass substrate and the second glass substrate 202, wherein the structures of the first glass substrate and the second glass substrate 202 are identical to the structure in the example shown in fig. 8, and detailed description is omitted in this example. As shown in fig. 15 and 16, for the PDMS interlayer 201, a strip-shaped through hole 2010 is formed in the PDMS interlayer 201, and after the first glass substrate, the PDMS interlayer 201, and the second glass substrate 202 are attached to each other, the first glass substrate, the second glass substrate 202, and the strip-shaped through hole 2010 form a reaction channel; in this example, reagent buffer areas 2011 are disposed at two ends of the strip-shaped through hole 2010, the reagent buffer areas 2011 are through holes disposed on the PDMS interlayer 201, the reagent inlet 101 is communicated with one of the reagent buffer areas 2011, the reagent outlet 102 is communicated with the other reagent buffer area 2011, a reaction reagent introduced through the reagent inlet 101 enters the reaction channel, before the reaction reagent enters the reaction channel, the reaction reagent firstly enters the reagent buffer area 2011 located at one end of the reaction channel, after the reagent buffer area 2011 is filled, the reaction reagent enters the reaction channel again, a gene sequencing reaction occurs with a sample to be detected in the reaction channel at the same time, then the reaction reagent flows out and enters the reagent buffer area 2011 at the other end of the reaction channel, and the reaction reagent is collected in the reagent buffer area 2011 and then discharged from a pipeline from which the reaction reagent is discharged. Because of the existence of the reagent buffer area 2011, the reagent buffer area 2011 can store a certain amount of reaction reagent, and before the reaction reagent is introduced into the reaction channel, the reaction reagent can flow into the reagent buffer area 2011, the reagent buffer area 2011 is beneficial to uniformly discharging air in each reaction channel, when the reaction reagent enters the reaction channel, the internal pressure is one, and the reaction reagent can uniformly pass through each reaction channel, so that the uniformity of the flow speed and the uniformity of the diffusivity of each reaction channel are ensured. Similarly, in this example, as shown in fig. 15 and 16, the PDMS interlayer 201 has a protrusion 40 integrally formed with the PDMS interlayer 201, the protrusion 40 is provided with a temperature measuring hole 401, and a temperature measuring probe of a temperature measuring device, which may be a temperature sensor or a thermocouple, extends into the temperature measuring hole 401. Due to the existence of the temperature measuring hole 401, the temperature measuring probe or the temperature sensor is arranged in the temperature measuring hole 401, so that the influence of air convection on the surface of the sequencing reaction chamber on temperature measurement can be well avoided, the measured temperature is closer to the temperature of a reaction reagent of gene sequencing reaction, and the accuracy of measuring the temperature of the gene sequencing reaction is improved.
The invention also discloses a sequencing reaction clamp comprising the sequencing reaction chamber, which comprises a sequencing reaction chamber 200, an upper chamber fixing clamp 300 and a lower chamber fixing clamp 400, wherein the sequencing reaction chamber 200 is arranged between the upper chamber fixing clamp 300 and the lower chamber fixing clamp 400; as shown in fig. 17, for the sequencing reaction fixture, the present invention provides an embodiment, a third groove 4001 for accommodating the sequencing reaction chamber 200 is disposed on the chamber lower fixing fixture 400, and the sequencing reaction chamber 200 is mounted in the third groove 4001 of the chamber lower fixing fixture 400 to fix the sequencing reaction chamber 200, in this embodiment, the depth of the third groove 4001 is slightly greater than the thickness of the sequencing reaction chamber 200; meanwhile, corresponding screw holes are formed in the cell upper fixing clamp 300 and the cell lower fixing clamp 400, and the cell upper fixing clamp 300 and the cell lower fixing clamp 400 are fastened through locking screws.
The invention also provides another example of the fixing clamp on the small chamber and the fixing clamp under the small chamber, wherein a fourth groove is arranged on the fixing clamp on the small chamber, correspondingly, a fifth groove is arranged on the fixing clamp under the small chamber, the shape and the size of the fourth groove are the same as those of the fifth groove, and the shape and the size of the fourth groove are the same as those of the sequencing reaction small chamber.
In the above example of the sequencing reaction clamp, it should be further explained that, for the fixing structure of the upper fixing clamp and the lower fixing clamp of the chamber, there are various implementable manners, for example, a hinge is installed on one side of the upper fixing clamp and the lower fixing clamp of the chamber, the upper fixing clamp and the lower fixing clamp of the chamber are rotatably connected through the hinge, meanwhile, a matched buckle is installed on the other side of the upper fixing clamp and the lower fixing clamp of the chamber, and the upper fixing clamp of the chamber is fixed on the lower fixing clamp of the chamber through the function of the buckle; when the sequencing reaction clamp needs to be opened, the buckle is unfastened, and after the fixing clamp on the small chamber is rotated, the sequencing reaction clamp can be opened, so that the sequencing reaction small chamber is operated.
On the basis of the example shown in FIG. 17, the present invention further provides an example of the sequencing reaction fixture, as shown in FIGS. 18 and 19, the difference between the example shown in FIG. 17 and the example in this example is that the lower surface of the chamber upper fixing fixture 300 is provided with a fourth groove 3001, the upper surface of the chamber lower fixing fixture 400 is correspondingly provided with a fifth groove 4002, and the sum of the depths of the fourth groove 3001 and the fifth groove 4002 is slightly greater than the thickness of the sequencing reaction chamber 200; a sixth groove 3002 is provided on the bottom surface of the fourth groove 3001, the width of the sixth groove 3002 is equal to the width of the fourth groove 3001, but the length of the sixth groove 3002 is shorter than the length of the fourth groove 3001; when the upper chamber holder 300 is fixed to the lower chamber holder 400, the sequencing reaction chamber 200 is positioned in the fourth recess 3001 of the upper chamber holder 300 and the fifth recess 4002 of the lower chamber holder 400, and the heating slide is positioned in the sixth recess 3002. Furthermore, a first threaded hole 3003 corresponding to the reagent inlet 101 is formed in the small chamber upper clamp 300, a second threaded hole 3004 corresponding to the reagent outlet 102 is formed in the small chamber upper clamp 300, and the first threaded hole 3003 and the second threaded hole 3004 are designed in such a structure that after a screw with a through hole is locked in the first threaded hole 3003, the top end of the through hole in the screw is communicated with a pipeline for introducing a reagent, and the bottom end of the through hole in the screw is pressed on the sequencing reaction small chamber and is communicated with the reagent inlet; similarly, after a screw with a through hole is locked into the second threaded hole 3004, the top end of the through hole on the screw is communicated with the pipeline for discharging the reagent, and the bottom end of the screw is pressed on the sequencing reaction chamber and is communicated with the reagent outlet, so that the introduction and outflow of the reaction reagent are realized; since the sum of the depths of the fourth recess 3001 and the fifth recess 4002 is slightly greater than the thickness of the sequencing reaction chamber 200, the sequencing reaction chamber 200 is in a movable state in the vertical direction between the chamber upper clamp 300 and the chamber lower clamp 400, and in this example, the screws locked into the first threaded hole 3003 and the second threaded hole 3004 are generally made of a material with a relatively soft texture, and when the screws are locked, the lower ends of the screws are pressed against the sequencing reaction chamber 200, so that good sealing is formed between the screws at the reagent inlet 101 and the reagent inlet 101 of the sequencing reaction chamber 200, and between the screws at the reagent outlet 102 and the reagent outlet 102, thereby avoiding the problem of leakage.
In addition, as shown in FIGS. 18 and 19, windows 3005 for photographing the sequencing reaction are provided on the upper chamber clamp 300 and the lower chamber clamp 400, and the windows 3005 provided on the upper chamber clamp 300 are through holes provided on the bottom surface of the fourth recess 3001, and the width of the windows 3005 is smaller than the width of the sixth recess 3002, but the length of the windows 3005 is greater than the length of the sixth recess. When a sequencing reaction is performed, the sequencing reaction in the reaction channel of the sequencing reaction chamber 200 is photographed in front of the window 3005 by the action of the image-taking device to obtain a fluorescent signal. Further, be provided with on the lateral wall of window 3005 and be convenient for conductive probe to pass the back contact the sunken groove 3006 of heating slide, through sunken groove 3006's design, make conductive probe can contact the heating slide on the one hand, on the other hand avoids conductive probe to cause the influence to the acquirement of fluorescence signal.
In the example shown in fig. 17 and the examples shown in fig. 18 and 19, it should be noted that the position of the first threaded hole 3003 corresponds to the position of the reagent inlet 101, and the position of the first threaded hole 3003 can be adjusted according to the position of the reagent inlet 101; the position of the second threaded hole 3004 corresponds to the position of the reagent outlet 102, and the position of the second threaded hole 3004 can be adjusted according to the position of the reagent outlet 102.
The invention also discloses a sequencing reaction device comprising the sequencing reaction clamp, wherein the sequencing reaction device comprises a cell fixing device and the optional sequencing reaction clamp, and the optional sequencing reaction cell is arranged in the sequencing reaction clamp, so that the structure of the sequencing reaction cell and the structure of the sequencing reaction clamp are not explained in detail in the example. As shown in FIG. 20, the chamber fixing means 500 comprises chamber mounting seat 5001 and chamber compressing member 5002, the chamber compressing member 5002 is fixed on chamber mounting seat 5001, chamber compressing member 5002 is used for compressing the sequencing reaction fixture 600 on chamber mounting seat 5001, and the sequencing reaction fixture 600 is fixed by the action of chamber compressing member 5002. The cell compresses tightly and is provided with ball plunger 5003 that is used for supporting on sequencing reaction anchor clamps 600 on the spare 5002, after sequencing reaction anchor clamps 600 pack into on cell mount pad 5001, ball plunger 5003 plays fixed effect to sequencing reaction anchor clamps 600, in addition, on the upper surface of sequencing reaction anchor clamps 600, is provided with the constant head tank corresponding to ball plunger 5003's position, after sequencing reaction anchor clamps 600 fix on cell mount pad 5001, ball plunger 5003 supports on the constant head tank, realizes sequencing reaction anchor clamps 600's location. In this example, through the effect of cell compact member 5002, bulb plunger 5003 and constant head tank, realized the dismantlement and fixed of sequencing reaction anchor clamps 600, made things convenient for sequencing reaction anchor clamps 600's change, improved gene sequencing reaction's efficiency.
For the sequencing reaction equipment, as shown in fig. 21, the present invention further provides an example, the sequencing reaction equipment further includes a temperature measuring device, the temperature measuring device includes a temperature measuring probe 700 and a data processing unit, the temperature measuring probe 700 is configured to be inserted into a temperature measuring hole of the sequencing reaction chamber, the temperature measuring probe 700 is connected to the data processing unit, the temperature measuring probe 700 is configured to detect a temperature of a reaction reagent in the reaction channel, the detected temperature is transmitted to the data processing unit, the data processing unit controls the heating slide according to the temperature detected by the temperature measuring probe 700, for example, the temperature of the gene sequencing reaction is set to be T1, the temperature measured by the temperature measuring probe 700 is T2, when the temperature T2 is less than T1, it is indicated that the temperature of the reaction reagent in the gene sequencing reaction channel is lower than the temperature required by the gene sequencing reaction, after the data processing unit receives a temperature measuring signal from the temperature measuring probe 700, the heating temperature of the slide is controlled to be increased. Further, in this example, a small chamber end cover (not shown in the figure) is rotatably mounted on the small chamber mounting seat, a second through hole is formed in the small chamber end cover corresponding to the window 3005 on the small chamber upper fixture 300 and the small chamber lower fixture 400, and the small chamber end cover is prevented from affecting the image acquisition of the image acquisition device through the arrangement of the second through hole; the cell end cover is provided with a third through hole corresponding to the concave groove 3006 on the cell upper clamp 300 and the cell lower fixing clamp 400, and the conductive probe penetrates through the third through hole to contact with the heating slide, and after the conductive probe is electrified, the heating slide generates heat, so that the sequencing reaction cell 200 is heated.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (14)
1. A sequencing reaction chamber, comprising: the device comprises a first substrate and a second substrate, wherein the lower surface of the first substrate is attached to the upper surface of the second substrate, and at least one hollow reaction channel is formed between the first substrate and the second substrate;
the first substrate or the second substrate is provided with a reagent inlet communicated with the reaction channel, and the first substrate or the second substrate is also provided with a reagent outlet communicated with the reaction channel;
a heating glass slide for heating the reagent in the reaction channel is attached to the upper surface of the first substrate; and a bulge is arranged on the second substrate corresponding to the reaction channel, a temperature measuring hole is arranged on the bulge, and the temperature measuring hole is used for accommodating a temperature measuring probe for detecting the temperature of the reaction reagent in the reaction channel.
2. The sequencing reaction chamber of claim 1, wherein: the two ends of the reaction channel are respectively provided with a reagent buffer zone for containing reaction reagents, the reagent inlet is communicated with one reagent buffer zone, the reagent outlet is communicated with the other reagent buffer zone, and the reagent buffer zone is a second groove which is arranged on the lower surface of the first substrate and is positioned at the two ends of the first groove or the reagent buffer zone is a second groove which is arranged on the upper surface of the second substrate and is positioned at the two ends of the first groove.
3. The sequencing reaction chamber of claim 1, wherein: the first substrate comprises a first glass substrate, the second substrate comprises a PDMS interlayer and a second glass substrate, and the PDMS interlayer is positioned between the first glass substrate and the second glass substrate; the PDMS interlayer is provided with a strip-shaped through hole, and the reaction channel is formed by enclosing a first glass substrate, the strip-shaped through hole and a second glass substrate; the PDMS interlayer is slightly longer than the second glass substrate, after the first glass substrate, the PDMS interlayer and the second glass substrate are jointed, the PDMS interlayer has a lower surface which is not shielded by the second glass substrate, and the protrusion is arranged on the lower surface of the PDMS interlayer which is not shielded.
4. The sequencing reaction chamber of claim 3, wherein: two ends of the reaction channel are respectively provided with a reagent buffer zone for containing a reaction reagent, and the reagent buffer zones are through holes arranged on the PDMS interlayer; the reagent inlet is communicated with one reagent buffer zone, and the reagent outlet is communicated with the other reagent buffer zone.
5. The sequencing reaction chamber of any one of claims 1 to 4, wherein: the extension direction of the temperature measuring hole on the bulge is vertical to the second substrate; or
The extending direction of the temperature measuring hole on the protrusion is parallel to the second substrate.
6. A sequencing reaction fixture comprising the sequencing reaction chamber of claim 1, wherein: the sequencing reaction clamp further comprises a small chamber upper fixing clamp and a small chamber lower fixing clamp, and the sequencing reaction small chamber is arranged between the small chamber upper fixing clamp and the small chamber lower fixing clamp.
7. The sequencing reaction fixture of claim 6, wherein: a third groove for accommodating the sequencing reaction chamber is arranged on the chamber upper fixing clamp or the chamber lower fixing clamp, or
Be provided with the fourth recess on the mounting fixture on the cell, be provided with the fifth recess under the cell on the mounting fixture, the sequencing reaction cell sets up in fourth recess and fifth recess.
8. The sequencing reaction fixture of claim 7, wherein: a first threaded hole corresponding to the reagent inlet is formed in the small chamber upper fixing clamp or the small chamber lower fixing clamp, a screw with a through hole is locked in the first threaded hole, the top end of the through hole in the screw is communicated with a pipeline for introducing a reagent, and the bottom end of the through hole in the screw is communicated with the reagent inlet;
and a second threaded hole corresponding to the reagent outlet is formed in the upper fixing clamp of the small chamber or the lower fixing clamp of the small chamber, a screw with a through hole is locked in the second threaded hole, the top end of the through hole in the screw is communicated with a pipeline for discharging the reagent, and the bottom end of the through hole in the screw is communicated with the reagent outlet.
9. The sequencing reaction fixture of claim 7, wherein: when a third groove for accommodating the sequencing reaction chamber is formed in the upper small chamber fixing clamp or the lower small chamber fixing clamp, a sixth groove for accommodating a heating slide of the sequencing reaction chamber is also formed in the bottom surface of the third groove; or
And when the fixing clamp is provided with a fifth groove, a sixth groove for accommodating the heating slide of the sequencing reaction chamber is also arranged on the bottom surface of the fourth groove or the fifth groove.
10. The sequencing reaction fixture of claim 9, wherein: the device comprises a fixed clamp and a glass slide, and is characterized in that windows for photographing sequencing reactions are arranged on the fixed clamp on the chamber and the fixed clamp under the chamber, and a concave groove for bringing convenience for a conductive probe to contact with the heating glass slide is arranged on the side wall of each window.
11. A sequencing reaction apparatus comprising the sequencing reaction jig of claim 6, wherein: also comprises a small chamber fixing device;
the small chamber fixing device comprises a small chamber mounting seat and a small chamber compressing piece, the small chamber compressing piece is fixed on the small chamber mounting seat, the sequencing reaction small chamber is mounted on the small chamber mounting seat, and the small chamber compressing piece is used for compressing the sequencing reaction small chamber on the small chamber mounting seat.
12. The sequencing reaction device of claim 11, wherein: and the small chamber pressing piece is provided with a ball plunger used for being pressed against the sequencing reaction small chamber.
13. The sequencing reaction device of claim 11, wherein: the sequencing reaction chamber is characterized by further comprising a temperature measuring device, wherein the temperature measuring device comprises a temperature measuring probe, and the temperature measuring probe is inserted into a temperature measuring hole of the sequencing reaction chamber.
14. The sequencing reaction device of claim 11, wherein: the cell mounting seat is rotatably provided with a cell end cover, a second through hole is formed in the cell end cover corresponding to a window on the cell fixing clamp and a window on the cell fixing clamp, and a third through hole is formed in a concave groove in the cell fixing clamp and the cell fixing clamp.
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| CN102517196A (en) * | 2011-12-31 | 2012-06-27 | 盛司潼 | Multi-channle sequencing reaction chamber and gene sequencing instrument |
| CN203811278U (en) * | 2014-03-10 | 2014-09-03 | 赵超 | Non-contact thermoscope used for measuring temperature of liquid in container |
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