CN111841669B - 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 PDF

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Publication number
CN111841669B
CN111841669B CN202010568460.XA CN202010568460A CN111841669B CN 111841669 B CN111841669 B CN 111841669B CN 202010568460 A CN202010568460 A CN 202010568460A CN 111841669 B CN111841669 B CN 111841669B
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channel
distribution
micro
channels
electrode
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CN111841669A (en
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吴骁伟
王麒
尹琎
旷婉
龚勋
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Tongji Medical College of Huazhong University of Science and Technology
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Tongji Medical College of Huazhong University of Science and Technology
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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
    • B01L3/502769—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
    • 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
    • B01L3/502738—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
    • 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
    • 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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0887—Laminated structure

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Dispersion Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Hematology (AREA)
  • Clinical Laboratory Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

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, 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 completely filled, correspondingly activates a micro valve in a distribution channel corresponding to the conveying channel, blocks continuous flow of liquid drops into 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 grade branch channels can be activated step by step to counteract the high cavity rate caused by channel non-uniformity caused by chip manufacturing process limitation and the distribution rate which is reduced.

Description

PCR chip for microorganism detection and liquid drop distribution method based on PCR chip
Technical Field
The invention relates to a microfluidic chip, in particular to a PCR chip for microorganism detection and a liquid drop distribution method based on the PCR chip.
Background
Polymerase Chain Reaction (PCR) detection is a technique for detecting a target nucleic acid by amplifying a specific DNA fragment, which enables accurate quantitative detection based on an extremely small amount of a sample, and is an important biological detection means at present. At present, a PCR chip combining a PCR technology and a microfluidic device is a research hot spot in the industry, and has the advantages of small required sample and reagent amount, high detection speed, large number of reaction units, high accuracy and the like.
One major difference of the PCR chip compared to the conventional PCR implementation is that the former mainly uses a single micro-droplet as one micro-reaction unit, and since the micro-droplet is small in size, 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, the fluorescence detection is carried out on the micro-droplet array area after the amplification reaction, then the proportion of micro-droplet units with fluorescent signals and without fluorescent signals is obtained by adopting a statistical method, 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 micro-droplet units with fluorescent signals and without fluorescent signals. The accuracy of this ratio depends on the exact distribution of the droplets as reaction units in the reaction zone. In practical application, such requirements are often difficult to meet. Taking a nested PCR chip as an example, micro-droplets need to be uniformly distributed into a plurality of micro-reaction cavities, which requires that each reaction cavity contains one droplet and cannot have a cavity; at the same time, there should be no residual droplets in the space of the array region other than the reaction chamber.
The existing liquid drop distribution means comprise two types of parallel connection type and serial connection type, wherein the cavity rate in the distribution process is lower due to the adoption of a single distribution path in the serial connection type distribution scheme, but the scheme takes too long time to complete liquid drop distribution of all micro-reaction cavities, which is not beneficial to realizing rapid detection; however, although the parallel distribution is fast, due to the limitation of the chip preparation level, the consistency of a plurality of parallel distribution channels is generally difficult to ensure, so that the actual liquid drop distribution amount in each parallel channel is not balanced, and therefore, a very high cavity rate often occurs when the distribution scheme is adopted, and the error of a detection result is larger. Therefore, there is a need to develop a technical means capable of rapidly and accurately performing micro-droplet dispensing.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a PCR chip for detecting microorganisms. The PCR chip can realize high distribution rate of parallel distribution and low cavity rate of serial distribution scheme at the same time; meanwhile, the PCR chip can also 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 microorganism detection, which comprises a cover plate layer 1 and a structural layer 4; wherein the structural layer 4 comprises a droplet dispensing zone 42, a micro-reaction chamber zone 41 and an outlet zone 43; the drop dispensing zone 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 dispensing channel 46 connected to the ends of the connecting channels 45; the micro-reaction chamber 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; also comprises a plurality of micro-reaction chambers 412 alternately arranged at two sides of the conveying channel 411 and used for accommodating micro-droplets 5; the outlet zone 43 comprises the same number and one-to-one correspondence of discharge channels 415 as the delivery channels 411; at least the portion of the cover sheet 1 corresponding to the micro-reaction chamber area 41 is made of transparent material.
Preferably, the microreaction chamber 412 is sized to receive only a single droplet 5, with both the front side wall 414 (the side wall that is closest to the direction of liquid flow) and the rear side wall being sloped at an acute angle, preferably no more than 30 degrees, relative to the direction of liquid flow within the transport channel 411.
Preferably, a flow blocking protrusion 413 extending into the conveying channel 411 is disposed on a rear sidewall of each micro reaction chamber 412; the minimum distance of the flow-blocking projections 413 from the opposite side wall of the conveying channel 411 is smaller than the diameter of the droplet 5, so that the droplet 5 needs to be deformed to pass the flow-blocking projections 413; the flow blocking protrusions 413 can block the liquid drops 5 in the conveying channels 411, so that the liquid drops are preferably filled in the micro-reaction cavities 412 of which the front ends of the corresponding flow blocking protrusions 413 are not filled yet; since the micro-reaction chamber 412 is only sized to accommodate a single droplet 5, the droplet 5 is forced to deform to a certain extent by the extrusion of the continuous phase fluid when passing the corresponding flow-blocking protrusion 413 of the filled micro-reaction chamber 412, and then passes through the flow-blocking protrusion 413 and continues to be conveyed downstream. The provision of the flow-blocking projections 413 ensures efficient filling of the several micro-reaction chambers 412.
Preferably, the vertical side wall of the flow-blocking protrusion 413 in the conveying passage 411 is curved, and the curvature of the upstream side is greater than that of the downstream side, so that the droplet 5 is prevented from being torn due to touching a sharp surface at the flow-blocking protrusion 413 or due to strong turbulence formed downstream of the flow-blocking protrusion 413.
Preferably, at the droplet dispensing area 42, except for the main channel 44, a lower film electrode 423 is disposed on the bottom surface of each of the connecting channel 45 and the dispensing channel 46 at the inlet thereof, and each of the lower film electrodes 423 is provided with a first contact electrode 421 electrically connected thereto; the corresponding lower film-covered 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 wire 422 located in the channel and the surface of the lower film-coated electrode 423 are covered with a hydrophobic film or a hydrophobic coating.
The PCR chip further 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 region 31 (which can be made of transparent materials or hollow structures) corresponding to the micro-reaction cavity region 41; the control layer 3 is also provided with a dispensing control region 32 corresponding to the dispensing of the droplet dispensing region 42; the distribution control region 32 is provided with the upper film-coated electrodes 323 which are the same in number as the lower control electrodes 423 and are located at the corresponding positions (refer to the same vertical projection positions when the structural layer 4 and the control layer 3 are correctly attached); and second contact electrodes 321 which are the same in number as the first contact electrodes 421 and are in corresponding positions; the upper film electrode 323 is not communicated with the second contact electrode 321, and each second contact electrode 321 is connected with one pole of an external power supply through an independent contact lead 322; each of the upper film covered electrodes 323 is connected to the other pole of the external power supply through a separate film covered lead 324. Therefore, when the structural layer 4 and the control layer 3 are properly bonded, the upper and lower film electrodes 323 and 423 corresponding to each other can be energized by the independent film-coated lead 324 and the contact lead 322, respectively, so that the wettability of the surfaces of the pair of film-coated electrodes to the droplet 5 can be changed. When a pair of covered electrodes is energized, the droplet 5 flowing therethrough will be trapped by the wettable covered electrode surface, thereby forming a microvalve that prevents further flow of the droplet 5 into the lower channel or the corresponding transport channel 411.
The outlet area 43 of the structural layer 4 further comprises strip electrodes 433 disposed on both sidewalls 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 through a second connection line 432; wherein the strip electrode 433 is exposed in the discharge passage 415; correspondingly, the control layer 3 is provided with an outlet detection area 33 corresponding to the outlet area 43; the outlet detection area 33 includes the same number of fourth contact electrodes 331 as the third contact electrodes 431 and at corresponding positions; each of the fourth contact electrodes 331 is connected to an external power source through a separate detection lead 332.
The above-described structural layer 4 and control layer 3 allow at least the following droplet control logic to be executed. After the structural layer 4 and the control layer 3 are properly attached, an external power supply is used for electrifying 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 432, when the liquid drops 5 of the water phase do not fill all the micro-reaction cavities 412 in the corresponding conveying channels 411, only the oil continuous phase passes through the discharge channel 415, and the circuit where the two strip electrodes 433 are positioned cannot be conducted; conversely, when all the reaction chambers 412 in the corresponding transport channels 411 have been filled, the droplets 5 with aqueous phase will flow out of the discharge channels 415, and at this time, the circuit in which the strip electrodes 433 are located will be turned on, so that an electrical signal of interval can be output outwards, and the occurrence of the electrical signal of interval means that the corresponding transport channels 411 have completed filling of droplets.
At this time, the logic controller energizes the upper and lower membrane electrodes 323 and 423 in the corresponding dispensing channels 46 of the delivery channel 411 to activate the micro-valves in the corresponding dispensing channels 46, preventing the droplets 5 from continuing to flow into the completed droplet-filled delivery channel 411; at this point, the droplet 5 will preferentially flow into the other dispensing channels 46 where the microvalve has not yet been activated, thereby increasing the overall dispensing efficiency and reducing the ineffective droplet dispensing process. When the micro valves in the two distribution channels 46 connected to the same connection channel 45 are activated, the micro valves in the upper channels of the two distribution channels 46 are further activated (i.e. the upper and lower membrane-covered electrodes in the upper channels are electrified), so as to further block the liquid drops 5 from flowing into the two water delivery channels 411 after the liquid drops are distributed; and so on until all of the micro-valves are activated, it is perceived that the filling of all of the micro-reaction chambers 412 in the micro-reaction chamber area 41 has been completed.
Preferably, the PCR chip further comprises a rinse layer 2 attached to the upper surface of the control layer 3; the rinse layer 2 has a rinse liquid channel corresponding to the distribution zone 42 of the structural layer 4, the rinse liquid channel comprising a rinse trunk 21, several levels of rinse connection channels 22 connected to the ends of the rinse trunk 21, and a rinse distribution channel 23 connected to the ends of the rinse connection channels 22; the number of flushing distribution channels 23 is equal to the number of distribution channels 46 and both are in corresponding positions; the flushing main channel 21, the flushing connection 22 and the flushing distribution channel 23 are all non-penetrating structures; 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; 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, after the micro valve in the distribution channel 46 is activated, to supply the oil-phase flushing liquid into the delivery channel 411 downstream of the distribution channel 46 to flush the liquid droplets 5 remaining in the delivery channel 411.
Preferably, the supply of the oil phase flushing liquid is started after all micro valves are activated.
Compared with the prior art, the invention has the following beneficial effects: 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 simultaneously, the PCR chip further 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, blocks continuous flow of liquid drops into the conveying channel, thereby accelerating liquid drop distribution speed of other channels which are not activated by the micro valve, and the micro valve in different grade branch channels can be activated step by step, further accelerating the flow speed of liquid drops into the conveying channels which are not filled, so that higher distribution speed can be obtained compared with the traditional parallel distribution channels, and the high cavity rate caused by non-uniformity of the channels due to the limitation of the chip manufacturing process and the distribution speed which is reduced can be counteracted; the PCR chip of the invention also comprises a flushing layer which allows oil phase flushing liquid to be supplied to all conveying channels through the flushing liquid channels connected to the downstream of the micro valves after all the micro valves are activated, and residual liquid drops remained in the conveying channels are flushed out of the micro reaction cavity areas, so that the micro reaction cavity areas are provided with single liquid drops independently filled in each micro reaction cavity after the PCR chip is filled, and the accuracy of detection results is ensured.
Drawings
FIG. 1 is a schematic view of a coversheet;
FIG. 2 is a schematic illustration of a rinse layer;
FIG. 3 is a schematic diagram of a control layer;
FIG. 4 is an enlarged view of a portion of the left circular area of FIG. 3;
FIG. 5 is an enlarged view of a portion of the right circular area of FIG. 3;
FIG. 6 is a schematic diagram of a structural layer;
FIG. 7 is an enlarged view of a portion of the left circular area of FIG. 6;
FIG. 8 is an enlarged view of a portion of the right circular area of FIG. 6;
FIG. 9 is a schematic view of the three-dimensional structure of FIG. 8;
FIG. 10 is a schematic illustration of the microreaction chamber region after filling with droplets;
FIG. 11 is an enlarged view of a portion of the circular area of FIG. 10;
fig. 12 is a schematic diagram of the stacking sequence of the layers of the present invention.
In the figure: 1 is a cover sheet layer, 2 is a flushing layer, 21 is a flushing trunk, 22 is a flushing connection channel, 23 is a flushing distribution channel, 24 is a through hole, 3 is a control layer, 31 is a transparent region, 32 is a distribution control region, 321 is a second contact electrode, 322 is a contact lead, 323 is an upper film covered electrode, 324 is a film covered lead, 33 is an outlet detection region, 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 region, 411 is a conveying channel, 412 is a micro-reaction chamber, 413 is a flow blocking protrusion, 414 is a front side wall, 415 is a discharge channel, 42 is a droplet distribution region, 421 is a first contact electrode, 422 is a first wiring, 423 is a lower film covered electrode, 431 is a third contact electrode, 432 is a second wiring, 433 is a strip electrode, 44 is a main channel, 45 is a connection channel, 46 is a distribution channel, and 5 is a droplet.
Detailed Description
In order to further illustrate the inventive concept, the following description of the inventive solution is provided in connection with the accompanying drawings.
Example 1
As shown in fig. 1, 6 to 11, a PCR chip for microorganism detection is provided, which includes a cover sheet 1 and a structural layer 4; wherein the structural layer 4 comprises a droplet dispensing zone 42, a micro-reaction chamber zone 41 and an outlet zone 43; the drop dispensing zone 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 dispensing channel 46 connected to the ends of the connecting channels 45; the micro-reaction chamber 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; also comprises a plurality of micro-reaction chambers 412 alternately arranged at two sides of the conveying channel 411 and used for accommodating micro-droplets 5; the outlet zone 43 comprises the same number and one-to-one correspondence of discharge channels 415 as the delivery channels 411; at least the portion of the cover sheet 1 corresponding to the micro-reaction chamber area 41 is made of transparent material. The microreaction chamber 412 is sized to accommodate only a single microdroplet 5, with both its front side wall 414 (the side wall that is closest to the direction of liquid flow) and its rear side wall being sloped at an acute angle, preferably no more than 30 degrees, relative to the direction of liquid flow within the transport channel 411. A flow blocking 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-blocking projections 413 from the opposite side wall of the conveying channel 411 is smaller than the diameter of the liquid droplets 5.
Example 2
As shown in fig. 3 to 9, unlike in embodiment 1, at the droplet dispensing section 42, except for the main passage 44, each of the connection passage 45 and the dispensing passage 46 is provided with a lower film electrode 423 at the passage bottom surface at the inlet thereof, and each of the lower film electrodes 423 is provided with a first contact electrode 421 electrically connected thereto; the corresponding lower film-covered 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 wire 422 located in the channel and the surface of the lower film-coated electrode 423 are covered with a hydrophobic film or a hydrophobic coating.
The PCR chip further 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 cavity area 41; the control layer 3 is also provided with a dispensing control region 32 corresponding to the dispensing of the droplet dispensing region 42; the distribution control region 32 is provided with upper film coating electrodes 323 which are the same in number as the lower control electrodes 423 and are positioned at corresponding positions; and second contact electrodes 321 which are the same in number as the first contact electrodes 421 and are in corresponding positions; the upper film electrode 323 is not communicated with the second contact electrode 321, and each second contact electrode 321 is connected with one pole of an external power supply through an independent contact lead 322; each of the upper film covered electrodes 323 is connected to the other pole of the external power supply through a separate film covered lead 324. When a pair of covered electrodes is energized, the droplet 5 flowing therethrough will be trapped by the wettable covered electrode surface, thereby forming a microvalve that prevents further flow of the droplet 5 into the lower channel or the corresponding transport channel 411.
The outlet area 43 of the structural layer 4 further comprises strip electrodes 433 disposed on both sidewalls 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 through a second connection line 432; wherein the strip electrode 433 is exposed in the discharge passage 415; correspondingly, the control layer 3 is provided with an outlet detection area 33 corresponding to the outlet area 43; the outlet detection area 33 includes the same number of fourth contact electrodes 331 as the third contact electrodes 431 and at corresponding positions; each of the fourth contact electrodes 331 is connected to an external power source through a separate detection lead 332.
Example 3
As shown in fig. 2 to 4, unlike example 2, the PCR chip further includes a rinse layer 2 attached to the upper surface of the control layer 3; the rinse layer 2 has a rinse liquid channel corresponding to the distribution zone 42 of the structural layer 4, the rinse liquid channel comprising a rinse trunk 21, several levels of rinse connection channels 22 connected to the ends of the rinse trunk 21, and a rinse distribution channel 23 connected to the ends of the rinse connection channels 22; the number of flushing distribution channels 23 is equal to the number of distribution channels 46 and both are in corresponding positions; the flushing main channel 21, the flushing connection 22 and the flushing distribution channel 23 are all non-penetrating structures; 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; 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, after the micro valve in the distribution channel 46 is activated, to supply the oil-phase flushing liquid into the delivery channel 411 downstream of the distribution channel 46 to flush the liquid droplets 5 remaining in the delivery channel 411. The supply of the oil phase flushing liquid is started after all micro valves are activated.
Example 4
There is provided a droplet dispensing method based on the PCR chip of example 3, comprising the steps of:
1) Attaching the cover plate 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 electrode 433 on the side wall of the discharge passage side wall 415, and at this time, the circuit where the strip electrode 433 is located is not turned on;
3) Connecting the main channel 44 to a droplet supply unit, providing droplets 5 to the micro-reaction chamber area 41; the droplets 5 are distributed substantially uniformly through the several stages of connecting channels 45 into a plurality of distribution channels 46;
4) The drops 5 entering the distribution channels 46 then enter the corresponding distribution channels 411 and touch the upstream side arc-shaped side walls of the flow-blocking projections 413, the flow of said drops 5 being blocked and thus preferentially entering the empty micro-reaction chambers 412 in front of the respective flow-blocking projections 413; whereas for the micro-reaction chamber 412 filled with the liquid droplets 5, since the accommodation space thereof is occupied, the liquid droplets 5 are slightly deformed by the extrusion of the oil continuous phase and the flow-blocking projections 413 and pass the corresponding flow-blocking projections 413;
5) Only the continuous oil phase is connected to the corresponding discharge channels 415 before the droplets 5 of aqueous phase have filled all the micro-reaction chambers 412 in the corresponding transport channels 411, so that the circuit in which the two strip electrodes 433 are located is still not conductive; when all the reaction chambers 412 in the corresponding transport channels 411 have been filled, the droplets 5 with aqueous phase flow out of the discharge channels 415, and at this time, the circuit of the strip electrode 433 is turned on, so that an electrical signal with a spacing can be output outwards, and the appearance of the electrical signal with a spacing means that the corresponding transport channels 411 have completed filling the droplets;
6) When receiving the electric signal output by a pair of electrodes 433 in a certain discharge channel 415, the logic controller energizes the upper and lower film-coated electrodes 323 and 423 in the corresponding distribution channel 46 of the discharge channel 415, and activates the micro valve in the corresponding distribution channel 46 to prevent the liquid drop 5 from continuing to flow into the completed liquid drop filled conveying channel 411;
7) When the micro-valves in both of the two distribution channels 46 connected to the same connection channel 45 are activated, the micro-valves in the previous stage channels of the two distribution channels 46 are further activated, thereby blocking the further flow of the liquid droplets 5 into the two completed liquid droplet distribution delivery channels 411; and so on until all micro valves are activated, determining that filling of all micro reaction chambers 412 in the micro reaction chamber area 41 is completed;
8) And the oil-phase flushing liquid is supplied into all the conveying channels 411 through the flushing layer 2, and residual liquid drops in the conveying channels 411 are flushed until all the electrodes 433 are not conducted any more, the supply of the flushing liquid is stopped, and the liquid drop distribution process is completed.
The above embodiments are merely examples of preferred embodiments of the technical concept of the present invention and should not be construed as limiting all possible embodiments of the present invention, which all embodiments obtained by substitution of conventional means by persons skilled in the art without inventive effort fall within the scope of possible embodiments of the present invention, the actual scope of which is defined in the claims.

Claims (4)

1. A PCR chip for microorganism detection comprises a cover plate layer (1) and a structural layer (4); the structural layer (4) comprises a liquid drop distribution area (42), a micro-reaction cavity area (41) and an outlet area (43); the drop dispensing zone (42) comprises a main channel (44), a plurality of stages of connecting channels (45) connected to the end of the main channel (44) and a dispensing channel (46) connected to the end of the connecting channels (45); the method is characterized in that: the micro-reaction chamber area (41) comprises conveying channels (411) the same 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 micro-reaction chambers (412) are alternately arranged at two sides of the conveying channel (411) and are used for accommodating liquid drops (5); the outlet zone (43) comprises discharge channels (415) which are connected in a one-to-one correspondence and are the same number as the conveying channels (411); at least the part of the cover plate layer (1) corresponding to the micro-reaction cavity area (41) is made of transparent materials; the microreaction chamber (412) is sized to accommodate only a single droplet (5), with both the front (414) and rear (rear) side walls being inclined at an acute angle relative to the direction of liquid flow within the delivery channel (411); a flow blocking protrusion (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 projections (413) from the opposite side wall of the conveying channel (411) is smaller than the diameter of the liquid drops (5); at the position of the liquid drop distribution area (42), except for the main channel (44), a lower film coating electrode (423) is arranged on the bottom surface of each connecting channel (45) and each distribution channel (46) at the inlet of the corresponding connecting channel, and each lower film coating electrode (423) is provided with a first contact electrode (421) electrically connected with the corresponding lower film coating electrode; the corresponding lower film-covered 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 part of the first wiring (422) positioned in the channel and the surface of the lower film-covered electrode (423) are covered with a hydrophobic film or a hydrophobic coating; the control layer (3) is 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 cavity area (41); a distribution control area (32) corresponding to the liquid drop distribution area (42) is further arranged on the control layer (3); the distribution control area (32) is provided with upper film coating electrodes (323) which are the same in number as the lower film coating electrodes (423) and are positioned at corresponding positions; and second contact electrodes (321) which are the same in number as the first contact electrodes (421) and are in corresponding positions; the upper film electrode (323) is not communicated with the second contact electrodes (321), and each second contact electrode (321) is connected with one pole of an external power supply through an independent contact lead (322); each upper film covered electrode (323) is connected with the other pole of the external power supply through an independent film covered lead wire (324); a pair of upper and lower membrane-coated electrodes (323, 423) corresponding to each other constitute a micro valve capable of closing a fluid flow path in the channel; the outlet area (43) of the structural layer (4) further comprises strip electrodes (433) arranged on two side walls of the discharge channel (415), and each strip electrode (433) is connected with a third contact electrode (431) positioned on the upper surface of the structural layer (4) through a second wiring (432); the strip electrode (433) is exposed in the discharge channel (415); an outlet detection area (33) corresponding to the outlet area (43) is arranged on the control layer (3); the outlet detection zone (33) comprises the same number of fourth contact electrodes (331) as the third contact electrodes (431) and in corresponding positions; each of said fourth contact electrodes (331) is connected to an external power source by a separate detection lead (332); the angle of the acute angle is not more than 30 degrees; the vertical side wall of the flow blocking protrusion (413) in the conveying channel (411) is curved, and the radian of the upstream side is larger than that of the downstream side.
2. The PCR chip for microbial detection as claimed in claim 1, wherein: the washing layer (2) is attached to the upper surface of the control layer (3); the rinsing layer (2) has rinsing liquid channels corresponding to the droplet distribution areas (42) of the structural layer (4), which at least comprise rinsing distribution channels (23) in a number equal to the number of distribution channels (46) and in a corresponding position thereto; 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).
3. The PCR chip for microbial detection as claimed in claim 2, wherein: a plurality of openings (34) corresponding to the through holes (24) are arranged on the control layer (3); the through-hole (24) corresponds to the downstream of the micro-valve in the distribution channel (46), which allows to supply the oil phase flushing liquid into the delivery channel (411) downstream of the distribution channel (46) after the micro-valve in the distribution channel (46) has been activated.
4. A method of droplet dispensing using the PCR chip of claim 3, wherein: the method comprises the following steps:
1), attaching the cover plate 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 electrode (433) on the side wall of the discharge passage (415), wherein the circuit where the strip electrode (433) is located is not turned on;
3) Connecting the main channel (44) to a droplet supply unit, providing droplets (5) to the micro-reaction chamber area (41); the droplets (5) are distributed substantially uniformly into a plurality of distribution channels (46) via a number of stages of connecting channels (45);
4) The liquid drops (5) entering the distribution channels (46) then enter the corresponding conveying channels (411) and touch the upstream arc-shaped side walls of the flow blocking protrusions (413), and the flow of the liquid drops (5) is blocked, so that the liquid drops enter the empty micro-reaction cavities (412) in front of the corresponding flow blocking protrusions (413) preferentially; whereas for the micro-reaction chamber (412) already filled with droplets (5), since its accommodation space is already occupied, the droplets (5) are slightly deformed under the extrusion of the oil continuous phase and the flow-blocking projections (413) and pass the corresponding flow-blocking projections (413);
5) Only the continuous oil phase is communicated with the corresponding discharge channel (415) before the droplets (5) of aqueous phase have not filled all the micro-reaction chambers (412) in the corresponding delivery channel (411), so that the circuit in which the two strip electrodes (433) are located is still not conducted; when all micro-reaction cavities (412) in the corresponding conveying channels (411) are filled, liquid drops (5) with water phase flow out of the discharge channels (415), at the moment, the circuit where the strip electrodes (433) are positioned is conducted, so that an interval electric signal can be output outwards, and the appearance of the interval electric signal means that the corresponding conveying channels (411) are filled with liquid drops;
6) When receiving the electric signals output by a pair of electrodes (433) in a certain discharge channel (415), the logic controller is used for electrifying an upper film-coated electrode (323) and a lower film-coated electrode (423) in a corresponding distribution channel (46) of the discharge channel (415), so as to activate a micro valve in the corresponding distribution channel (46) and prevent the liquid drops (5) from continuing to flow into the conveying channel (411) with finished liquid drop filling;
7) When the micro-valves in the two distribution channels (46) connected to the same connection channel (45) are activated, the micro-valves in the previous channels of the two distribution channels (46) are further activated, and further the liquid drops (5) are blocked from flowing into the two conveying channels (411) with the liquid drop distribution completed; and so on until all the micro valves are activated, judging that filling of all the micro reaction cavities (412) in the micro reaction cavity area (41) is completed;
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.
CN202010568460.XA 2020-06-19 2020-06-19 PCR chip for microorganism detection and liquid drop distribution method based on PCR chip Active CN111841669B (en)

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