CN113210024B - Continuous liquid inlet device based on PCR - Google Patents
Continuous liquid inlet device based on PCR Download PDFInfo
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- CN113210024B CN113210024B CN202110621774.6A CN202110621774A CN113210024B CN 113210024 B CN113210024 B CN 113210024B CN 202110621774 A CN202110621774 A CN 202110621774A CN 113210024 B CN113210024 B CN 113210024B
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- 238000005336 cracking Methods 0.000 claims description 96
- 239000006166 lysate Substances 0.000 claims description 43
- 238000000746 purification Methods 0.000 claims description 31
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L7/00—Heating or cooling apparatus; Heat insulating devices
- B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
Abstract
The invention relates to a continuous liquid inlet device based on PCR (polymerase chain reaction), which comprises a sample adding layer and a motor pump; the sample adding layer is connected with a motor pump, and the motor pump is used for adding the reagent into the pipeline in the pipeline layer through the sample adding layer; the sample adding layer comprises a reagent tube and a piston, one end of the reagent tube is provided with a sealing structure, the other end of the reagent tube is provided with the piston, and a piston rod of the piston is fixedly connected with a connecting shaft of the motor pump; the motor pump comprises a motor set and a connecting shaft, and an output shaft of the motor set is fixedly connected with the connecting shaft. Through the drive effect of motor pump, the connecting axle that control motor group connects drives the intraductal piston of reagent along pipe wall reciprocating motion, realizes pushing reagent and suction reagent, and the motor group is including the multiunit motor, and corresponding reagent pipe is also including a plurality of, and in the use, the liquid in every reagent pipe is different, and its chronogenesis that pushes or the suction is also different, realizes the continuity of reaction, also can realize adding the continuity of different reagents.
Description
Technical Field
The invention relates to the field of biological detection, in particular to a continuous liquid inlet device based on PCR.
Background
In scientific experiments of biology, chemistry, materials and the like, operations on fluid are often required, such as preparation of sample DNA, liquid chromatography, PCR reaction, electrophoresis detection and the like are all performed in a liquid phase environment. If the steps of sample preparation, biochemical reaction, result detection, etc. are integrated on a biochip, the amount of fluid used for the experiment is reduced from milliliter, micro-liter to nanoliter or picoliter, and a powerful microfluidic device is necessary. Therefore, with the development of biochip technology, microfluidics technology has gained more and more attention as a key supporting technology of biochip.
The purification and recovery of nucleic acid is a routine operation of nucleic acid detection test, and is also a key step in the gene analysis process. In the gene analysis process, specific nucleic acid fragments are often required to be separated and extracted from a mixed sample for subsequent PCR amplification, so that the purification and recovery effects of nucleic acid directly influence the progress and the result of the whole gene analysis process.
In the related art, a pipeline structure is usually arranged to separate ions in a reaction solution under the action of an electric field by adding an electrode, so as to complete subsequent experiments. The mode of combined action of the pipeline structure and the electrodes is adopted, so that the difficulty is increased for manufacturing the pipeline layer due to the complex structure, the arrangement of the electrodes is more complicated, the consideration factors are more, and the realization is difficult. Meanwhile, the internal pipelines of the chip are not integrated, reaction liquid is difficult to mix and test according to expectation, and usually, the extraction, purification and amplification of nucleic acid are completed by using different instruments, so that the operation is complicated.
Disclosure of Invention
Therefore, the continuous liquid feeding device based on PCR provided by the invention can realize continuous liquid feeding in the nucleic acid detection process and realize uninterrupted extraction, purification and amplification.
In order to achieve the above object, the present invention provides a continuous liquid feeding device based on PCR, comprising: a sample adding layer and a motor pump;
the sample adding layer is connected with the motor pump, the motor pump is used for adding a reagent into a pipeline in the pipeline layer through the sample adding layer, a sample adding port is formed in the sample adding layer, and a liquid level height meter is arranged in the sample adding port and used for detecting the height of a sample added from the sample adding port;
the sample adding layer comprises a reagent tube and a piston, one end of the reagent tube is provided with a sealing structure, the other end of the reagent tube is provided with the piston, and a piston rod of the piston is fixedly connected with a connecting shaft of the motor pump;
the motor pump comprises a motor set, a connecting shaft and a screw rod, an output shaft of the motor set is fixedly connected with the connecting shaft, the connecting shaft is used for pushing a reagent in the reagent tube into a pipeline or sucking the reagent out of the pipeline through a piston arranged on the screw rod, and the motor set comprises a second motor, a third motor, a fourth motor, a fifth motor and a sixth motor;
the central control unit is respectively connected with a second motor, a third motor and a liquid level height meter in the motor set and used for adjusting the liquid inlet speed v20 and the liquid inlet time t0 of the third motor according to the height of the liquid level height meter, wherein a first liquid level height h10, a second standard level height h20 and a third liquid level height h30 are arranged in the central control unit, and the first liquid level height h10 is less than the second standard level height h20 and less than the third liquid level height h 30;
if the height of the sample detected by the liquid level meter is less than or equal to the first liquid level height h10, the liquid inlet speed when the lysate is injected into the pipeline layer is v20 and the liquid inlet time is t 0;
if the second standard level height h20 is not less than the height of the sample detected by the liquid level meter and is greater than the first liquid level height h10, the liquid inlet speed when the lysate is injected into the pipeline layer is 1.1 xv 20, and the liquid inlet time is t 0;
if the third standard level height h30 is not less than the height of the sample detected by the liquid level meter and is greater than the second liquid level height h20, the liquid inlet speed when the lysate is injected into the pipeline layer is v20 and the liquid inlet time is 1.1 × t 0;
if the height of the sample detected by the liquid level meter is larger than the third standard height h30, the liquid inlet speed when injecting the lysate into the pipeline layer is 1.1 Xv 20 and the liquid inlet time length is 1.1 Xt 0.
Furthermore, a counter and a preset standard number n0 are arranged in the central control unit, the counter is used for counting the starting number of the second motor, the central control unit judges the cracking degree of the sample according to the starting number of the second motor, and the liquid inlet speed V40 of the fourth motor and the liquid inlet speed V50 of the fifth motor are selected according to the cracking degree.
Further, the judging the degree of sample lysis according to the number of times of starting the second motor comprises:
a first cracking degree a1, a second cracking degree a2 and a third cracking degree a3 are arranged in the central control unit, wherein the first cracking degree a1 indicates that the cracking degree of the sample is more than or equal to a preset cracking degree standard s0, the second cracking degree a2 indicates that the cracking degree of the sample is more than the cracking degree standard s0 and less than or equal to 1.1 × s0, and the third cracking degree a3 indicates that the = cracking degree of the sample is more than 1.1 × s 0;
if the starting times of the second motor are more, the sample and the lysate are fully mixed, the protein molecules in the sample are small, and the cracking degree of the sample is a third cracking degree;
if the starting frequency of the second motor is less, the sample and the lysate are mixed but not sufficiently mixed, the protein molecules in the sample are large, and the cracking degree of the sample is the first cracking degree;
if the starting frequency of the second motor is moderate, the protein molecules in the sample have the size, and the cracking degree of the sample is the second cracking degree.
Further, the liquid inlet speed of adjusting the fourth motor and the liquid inlet speed of the fifth motor according to the cracking degree comprises:
in the purification process, if the cracking degree of the substances in the purification bin belongs to the first cracking degree, the liquid inlet speed of the fourth motor and the liquid inlet speed of the fifth motor are increased;
if the cracking degree of the substances in the purification bin belongs to the second cracking degree, the liquid inlet speeds of the fourth motor and the fifth motor are kept;
if the cracking degree of the substances in the purification bin belongs to the third cracking degree, the liquid inlet speed of the fourth motor and the liquid inlet speed of the fifth motor are reduced.
Furthermore, the motor set also comprises a speed reduction device set, and the speed reduction device set is arranged in the motor set and used for controlling the rotating speed of the motor set.
Further, still include the circuit control board, the circuit control board sets up the upside of motor train, the circuit control board with the motor train electricity is connected, the circuit control board is used for controlling the operating condition of motor train, well accuse unit sets up on the circuit control board.
Furthermore, a zero piece and a photoelectric sensor are arranged at the upper end of the transfer shaft, the photoelectric sensor is electrically connected with the zero piece, the photoelectric sensor is used for receiving a signal of reaction completion, and then the rotating position of the connecting shaft is reset to zero through the zero piece.
Furthermore, the second motor, the third motor, the fourth motor, the fifth motor and the sixth motor are arranged in parallel, the end parts of connecting shafts connected with the second motor, the third motor, the fourth motor, the fifth motor and the sixth motor are provided with concave grooves, the concave grooves are used for connecting the piston rod,
a sealing ring is arranged at the end part of a piston rod of the piston and used for sealing; the piston reciprocates along the tube wall to push the reagent in the piston to flow out or be drawn back to the reagent outlet.
Furthermore, the piston rod is also provided with a nut which is in threaded connection with the nut, a guide sleeve is sleeved outside the nut, and a corresponding shaft shoulder is arranged on the inner side of the pipe wall and used for positioning and fixing the guide sleeve; the outside of uide bushing still is provided with the sheath for protect piston rod, nut and uide bushing the below on application of sample layer is provided with the second buckle, is used for preventing application of sample layer slides, still is provided with the snap ring in the both ends outside of uide bushing, is used for blocking corresponding uide bushing.
Furthermore, the number of the reagent tubes is 5, the second reagent tube connected with the second motor is empty, the third reagent tube connected with the third motor is used for containing lysis solution, the fourth reagent tube connected with the fourth motor is used for containing first cleaning solution, the fifth reagent tube connected with the fifth motor is used for containing second cleaning solution, and the sixth reagent tube connected with the sixth motor is used for containing eluent.
Compared with the prior art, the liquid level height of the sample is detected, and the liquid injection speed and the liquid injection time of the lysate are adjusted according to the liquid level height, so that the content of the sample is matched with the content of the lysate, the sample is fully cracked, the continuous liquid feeding is realized in the experimental process, the matching of the injection amount of the sample and the lysate is further improved, the sample is fully cracked, and the precision of subsequent experiments is improved.
Especially, the starting frequency of the second motor is determined through a counter in the central control unit, the cracking degree of the sample is judged according to the starting frequency of the second motor, in the actual reaction process, if the cracking degree of the sample is high, the separation of nucleic acid and protein is easier during cleaning, the liquid inlet speeds of the fourth motor and the fifth motor can be properly increased to reduce the cleaning time, and the cracking degree of the sample is low, so that the separation of nucleic acid and protein is not easy during cleaning, and at the moment, the liquid inlet speeds of the fourth motor and the fifth motor need to be greatly increased, so that the cleaning of the nucleic acid in the sample is completed as soon as possible, and the experimental efficiency is improved.
Especially, the degree of reaction of the sample in the lysis solution is defined by the size of protein molecules in the sample, the central control unit determines the cracking degree for the starting times of the motor, the cracking degree of the sample is defined more accurately, and the rotating speeds of the fourth motor and the fifth motor are convenient to adjust according to the cracking degree, so that the experimental efficiency is improved.
Especially, through the schizolysis degree that sets up three different degree in well accuse unit, and then adjust the inlet liquid speed of fourth motor and fifth motor according to the schizolysis degree of difference for at the in-process that carries out the purification, purification speed is faster, realizes the high-efficient utilization of motor and lysate, avoids the energy consumption of motor, improves experimental efficiency. Furthermore, by adjusting the rotating speed of the motor, when the molecular group after protein cracking is large, the liquid inlet speed is increased, the molecular group is flushed away by the pressure of the cleaning liquid of the liquid inlet, better cleaning is realized, nucleic acid substances in the molecular group are prevented from being unclean, the cleaning efficiency is further improved, and the purity of the nucleic acid is improved.
Especially, through the drive effect of motor pump, the connecting axle that control motor assembly connects drives the intraductal piston of reagent along pipe wall reciprocating motion, realizes pushing reagent and suction reagent, and the motor assembly is including the multiunit motor, and corresponding reagent pipe also is including a plurality of, and in the use, the intraductal liquid of every reagent is different, and its chronogenesis of pushing or suction is also different, realizes the continuity of reaction, also can realize adding the continuity of different reagents.
Especially, the setting of decelerator group makes the injection velocity of reagent in the reagent pipe or the velocity of flow in the pipeline layer all can have corresponding slowing down or reducing, through the control to reagent injection velocity, realizes the effective control to the experimentation, is convenient for master experiment rhythm.
Particularly, the photoelectric sensor and the zero position sheet are matched for use, can be used as a counting function, and can record the number of cycles of movement and the number of turns of a motor pump connected with each motor, so that the accuracy of liquid adding quantity can be simply corrected.
In particular, in the experiment process, the working time of each motor and whether the motor rotates forwards or backwards depends on other motors, that is, the working state of each motor is closely related to the working state of other motors or the current experiment progress, in order to further ensure that the working state of each motor is normal and the working time sequence of each motor is correct in the whole experiment process, the whole planning and planning of the motor needs to be carried out, and the control of each motor is simple and convenient through the arrangement of the circuit control board.
Particularly, according to the continuous liquid inlet device based on the PCR provided by the embodiment of the invention, liquid inlet of the corresponding reagent tube is controlled by the motor pump or liquid inlet is matched according to the experiment purpose, so that continuous liquid inlet is realized, the whole process is uninterrupted, the experiment efficiency is improved, and the experiment time is shortened.
Drawings
FIG. 1 is a schematic structural diagram of a motor pump in a PCR-based continuous liquid feeding device according to an embodiment of the present invention;
FIG. 2 is a schematic perspective view of a motor pump in a PCR-based continuous liquid feeding apparatus according to an embodiment of the present invention;
FIG. 3 is a schematic side view of a motor pump in the PCR-based continuous liquid feeding apparatus according to the embodiment of the present invention;
FIG. 4 is a schematic structural diagram of a sample addition layer in a PCR-based continuous liquid feeding device according to an embodiment of the present invention.
Detailed Description
In order that the objects and advantages of the invention will be more clearly understood, the invention is further described below with reference to examples; it should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Preferred embodiments of the present invention are described below with reference to the accompanying drawings. It should be understood by those skilled in the art that these embodiments are only for explaining the technical principle of the present invention, and do not limit the scope of the present invention.
It should be noted that in the description of the present invention, the terms of direction or positional relationship indicated by the terms "upper", "lower", "left", "right", "inner", "outer", etc. are based on the directions or positional relationships shown in the drawings, which are only for convenience of description, and do not indicate or imply that the device or element must have a specific orientation, be constructed in a specific orientation, and be operated, and thus, should not be construed as limiting the present invention.
Furthermore, it should be noted that, in the description of the present invention, unless otherwise explicitly specified or limited, the terms "mounted," "connected," and "connected" are to be construed broadly, and may be, for example, fixedly connected, detachably connected, or integrally connected; can be mechanically or electrically connected; they may be connected directly or indirectly through intervening media, or they may be interconnected between two elements. The specific meanings of the above terms in the present invention can be understood by those skilled in the art according to specific situations.
Referring to fig. 1-4, a continuous liquid feeding device based on PCR according to an embodiment of the present invention includes a sample adding layer and a motor pump 1, wherein the sample adding layer is connected to the motor pump, the motor pump is configured to add a reagent into a tube in a tube layer through the sample adding layer, the sample adding layer is provided with a sample adding port, and a liquid level height gauge is disposed in the sample adding port for measuring a height of a sample added from the sample adding port;
the sample adding layer comprises a reagent tube and a piston, one end of the reagent tube is provided with a sealing structure, the other end of the reagent tube is provided with the piston, and a piston rod of the piston is fixedly connected with a connecting shaft of the motor pump; the motor pump 1 comprises a motor set 10 and a connecting shaft 11, an output shaft of the motor set 10 is fixedly connected with the connecting shaft 11, the motor set 10 drives the connecting shaft 11 to push or suck out the liquid in the pipeline layer when the valve head is downward or upward, the motor set comprises a second motor, a third motor, a fourth motor, a fifth motor and a sixth motor, the connecting shaft 11 is used for connecting a reagent tube with a liquid inlet through a sample adding layer, one end of the reagent tube provided with a sealing structure is connected with the liquid inlet, the sealing structure is punctured by a probe when the reagent tube is used, then the piston pushes the reagent liquid in the reagent tube into the liquid inlet under the action of the motor pump, and then the flow of the reagent in the pipeline layer is realized under the action of the cooperation of the pump valve, so that the extraction, the purification and the amplification are realized.
Specifically, the central control unit is respectively connected with a second motor, a third motor and a liquid level height meter in the motor set and used for adjusting the liquid inlet speed v20 and the liquid inlet time t0 of the third motor according to the height of the liquid level height meter, wherein a first liquid level height h10, a second standard position height h20 and a third liquid level height h30 are arranged in the central control unit, and the first liquid level height h10 is greater than the second standard position height h20 and is less than the third liquid level height h 30;
if the height of the sample detected by the liquid level meter is less than or equal to the first liquid level height h10, the liquid inlet speed when the lysate is injected into the pipeline layer is v20 and the liquid inlet time is t 0;
if the second standard level height h20 is not less than the height of the sample detected by the liquid level meter and is greater than the first liquid level height h10, the liquid inlet speed when the lysate is injected into the pipeline layer is 1.1 xv 20, and the liquid inlet time is t 0;
if the third standard level height h30 is not less than the height of the sample detected by the liquid level meter and is greater than the second liquid level height h20, the liquid inlet speed when the lysate is injected into the pipeline layer is v20 and the liquid inlet time is 1.1 × t 0;
if the height of the sample detected by the liquid level meter is larger than the third standard height h30, the liquid inlet speed when injecting the lysate into the pipeline layer is 1.1 Xv 20 and the liquid inlet time length is 1.1 Xt 0.
Specifically, the liquid level height of the sample is detected, and the liquid injection speed and the liquid injection duration of the lysate are adjusted according to the liquid level height, so that the content of the sample is matched with the content of the lysate, the sample is fully cracked, in the experimental process, continuous liquid injection is realized, meanwhile, the matching of the injection amount of the sample and the lysate is further improved, the sample is fully cracked, and the precision of subsequent experiments is improved.
Specifically, a counter and a preset standard number n0 are arranged in the central control unit, the counter is used for counting the starting times of the second motor, the central control unit judges the cracking degree of the sample according to the starting times of the second motor, and the liquid inlet speed V40 of the fourth motor and the liquid inlet speed V50 of the fifth motor are selected according to the cracking degree.
Specifically, the starting frequency of the second motor is determined by a counter in the central control unit, the sample cracking degree is determined according to the starting frequency of the second motor, in the actual reaction process, if the sample cracking degree is high, the nucleic acid and the protein are more easily separated during cleaning, the liquid inlet speed of the fourth motor and the liquid inlet speed of the fifth motor can be properly increased to reduce the cleaning time, and the sample cracking degree is low, so that the nucleic acid and the protein are not easily separated during cleaning, and at the moment, the liquid inlet speed of the fourth motor and the liquid inlet speed of the fifth motor need to be greatly increased, so that the nucleic acid in the sample is cleaned as soon as possible, and the experimental efficiency is improved.
Specifically, the judging the degree of sample lysis according to the number of times of starting the second motor includes:
a first cracking degree a1, a second cracking degree a2 and a third cracking degree a3 are arranged in the central control unit, wherein the first cracking degree a1 represents that the cracking degree of the sample is more than or equal to a preset cracking degree standard s0, the second cracking degree a2 represents that the cracking degree of the sample is more than the cracking degree standard s0 and less than or equal to 1.1 × s0, and the third cracking degree a3 represents that the cracking degree of the sample is more than 1.1 × s 0;
if the starting times of the second motor are more, the sample and the lysate are fully mixed, the protein molecules in the sample are small, and the cracking degree of the sample is a third cracking degree;
if the starting frequency of the second motor is less, the sample and the lysate are mixed but not sufficiently mixed, the protein molecules in the sample are large, and the cracking degree of the sample is the first cracking degree;
if the starting frequency of the second motor is moderate, the protein molecules in the sample have the size, and the cracking degree of the sample is the second cracking degree.
Specifically, the degree of reaction of the sample in the lysis solution is defined by the size of protein molecules in the sample, the central control unit determines the cracking degree for the starting times of the motor, the cracking degree of the sample is defined more accurately, and the rotating speeds of the fourth motor and the fifth motor are conveniently adjusted according to the cracking degree, so that the experimental efficiency is improved.
Specifically, the liquid inlet speed of adjusting the fourth motor and the liquid inlet speed of the fifth motor according to the cracking degree comprises:
in the purification process, if the cracking degree of the substances in the purification bin belongs to the first cracking degree, the liquid inlet speed of the fourth motor and the liquid inlet speed of the fifth motor are increased;
if the cracking degree of the substances in the purification bin belongs to the second cracking degree, the liquid inlet speeds of the fourth motor and the fifth motor are kept;
if the cracking degree of the substances in the purification bin belongs to the third cracking degree, the liquid inlet speed of the fourth motor and the liquid inlet speed of the fifth motor are reduced.
Specifically, according to the embodiment of the invention, three cracking degrees with different degrees are arranged in the central control unit, and then the liquid inlet speeds of the fourth motor and the fifth motor are adjusted according to the different cracking degrees, so that the purification speed is higher in the purification process, the motors and the cracking liquid are efficiently utilized, the energy consumption of the motors is avoided, and the experiment efficiency is improved. Furthermore, by adjusting the rotating speed of the motor, when the molecular group after protein cracking is large, the liquid inlet speed is increased, the molecular group is flushed away by the pressure of the cleaning liquid of the liquid inlet, better cleaning is realized, nucleic acid substances in the molecular group are prevented from being unclean, the cleaning efficiency is further improved, and the purity of the nucleic acid is improved.
According to the continuous liquid inlet device based on PCR provided by the embodiment of the invention, the connecting shaft connected with the motor set is controlled to drive the piston in the reagent tube to reciprocate along the tube wall under the driving action of the motor pump, so that the reagent is pushed in and sucked out, the motor set comprises a plurality of groups of motors, the corresponding reagent tubes also comprise a plurality of reagent tubes, and in the using process, the liquid in each reagent tube is different, the pushing-in or sucking-out time sequence is also different, so that the reaction continuity is realized, and the continuity of adding different reagents can also be realized.
The following description will be given by taking a process of injecting a lysis solution as an example, when a lysis solution is injected into a pipeline layer, a person skilled in the art can understand that the purpose of injecting the lysis solution is to crack a sample, so as to split nucleic acid substances and proteins in the sample, before injecting the lysis solution, the sample is added from a sample adding hole formed in a sample adding layer, at this time, the double valve is closed, the second single valve is closed, the first single valve is opened, the first single valve is arranged between the sample inlet and the lysis solution inlet, a valve head connected with the first single valve does not need to be pressed down, so as to open the first single valve, at this time, the valve heads of the second single valve and the double valve need to move down under the action of the first motor to apply pressure to the pipeline layer, so as to achieve shutoff, and the lysis solution and the sample are mixed in a pipeline in the pipeline layer after being injected.
In order to promote the mixing to be sufficient even, second motor 102 and third motor 103 can cooperate to work this moment, when third motor 103 pushed in, second motor 102 outwards imbibition, or second motor 102 pushes away the liquid, third motor 103 imbibition, and then realized sample and lysate intensive mixing reaction between sample inlet and lysate filling opening, after the two mixes evenly fully, just need push it in the purification storehouse, carry out subsequent purification reaction.
Specifically, how the lysate is pushed into the pipeline is further described below, the output shaft of the third motor 103 is connected to the connecting shaft 11, the connecting shaft 11 is connected to the liquid injection port through the liquid injection device, the liquid injection device includes a reagent tube and a piston, the piston can move back and forth in the reagent tube, the number of revolutions of the motor is converted into the transverse movement distance of the piston in the reagent tube, and the lysate in the reagent tube is injected into the pipeline layer. In practical application, if the lysate needs to be pushed in, the third motor 103 rotates forwards to realize the previous movement of the piston in the reagent tube, the lysate is injected, if the liquid needs to be pumped out, the control point controls the third motor 103 to rotate backwards, at this time, in order to match with the liquid pumping action, the second motor 102 needs to rotate forwards to perform the pushing-in action, and the lysate and the sample are fully mixed through the forward rotation or the reverse rotation of the second motor 102 and the third motor 103.
Specifically, motor pump 1 is connected with the inlet on the pipeline layer for to adding reagent in the pipeline layer, at different reaction stages, the reagent that adds is different, and the inlet that the reagent was injected into is also different, can be the lysate in the reagent pipe that motor pump 1 is connected, can be the washing liquid, can also be the eluant, is different according to the experiment purpose and selects. In practical application, the motor pump 1 and the liquid path valve cooperate to control the flow direction of liquid in the pipeline layer, the process of injecting lysis solution is taken as an example for explanation, when injecting the lysis solution into the pipeline layer, it can be understood by those skilled in the art that the purpose of injecting the lysis solution is to crack the sample, so as to realize the cleavage of nucleic acid substances and proteins in the sample, before injecting the lysate, the sample is added into the sample inlet from the sample injection port, the double valves are closed, the second single valve is closed, the first single valve is opened, the first single valve is arranged between the sample inlet and the lysate inlet, the valve head connected with the first single valve does not need to be pressed down, the opening of the first single valve is realized, and at the moment, the valve heads of the second single valve and the double valve need to move downwards under the action of the first motor to apply pressure to the pipeline layer, and then the cut-off is realized, and the cracking liquid and the sample are mixed in the pipeline layer after being injected.
In order to promote the mixing to be sufficient even, second motor 102 and third motor 103 can cooperate to work this moment, when third motor 103 pushed in, second motor 102 outwards imbibition, or second motor 102 pushes away the liquid, third motor 103 imbibition, and then realized sample and lysate intensive mixing reaction between sample inlet and lysate filling opening, after the two mixes evenly fully, just need push it in the purification storehouse, carry out subsequent purification reaction.
Specifically, how the lysate is pushed into the pipeline is further described below, the output shaft of the third motor 103 is connected to the connecting shaft 11, the connecting shaft 11 is connected to the liquid injection port through the liquid injection device, the liquid injection device includes a reagent tube and a piston 308, the piston 308 can move back and forth in the reagent tube, the number of revolutions of the motor is converted into the lateral movement distance of the piston in the reagent tube, and the lysate in the reagent tube is injected into the pipeline layer. In practical application, if the lysate needs to be pushed in, the third motor 103 rotates forwards to realize that the piston moves forwards in the reagent tube and injects the lysate, if the liquid needs to be pumped out, the third motor 103 is controlled to rotate backwards, at the moment, in order to match with the liquid pumping action, the second motor 102 needs to rotate forwards to perform the pushing-in action, the lysate and the sample are fully mixed through the forward rotation or the reverse rotation of the second motor 102 and the third motor 103, the piston moves forwards and backwards through the transmission action of the screw rod due to the rotation of the motors, and then the injection and the suction of the reagent are realized.
Through the cooperation of motor pump 1 and liquid way valve, realize the injection of reagent, carry out relevant experiment reaction on the pipeline layer, and utilize motor pump 1 and liquid way valve cooperation to realize in the intraformational reaction of pipeline and shift to nucleic acid material, and carry out corresponding operation in purification storehouse or amplification storehouse, realize the continuity of experiment, and the progress and the flow of experiment have been controlled through the cooperation of motor pump 1 and liquid way valve, realize the feed liquor in succession, and is simple and convenient, and easy to realize.
Can also adjust the motor slew velocity in motor group 10 through decelerator group 13 in practical application, decelerator group 13's setting makes the injection velocity of reagent in the reagent pipe or all can have corresponding slowing down or reducing at the intraformational flow velocity of pipeline, through the control to reagent injection velocity, realizes the effective control to the experimentation, is convenient for master the experiment rhythm.
After the experiment is finished, the upper end of the adapter shaft is provided with a zero piece 15 and a photoelectric sensor 16, the photoelectric sensor 16 is electrically connected with the zero piece 15, the photoelectric sensor 16 is used for receiving a signal of finishing the reaction, and then the rotating position of the connecting shaft 11 is returned to 0 through the zero piece 15. The photoelectric sensor 16 and the zero position sheet 15 are used in a matched mode, after the reaction is finished, the screw rod on the chip returns to the zero position, the chip can be taken out, the structure for placing the chip limits, the other function can be used as a counting function, the number of cycles of movement and the number of turns of the motor pump 1 connected with each motor can be recorded, and therefore the accuracy of the liquid adding amount can be simply corrected. The rotation position of the connecting shaft 11 is returned to 0, so that the chip can be conveniently taken out, and the next experiment process of a sample to be detected is carried out.
Specifically, the continuous liquid inlet device based on PCR provided by the embodiment of the present invention further includes a circuit control board 14, where the circuit control board 14 is disposed at one side of the motor set 10, the circuit control board 14 is electrically connected to the motor set 10, and the circuit control board 14 is configured to control an operating state of the motor set 10. Through circuit control board 14, the realization carries out overall control to the operating condition of motor group 10, because in the experimentation, the operating time of every motor, and it is corotation or reversal all to rely on other motors, that is to say, the operating condition of every motor all is closely relevant with the operating condition of other motors or current experiment progress, it is normal in order to further guarantee the operating condition of every motor, and the chronogenesis of its work is correct in whole experimentation, consequently, need carry out whole overall planning to it, through circuit control board 14's setting, the control with every motor becomes simple, and control is convenient.
Specifically, the motor set 10 includes a second motor 102, a third motor 103, a fourth motor 104, a fifth motor 105 and a sixth motor 106, the second motor 102, the third motor 103, the fourth motor 104, the fifth motor 105 and the sixth motor 106 are arranged in parallel, and a concave groove is formed in an end of a connecting shaft 11 connected to the second motor 102, the third motor 103, the fourth motor 104, the fifth motor 105 and the sixth motor 106, and is used for connecting the liquid pushing and sucking device. The end of the connecting shaft 11 is provided with a concave groove, the corresponding piston tube connected with the connecting shaft 11 is flat and is just embedded in the concave groove, the corresponding piston is driven to reciprocate in the reagent tube under the action of each motor, so that liquid injection or liquid suction is realized, and the motors are arranged in parallel, so that the motor set 10 can be ensured to have smaller volume in the whole structure and is easy to realize.
The sample adding hole 302 of the present embodiment is provided with a sample adding bin below, the sample adding hole 302 is provided with a sample adding hole cover 303 for sealing, the sample adding bin can be connected with a reagent tube for loading a reagent or a sample, the lower part of the sample adding bin is provided with a reagent outlet 312, a sealing structure 313 is arranged between the reagent outlet 312 and the sample adding bin for sealing, and when the reagent is required to be added, the puncture needle can puncture 313 so that the reagent enters the fluid pipeline along the reagent outlet 312. A pressurizing structure is further arranged on one side of the sample adding bin, the pressurizing structure comprises a tube wall 305, a piston 308 is arranged in the tube wall 305, the piston 308 moves towards the sample adding bin, the piston 308 further comprises a piston head 304 and a piston rod, and the piston pushes the reagent in the piston to flow out to a reagent outlet 312; of course, when the reagent is required to be withdrawn, the piston 308 may also withdraw the reagent or other waste liquid, and a sealing ring 311 is provided at the end of the piston rod of the piston 308 for sealing.
Continuing to refer to fig. 4, the piston rod of this embodiment is further provided with a nut 307, which is in threaded connection with the nut 307 to realize relative rotation, and correspondingly, an output structure, such as an air cylinder and an oil cylinder, is provided at one end of the piston rod, or connected to the piston rod by rotating the output structure, such as a motor and a lead screw, at this time, the piston rod rotates, and only the reagent needs to be pushed to flow out of the reagent outlet. Correspondingly, a guide sleeve 306 is sleeved outside the nut 307, and a corresponding shaft shoulder is arranged inside the pipe wall 305 to position and fix the guide sleeve 306; snap rings 314 are further provided at the outer sides of both ends of the guide sleeve 306 to catch the corresponding guide sleeve 306. A sheath 309 is also provided outside the guide sleeve 306 to protect the piston rod, the nut 307 and the guide sleeve 306. When reagent is injected into the pipeline layer 101, the piston moves toward the sample loading bin to increase the pressure therein, so as to push the reagent to flow toward the reagent outlet 312, thereby realizing reagent injection. With reference to fig. 1, in the embodiment of the present invention, a plurality of sets of reagent tubes are provided, and in the embodiment, five sets of reagent tubes are provided, and different or the same reagents are sequentially applied to the pipeline layer according to the experiment requirement, so that the use efficiency can be greatly improved.
As shown in fig. 4, a second buckle 310 is disposed below the sample-adding layer 3, and the second buckle 310 is disposed on a side opposite to the first buckle to prevent the sample-adding layer from sliding.
It can be seen that this embodiment is integrated at chip pipeline layer to complicated experimentation, can control the liquid trend to can improve work efficiency effectively.
Specifically, the following description is made of the operation sequence processes of the second motor, the third motor, the fourth motor, the fifth motor and the sixth motor, where the second motor, the third motor, the fourth motor, the fifth motor and the sixth motor are arranged in parallel, and the end of the connecting shaft connected to the second motor, the third motor, the fourth motor, the fifth motor and the sixth motor is provided with a concave groove for connecting the piston rod. In practical application, the reagent pipe that the second motor is connected is empty, play the mating reaction, after sample and lysate mixing reaction, close first single valve, open the second single valve, then the imbibition of third motor, the second motor pushes away the liquid, introduce the mixture in the purification storehouse, introduce the mixture behind the purification storehouse, need wash it, the fourth motor pushes away first washing liquid this moment, second motor or third motor inhale, make the washing liquid reach the purification storehouse along the pipeline, wash the mixture in the purification storehouse, technical personnel in the field can understand, in order to improve nucleic acid concentration, can wash once more, the cleaning process is the same, no longer give unnecessary details. After the washing, utilize the sixth motor to push into the eluant, the cooperation that needs the second motor this moment, it shifts to the storehouse that amplifies to need elute the nucleic acid material from the magnetic bead after, the bivalve opens this moment, first single valve and second single valve are closed, technical personnel in the art can understand, the bivalve also carries out control operation by the corresponding motor with the control of first single valve and second single valve, the second motor reversal carries out the imbibition, lead the nucleic acid material in the purification storehouse to the storehouse of amplifying along the pipeline in, thereby carry out subsequent amplification reaction.
Therefore, according to the continuous liquid inlet device based on the PCR, liquid inlet of the corresponding reagent tube is controlled by the motor pump or liquid inlet is matched according to the experiment purpose, continuous liquid inlet is achieved, the whole process is uninterrupted, the experiment efficiency is improved, and the experiment time is shortened.
So far, the technical solutions of the present invention have been described in connection with the preferred embodiments shown in the drawings, but it is easily understood by those skilled in the art that the scope of the present invention is obviously not limited to these specific embodiments. Equivalent changes or substitutions of related technical features can be made by those skilled in the art without departing from the principle of the invention, and the technical scheme after the changes or substitutions can fall into the protection scope of the invention.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention; various modifications and alterations to this invention will become apparent to those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (9)
1. A continuous liquid inlet device based on PCR is characterized by comprising a sample adding layer and a motor pump;
the sample adding layer is connected with the motor pump, the motor pump is used for adding a reagent into a pipeline in the pipeline layer through the sample adding layer, a sample adding port is formed in the sample adding layer, and a liquid level height meter is arranged in the sample adding port and used for detecting the height of a sample added from the sample adding port;
the sample adding layer comprises a reagent tube and a piston, one end of the reagent tube is provided with a sealing structure, the other end of the reagent tube is provided with the piston, and a piston rod of the piston is fixedly connected with a connecting shaft of the motor pump;
the motor pump comprises a motor set, a connecting shaft and a screw rod, an output shaft of the motor set is fixedly connected with the connecting shaft, the connecting shaft is used for pushing a reagent in the reagent tube into a pipeline or sucking the reagent out of the pipeline through a piston arranged on the screw rod, and the motor set comprises a second motor, a third motor, a fourth motor, a fifth motor and a sixth motor;
the central control unit is respectively connected with a second motor, a third motor and a liquid level height meter in the motor set and used for adjusting the liquid inlet speed v20 and the liquid inlet time t0 of the third motor according to the height of the liquid level height meter, wherein a first liquid level height h10, a second standard level height h20 and a third liquid level height h30 are arranged in the central control unit, and the first liquid level height h10 is less than the second standard level height h20 and less than the third liquid level height h 30;
if the height of the sample detected by the liquid level meter is less than or equal to the first liquid level height h10, the liquid inlet speed when the lysate is injected into the pipeline layer is v20 and the liquid inlet time is t 0;
if the second standard level height h20 is not less than the height of the sample detected by the liquid level meter and is greater than the first liquid level height h10, the liquid inlet speed when the lysate is injected into the pipeline layer is 1.1 xv 20, and the liquid inlet time is t 0;
if the third standard level height h30 is not less than the height of the sample detected by the liquid level meter and is greater than the second liquid level height h20, the liquid inlet speed when the lysate is injected into the pipeline layer is v20 and the liquid inlet time is 1.1 × t 0;
if the height of the sample detected by the liquid level meter is greater than the third standard height h30, the liquid inlet speed when the lysate is injected into the pipeline layer is 1.1 Xv 20 and the liquid inlet time length is 1.1 Xt 0;
the central control unit is internally provided with a counter and a preset standard number n0, the counter is used for counting the starting number of the second motor, the central control unit judges the cracking degree of the sample according to the starting number of the second motor, and the liquid inlet speed V40 of the fourth motor and the liquid inlet speed V50 of the fifth motor are selected according to the cracking degree.
2. The continuous inlet means for PCR-based assays according to claim 1, wherein the determining the extent of sample lysis according to the number of times the second motor is activated comprises:
a first cracking degree a1, a second cracking degree a2 and a third cracking degree a3 are arranged in the central control unit, wherein the first cracking degree a1 represents that the cracking degree of the sample is more than or equal to a preset cracking degree standard s0, the second cracking degree a2 represents that the cracking degree of the sample is more than the cracking degree standard s0 and less than or equal to 1.1 × s0, and the third cracking degree a3 represents that the cracking degree of the sample is more than 1.1 × s 0;
if the starting times of the second motor are more, the sample and the lysate are fully mixed, the protein molecules in the sample are small, and the cracking degree of the sample is a third cracking degree;
if the starting frequency of the second motor is less, the sample and the lysate are mixed but not sufficiently mixed, the protein molecules in the sample are large, and the cracking degree of the sample is the first cracking degree;
if the starting frequency of the second motor is moderate, the protein molecules in the sample have the size, and the cracking degree of the sample is the second cracking degree.
3. The continuous inlet device for PCR-based samples according to claim 2, wherein said adjusting the inlet speed of the fourth motor and the inlet speed of the fifth motor according to the cracking degree comprises:
in the purification process, if the cracking degree of the substances in the purification bin belongs to the first cracking degree, the liquid inlet speed of the fourth motor and the liquid inlet speed of the fifth motor are increased;
if the cracking degree of the substances in the purification bin belongs to the second cracking degree, the liquid inlet speeds of the fourth motor and the fifth motor are kept;
if the cracking degree of the substances in the purification bin belongs to the third cracking degree, the liquid inlet speed of the fourth motor and the liquid inlet speed of the fifth motor are reduced.
4. The continuous liquid inlet device based on PCR of claim 3, wherein the motor set further comprises a speed reducer set, and the speed reducer set is disposed in the motor set and used for controlling the rotation speed of the motor set.
5. The continuous liquid inlet device based on PCR as claimed in claim 4, further comprising a circuit control board disposed at the upper side of the motor set, the circuit control board being electrically connected to the motor set, the circuit control board being configured to control the operating state of the motor set, and a central control unit disposed on the circuit control board.
6. The continuous liquid inlet device based on PCR as claimed in claim 5, wherein a zero piece and a photoelectric sensor are arranged at the upper end of the connection shaft, the photoelectric sensor is electrically connected with the zero piece, the photoelectric sensor is used for receiving a signal indicating that the reaction is finished, and the rotation position of the connection shaft is reset to zero by the zero piece.
7. The continuous liquid inlet device based on PCR as claimed in claim 6, wherein the second motor, the third motor, the fourth motor, the fifth motor and the sixth motor are arranged in parallel, and the end of the connecting shaft connected with the second motor, the third motor, the fourth motor, the fifth motor and the sixth motor is provided with a concave groove for connecting the piston rod,
a sealing ring is arranged at the end part of a piston rod of the piston and used for sealing; the piston reciprocates along the tube wall of the reagent tube to push the reagent in the piston to flow out of or be drawn back to the reagent outlet.
8. The continuous liquid inlet device based on PCR as claimed in claim 7, wherein the piston rod is further provided with a nut, the nut is in threaded connection with the nut, a guiding sleeve is sleeved outside the nut, and a corresponding shaft shoulder is arranged inside the pipe wall for positioning and fixing the guiding sleeve; the outside of uide bushing still is provided with the sheath for protect piston rod, nut and uide bushing the below on application of sample layer is provided with the second buckle, is used for preventing application of sample layer slides, still is provided with the snap ring in the both ends outside of uide bushing, is used for blocking corresponding uide bushing.
9. The continuous liquid inlet device based on PCR as claimed in claim 8, wherein the number of the reagent tubes is 5, the second reagent tube connected to the second motor is empty, the third reagent tube connected to the third motor is used for containing the lysis solution, the fourth reagent tube connected to the fourth motor is used for containing the first cleaning solution, the fifth reagent tube connected to the fifth motor is used for containing the second cleaning solution, and the sixth reagent tube connected to the sixth motor is used for containing the elution solution.
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| CN113462547B (en) * | 2021-07-06 | 2022-05-27 | 北京中科生仪科技有限公司 | Reagent dosage adjusting device based on PCR |
| CN113846014A (en) * | 2021-10-29 | 2021-12-28 | 青岛全诊生物技术有限公司 | Nucleic acid extraction and amplification device |
| CN114984896B (en) * | 2022-03-30 | 2023-08-18 | 北京擎科生物科技股份有限公司 | Oligonucleotide synthesis device and oligonucleotide synthesis method |
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Application publication date: 20210806 Assignee: Beijing Linke Biotechnology Co.,Ltd. Assignor: BEIJING INTEGRATED BIOSYSTEMS Co.,Ltd. Contract record no.: X2022990000932 Denomination of invention: Continuous liquid feeding device based on PCR Granted publication date: 20220408 License type: Common License Record date: 20221114 |
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Application publication date: 20210806 Assignee: Beijing Linke Biotechnology Co.,Ltd. Assignor: BEIJING INTEGRATED BIOSYSTEMS Co.,Ltd. Contract record no.: X2022990000932 Denomination of invention: PCR based continuous liquid feeding device Granted publication date: 20220408 License type: Common License Record date: 20221114 |