CN111841669A - PCR chip for microorganism detection and liquid drop distribution method based on PCR chip - Google Patents
PCR chip for microorganism detection and liquid drop distribution method based on PCR chip Download PDFInfo
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- 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
- B01L3/502769—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 characterised by multiphase flow arrangements
- B01L3/502784—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 characterised by multiphase flow arrangements specially adapted for droplet or plug flow, e.g. digital microfluidics
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- 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
- B01L3/502738—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 characterised by integrated valves
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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
- B01L3/502746—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 characterised by the means for controlling flow resistance, e.g. flow controllers, baffles
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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
- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
- B01L2200/14—Process control and prevention of errors
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- B—PERFORMING OPERATIONS; TRANSPORTING
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Abstract
The invention relates to a PCR chip for detecting microorganisms and a liquid drop distribution method based on the PCR chip, wherein the PCR chip combines the advantages of serial distribution and parallel distribution, has high liquid drop distribution rate and low cavity rate, is suitable for executing rapid nucleic acid detection, and ensures the accuracy of detection results, and also comprises a control layer which can automatically sense whether a micro reaction cavity in a single conveying channel is filled, correspondingly activates a micro valve in a distribution channel corresponding to the conveying channel, and blocks the continuous flow of liquid drops to the conveying channel, thereby accelerating the liquid drop distribution speed of other channels which are not activated by the micro valve, and the micro valves in different levels of branch channels can be activated step by step to offset the high cavity rate and the reduced distribution rate caused by the non-uniformity of the channels due to the limitation of chip manufacturing process.
Description
Technical Field
The invention relates to a micro-fluidic chip, in particular to a PCR chip for detecting microorganisms and a liquid drop distribution method based on the PCR chip.
Background
Polymerase Chain Reaction (PCR) detection, which is a technique for detecting a target nucleic acid by amplifying a specific DNA fragment, is capable of performing accurate quantitative detection based on a very small amount of a sample, and is currently an important biological detection means. At present, a PCR chip combining a PCR technology and a microfluidic device is a research hotspot in the industry, and has the advantages of less required sample and reagent amount, high detection speed, more reaction units, high accuracy and the like.
One of the main differences of the PCR chip compared to the conventional PCR implementation is that the former mainly uses a single micro-droplet as a micro-reaction unit, and since the size of the micro-droplet is small, one PCR chip can accommodate many micro-droplets at the same time, which makes the amplification reaction performed based on the PCR chip have much higher accuracy compared to the conventional PCR implementation.
At present, the output of the detection result of the PCR chip mainly depends on fluorescence test, namely, fluorescence detection is carried out on a micro-droplet array region after amplification reaction, then the proportion of micro-droplet units with fluorescence signals and without fluorescence signals is obtained by adopting a statistical approach, and the original concentration of target DNA is finally obtained by combining the amplification multiplying power. Therefore, the accuracy of the detection result of the PCR chip mainly depends on the proportion of the micro-droplet units with fluorescence signals and without fluorescence signals. The accuracy of this ratio depends on the exact distribution of the droplets as reaction units in the reaction area. In practical application, such requirements are often difficult to meet. Taking the nested PCR chip as an example, the micro-droplets need to be uniformly distributed into a plurality of micro-reaction chambers, which requires that each reaction chamber contains one droplet and no cavity can be formed; meanwhile, the space of the array region other than the reaction chamber should not have residual droplets.
The existing droplet distribution means comprises a parallel type and a serial type, wherein the serial type distribution scheme adopts a single distribution path, so that the cavity rate in the distribution process is low, but the scheme takes too long time for completing the droplet distribution of all micro reaction chambers, and is not beneficial to realizing quick detection; although the distribution rate of parallel distribution is high, the consistency of a plurality of parallel distribution channels is difficult to guarantee due to the limitation of the chip preparation level, so that the actual liquid drop distribution amount in each parallel channel is not balanced, and the distribution scheme often has high cavity rate, thereby causing large error of detection results. Therefore, it is necessary to develop a technical means capable of rapidly and accurately performing micro-droplet dispensing.
Disclosure of Invention
In order to solve the above problems in the prior art, the present invention provides a PCR chip for detecting microorganisms. The PCR chip can simultaneously realize high distribution rate of parallel distribution and low cavity rate of a serial distribution scheme; meanwhile, the PCR chip can reduce the residual quantity of liquid drops in the array area of the reaction cavity.
In order to achieve the above object, the present invention specifically provides the following solutions:
a PCR chip for detecting microorganisms comprises a cover sheet layer 1 and a structural layer 4; wherein the structural layer 4 comprises a droplet distribution region 42, a micro-reaction chamber region 41 and an outlet region 43; the droplet distribution section 42 includes a main channel 44, a plurality of stages of connecting channels 45 connected to the ends of the main channel 44, and a distribution channel 46 connected to the ends of the connecting channels 45; the micro-reaction chamber region 41 comprises the same number of conveying channels 411 as the distribution channels 46, and the inlet of each conveying channel 411 is correspondingly connected with the outlet of one distribution channel 46; the device also comprises a plurality of micro reaction cavities 412 which are alternately arranged at two sides of the conveying channel 411 and are used for accommodating micro liquid drops 5; the outlet area 43 comprises the same number of discharge channels 415 as the conveying channels 411, which are connected in a one-to-one correspondence; at least the part of the cover plate layer 1 corresponding to the micro-reaction cavity area 41 is made of transparent material.
Preferably, the micro-reaction chamber 412 is sized to accommodate only a single micro-droplet 5, and both the front side wall 414 (a side wall close to the incoming flow direction of the liquid) and the rear side wall are inclined planes inclined at an acute angle, preferably not more than 30 degrees, with respect to the flow direction of the liquid in the transport channel 411.
Preferably, a flow-resisting protrusion 413 extending into the conveying channel 411 is disposed on a rear side wall of each micro-reaction chamber 412; the minimum distance from the flow-obstructing protrusion 413 to the opposite side wall of the conveying channel 411 is smaller than the diameter of the liquid droplet 5, so that the liquid droplet 5 needs to be deformed to pass through the flow-obstructing protrusion 413; the flow-resisting protrusions 413 can block the liquid drops 5 in the conveying channel 411, so that the liquid drops are preferably filled in the micro-reaction chambers 412 which are not filled at the front ends of the corresponding flow-resisting protrusions 413; since the size of the micro-reaction chamber 412 only allows for accommodating a single droplet 5, when the droplet 5 passes through the flow-obstructing protrusion 413 corresponding to the filled micro-reaction chamber 412, the droplet is forced to deform to some extent due to the extrusion of the continuous phase fluid, and then passes through the flow-obstructing protrusion 413, and is transported further downstream. The provision of the flow-blocking protrusions 413 ensures effective filling of the plurality of micro-reaction chambers 412.
Preferably, the vertical side wall of the flow-obstructing protrusion 413 within the conveying passage 411 is curved, and the arc of the upstream side is greater than that of the downstream side, thereby preventing the droplet 5 from being torn due to the contact of a sharp surface at the flow-obstructing protrusion 413 or due to strong turbulence formed downstream of the flow-obstructing protrusion 413.
Preferably, at the droplet distribution region 42, in addition to the main channel 44, each of the connecting channel 45 and the distribution channel 46 is provided with a lower coated electrode 423 on the channel bottom surface at the inlet thereof, and each of the lower coated electrodes 423 is provided with a first contact electrode 421 electrically connected thereto; the corresponding lower coated film electrode 423 and the first contact electrode 421 are electrically connected through a first wiring 422; the first contact electrode 421 is located on the upper surface of the structural layer 4; the portion of the first wiring 422 located within the passage and the surface of the lower film-covered electrode 423 are covered with a hydrophobic film or a hydrophobic coating.
The PCR chip also comprises a control layer 3 directly attached to the upper surface of the structural layer 4; a transparent area 31 (which can be made of transparent materials or hollow structures) corresponding to the micro-reaction cavity area 41 is arranged on the control layer 3; the control layer 3 is also provided with a dispensing control area 32 corresponding to the dispensing area 42 for dispensing the liquid drops; the distribution control area 32 is provided with the upper membrane electrodes 323 which are the same as the lower control electrodes 423 in number and are in corresponding positions (namely, in the same vertical projection positions when the structural layer 4 and the control layer 3 are correctly attached); and the number of the second contact electrodes 321 is the same as the number of the first contact electrodes 421 and is in the corresponding position; the upper membrane electrode 323 is not communicated with the second contact electrodes 321, and each second contact electrode 321 is communicated with one electrode of an external power supply through an independent contact lead 322; each of the overlying membrane electrodes 323 is connected to the other pole of the external power source by a separate lamination lead 324. Therefore, after the structural layer 4 and the control layer 3 are correctly bonded, electricity can be respectively supplied to the corresponding upper film electrode 323 and the corresponding lower film electrode 423 through the independent film-covered lead 324 and the independent contact lead 322, so that the wettability of the surfaces of the pair of film-covered electrodes to the liquid drops 5 is changed. When a certain pair of film-covered electrodes is energized, the droplets 5 flowing therethrough will be retained by the wettable film-covered electrode surface, thereby forming a microvalve that prevents further flow of droplets 5 into the next level channel or corresponding delivery channel 411.
The exit region 43 of the structural layer 4 further comprises strip electrodes 433 arranged on both sidewalls of the discharge channel 415, each of the strip electrodes 433 being connected to a third contact electrode 431 located on the upper surface of the structural layer 4 by a second connection 432; wherein the strip electrodes 433 are disposed in the discharge passage 415 exposed; correspondingly, an outlet detection area 33 corresponding to the outlet area 43 is arranged on the control layer 3; the exit detection area 33 includes the same number of fourth contact electrodes 331 as the third contact electrodes 431 in corresponding positions; each of the fourth contact electrodes 331 is connected to an external power source through an independent sensing lead 332.
The above-described structural layer 4 and control layer 3 allow at least the following droplet control logic to be performed. When the structural layer 4 and the control layer 3 are correctly attached, an external power supply energizes the strip electrodes 433 positioned at two sides of the discharge channel 415 through the detection lead 332, the fourth contact electrode 331, the third contact electrode 431 and the second connection wire 432, and when the droplets 5 of the water phase do not fill all the micro-reaction cavities 412 in the corresponding delivery channel 411, only the oil continuous phase passes through the discharge channel 415, and the circuits where the two strip electrodes 433 are positioned cannot be conducted; on the contrary, when all the reaction chambers 412 in the corresponding transport channels 411 are filled, the droplets 5 with the water phase flow out from the discharge channel 415, and at this time, the circuit where the strip electrode 433 is located is conducted, so that an interval electrical signal can be outputted outwards, and the occurrence of the interval electrical signal means that the corresponding transport channels 411 have completed the droplet filling.
At this time, the logic controller energizes the upper membrane electrode 323 and the lower membrane electrode 423 in the distribution channel 46 corresponding to the delivery channel 411, and activates the micro valve in the corresponding distribution channel 46 to prevent the droplet 5 from continuously flowing into the delivery channel 411 which is filled with the droplet; at this point, the droplets 5 will preferentially flow into the other dispensing channels 46 where the microvalve has not been activated, thereby increasing overall dispensing efficiency and reducing inefficient droplet dispensing processes. When the micro valves in the two distribution channels 46 connected to the same connecting channel 45 are activated, the micro valves in the upper-stage channels of the two distribution channels 46 are further activated (i.e., the upper and lower film-coated electrodes in the upper-stage channels are electrified), so as to block the droplets 5 from further flowing into the two water delivery channels 411 where the droplet distribution is completed; and so on until all the microvalves are activated, it is sensed that the filling of all the micro reaction chambers 412 in the micro reaction chamber region 41 has been completed.
Preferably, the PCR chip further comprises a washing layer 2 attached to the upper surface of the control layer 3; the flushing layer 2 is provided with a flushing liquid channel corresponding to the distribution area 42 of the structural layer 4, and the flushing liquid channel comprises a flushing main channel 21, a plurality of stages of flushing connecting channels 22 connected to the tail end of the flushing main channel 21 and flushing distribution channels 23 connected to the tail end of the flushing connecting channels 22; the number of said flush distribution channels 23 is equal to the number of distribution channels 46 and in corresponding positions; the flushing trunk 21, the flushing connection 22 and the flushing distribution channel 23 are all non-penetrating structures; the tail end of each flushing distribution channel 23 is provided with a through hole 24 correspondingly connected to the tail end of the distribution channel 46; the control layer 3 is provided with a plurality of openings 34 corresponding to the through holes 24; the through-hole 24 corresponds to the downstream of the micro-valve in the distribution channel 46, which allows supplying an oil-phase flushing liquid into the delivery channel 411 downstream of the distribution channel 46 after the micro-valve in the distribution channel 46 is activated, to flush out the residual droplets 5 remaining in the delivery channel 411.
Preferably, the supply of the oil phase flushing fluid is started after all the microvalves have been activated.
Compared with the prior art, the invention can at least obtain the following beneficial effects: the PCR chip of the invention combines the advantages of serial distribution and parallel distribution, has high liquid drop distribution rate and low cavity rate, is suitable for executing rapid nucleic acid detection, and the accuracy of the detection result is ensured, meanwhile, the PCR chip of the invention also comprises a control layer, which can automatically sense whether the micro-reaction cavity in a single conveying channel is filled or not, correspondingly activate the micro-valve in the distribution channel corresponding to the conveying channel, block the continuous flow of liquid drops to the conveying channel, thereby accelerating the distribution speed of the liquid drops of other channels which are not activated by the micro valve, the micro valves positioned in the branch channels of different grades can be activated step by step, further accelerating the flow speed of the liquid drops to the conveying channel which is not filled, thus, a higher distribution rate can be achieved compared to a conventional parallel distribution channel, counteracting the high cavity rate and hence the reduced distribution rate due to channel non-uniformity caused by chip manufacturing process limitations; the PCR chip of the invention also comprises a flushing layer which allows an oil-phase flushing fluid to be supplied into all the conveying channels through a flushing fluid channel connected to the downstream of the micro valves after all the micro valves are activated, and residual droplets remained in the conveying channels are flushed out of the micro reaction cavity area, so that after the PCR chip of the invention is filled, the micro reaction cavity area has only a single droplet independently filled in each micro reaction cavity, and the accuracy of a detection result is ensured.
Drawings
FIG. 1 is a schematic view of a cover sheet layer;
FIG. 2 is a schematic view of a strike layer;
FIG. 3 is a schematic diagram of a control layer;
FIG. 4 is an enlarged fragmentary view of the left circular area of FIG. 3;
FIG. 5 is an enlarged partial view of the right circular area of FIG. 3;
FIG. 6 is a schematic view of a structural layer;
FIG. 7 is an enlarged fragmentary view of the left circular area of FIG. 6;
FIG. 8 is an enlarged partial view of the right circular area of FIG. 6;
FIG. 9 is a schematic three-dimensional structure of FIG. 8;
FIG. 10 is a schematic view of a micro-reaction chamber region after being filled with droplets;
FIG. 11 is an enlarged fragmentary view of the circled area of FIG. 10;
fig. 12 is a schematic view showing the stacking sequence of the layers according to the present invention.
In the figure: 1 is a cover plate layer, 2 is a washing layer, 21 is a washing trunk, 22 is a washing connecting channel, 23 is a washing distribution channel, 24 is a through hole, 3 is a control layer, 31 is a transparent area, 32 is a distribution control area, 321 is a second contact electrode, 322 is a contact lead, 323 is an overlying membrane electrode, 324 is a laminating lead, 33 is an outlet detection area, 331 is a fourth contact electrode, 332 is a detection lead, 34 is an opening, 4 is a structural layer, 41 is a micro-reaction chamber area, 411 is a conveying channel, 412 is a micro-reaction chamber, 413 is a flow-resisting bulge, 414 is a front side wall, 415 is a discharge channel, 42 is a droplet distribution area, 421 is a first contact electrode, 422 is a first wiring, 423 is a lower laminating electrode, 431 is a third contact electrode, 432 is a second wiring, 433 is a bar electrode, 44 is a main channel, 45 is a connecting channel, 46 is a distribution channel, and 5 is a droplet.
Detailed Description
To further illustrate the concept of the present invention, the following description will be made with reference to the accompanying drawings.
Example 1
As shown in FIGS. 1, 6 to 11, there is provided a PCR chip for microorganism detection, which comprises a cover sheet layer 1 and a structure layer 4; wherein the structural layer 4 comprises a droplet distribution region 42, a micro-reaction chamber region 41 and an outlet region 43; the droplet distribution section 42 includes a main channel 44, a plurality of stages of connecting channels 45 connected to the ends of the main channel 44, and a distribution channel 46 connected to the ends of the connecting channels 45; the micro-reaction chamber region 41 comprises the same number of conveying channels 411 as the distribution channels 46, and the inlet of each conveying channel 411 is correspondingly connected with the outlet of one distribution channel 46; the device also comprises a plurality of micro reaction cavities 412 which are alternately arranged at two sides of the conveying channel 411 and are used for accommodating micro liquid drops 5; the outlet area 43 comprises the same number of discharge channels 415 as the conveying channels 411, which are connected in a one-to-one correspondence; at least the part of the cover plate layer 1 corresponding to the micro-reaction cavity area 41 is made of transparent material. The micro-reaction chamber 412 is sized to accommodate only a single micro-droplet 5, and has a front side wall 414 (a side wall close to the incoming flow direction of the liquid) and a rear side wall each having an inclined surface inclined at an acute angle, preferably not more than 30 degrees, with respect to the flow direction of the liquid in the transport channel 411. A flow-resisting protrusion 413 extending into the conveying channel 411 is arranged on the rear side wall of each micro-reaction chamber 412; the minimum distance of the flow-obstructing protrusion 413 to the opposite side wall of the conveying channel 411 is smaller than the diameter of the liquid droplet 5.
Example 2
As shown in fig. 3 to 9, differing from example 1, at the droplet dispensing region 42, in addition to the main channel 44, each of the connecting channel 45 and the dispensing channel 46 is provided with a lower coated electrode 423 at a channel bottom surface at an entrance thereof, and each of the lower coated electrodes 423 is provided with a first contact electrode 421 electrically connected thereto; the corresponding lower coated film electrode 423 and the first contact electrode 421 are electrically connected through a first wiring 422; the first contact electrode 421 is located on the upper surface of the structural layer 4; the portion of the first wiring 422 located within the passage and the surface of the lower film-covered electrode 423 are covered with a hydrophobic film or a hydrophobic coating.
The PCR chip also comprises a control layer 3 directly attached to the upper surface of the structural layer 4; the control layer 3 is provided with a transparent area 31 corresponding to the micro-reaction chamber area 41; the control layer 3 is also provided with a dispensing control area 32 corresponding to the dispensing area 42 for dispensing the liquid drops; the distribution control area 32 is provided with upper membrane electrodes 323 which are the same as the lower control electrodes 423 in number and are located at corresponding positions; and the number of the second contact electrodes 321 is the same as the number of the first contact electrodes 421 and is in the corresponding position; the upper membrane electrode 323 is not communicated with the second contact electrodes 321, and each second contact electrode 321 is communicated with one electrode of an external power supply through an independent contact lead 322; each of the overlying membrane electrodes 323 is connected to the other pole of the external power source by a separate lamination lead 324. When a certain pair of film-covered electrodes is energized, the droplets 5 flowing therethrough will be retained by the wettable film-covered electrode surface, thereby forming a microvalve that prevents further flow of droplets 5 into the next level channel or corresponding delivery channel 411.
The exit region 43 of the structural layer 4 further comprises strip electrodes 433 arranged on both sidewalls of the discharge channel 415, each of the strip electrodes 433 being connected to a third contact electrode 431 located on the upper surface of the structural layer 4 by a second connection 432; wherein the strip electrodes 433 are disposed in the discharge passage 415 exposed; correspondingly, an outlet detection area 33 corresponding to the outlet area 43 is arranged on the control layer 3; the exit detection area 33 includes the same number of fourth contact electrodes 331 as the third contact electrodes 431 in corresponding positions; each of the fourth contact electrodes 331 is connected to an external power source through an independent sensing lead 332.
Example 3
As shown in fig. 2 to 4, unlike example 2, the PCR chip further includes a washing layer 2 attached to the upper surface of the control layer 3; the flushing layer 2 is provided with a flushing liquid channel corresponding to the distribution area 42 of the structural layer 4, and the flushing liquid channel comprises a flushing main channel 21, a plurality of stages of flushing connecting channels 22 connected to the tail end of the flushing main channel 21 and flushing distribution channels 23 connected to the tail end of the flushing connecting channels 22; the number of said flush distribution channels 23 is equal to the number of distribution channels 46 and in corresponding positions; the flushing trunk 21, the flushing connection 22 and the flushing distribution channel 23 are all non-penetrating structures; the tail end of each flushing distribution channel 23 is provided with a through hole 24 correspondingly connected to the tail end of the distribution channel 46; the control layer 3 is provided with a plurality of openings 34 corresponding to the through holes 24; the through-hole 24 corresponds to the downstream of the micro-valve in the distribution channel 46, which allows supplying an oil-phase flushing liquid into the delivery channel 411 downstream of the distribution channel 46 after the micro-valve in the distribution channel 46 is activated, to flush out the residual droplets 5 remaining in the delivery channel 411. The supply of the oil phase flushing fluid is started after all the microvalves have been activated.
Example 4
A method for dispensing droplets based on the PCR chip of embodiment 3 is provided, which comprises the following steps:
1) laminating the cover layer 1, the flushing layer 2, the control layer 3 and the structural layer 4 layer by layer from top to bottom;
2) energizing the strip electrodes 433 on the side wall 415 of the discharge channel, wherein the circuit where the strip electrodes 433 are located is not energized;
3) connecting the main channel 44 to a droplet supply unit, supplying the droplets 5 to the micro-reaction chamber region 41; the droplets 5 are substantially uniformly distributed into a plurality of distribution channels 46 via a number of stages of connecting channels 45;
4) the liquid droplets 5 entering the distribution channel 46 then enter the corresponding distribution channel 411 and touch the arc-shaped side wall on the upstream side of the flow-blocking protrusion 413, and the flow of the liquid droplets 5 is blocked, so that the liquid droplets preferentially enter the micro-reaction chamber 412 which is vacant on the front side of the corresponding flow-blocking protrusion 413; as for the micro-reaction chamber 412 filled with the liquid droplets 5, since the accommodating space thereof is occupied, the liquid droplets 5 are slightly deformed and pass through the corresponding flow-resisting protrusions 413 under the squeezing of the oil continuous phase and the flow-resisting protrusions 413;
5) before the droplets 5 of the aqueous phase do not fill all the micro-reaction chambers 412 in the corresponding conveying channels 411, only the oil continuous phase passes through the corresponding discharge channel 415, so that the circuits where the two strip electrodes 433 are located cannot be conducted; when all the reaction chambers 412 in the corresponding delivery channels 411 are filled, the liquid drops 5 with water phase flow out from the discharge channel 415, and at this time, the circuit where the strip electrodes 433 are located is conducted, so that an interval electrical signal can be output outwards, and the occurrence of the interval electrical signal means that the corresponding delivery channels 411 have completed the liquid drop filling;
6) When receiving the electric signal output by a pair of strip electrodes 433 in a certain discharge channel 415, energizing the upper membrane electrode 323 and the lower membrane electrode 423 in the distribution channel 46 corresponding to the discharge channel 415 through the logic controller, activating the micro valve in the corresponding distribution channel 46, and preventing the liquid droplet 5 from continuously flowing into the delivery channel 411 which is filled with the liquid droplet;
7) when the microvalves in both the two distribution channels 46 connected to the same connecting channel 45 are activated, the microvalve in the channel of the previous stage of the two distribution channels 46 is further activated, thereby blocking further flow of the droplet 5 into the two delivery channels 411 where droplet distribution has been completed; analogizing in sequence until all the micro valves are activated, and then judging that the filling of all the micro reaction cavities 412 in the micro reaction cavity area 41 is finished;
8) and supplying oil-phase flushing liquid into all the conveying channels 411 through the flushing layer 2, flushing residual liquid drops in the conveying channels 411 until all the strip electrodes 433 are not conducted any more, stopping supplying the flushing liquid, and completing the liquid drop distribution process.
The above embodiments are only examples of the preferred embodiments of the technical idea of the present invention, which should not be construed as limiting all possible embodiments of the present invention, and all embodiments obtained by the replacement of conventional means by those skilled in the art are within the possible scope of the present invention without creative efforts, and the actual protection scope of the present invention is subject to the limitation of the claims.
Claims (10)
1. A PCR chip for detecting microorganisms comprises a cover sheet layer (1) and a structural layer (4); the structural layer (4) comprises a droplet distribution region (42), a micro-reaction cavity region (41) and an outlet region (43); the droplet distribution region (42) comprising a main channel (44), several stages of connecting channels (45) connected to the ends of the main channel (44) and a distribution channel (46) connected to the ends of the connecting channels (45); the method is characterized in that: the micro-reaction cavity area (41) comprises the same number of conveying channels (411) as the distribution channels (46), and the inlet of each conveying channel (411) is correspondingly connected with the outlet of one distribution channel (46); the device also comprises a plurality of micro-reaction cavities (412) which are alternately arranged at two sides of the conveying channel (411) and used for containing liquid drops (5); the outlet area (43) comprises discharge channels (415) which are the same in number as the conveying channels (411) and are connected in a one-to-one correspondence manner; at least the part of the cover plate layer (1) corresponding to the micro-reaction cavity area (41) is made of transparent materials; the micro-reaction chamber (412) has dimensions that allow only a single droplet (5) to be accommodated, the front (414) and rear (411) side walls of which are slopes inclined at an acute angle with respect to the direction of liquid flow in the transport channel; a flow-resisting bulge (413) extending into the conveying channel (411) is arranged on the rear side wall of each micro-reaction cavity (412); the smallest distance of the flow-blocking projection (413) to the opposite side wall of the conveying channel (411) is smaller than the diameter of the droplet (5).
2. The PCR chip for microorganism detection according to claim 1, wherein: the angle of the acute angle is preferably no more than 30 degrees; the vertical side wall of the flow blocking bulge (413) in the conveying channel (411) is a curve, and the radian of the upstream side is larger than that of the downstream side.
3. The PCR chip for microorganism detection according to claim 1, wherein: at the droplet distribution region (42), except for the main channel (44), each connecting channel (45) and each distribution channel (46) are provided with a lower film-coated electrode (423) at the channel bottom surface at the inlet of the connecting channel and the distribution channel, and each lower film-coated electrode (423) is provided with a first contact electrode (421) electrically connected with the lower film-coated electrode; the corresponding lower coated film electrode (423) and the first contact electrode (421) are electrically connected through a first wiring (422); the first contact electrode (421) is positioned on the upper surface of the structural layer (4); the portion of the first wiring (422) located within the channel and the surface of the lower film-coated electrode (423) are covered with a hydrophobic film or a hydrophobic coating.
4. The PCR chip for detecting microorganisms according to claim 3, wherein: the control layer (3) is directly attached to the upper surface of the structural layer (4); a transparent area (31) corresponding to the micro-reaction cavity area (41) is arranged on the control layer (3); the control layer (3) is also provided with a distribution control area (32) corresponding to the liquid drop distribution area (42); the distribution control area (32) is provided with overlying membrane electrodes (323) which are the same as the lower membrane-covered electrodes (423) in number and are in corresponding positions; and a number of second contact electrodes (321) which are the same as the number of the first contact electrodes (421) and are in corresponding positions; the upper membrane electrode (323) is not communicated with the second contact electrodes (321), and each second contact electrode (321) is communicated with one pole of an external power supply through an independent contact lead (322); each overlying membrane electrode (323) is connected with the other pole of an external power supply through an independent film-covered lead wire (324); a pair of upper and lower membrane electrodes (423, 323) corresponding to each other form a microvalve that can close a fluid flow path in the channel.
5. The PCR chip for detecting microorganisms according to claim 4, wherein: the outlet region (43) of the structural layer (4) further comprises strip electrodes (433) arranged on both side walls of the discharge channel (415), each strip electrode (433) being connected to a third contact electrode (431) located on the upper surface of the structural layer (4) by a second connection (432); the strip electrode (433) is exposed in the discharge passage (415).
6. The PCR chip for detecting microorganisms according to claim 5, wherein: an outlet detection area (33) corresponding to the outlet area (43) is arranged on the control layer (3); the outlet detection area (33) comprises fourth contact electrodes (331) which are the same in number and in corresponding positions as the third contact electrodes (431); each of the fourth contact electrodes (331) is connected to an external power source through an independent sensing lead (332).
7. The PCR chip for detecting microorganisms according to any one of claims 4 to 6, wherein: the washing layer (2) is attached to the upper surface of the control layer (3); the flushing layer (2) having flushing liquid passages corresponding to the distribution area (42) of the structural layer (4), said flushing liquid passages comprising at least a number of flushing distribution passages (23) equal to the number of distribution passages (46) and in corresponding positions therewith; the flushing distribution channel (23) is of a non-penetrating structure; the end of each flushing distribution channel (23) is provided with a through hole (24) correspondingly connected to the end of the distribution channel (46).
8. The PCR chip for detecting microorganisms according to claim 7, wherein: the control layer (3) is provided with a plurality of openings (34) corresponding to the through holes (24); the through-going opening (24) corresponds to the downstream of the microvalve in the distribution channel (46) and allows the supply of an oil phase flushing liquid into the delivery channel (411) downstream of the distribution channel (46) after the microvalve in the distribution channel (46) has been activated.
9. A method of droplet dispensing using the PCR chip of claim 8, wherein: the method comprises the following steps:
1) laminating the cover sheet layer (1), the flushing layer (2), the control layer (3) and the structural layer (4) layer by layer from top to bottom;
2) energizing the strip electrodes (433) on the side wall of the discharge channel side wall (415), wherein the circuit where the strip electrodes (433) are located is not turned on;
3) connecting the main channel (44) to a droplet supply unit, supplying droplets (5) to the micro-reaction-chamber region (41); the droplets (5) are distributed substantially uniformly through several stages of connecting channels (45) into a plurality of distribution channels (46);
4) the liquid drops (5) entering the distribution channel (46) then enter the corresponding distribution channel (411) and touch the arc-shaped side wall on the upstream side of the flow-resisting bulge (413), and the flow of the liquid drops (5) is blocked, so that the liquid drops preferentially enter the micro-reaction cavity (412) which is vacant on the front side of the corresponding flow-resisting bulge (413); and for the micro-reaction chamber (412) filled with the liquid drops (5), because the accommodating space of the micro-reaction chamber is occupied, the liquid drops (5) are slightly deformed under the extrusion of the oil continuous phase and the flow resisting bulges (413) and pass through the corresponding flow resisting bulges (413);
5) Before the droplets (5) of the aqueous phase are not filled in all the micro-reaction cavities (412) in the corresponding conveying channels (411), only the oil continuous phase passes through the corresponding discharge channels (415), so that the circuits where the two strip electrodes (433) are located cannot be conducted; when all the reaction chambers (412) in the corresponding conveying channels (411) are filled, the liquid drops (5) with water phase flow out of the discharge channel (415), at the moment, the circuit where the strip electrodes (433) are located is conducted, and therefore, interval electric signals can be output outwards, and the appearance of the interval electric signals means that the corresponding conveying channels (411) are filled with the liquid drops.
10. A method of droplet dispensing as claimed in claim 9, wherein: further comprising the steps of:
6) when receiving an electric signal output by a pair of strip electrodes (433) in a certain discharge channel (415), electrifying an upper membrane electrode (323) and a lower membrane electrode (423) in a distribution channel (46) corresponding to the discharge channel (415) through a logic controller, activating a micro valve in the corresponding distribution channel (46), and preventing the liquid drops (5) from continuously flowing into the conveying channel (411) filled with the liquid drops;
7) when the micro valves in the two distribution channels (46) connected to the same connecting channel (45) are activated, the micro valves in the upper-stage channels of the two distribution channels (46) are further activated, and further liquid drops (5) are blocked from flowing into the two delivery channels (411) which are completely distributed; analogizing in sequence until all the micro valves are activated, and judging that the filling of all the micro reaction cavities (412) in the micro reaction cavity area (41) is finished;
8) And supplying oil-phase flushing liquid into all the conveying channels (411) through the flushing layer (2), flushing residual liquid drops in the conveying channels (411) until all the strip electrodes (433) are not conducted any more, stopping supplying the flushing liquid, and completing the liquid drop distribution process.
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