CN109746063B - Micro droplet detection system - Google Patents
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- CN109746063B CN109746063B CN201711074989.0A CN201711074989A CN109746063B CN 109746063 B CN109746063 B CN 109746063B CN 201711074989 A CN201711074989 A CN 201711074989A CN 109746063 B CN109746063 B CN 109746063B
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Abstract
本发明提供一种微液滴检测系统，所述微液滴检测系统包括第一部件、第二部件和第三部件；所述第一部件是微液滴检测时用于间隔油和上浮油的加样以及检测后废液收集的装置；所述第二部件是微液滴检测系统，所述微液滴检测系统包括有一中心孔，所述中心孔用于所述微液滴检测系统制备过程中注入注塑料以及批量化生产过程中和/或批量化荧光检测过程中微液滴检测系统的转运；和所述第三部件是装有微液滴进行微液滴PCR扩增的容器。该微液滴检测系统，可以快速、可靠、便捷、以及无污染的检测微液滴样品的荧光信号；该微液滴检测系统的材料和加工成本低，有利于临床检测和其他生物检测的广泛应用。
The present invention provides a micro-droplet detection system, the micro-droplet detection system comprises a first component, a second component and a third component; the first component is a device for adding spacer oil and floating oil during micro-droplet detection and collecting waste liquid after detection; the second component is a micro-droplet detection system, the micro-droplet detection system comprises a central hole, the central hole is used for injecting injection plastic during the preparation process of the micro-droplet detection system and for transporting the micro-droplet detection system during batch production and/or batch fluorescence detection; and the third component is a container containing micro-droplets for micro-droplet PCR amplification. The micro-droplet detection system can detect the fluorescence signal of micro-droplet samples quickly, reliably, conveniently and without pollution; the material and processing costs of the micro-droplet detection system are low, which is conducive to the wide application of clinical detection and other biological detection.
Description
Technical Field
The invention relates to the technical field of micro-droplet digital PCR, in particular to a micro-droplet detection system.
Background
The micro-droplet digital PCR technique (droplet DIGITAL PCR, DDPCR) is a nucleic acid absolute quantitative analysis technique based on single molecule PCR. The micro-droplet digital PCR technology is becoming the next revolutionary technology in the industry with the advantage of high sensitivity and high accuracy. In recent years, with the development of micro-nano manufacturing technology and micro-fluidic technology (micro-nanofabrication and microfluidics), micro-droplet digital PCR technology encounters an optimal opportunity to break through the technical bottleneck. The technology generates liquid drops with diameters of several micrometers to hundreds of micrometers by means of a microfluidic chip; the micro-droplets wrap single molecules or single cells, so that the reaction and detection are fully closed and fully integrated. The working principle of the micro-droplet digital PCR system is as follows: firstly, a sample to be detected is uniformly divided into a large number of nano-scaled (diameter is several micrometers to hundreds of micrometers) water-in-oil micro-droplets by a special micro-droplet generator, and the number of the micro-droplets is in the millions. Because the number of the micro-droplets is enough, the micro-droplets are mutually isolated by an oil layer, each micro-droplet is equivalent to a micro-reactor, and only DNA single molecules of a sample to be detected are contained in the micro-droplets; these micro-droplets were then transferred to an EP tube and reacted in a conventional PCR instrument. The micro-droplets subjected to PCR amplification reaction are detected by a special micro-droplet analyzer one by one, and the droplets with fluorescent signals are interpreted as 1 and the droplets without fluorescent signals are interpreted as 0. Finally, the target DNA molecule number of the sample to be detected can be obtained according to the Poisson distribution principle and the number and proportion of the positive microdroplets, so that absolute quantification of the nucleic acid sample is realized. One key step of the micro-droplet digital PCR technology is to rapidly and reliably judge the identification of fluorescent signals of micro-droplet samples and count the number and proportion of positive micro-droplets.
Determination of fluorescent signals of a sample of microdroplets depends on a core technique: the design and processing of the micro-droplet fluorescence detection device utilizes the fluorescence signal of the product in the laser excited micro-droplet to distinguish the negative micro-droplet and the positive micro-droplet. The conventional flow of the microdroplet digital PCR technique is: the resulting microdroplets were transferred to an EP tube and reacted in a conventional PCR instrument. The micro-droplets subjected to PCR amplification reaction are injected into a micro-droplet fluorescence detection device, and are matched with a special micro-droplet analyzer to carry out fluorescence signal detection. The micro-droplet fluorescence detection device is widely applied and clinically detected, and the micro-droplet fluorescence detection device is required to have the following principles: (1) The EP tube containing the micro-droplets subjected to PCR amplification reaction is hermetically connected with the micro-droplet fluorescence detection device, so that the transfer is not needed, and the possible cross contamination is reduced; (2) The micro-droplet is injected into the micro-droplet fluorescence detection device under the action of a special micro-droplet analyzer; the micro liquid drops flow through the laser detection area, are arranged in a single row in order, so that the accurate detection of fluorescent signals is facilitated; (3) The detected micro-droplets are collected in a closed storage container, so that possible cross contamination is reduced; (4) The micro-droplet fluorescence detection device is disposable, the material and processing cost is low, and (5) the micro-droplet fluorescence detection device is convenient to operate. Aiming at the principle, the micro-droplet detection system based on the micro-fluidic technology has a great application prospect.
Currently, polydimethylsiloxane (PDMS) based microfluidic chips have been widely used for detecting micro-droplets. First, researchers process PDMS microdroplet chips with a micrometer scale using a soft lithography process (manual operation). And after the PDMS micro-droplet chip is successfully prepared, punching holes are formed in a sample inlet and a micro-droplet generation outlet of the PDMS micro-droplet chip by using a machining process, and assembling a sample inlet pipe and a sample outlet pipe. The "oil phase" sample in the EP tube, the "microdroplet" sample, was manually aspirated into the syringe. Then, the "oil phase" sample and the "microdroplet" sample are injected into the PDMS microdroplet chip through the sample injection tube by an external syringe pump. In a pre-designed flow channel area, the optical detection system detects fluorescent signals of the liquid drops one by one. Finally, the detected microdroplets are collected via a sample tube into a conventional assay consumable, such as an EP tube. Although PDMS micro-droplet chip materials have low research and development cost and simple laboratory processing technology, the defects include:
(1) The PDMS microdroplet detection system is in open connection with the EP tube containing the "microdroplet" sample, which is prone to cross contamination.
(2) The detected micro-droplets were collected in an open EP tube and cross-contamination was easily caused.
(3) PDMS is a thermo-elastic polymer material, and the material is not suitable for industrial injection molding and packaging processes. The reliability of the hand-processed PDMS microdroplet chip is poor. The batch processing cost of PDMS micro-droplet chip is high.
(4) The PDMS micro-droplet chip sample injection and droplet collection are manual operation procedures with complicated processes, and are not suitable for clinical examination application.
Aiming at the defects of the PDMS micro-droplet chip, a micro-droplet detection system based on a micro-fluidic technology is designed and processed. The micro-droplet detection system can be used for detecting fluorescent signals of micro-droplet samples rapidly, reliably, conveniently and pollution-free; the micro-droplet detection system has low material and processing cost and is favorable for wide application of clinical detection.
Disclosure of Invention
In order to overcome the above-described problems, the present invention provides a micro droplet detection system including a first member, a second member, and a third member; the first component is a device for adding samples of spacer oil and floating oil and collecting waste liquid after detection during micro-droplet detection; the second component is a micro-droplet detection chip, and the micro-droplet detection system comprises a central hole, wherein the central hole is used for injecting plastic material in the preparation process of the micro-droplet detection system and transferring the micro-droplet detection system in the mass production process and/or the mass fluorescence detection process; and the third component is a container filled with micro-droplets for performing micro-droplet PCR amplification; the second component is fixedly connected to the upper surface of the first component; and the third component is detachably and fixedly connected to the lower surface of the first component.
In one embodiment, the first component and the second component are fixedly connected in a sealing manner by a dispensing manner or an ultrasonic welding manner.
In one embodiment, the first and second parts are each formed by integral injection molding.
In one embodiment, the first and second parts are thermoplastic materials, preferably polycarbonate materials, cyclic olefin copolymers or polymethyl methacrylate, polypropylene.
In one embodiment, the first member is provided with a detent for securing the third member.
In one embodiment, the third component comprises a micro-droplet container and a micro-droplet container upper cover, wherein the micro-droplet container is made of a hard plastic material, and the micro-droplet container upper cover is made of a soft plastic material; when the micro-droplet container is fixed, the upper cover of the micro-droplet container is matched with the clamping groove, so that the first component and the third component are detachably and fixedly connected.
In one embodiment, a puncture needle protruding from the lower surface of the first member is provided in the click groove, the puncture needle being hollow; when the first component and the third component are detached and fixed, the puncture needle penetrates through the upper cover of the micro-droplet container.
In one embodiment, the first component is provided with at least one sampling of spacer oil and up-floating oil and a collection unit of post-detection waste liquid, preferably four, eight and twelve; the second component is provided with at least one micro-droplet detection unit, preferably four, eight and twelve; the third component is a kit comprising at least one, preferably four, eight and twelve containers containing microdroplets for microdroplet PCR amplification; and the sampling of each spacer oil and the floating oil and the collection unit of the waste liquid after detection are matched with each corresponding micro-droplet detection unit and each container filled with micro-droplets for performing micro-droplet PCR amplification, so that the detection of the amplified micro-droplets is completed.
In one embodiment, the collection unit for the oil and oil-up sample and the waste liquid after detection comprises an oil-up sample adding groove, an oil-up sample adding through hole, an oil-up sample adding groove and an oil-up sample adding through hole which are arranged on the upper surface above the first component, wherein the oil-up sample adding through hole and the oil-up sample adding through hole are respectively arranged at the bottoms of the oil-up sample adding groove and the oil-up sample adding groove; and an upper floating oil sample injection pipeline connected with the upper floating oil sample injection through hole on the lower surface of the first component, and a spacer oil sample injection pipeline connected with the spacer oil sample injection through hole on the lower surface of the first component.
In one embodiment, a waste liquid tank is provided on the lower surface below the first member for collecting all waste liquid after detection.
In one embodiment, a structure for discharging the residual air in the waste liquid tank is provided below the second member.
In one embodiment, the volumes of the floating oil loading well and the spacer oil loading well are each 1 to 900 microliters, preferably 5 to 500 microliters, and more preferably 100 to 200 microliters.
In one embodiment, the micro-droplet detection unit comprises a micro-droplet detection pipeline, a spacer oil inlet, a spacer oil pipeline, a micro-droplet container, a micro-droplet floating hole and a micro-droplet pipeline; spacer oil enters the spacer oil pipeline from the spacer oil inlet, and micro-droplets enter the micro-droplet pipeline from the micro-droplet container through micro-droplet floating holes; the spacer oil pipeline and the micro-droplet pipeline form a crisscross structure in front of the micro-droplet detection pipeline, so that the spacer oil separates micro-droplets in the micro-droplet pipeline.
In one embodiment, the crisscross configuration is formed by two of the spacer oil lines and one of the micro-droplet lines.
In one embodiment, a flow resistance region of a loop type is arranged in a pipeline behind the oil spacer inlet; and/or a spacer oil filtering area is arranged in the spacer oil pipeline.
In one embodiment, after the spacer oil flows through the loop-shaped flow resistance area, the spacer oil is divided into two paths to enter the two spacer oil pipelines respectively.
In one embodiment, the micro-droplet detection unit further comprises an upper oil-floating inlet, an upper oil-floating pipeline and an upper oil-floating connecting hole, wherein the upper oil-floating is injected from the upper oil-floating inlet, passes through the upper oil-floating pipeline, and enters the micro-droplet container from the upper oil-floating connecting hole, and extrudes micro-droplets in the micro-droplet container, so that the micro-droplets float up into the micro-droplet pipeline through the micro-droplet floating hole.
In one embodiment, an oil-up filter zone is disposed in the oil-up pipeline.
In one embodiment, a positioning auxiliary channel is provided alongside the microdroplet detection circuit.
In one embodiment, the second component is further provided with an observation window, which is used for monitoring the generated micro-droplets in real time in cooperation with an optical system.
In one embodiment, the micro-droplet detection system further comprises a fourth component, and external pressure is applied to the spacer oil and the up-floating oil in the first component by the fourth component.
In one embodiment, the first component and the second component are respectively provided with a positioning hole which is convenient for the fixed connection of the first component and the second component to position.
The micro-droplet detection system of the invention can achieve the following effects: (1) And (3) rapidly, reliably and parallelly detecting fluorescent signals of the micron-sized water-in-oil micro-droplet sample. (2) The first part and the second part of the device are processed by thermoplastic materials (such as PC, COP, PMMA), the material and batch processing cost is low, (3) the detection speed of the micro-droplet sample is high-speed controllable by matching with an external pressure source. (4) The integrated micro-droplet chip is designed, cross contamination is not easy to occur in the whole process, an EP tube for containing a micro-droplet sample after PCR is directly connected with the chip, a cover is not required to be opened, waste liquid after detection can be collected into a waste liquid collecting tank, and cross contamination is not easy to occur in the whole process. And (5) the micro-droplet fluorescence detection chip is convenient to operate. The whole detection process can be conveniently finished only by manually pre-adding the spacing oil and the floating oil and then matching with an optical detection instrument.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments described in the present application, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram of a micro-droplet detection system according to an embodiment of the present invention;
FIG. 2 is a schematic view of a first component configuration of an embodiment of the present invention;
FIG. 3 is a schematic view showing a structure in which a first member is connected to a third member through a detent;
FIG. 4 is a schematic view of a third component configuration of an embodiment of the present invention;
FIG. 5 is a schematic diagram of a second component configuration of an embodiment of the present invention;
FIG. 6 is a schematic view of a spacer oil return flow resistance region according to one embodiment of the present invention;
FIG. 7 is a schematic diagram of a spacer oil pipeline filtration zone according to one embodiment of the present invention;
FIG. 8 is a schematic diagram of a floating oil driven micro-droplet floating structure according to an embodiment of the present invention;
FIG. 9 is a schematic representation of a crisscross configuration of one embodiment of the present invention; and
FIG. 10 is a schematic diagram of a detection pipeline according to an embodiment of the present invention.
Detailed Description
In order that those skilled in the art will better understand the technical solutions of the present application, the present application will be further described with reference to examples, and it is apparent that the described examples are only some of the examples of the present application, not all the examples. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, shall fall within the scope of the application. The application is further described below with reference to the drawings and examples.
Micro-droplet detection system structure
As shown in fig. 1, the micro-droplet generation device of the present invention includes: the device comprises a first component 1, a second component 2 and a third component 3, wherein the first component 1 is a device for adding and collecting the spacer oil and the floating oil and the waste liquid after detection during micro-droplet detection, the second component 2 is a micro-droplet detection system, and the third component 3 is a container for micro-droplet PCR amplification of micro-droplets. The second component 2 is fixedly connected to the upper surface of the first component 1; the third member 3 is detachably and fixedly attached to the lower surface of the first member 1.
As shown in fig. 1, in some embodiments, the micro-droplet generation device of the present invention further comprises a fourth member 4, the fourth member 4 being a spacer oil loading groove and a floating oil loading groove for sealing the first member 1. The fourth part 4 is for example a water-tight rubber gasket, for example made of silica gel, which may be provided with air holes for exerting pressure therethrough, which functions to ensure a crimp tightness between the external pressure source and the first part and to facilitate the application of external pressure.
In some embodiments, as shown in fig. 1, locating holes are machined into the first and/or second components 2 to facilitate mating with one another.
As shown in fig. 2, the first component 1 is to realize the application of spacer oil and floating oil during the detection of micro-droplets and the collection of waste liquid after the detection. As shown in fig. 2, in this embodiment, the first component 1 is provided with 8 sampling units of spacer oil and upper oil slick and collecting units of waste liquid after detection in parallel, from left to right, the sampling units of spacer oil and upper oil slick and the collecting units of waste liquid after detection are arranged on the first component 1 in parallel at equal intervals above the upper surface of the first component 1, and each collecting unit of spacer oil and upper oil slick and each collecting unit of micro liquid drop after detection is matched with each micro liquid drop detecting unit on the second component 2, so as to be used for adding spacer oil and oil sample of the micro liquid drop detecting units and collecting micro liquid drop waste liquid after detection. The upper surface above the first component 1 is provided with an upper floating oil sample adding groove 111, an upper floating oil sample adding through hole 112, a spacer oil sample adding groove 113 and a spacer oil sample adding through hole 114, wherein the upper floating oil sample adding through hole 112 and the spacer oil sample adding through hole 114 are respectively arranged at the centers of the bottoms of the upper floating oil sample adding groove 111 and the spacer oil sample adding groove 113; and an upper floating oil sample injection pipe 115 connected to the lower surface of the first member 1 at the upper floating oil sample injection through hole 112 of the first member 1, and a spacer oil sample injection pipe 116 connected to the spacer oil sample injection through hole 114 of the first member 1.
In some embodiments, the floating oil sampling pipe 115 and the spacer oil sampling pipe 116 are pipes machined on the lower surface above the first component 1, and then encapsulated with an encapsulation sheet to form a closed pipe.
As shown in fig. 3, a clamping groove 117 of the third component 3 is arranged below the upper floating oil sampling pipeline 115 and the spacer oil sampling pipeline 116 on the lower surface of the first component 1, and the clamping groove 117 is detachably and fixedly connected with the third component 3; a puncture needle 118 protruding from the lower surface of the first member 1 is provided in the positioning groove 117 of the first member 1, the puncture needle 118 being hollow, through which the floating oil enters the third member 3.
A waste liquid tank 119 is provided on the lower surface of the first member 1, and is configured to collect all the waste liquid after detection, and to hermetically collect the micro droplets after fluorescence detection.
As shown in fig. 4, the third member 3 includes a micro-droplet container 31 for containing micro-droplets and an upper cover 32 that can be pierced by a piercing needle 118 provided on the first member, the micro-droplet container 31 is typically made of a hard plastic material, for example, the micro-droplet container 31 is a conventional centrifuge tube (EP tube), the upper cover 32 is typically made of a soft plastic material, and piercing by the piercing needle 118 is facilitated, for example, the upper cover 32 is a centrifuge tube cover made of silicone rubber. In some embodiments, each micro-droplet receptacle 31 cooperates with a collection unit for each spacer and up-floating oil loading and post-detection waste liquid of the component 1. In some embodiments, each micro-droplet container 31 is connected together. In the present embodiment, 8 micro droplet containers 31 are connected together, and 8 covers 32 are connected together; they are respectively integrally processed.
As shown in fig. 5, in a standard optical disc having an outer diameter of 118mm and an inner diameter of 22mm, 8 identical microdroplet detection units 21 for parallel fluorescence detection of microdroplets are arranged in one standard optical disc at equal intervals from left to right.
A central hole 22 is provided in the center of the chip, and the central hole 22 is derived from the optical disc processing technology, and is used for injecting plastic and transferring substrates in the mass production process and for transferring micro-droplet detection systems in the mass fluorescence detection process. The traditional round optical disc structure is not easy to position, the chip is processed into an octagonal structure, and two positioning holes 23 are processed, so that the micro-droplet chip is convenient to be matched with related equipment in a positioning way. 4 identical micro-droplet detection units 21 are respectively arranged at equal intervals on two sides of the central hole and are used for detecting micro-droplets in parallel.
As shown in fig. 5, each droplet detection unit 1 includes, from top to bottom: a spacer oil inlet 2111, a flow resistance region 2112 of a loop type, two spacer oil lines 2113, two spacer oil filtration regions 2114, an upper floating oil inlet 2121, one upper floating oil filtration region 2122, an upper floating oil line 2123, an upper floating oil connection hole 2124, a micro-droplet upper floating hole 213, a micro-droplet line 214, a micro-droplet detection line 215, and a micro-droplet waste liquid port 2126; the floating oil connection hole 2124 is a through hole connecting the floating oil pipe 2123 and the third member 3 (not shown below). The micro-droplet detection system shown in fig. 1 corrects the standard structure of a conventional optical disc, so that the space of the optical disc can be utilized to the maximum extent, and the micro-droplet detection channels are arranged in parallel. Meanwhile, a chip processed by a precise injection molding process is combined with the design of a flow resistance area and a filtering area, and uniform micron-sized water-in-oil micro-droplets are rapidly and reliably detected by fluorescence.
In one embodiment, in the 8 droplet detection unit 1 of fig. 5, the distance between the individual spacer oil inlets 111, the distance between the floating oil inlets 121 and the distance between the micro-droplet floating holes 13 are equal, which is equal to the distance between standard eight-channel pipette tips.
As shown in fig. 6, spacer oil is first injected into the spacer oil inlet 2111 using an external air pump or peristaltic pump. In order to precisely control the amount of oil phase sample introduced at intervals, in one embodiment, a flow resistance region 2112 is provided, precisely controlling the amount of oil phase sample introduced at intervals. The spacer oil wets the surface of the polymer material and automatically flows into the micro-pipe through capillary action under the condition that no pressure is applied. In extreme cases, the spacer oil continues to flow under capillary action. The purpose of the design of the loop-shaped flow resistance region 2112 is to precisely control the spacer oil injection amount, and minimize the continuous flow of spacer oil in the micro-channel under capillary action, so that the spacer oil injection amount is controlled only by an external air pump or peristaltic pump.
Then, the spacer oil passes through an oil phase diversion inlet and enters a spacer oil pipeline 2113 with the same design in a split way, and the spacer oil and the floating liquid drop are intersected at a cross part to push the micro liquid drop to move; and the floating liquid drop is extruded to the center of the flow channel by means of the sheath flow effect, so that fluorescent signals in the micro liquid drop can be conveniently detected. As shown in fig. 7, two paths of spacer oil enter each spacer oil filtering area 2114, and the filtering areas 2114 are a group of columnar array structures, and as shown in fig. 7, the columnar array structures are formed by staggered rows of columnar arrays. Impurities (particles, flocked fibers, etc.) present in the spacer oil are blocked at the set of columnar structures, eliminating the effect of the impurities on the fluorescence detection of the droplets.
As shown in fig. 8, the floating oil flows into the floating oil pipe 2123 from the floating oil inlet 2121 under the action of the external air pressure, flows down at the floating oil connecting hole 2124, flows into the third member 3 (not shown below), and the micro-droplets in the third member 3 float up from the micro-droplet floating holes 13 under the action of the floating oil. The third member 3 is placed under the upper oil bath 2124 and the micro-droplet floating hole 213, and the pitch between the upper oil bath 2124 and the micro-droplet floating hole 213 is designed with reference to the size of the third member 3, and if the third member 3 is an EP pipe, the pitch between the upper oil bath 2124 and the micro-droplet floating hole 213 is smaller than the width of the EP pipe.
As shown in fig. 9, two spacer oil lines 2113 and one micro-droplet line 214 form a crisscross structure in order to space the floating closely arranged micro-droplets by spacer oil on both sides and to reduce signal interference between the micro-droplets; meanwhile, the distance between the micro-droplet monolayer arrangement can be controlled by controlling the air pressure of the spacer oil at two sides through the design of the cross structure. In the micro-droplet detection pipeline behind the cross structure, after the micro-droplets are separated, the flowing through of the straight-line-shaped discharge flow is optically detected.
After the cross structure shown in fig. 10, the micro-droplets are arranged at equal intervals in a single row of the micro-droplet detecting lines, and a closed auxiliary channel 2151 is provided at a position of a fixed interval beside the micro-droplet detecting line to be detected. No oil phase, water phase and micro-droplet sample flow in the closed channel, and the imaging, light transmission/light scattering properties of the region are stable, so that the light path detection system is conveniently positioned at the center of the micro-droplet detection pipeline 215.
Following detection of a plurality of microdroplets, the detected microdroplets flow out of the microdroplet waste port 216 into an external closed collection tank inlet. The purpose of the collection tank closure is to prevent cross-contamination by micro-droplet waste. As shown in fig. 5, in some embodiments, a collection tank vent inlet 2171, vent line 2172, and vent outlet 2173 are provided in the microdroplet detection system, the vent inlet 2171 being connected to the closed collection tank outlet and connected to the vent outlet 2173 via the vent line 2172 in order to relieve pressure in the collection tank from the continuous inflow of liquid while reducing cross-contamination.
2. Micro-droplet detection process
The micro-droplet detection working process of the invention is as follows. First, the generated microdroplets are collected in a microdroplet container 31, such as an EP tube. Then, the EP tube was fitted with a closed EP tube cap 32, and placed in a conventional PCR apparatus to perform PCR amplification reaction. Then, the third member containing the amplified sample is assembled and connected with the first member 1 through the detent 117, and the puncture needle 118 completes the puncture of the upper cover 32 during the assembly and connection.
The floating oil is preliminarily loaded in the floating oil loading tank 111 of the first member 1. Under the action of external pressure, the floating oil flows into the floating oil sample injection pipeline 115 from the sample injection through hole 112, then enters the floating oil filtering region 2122, the floating oil pipeline 2123 and the floating oil connecting hole 2124 from the floating oil inlet 2121, and then enters the micro-droplet container 31 through the puncture needle 118. Since the density of the oil is greater than the density of the droplets in the droplet receptacle 31, the droplets are always above the liquid surface.
The oil up line 1222 then passes through the oil up hole 224 and the hollow of the spike 118 into the micro-droplet container 31. The first component 1 is hermetically connected with the upper cover 32 of the third component 3, and the upper oil floating hole 224 is arranged at the hollow part corresponding to the puncture needle 118; the oil can only flow from the hollow of the puncture needle 118 into the third member 31. Since the density of the oil is greater than the density of the droplets in the third part 331, the droplets are always above the liquid level. In the process that the puncture needle 118 punctures the upper cover 32, a gap is formed around the puncture needle 118 penetrating through the upper cover 32, and the micro-droplets in the third component 3 float upwards through the gap around the puncture needle 118 under the action of the floating oil, and as the first component 1 is tightly connected with the second component 2, the floating droplets can only enter the micro-droplet floating holes 213 on the second component 2 and then enter the micro-droplet pipeline 214.
The spacer oil passes through an oil phase diversion inlet and enters a spacer oil pipeline 2113 with the same design in a split way, and the spacer oil and the floating liquid drop are intersected at a cross part to push the micro liquid drop to move; and the floating liquid drop is extruded to the center of the flow channel by means of the sheath flow effect, so that fluorescent signals in the micro liquid drop can be conveniently detected. Two paths of spacer oil respectively enter a spacer oil filtering area 2114, and the filtering area 2114 is of a group of columnar array structures, and the columnar array structures are formed by staggered rows of columnar arrays. Impurities (particles, flocked fibers, etc.) present in the spacer oil are blocked at the set of columnar structures, eliminating the effect of the impurities on the fluorescence detection of the droplets.
Two spacer oil lines 2113 and one micro-droplet line 214 form a crisscross structure in order to space the floating closely arranged micro-droplets by the spacer oil on both sides and to reduce signal interference between the micro-droplets; meanwhile, the distance between the micro-droplet monolayer arrangement can be controlled by controlling the air pressure of the spacer oil at two sides through the design of the cross structure. In the micro-droplet detection pipeline behind the cross structure, after the micro-droplets are separated, the flowing through of the straight-line-shaped discharge flow is optically detected.
After the cross structure, the micro drops are arranged at equal intervals in a single row of the micro drop detection pipeline, and a closed auxiliary channel 2151 is arranged at a position with fixed intervals beside the micro drop detection pipeline to be detected. No oil phase, water phase and micro-droplet sample flow in the closed channel, and the imaging, light transmission/light scattering properties of the region are stable, so that the light path detection system is conveniently positioned at the center of the micro-droplet detection pipeline 215.
Following detection of a plurality of microdroplets, the detected microdroplets flow out of the microdroplet waste port 216 into an external closed collection tank inlet. The purpose of the collection tank closure is to prevent cross-contamination by micro-droplet waste.
It is to be understood that this invention is not limited to the particular methodology, protocols, and materials described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.
Those skilled in the art will also recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are also encompassed by the appended claims.
Claims (23)
1. A micro-droplet detection system, characterized by:
The micro-droplet detection system includes a first component, a second component, and a third component;
The first component is a device for adding samples of spacer oil and floating oil and collecting waste liquid after detection during micro-droplet detection;
The second component is a micro-droplet detection chip, and the micro-droplet detection system comprises a central hole, wherein the central hole is used for injecting plastic material in the preparation process of the micro-droplet detection system and transferring the micro-droplet detection system in the mass production process and/or the mass fluorescence detection process;
And the third component is a container filled with micro-droplets for performing micro-droplet PCR amplification;
The second component is fixedly connected to the upper surface of the first component; and the third component is detachably and fixedly connected to the lower surface of the first component;
The first component is provided with at least one sampling unit for adding spacer oil and floating oil and collecting waste liquid after detection;
The collection unit of the oil removal and oil removal waste liquid after detection comprises an oil removal and sample addition groove, an oil removal and sample addition through hole, an oil removal and sample addition groove and an oil removal and sample addition through hole which are arranged on the upper surface above the first component, wherein the oil removal and sample addition through hole and the oil removal and sample addition through hole are respectively arranged at the bottom of the oil removal and sample addition groove and the oil removal and sample addition through hole which are connected with each other at the upper oil removal and sample addition through hole on the lower surface of the first component, and the oil removal and sample addition through hole is connected with the oil removal and sample addition through hole on the lower surface of the first component;
The micro-droplet detection system further comprises a fourth component, wherein the fourth component is a spacer oil sample adding groove and an upper floating oil sample adding groove for sealing the first component, and external pressure is applied to the spacer oil and the upper floating oil in the first component through the spacer oil sample adding groove and the upper floating oil sample adding groove;
and positioning holes which are convenient for the fixed connection of the first component and the second component to position are also respectively arranged on the first component and the second component.
2. The micro-droplet detection system of claim 1, wherein: the first component and the second component are fixedly connected in a sealing way through a dispensing way or an ultrasonic welding way.
3. The micro-droplet detection system of claim 1, wherein: the first part and the second part are respectively formed by integral injection molding.
4. The micro-droplet detection system of claim 1, wherein: the first and second components are thermoplastic materials.
5. The micro-droplet detection system of claim 4, wherein: the thermoplastic material is polycarbonate material, cycloolefin copolymer, polymethyl methacrylate or polypropylene.
6. The micro-droplet detection system of claim 1, wherein: the first component is provided with a clamping groove for fixing the third component.
7. The micro-droplet detection system of claim 6, wherein: the third component comprises a micro-droplet container and a micro-droplet container upper cover, wherein the micro-droplet container is made of hard plastic materials, and the micro-droplet container upper cover is made of soft plastic materials; when the micro-droplet container is fixed, the upper cover of the micro-droplet container is matched with the clamping groove, so that the first component and the third component are detachably and fixedly connected.
8. The micro-droplet detection system of claim 7, wherein: a puncture needle protruding from the lower surface of the first member is provided in the click groove, the puncture needle being hollow; when the first component and the third component are detached and fixed, the puncture needle penetrates through the upper cover of the micro-droplet container.
9. The micro-droplet detection system of claim 1, wherein:
the second component is provided with at least one micro-droplet detection unit;
The third component comprises at least one container filled with micro-droplets for performing micro-droplet PCR amplification;
And the sampling of each spacer oil and the floating oil and the collection unit of the waste liquid after detection are matched with each corresponding micro-droplet detection unit and each container filled with micro-droplets for performing micro-droplet PCR amplification, so that the detection of the amplified micro-droplets is completed.
10. The micro-droplet detection system of claim 9, wherein:
the sample adding and floating oil collecting units of the spacer oil and the floating oil are four, eight or twelve;
the micro-droplet detection units are four, eight or twelve;
The containers for carrying out micro-droplet PCR amplification are four, eight or twelve.
11. The micro-droplet detection system of claim 1, wherein: a waste liquid tank is arranged on the lower surface below the first component and is used for collecting all waste liquid after detection.
12. The micro-droplet detection system of claim 11, wherein: and a structure for discharging the residual air in the waste liquid tank is arranged below the second component.
13. The micro-droplet detection system of claim 11, wherein: the volumes of the floating oil sample adding groove and the interval oil sample adding groove are respectively 1-900 microliters.
14. The micro drop detection system of claim 13, wherein: the volumes of the floating oil sample adding groove and the interval oil sample adding groove are 5-500 microliters respectively.
15. The micro drop detection system of claim 14, wherein: the volumes of the floating oil sample adding groove and the interval oil sample adding groove are respectively 100-200 microliters.
16. The micro-droplet detection system of claim 9, wherein: the micro-droplet detection unit comprises a micro-droplet detection pipeline, a spacer oil inlet, a spacer oil pipeline, a micro-droplet container, a micro-droplet floating hole and a micro-droplet pipeline; spacer oil enters the spacer oil pipeline from the spacer oil inlet, and micro-droplets enter the micro-droplet pipeline from the micro-droplet container through micro-droplet floating holes; the spacer oil pipeline and the micro-droplet pipeline form a crisscross structure in front of the micro-droplet detection pipeline, so that the spacer oil separates micro-droplets in the micro-droplet pipeline.
17. The micro drop detection system of claim 16, wherein: and forming the crisscross structure through two interval oil pipelines and one micro-droplet pipeline.
18. The micro drop detection system of claim 16, wherein: a loop-shaped flow resistance area is arranged in the pipeline behind the oil spacer inlet; and/or a spacer oil filtering area is arranged in the spacer oil pipeline.
19. The micro drop detection system of claim 18, wherein: after flowing through the loop-shaped flow resistance area, the spacer oil is divided into two paths and enters the two spacer oil pipelines respectively.
20. The micro drop detection system of claim 16, wherein: the micro-droplet detection unit further comprises an upper floating oil inlet, an upper floating oil pipeline and an upper floating oil connecting hole, wherein upper floating oil is injected from the upper floating oil inlet, passes through the upper floating oil pipeline, enters the micro-droplet container from the upper floating oil connecting hole, and extrudes micro-droplets in the micro-droplet container, so that the micro-droplets float upwards through the micro-droplet floating hole and enter the micro-droplet pipeline.
21. The micro drop detection system of claim 20, wherein: an upper floating oil filtering area is arranged in the upper floating oil pipeline.
22. The micro drop detection system of claim 16, wherein: and a positioning auxiliary channel is arranged beside the micro-droplet detection pipeline.
23. The micro drop detection system according to any one of claims 1-22, wherein: and the second component is also provided with an observation window for matching with an optical system to monitor the generated micro-droplets in real time.
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
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| CN201711074989.0A CN109746063B (en) | 2017-11-06 | 2017-11-06 | Micro droplet detection system |
| PCT/CN2018/113852 WO2019086019A1 (en) | 2017-11-06 | 2018-11-03 | Droplet detection apparatus |
| EP18872941.2A EP3674393A4 (en) | 2017-11-06 | 2018-11-03 | Droplet detection apparatus |
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| CN201711074989.0A CN109746063B (en) | 2017-11-06 | 2017-11-06 | Micro droplet detection system |
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| CN109746063B true CN109746063B (en) | 2024-05-24 |
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| CN113846002B (en) * | 2021-10-15 | 2023-06-06 | 西安交通大学 | Totally-enclosed pollution-free digital nucleic acid detection chip and detection method |
| CN114196741B (en) * | 2021-11-20 | 2023-12-29 | 新羿制造科技（北京）有限公司 | Digital PCR method of integrated micro-droplet chip |
| CN114085762B (en) * | 2021-11-20 | 2025-02-28 | 新羿制造科技（北京）有限公司 | Integrated digital PCR instrument and control method thereof |
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