CN113846002B

CN113846002B - Totally-enclosed pollution-free digital nucleic acid detection chip and detection method

Info

Publication number: CN113846002B
Application number: CN202111204315.4A
Authority: CN
Inventors: 彭年才; 梁焱; 赵书豪; 胡飞; 张增明; 姜颖涛; 刘振; 李希晨; 路朝昌; 郭晓牛
Original assignee: Xian Jiaotong University
Current assignee: Xian Jiaotong University
Priority date: 2021-10-15
Filing date: 2021-10-15
Publication date: 2023-06-06
Anticipated expiration: 2041-10-15
Also published as: CN113846002A

Abstract

The invention discloses a totally-enclosed pollution-free digital nucleic acid detection chip, which comprises a chip body, a first puncture needle and a second puncture needle, wherein the chip body is made of transparent materials, a plurality of oil inlet through holes are formed in the chip body, the outlets of the oil inlet through holes are connected with an oil inlet groove, one end opening of the first puncture needle is connected with the oil inlet groove, the other end opening of the first puncture needle extends into a test tube, the test tube is used for receiving an oil phase conveyed by the oil inlet through holes, one end opening of the second puncture needle is connected into a liquid inlet groove formed in an inner cavity of the chip body, the outlet of the liquid inlet groove is connected with a tiling cavity, the bottom plane of the tiling cavity is in the horizontal direction, and the other end opening of the second puncture needle extends into the test tube; the other end opening of the first puncture needle always stretches into the oil phase in the test tube, and when the oil phase is full of the test tube, the other end opening of the second puncture needle contacts with the top liquid level of the oil phase. The invention avoids the risk of cross contamination, and has simple operation and short time consumption.

Description

Totally-enclosed pollution-free digital nucleic acid detection chip and detection method

Technical Field

The invention belongs to the technical field of microfluidic liquid drop digital polymerase chain reaction, and particularly relates to a totally-enclosed pollution-free digital nucleic acid detection chip and a detection method.

Background

With the transition of medical modes and the continuous development of personalized medicine, the medical inspection industry urgently needs a rapid and accurate detection means, and molecular detection brings out unique advantages. At present, the molecular detection technology mainly comprises nucleic acid molecular hybridization, polymerase Chain Reaction (PCR), biochip technology and the like, and the molecular detection product is mainly applied to detection of clinical various departments such as tumor, infection, genetics, prenatal screening and the like, and has important roles in detection of novel coronaviruses in aspects such as physical examination centers, technical service centers, third-party detection mechanisms, fast detection markets of microorganisms and the like. The Digital PCR (dPCR) technique is a technique for amplifying and absolute quantifying nucleic acid molecules, which is based on the single-molecule PCR method for quantifying nucleic acids. The liquid drop digital PCR based on the liquid drop micro-fluidic technology is provided with: the advantages of providing a small volume, high throughput microreactor are more readily addressed.

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; then, PCR amplification reaction was performed on each of these microdroplets, and the fluorescent signal of the droplet was detected one by a microdroplet analyzer, and the droplet with the fluorescent signal interpreted as 1 and the droplet without the fluorescent signal 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. 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.

Currently common instruments such as BIO-RAD QX200 ^TM By combining water-in-oil emulsion droplet technology with microfluidic technology, 20000 homogeneous nanoliter droplets can be generated per sample by means of a droplet generator, the fragments of interest and the background sequences being randomly distributed in the droplets, each droplet being a separate reactor. After PCR using a thermal cycler, the QX200 droplet analyzer will analyze droplets in each sample individually. After the droplets are sucked, the droplets sequentially pass through a bicolor optical detection system under the action of sheath fluid oil. The system will count the number of positive and negative droplets to perform absolute quantitative analysis of the target DNA in a digital format.

For micro-droplet observation of a digital PCR detector, a droplet array microscopic imaging mode is adopted, so that the forms of all droplets can be clearly obtained, interference factors such as nonspecific amplification and the like are reduced, and the reliability of detection results is improved, but meanwhile, the traditional mode of transferring and tiling generated droplets is that negative pressure or positive pressure is generated by utilizing peripheral driving components or driving structures such as a negative pressure pump and the like, and the operation process is increased, the time is consumed, the possibility of polluting the droplets is also existed, and the inaccuracy of the results is finally caused, so that the design and the manufacture of the integrated detection chip and the system thereof capable of realizing automatic full-closed pollution-free are particularly critical.

Patent document CN109536380a (a droplet microfluidic chip for high-sensitivity detection of nucleic acid and a method for using the same) discloses a droplet microfluidic chip for high-sensitivity detection of nucleic acid and a method for using the same, which are used for separating functional areas and respectively completing three links of digital PCR, droplet preparation, PCR amplification and tiling statistical techniques in independent areas. The liquid drop preparation, PCR amplification and tiling statistical areas are in the same plane, so that the area of the chip is larger, and the detection flux is greatly reduced; the preparation process of the liquid drops is difficult to realize in practice, and the size of the generated liquid drops is not easy to control; the structure of the amplified and transferred part is complex, and strict sealing is difficult to achieve; the oil phase has higher requirement, two oil phases which are mutually insoluble and do not influence subsequent reaction and observation results are needed to be used, so that the experimental difficulty is increased, and the experimental cost is increased.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides a fully-closed pollution-free digital nucleic acid detection chip and a detection method, and solves the problems that the pipetting operation of the existing liquid drops is complex, time is consumed and cross pollution is easy to generate.

In order to achieve the above purpose, the present invention provides the following technical solutions: the utility model provides a totally closed pollution-free digital nucleic acid detection chip, includes chip body, first pjncture needle and second pjncture needle, the chip body is transparent material, be equipped with a plurality of oil feed through-hole on the chip body, the exit linkage oil inlet groove of oil feed through-hole, be connected with the one end opening of first pjncture needle on the oil inlet groove, the other end opening of first pjncture needle stretches into the test tube, the test tube is used for receiving the oil phase that oil feed through-hole carried, the one end opening of second pjncture needle is connected in the feed tank of seting up in chip body inner chamber, the exit linkage of feed tank has the tiling cavity, the bottom plane of tiling cavity is in the horizontal direction, the other end opening of second pjncture needle stretches into in the test tube;

the other end opening of the first puncture needle always stretches into the oil phase in the test tube, and when the oil phase is full of the test tube, the other end opening of the second puncture needle contacts with the top liquid level of the oil phase.

Further, the chip body comprises a first chip, a second chip and a third chip which are sequentially stacked, a first connecting surface on the first chip is used for connecting the second chip, an oil inlet groove and a liquid inlet groove are formed in the first connecting surface, one side wall of the oil inlet groove is communicated to the side wall of an oil inlet through hole, the oil inlet through hole penetrates through the second chip and the third chip, the oil inlet groove is coaxially communicated with an oil outlet through hole, the oil outlet through hole penetrates through the second chip and the third chip, and the oil outlet through hole is in interference fit with the first puncture needle; the liquid inlet tank is characterized in that a smooth flow passage is communicated with one side wall of the liquid inlet tank, an outlet of the smooth flow passage is communicated with a tiling cavity, the diameter from the inlet diameter of the smooth flow passage to the outlet diameter of the smooth flow passage is gradually reduced by 3-5 times of the diameter of the micro-droplet, the liquid inlet tank is coaxially communicated with a liquid inlet through hole, the liquid inlet through hole penetrates through the second chip and the third chip, the liquid inlet through hole is in interference fit with the second puncture needle, and the area of the tiling cavity is 1.2 times of the tiling area of the total micro-droplet in the test tube.

Further, the first chip, the second chip and the third chip are also communicated with an exhaust through hole, the tail end of the flat cavity is communicated to the side wall of the exhaust through hole, and the diameter of the tail end of the flat cavity is smaller than the diameter of the micro liquid drop;

and a waste liquid cavity is arranged in the inner cavity of the third chip, and an inlet of the waste liquid cavity is communicated with one end of the exhaust through hole.

Further, the second connection face on the third chip is used for connecting the test tube, be equipped with the screens thin wall on the second connection face, the screens thin wall is used for the test tube upper cover of block test tube, advance oil through-hole and play oily through-hole and be located the region that the screens thin wall encloses, integrated into one piece has the cap on the second connection face, it has the cap all coaxial coupling on advance oil through-hole and the play oily through-hole, the cap is protruding structure, cap and first pjncture needle and second pjncture needle are interference fit, cap and screens thin wall are the silica gel material.

Furthermore, a tiling cavity supporting column is integrated on the first connecting surface, and the tiling cavity supporting column is contacted with the second chip.

Further, the height of the tiling cavity is 0.5-2 times of the diameter of the micro-liquid drops from the second chip to the first chip;

the inlet diameter of the smooth flow channel is 1mm, the outlet diameter of the smooth flow channel is 0.3mm, the height of the inlet of the smooth flow channel is 0.3mm, and the height of the outlet of the smooth flow channel is 0.1mm.

Furthermore, a plurality of positioning marks are integrated on the first chip, and the positioning marks are positioned between adjacent tiling cavities, and the shapes of the adjacent positioning marks between the same tiling cavities are inconsistent.

Further, the first puncture needle and the second puncture needle are long puncture needles or short puncture needles, and the distance from the opening of the long puncture needles to the opening of the test tube is larger than the distance from the opening of the short puncture needles to the opening of the test tube;

the long puncture needle and the short puncture needle are provided with a first connecting surface, a boss is uniformly formed on the opening at one end of the long puncture needle and the opening at one end of the short puncture needle, and the boss is propped against the wall surface of the first chip.

Further, the oil supply device comprises an oil supply device and a bottom support, wherein an outlet of the oil supply device is connected with a rubber tube, and the rubber tube is in interference fit with the oil inlet through hole;

the bottom support is provided with a test tube groove for placing a test tube, and the bottom support is also integrally formed with a support column;

when the bottom support is placed at the bottom of the chip body, the support column is in contact with the bottom wall surface of the chip body.

The invention also provides a detection method of the totally-enclosed pollution-free digital nucleic acid chip, which comprises the following steps:

the test tube with the micro-droplets is fixed on the chip body, the first puncture needle stretches into the test tube, the oil inlet through hole is connected with the oil phase, the oil phase enters the oil inlet groove through the oil inlet through hole, the oil phase in the oil inlet groove enters the test tube through the first puncture needle, and the micro-droplets float at the liquid level of the oil phase;

when the oil phase is full of the inner cavity of the test tube, the liquid level of the oil phase contacts with the second puncture needle, the oil phase continuously enters the test tube from the first puncture needle, micro-droplets on the liquid level of the oil phase are pushed into the second puncture needle, the micro-droplets sequentially pass through the second puncture needle, the liquid inlet groove and the paving chamber, the micro-droplets continuously enter the paving chamber, the paving is gradually completed, and after the paving is completed, the chip body is moved to the optical detection platform for detection;

and obtaining a detection image through an optical detection platform, splicing and enhancing the plurality of images to obtain the original nucleic acid copy number and the confidence interval, and finishing the detection of the digital nucleic acid chip.

Compared with the prior art, the invention has at least the following beneficial effects:

the invention provides a totally-enclosed pollution-free digital nucleic acid detection chip, transparent materials are used as a chip body, real-time observation is facilitated, oil inlet through holes, oil inlet grooves and first puncture needles are formed in the chip body, oil phases enter a test tube with micro-droplets, the density of the micro-droplets is smaller than that of the oil phases, the micro-droplets are always positioned at the liquid level of the oil phases, therefore, when the oil phases are full of the test tube, the oil phases are continuously filled into the test tube, the micro-droplets on the liquid level of the oil phases are passively pushed into a second puncture needle and enter a tiling cavity through the liquid outlet grooves, tiling is performed in the tiling cavity, and then optical detection operation is performed to obtain fluorescent information of amplified droplets.

Further, the diameter from the inlet to the outlet of the smooth flow channel is uniformly reduced, so that the liquid inlet tank and the paving cavity can be smoothly connected, the liquid drops are ensured to smoothly enter the flow channel, the liquid drops are prevented from being fused due to extrusion collision of the liquid drops in the flow channel, the micro liquid drops are prevented from being broken or seriously deformed, and the quality of the micro liquid drops after liquid transfer is ensured.

Further, the exhaust through holes penetrating through the first chip, the second chip and the third chip are convenient for exhausting gas, and are beneficial to the micro-droplet tiling process.

Further, the clamping thin wall can be more tightly connected with the test tube, the cap can be more tightly connected with the first puncture needle and the second puncture needle, and the clamping and sealing functions are achieved.

Further, the support column of the flat-laying cavity can support the flat-laying cavity, collapse of the hot-press bonding is avoided, and the liquid drop flat-laying effect is guaranteed.

Furthermore, the diameter and the height of the smooth flow channel can be designed to ensure that liquid drops with specific volumes, such as liquid drops with the diameter of 20 um-200 um, are generated, the volumes of micro liquid drops are controllable, and the height of the tiling cavity is 0.5-2 times of the diameter of the micro liquid drops, so that the tiling quality of the micro liquid drops can be ensured.

Furthermore, after the image processing is finished, each picture can be spliced correspondingly through the positioning mark of each tiling cavity, so that the tiling state of the micro liquid drops can be conveniently observed.

Furthermore, the bosses of the long puncture needle and the short puncture needle are beneficial to the oil phase and micro liquid drops to enter the cavity along the pipeline, and the structure is simple, and complex structures and manufacturing processes are avoided.

Further, the support column can support the chip body, so that the level of the chip body is guaranteed, and the quality of micro liquid drops after pipetting is guaranteed.

The invention also provides a detection method of the fully-enclosed pollution-free digital nucleic acid chip, which adopts an integrated design to realize functions of micro-droplet tiling and imaging analysis. After PCR thermal cycle amplification of micro-droplets is completed in a test tube, the micro-droplets are assembled and matched with the chip of the invention, the oil phase is pressed into the oil inlet through hole of the chip, the oil phase enters the test tube through the first puncture needle, the micro-droplets in the test tube float upwards under the buoyancy effect, the micro-droplets enter the tiling cavity of the chip through the second puncture needle, and then flow into the tiling cavity to realize single-layer tiling of the droplets, and then fluorescent information of amplified droplets can be obtained through optical detection operation, so that cross contamination risks possibly brought by uncapping operation are avoided, and the operation is simple.

Drawings

FIG. 1 is a schematic overall flow chart of the present invention;

FIG. 2 is a schematic diagram of the overall structure of the present invention;

FIG. 3 is a schematic diagram of the structure of the chip body;

FIG. 4 is a schematic bottom view of the first chip;

FIG. 5 is a schematic view of the cross-sectional C-C structure of FIG. 4;

FIG. 6 is an enlarged partial schematic view of H of FIG. 5;

FIG. 7 is an isometric view of a second chip;

FIG. 8 is a schematic view of the structure of the first and second puncture needles;

FIG. 9 is a schematic bottom view of the third chip;

FIG. 10 is a schematic view of the cross-sectional structure B-B of FIG. 9;

FIG. 11 is a schematic view of a four-row test tube configuration;

FIG. 12 is a schematic view of the mating structure of the bottom bracket and test tube;

FIG. 13 is a schematic view of the D-D cross-sectional structure of FIG. 12;

fig. 14 is a process diagram of filling the oil phase into the test tube, fig. 14a is a schematic diagram of an initial state of the test tube, fig. 14b is a schematic diagram of a state of filling the oil phase into the test tube, fig. 14c is a schematic diagram of an initial state of the test tube, and fig. 14d is a schematic diagram of a state of filling the oil phase into the test tube;

FIG. 15 is a schematic illustration of tiling of micro-droplets under a 10 objective lens;

fig. 16 is a schematic illustration of tiling of microdroplets under a 5 x objective lens.

In the accompanying drawings: 1-chip body, 1100-first chip, 1110-oil inlet groove, 1120-liquid inlet groove, 1130-smooth runner, 1140-smooth runner outlet, 1150-tiling chamber, 1160-positioning mark, 1170-tiling chamber support column, 1200-second chip, 1210-oil inlet through hole, 1220-oil outlet through hole, 1230-liquid inlet through hole, 1300-third chip, 1310-cap, 1320-clamping thin wall, 1330-waste liquid cavity, 1400-exhaust through hole, 1500-positioning pin through hole, 1600-short puncture needle, 1700-long puncture needle, 1800-first puncture needle, 1900-second puncture needle, 2-test tube upper cover, 3-test tube, 4-bottom support, 401-test tube groove, 402-support column, 5-oil supply device.

Detailed Description

The invention is further described below with reference to the drawings and the detailed description.

As shown in fig. 2 and 3, the invention provides a totally-enclosed pollution-free digital nucleic acid detection chip, which is characterized in that PCR amplification and subsequent detection are independent, the complexity of the chip is reduced, the restriction of the detection link on the reaction flux released by the PCR amplification is avoided, the liquid drop tiling can be quickly and effectively promoted, two oil phases are not needed, the consumption of the chip is less, the cost is lower, and especially the cross contamination risk possibly brought by the uncapping operation is avoided in the pipetting process of the integrated chip.

In the embodiment, the fully-closed pollution-free digital nucleic acid detection chip comprises four parts, namely an oil supply device 5, a micro-droplet tiling and detecting micro-fluidic chip body 1, a test tube 3 loaded with micro-droplets, a fixed test tube 3 and a bottom support 4 for supporting the chip; the chip body 1 includes an oil inlet hole 1210, an oil inlet groove 1110, an oil outlet hole 1220, an oil inlet groove 1120, a cap 1310 and a clamping thin wall 1320 which are matched with the test tube 3, a smooth flow passage 1130, a flat cavity 1150, a flat cavity support column 1170, an exhaust hole 1400, a waste liquid cavity 1330 and the like. The chip body 1 is made of transparent materials, a plurality of oil inlet through holes 1210 are formed in the chip body 1, an outlet of each oil inlet through hole 1210 is connected with an oil inlet groove 1110, one end opening of each first puncture needle 1800 is connected to each oil inlet groove 1110, the other end opening of each first puncture needle 1800 extends into a test tube 3, the test tubes 3 are used for receiving oil phases conveyed by the oil inlet through holes 1210, one end opening of each second puncture needle 1900 is connected into a liquid inlet groove 1120 formed in an inner cavity of the chip body 1, an outlet of each liquid inlet groove 1120 is connected with a flat-laying cavity 1150, the bottom plane of each flat-laying cavity 1150 is in the horizontal direction, and the other end opening of each second puncture needle 1900 extends into each test tube 3;

the other end opening of the first puncture needle 1800 always extends into the oil phase in the test tube 3, and when the oil phase is full of the test tube 3, the other end opening of the second puncture needle 1900 contacts the top liquid surface of the oil phase.

Specifically, the transparent material of the chip body 1 is transparent glass, and may be made of a high polymer material such as polymethyl methacrylate (PMMA) or Polycarbonate (PC). The chip body comprises three layers, namely a first chip 1100, a second chip 1200 and a third chip 1300, wherein the first chip 1100, the second chip 1200 and the third chip 1300 are sequentially laminated, and the first chip 1100 and the second chip 1200 are formed by hot-press bonding after injection molding of polymer materials or are manufactured by transparent glass etching, so that a high-precision cavity and a matching relationship are obtained.

As shown in fig. 4, fig. 5 and fig. 6, specifically, the first chip 1100 is in contact connection with the second chip 1200 through a first connection surface, an oil inlet groove 1110 and an oil inlet groove 1120 are formed on the first connection surface, a side wall of the oil inlet groove 1110 is communicated with a side wall of an oil inlet through hole 1210, wherein the oil inlet through hole 1210 penetrates through the second chip 1200 and the third chip 1300, the oil inlet groove 1110 is coaxially communicated with an oil outlet through hole 1220, the oil outlet through hole 1220 penetrates through the second chip 1200 and the third chip 1300, and the oil outlet through hole 1220 is in interference fit with the first puncture needle 1800; a smooth flow channel 1130 is communicated with one side wall of the liquid inlet tank 1120, an outlet of the smooth flow channel 1130 is communicated with a tiling cavity 1150, the diameter from an inlet diameter of the smooth flow channel 1130 to an outlet 1140 of the smooth flow channel is uniformly reduced by 3-5 times of the diameter of the micro-droplets, the liquid inlet tank 1120 is coaxially communicated with a liquid inlet through hole 1230, the liquid inlet through hole 1230 penetrates through the second chip 1200 and the third chip 1300, the liquid inlet through hole 1230 is in interference fit with the second puncture needle 1900, and the area of the tiling cavity 1150 is 1.2 times of the single-layer tiling area of the total micro-droplets in the test tube 3. The volumes of the oil inlet tank 1110 and the oil inlet tank 1120 can be adjusted according to the size of the tiling chamber 1150 or the array PCR reaction chamber. The diameter of the oil inlet groove 1110 on the first chip 1100 is 3mm, the height is a circular chamber with 0.3mm, the width of the oil inlet through hole 1210 is 1mm, the height is 0.3mm, the liquid inlet groove 1120 is a circular chamber with 3mm diameter and 0.3mm height, the liquid inlet groove 1120 passes through the smooth flow passage 1130, the height of the smooth flow passage 1130 is gradually reduced from 0.3mm to 0.1mm, and the width is also gradually reduced from 1mm to 0.3 mm. The liquid inlet tank 1120 is smoothly connected with the smooth flow passage 1130 entering the flat chamber 1150, the height of the smooth flow passage 1130 is smoothly reduced, micro liquid drops are ensured to smoothly enter the smooth flow passage 1130, liquid drop fusion caused by extrusion collision of the liquid drops in the smooth flow passage 1130 is avoided, micro liquid drops are prevented from being broken or seriously deformed, and the unique design of the smooth flow passage 1130 enables the micro liquid drop generating device to generate micro liquid drops with specific volumes, such as micro liquid drops with diameters of 20um to 200 um.

Further, the height of the tiling chamber 1150 is 0.5 to 2 times the diameter of the micro-droplets, preferably 1.25 times the diameter of the droplets, wherein the area of the tiling chamber 1150 is greater than about 20% of the total area of a single layer of micro-droplets.

In this embodiment, the end of the tiling chamber 1150 is connected to the sidewall of the exhaust through hole 1400, and the exhaust through hole 1400 penetrates the first chip 1100, the second chip 1200 and the third chip 1300, and the exhaust through hole 1400 is used for exhausting gas, which is beneficial to the micro-droplet tiling process. The width of the end flow channel of the tiling chamber 1150 is smaller than the diameter of the micro-droplet, and the end of the flow channel is connected with the exhaust through hole 1400 for exhausting, which is beneficial to smooth tiling process. The inlet of the exhaust through hole 1400 is connected with the upper end of the waste liquid cavity 1330, wherein the waste liquid cavity 1330 is located in the inner cavity of the third chip 1300, the lower surface of the third chip 1300 is hollowed out, the waste liquid cavity 1330 is used for collecting waste liquid, the environment is prevented from being polluted, and the purpose is to release pressure generated by continuously flowing in the liquid collecting and tiling cavity 1150, and meanwhile cross contamination is reduced.

As shown in fig. 7, in the present embodiment, the second chip 1200 is further provided with a positioning pin through hole 1500, and the positioning pin through hole 1500 penetrates the first chip 1100, the second chip 1200 and the third chip 1300, and is used for precise matching during manufacturing of the chip body 1 through the positioning pin through hole 1500.

Specifically, there is the tiling chamber support column 1170 still integrally on the first junction surface, tiling chamber support column 1170 and second chip 1200 contact, and tiling chamber support column 1170 plays the effect of supporting tiling chamber 1150, avoids hot pressing bonding to appear collapsing, guarantees the liquid drop tiling effect.

In this embodiment, positioning marks are disposed between adjacent tile chambers 1150, the positioning marks 1160 and the first chip 1100 are integrally formed, shapes of the adjacent positioning marks 1160 between the same tile chambers 1150 are inconsistent, such as rectangle, triangle, circle or square, when images of each tile chamber 1150 are obtained, the image is spliced by aligning each positioning mark 1160, so that the splicing operation is convenient during image processing.

As shown in fig. 9 and fig. 10, specifically, the third chip 1300 is provided with a second connection surface, the second connection surface is a surface of the third chip 1300 close to a test tube, the second connection surface is provided with a clamping thin wall 1320, the clamping thin wall 1320 is used for being clamped in the test tube upper cover 2, the effects of strict clamping and sealing are achieved, the oil inlet through hole 1210 and the oil outlet through hole 1220 are located in an area surrounded by the clamping thin wall 1320, the second connection surface is integrally formed with a cap 1310, the oil inlet through hole 1210 and the oil outlet through hole 1220 are both coaxially connected with the cap 1310, the cap 1310 is of a convex structure, the first puncture needle 1800 penetrates through the oil inlet through hole 1210 and the cap 1310 and then penetrates through the test tube upper cover 2 to enter the test tube 3, the second puncture needle 1900 penetrates through the oil outlet through hole 1220 and the cap 1310 and then penetrates through the test tube upper cover 2, the first puncture needle 1800 and the second puncture needle 1800 are fixed through the cap 1310, the cap 1310 and the cap 1310 are in interference fit, the stability of connection between the puncture needle and the chip body 1 is increased, and the cap 1310 are in interference fit, and the interference fit between the cap 1310 and the cap and the silicone body 1 are both in order to achieve the sealing effect by using the compression effect.

As shown in fig. 8, specifically, first puncture needle 1800 and second puncture needle 1900 are long puncture needle 1700 or short puncture needle 1600, and the distance from the opening of long puncture needle 1700 to the opening of test tube 3 is greater than the distance from the opening of short puncture needle 1600 to the opening of test tube 3; preferably, the openings at one ends of the long puncture needle 1700 and the short puncture needle 1600 are flush with the first connecting surface, bosses are uniformly formed on the openings at one ends of the long puncture needle 1700 and the short puncture needle 1600, and the bosses are pressed on the wall surface of the first chip 1100, so that the oil phase and the micro liquid drops can enter the cavity along the pipeline, and the structure is simple, and the complex structure and manufacturing process are avoided. In this embodiment, the first puncture needle 1800 is a long puncture needle 1700, the second puncture needle 1900 is a short puncture needle 1600, the upper end of the short puncture needle 1600 is connected with the liquid inlet tank 1120, the stability of the cooperation is increased by the cap 1310 structure at the lower end of the third chip 1300, the lower end of the short puncture needle 1600 extends into the test tube 3, wherein the lower end of the short puncture needle 1600 is higher than the lower end of the long puncture needle 1700, and the short puncture needle 1600 is above the emulsion liquid level in the initial state so as to ensure that micro droplets can smoothly enter the short puncture needle.

As shown in fig. 11, 12 and 13, in this embodiment, the test tube 3 is a standard PCR tube, the test tube upper cover 2 is made of soft silica gel material, the tube body can directly adopt a 0.2mL PCR eight-way tube to reduce the consumable cost, the test tube 3 is fixed by the bottom support 4, wherein the test tube groove 401 is provided thereon, the test tube groove 401 is used for placing the test tube 3, the bottom support 4 is further integrally formed with a pillar 402, the circular pillar 402 is used for supporting the chip body 1, so as to prevent the observation due to the chip inclination, and when the bottom support 4 is placed at the bottom of the chip body 1, the pillar 402 contacts with the bottom wall surface of the chip body 1.

In this embodiment, the oil supply device further comprises an oil supply device 5, an outlet of the oil supply device 5 is connected with an oil outlet pipe, in this embodiment, the oil outlet pipe adopts a rubber tube, and the rubber tube is in interference fit with the oil inlet through hole 1210, so that oil phase leakage is prevented.

In this embodiment, the connection parts of the puncture needle, the oil outlet pipe, the chip body 1, the oil supply device 5 and the test tube 3 can be sealed by a silica gel plug with a Chinese character 'mi' or a cross-shaped notch, so that cross contamination is effectively avoided.

The invention adopts an integrated design to realize the functions of micro-droplet tiling and imaging analysis. After PCR thermal cycle amplification of the micro-droplets is completed in the PCR tube, the micro-droplets are assembled and matched with the chip of the invention, the oil phase is pressed into the chip oil inlet 1210, the oil phase enters the PCR tube through the long puncture steel needle 1700, the micro-droplets in the PCR tube float upwards under the buoyancy effect, the micro-droplets enter the flat-laying cavity of the chip through the short puncture steel needle 1600, and then flow into the flat-laying cavity to realize single-layer flat-laying of the droplets, and then optical detection operation is carried out to obtain fluorescence information of the amplified droplets.

In another embodiment of the present invention, a method for detecting a fully-enclosed, pollution-free, digital nucleic acid chip is provided, comprising the steps of:

the test tube 3 filled with micro-droplets is fixed on the chip body 1, the first puncture needle 1800 extends into the test tube 3, the oil inlet through hole 1210 is connected with the oil phase, the oil phase enters the oil inlet groove 1110 through the oil inlet through hole 1210, the oil phase in the oil inlet groove 1110 enters the test tube 3 through the first puncture needle 1800, and the micro-droplets float at the liquid level of the oil phase;

when the oil phase is full of the inner cavity of the test tube 3, the liquid level of the oil phase contacts the second puncture needle 1900, the oil phase continuously enters the test tube 3 from the first puncture needle 1800, micro-droplets on the liquid level of the oil phase are pushed into the second puncture needle 1900, the micro-droplets sequentially pass through the second puncture needle 1900, the liquid inlet tank 1120 and the flat-laying chamber 1150, the micro-droplets continuously enter the flat-laying chamber 1150, the flat-laying is gradually completed, and after the flat-laying is completed, the chip body 1 is moved to the optical detection platform for detection;

obtaining detection images through an optical detection platform, performing splicing and enhancement processing on a plurality of images, obtaining total liquid drop number through bright field image processing, obtaining positive liquid drop number through fluorescent field image processing, identifying liquid drop fusion and breakage, removing false positive and false negative liquid drops, further deriving original nucleic acid copy number and confidence interval through a poisson probability model, and finishing digital nucleic acid chip detection.

As shown in fig. 14a and 14b of fig. 1 and 14, the first puncture needle is a long puncture needle, the second puncture needle is a short puncture needle, specifically, the first step: the PCR tube with micro drop is fixed by the upper cover of the PCR tube and the clamping thin wall of the chip body, and the long and short puncture steel needles enter the PCR tube.

And a second step of: the oil supply device is used for injecting oil from the oil inlet guide pipe, and the oil phase enters the PCR tube through the oil inlet through hole, the oil inlet groove, the oil outlet through hole and the long puncture steel needle to control the flow rate.

And a third step of: because the density of the micro-droplets is smaller than that of the added oil phase, the micro-droplets float upwards along with the continuous inflow of the oil phase until reaching the lower end of the short puncture steel needle, and then enter the chip along the short steel needle.

Fourth step: micro-droplets enter the flat cavity along the flow channel along the short puncture steel needle, the liquid inlet groove and the smooth flow channel, and gas is discharged from the vent hole at the tail end.

Fifth step: the micro-droplets continuously enter the tiling cavity to gradually finish tiling, and after the tiling process is finished, the chip is moved to the optical detection platform for detection.

Sixth step: and performing processing such as splicing and enhancing on the images to obtain analysis results.

As shown in fig. 14c and 14d of fig. 14, the first puncture needle is a short puncture needle, the second puncture needle is a long puncture needle, specifically, the first step: the PCR tube with micro drop is fixed by the upper cover of the PCR tube and the clamping thin wall of the chip body, and the long and short puncture steel needles enter the PCR tube.

And a second step of: the oil supply device is used for injecting oil from the oil inlet guide pipe, and the oil phase enters the PCR tube through the oil inlet through hole, the oil inlet groove, the oil outlet through hole and the short puncture steel needle to control the flow rate.

And a third step of: because the density of the micro-droplets is smaller than that of the added oil phase, the micro-droplets float upwards along with the continuous inflow of the oil phase until reaching the opening of the long puncture steel needle, and then enter the chip along the long puncture needle.

Fourth step: the micro liquid drops enter the laying cavity along the long puncture needle, the liquid inlet groove and the smooth flow passage, and the gas is discharged from the vent hole at the tail end.

As shown in fig. 15 and 16, it is illustrated that uniform, well-formed microdroplets can be produced by the apparatus for preparing droplets in a tube of the present invention.

Finally, it should be noted that: the above examples are only specific embodiments of the present invention, and are not intended to limit the scope of the present invention, but it should be understood by those skilled in the art that the present invention is not limited thereto, and that the present invention is described in detail with reference to the foregoing examples: any person skilled in the art may modify or easily conceive of the technical solution described in the foregoing embodiments, or perform equivalent substitution of some of the technical features, while remaining within the technical scope of the present disclosure; such modifications, changes or substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention, and are intended to be included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Claims

1. The utility model provides a totally closed pollution-free digital nucleic acid detection chip which is characterized in that, including chip body (1), first pjncture needle (1800) and second pjncture needle (1900), chip body (1) is transparent material, be equipped with a plurality of oil feed through-hole (1210) on chip body (1), the exit linkage oil feed groove (1110) of oil feed through-hole (1210), be connected with the one end opening of first pjncture needle (1800) on oil feed groove (1110), the other end opening of first pjncture needle (1800) stretches into test tube (3), test tube (3) are used for receiving the oil phase that oil feed through-hole (1210) carried, and the one end opening of second pjncture needle (1900) is connected in feed tank (1120) of seting up in chip body (1), the exit linkage of feed tank (1120) has tiling cavity (1150), the bottom plane of tiling cavity (1150) is in the horizontal direction, the other end opening of second pjncture needle (1900) stretches into in test tube (3);

the opening at the other end of the first puncture needle (1800) always stretches into the oil phase in the test tube (3), and when the oil phase is full of the test tube (3), the opening at the other end of the second puncture needle (1900) contacts the top liquid level of the oil phase;

the chip body (1) comprises a first chip (1100), a second chip (1200) and a third chip (1300) which are sequentially stacked, a first connecting surface on the first chip (1100) is used for connecting the second chip (1200), an oil inlet groove (1110) and a liquid inlet groove (1120) are formed in the first connecting surface, one side wall of the oil inlet groove (1110) is communicated with the side wall of an oil inlet through hole (1210), the oil inlet through hole (1210) penetrates through the second chip (1200) and the third chip (1300), the oil inlet groove (1110) is coaxially communicated with an oil outlet through hole (1220), the oil outlet through hole (1220) penetrates through the second chip (1200) and the third chip (1300), and the oil outlet through hole (1220) is in interference fit with the first puncture needle (1800); a smooth flow channel (1130) is communicated with one side wall of the liquid inlet groove (1120), an outlet of the smooth flow channel (1130) is communicated with the flat cavity (1150), the liquid inlet groove (1120) is coaxially communicated with a liquid inlet through hole (1230), the liquid inlet through hole (1230) penetrates through the second chip (1200) and the third chip (1300), the liquid inlet through hole (1230) is in interference fit with the second puncture needle (1900),

the first puncture needle (1800) and the second puncture needle (1900) are long puncture needles (1700) or short puncture needles (1600), and the distance from the opening of the long puncture needles (1700) to the opening of the test tube (3) is larger than the distance from the opening of the short puncture needles (1600) to the opening of the test tube (3);

one end openings of the long puncture needle (1700) and the short puncture needle (1600) are flush with the first connecting surface, bosses are uniformly formed on one end openings of the long puncture needle (1700) and the short puncture needle (1600), and the bosses are propped against the wall surface of the first chip (1100);

a tiled cavity supporting column (1170) is also integrated on the first connecting surface, and the tiled cavity supporting column (1170) is contacted with the second chip (1200);

the first chip (1100) is also integrated with a plurality of positioning marks (1160), the positioning marks (1160) are positioned between adjacent tiling chambers (1150), and the shapes of the adjacent positioning marks (1160) between the same tiling chambers (1150) are inconsistent.

2. The fully-enclosed, pollution-free, digital nucleic acid detection chip of claim 1, wherein the entrance diameter to the exit diameter of the smooth flow channel (1130) is gradually reduced from 3-5 times the diameter of the microdroplet, and the area of the tiling chamber (1150) is 1.2 times the tiling area of the total microdroplet monolayer in the test tube (3).

3. The totally enclosed pollution-free digital nucleic acid detecting chip according to claim 1, wherein the first chip (1100), the second chip (1200) and the third chip (1300) are further provided with through-holes (1400) for exhaust, the ends of the flat chambers (1150) are connected to the side walls of the through-holes (1400), and the diameters of the ends of the flat chambers (1150) are smaller than the diameters of the micro-droplets;

a waste liquid cavity (1330) is arranged in the inner cavity of the third chip (1300), and an inlet of the waste liquid cavity (1330) is communicated with one end of the exhaust through hole (1400).

4. The fully-enclosed pollution-free digital nucleic acid detection chip according to claim 1, wherein a second connection surface on the third chip (1300) is used for connecting a test tube (3), a clamping thin wall (1320) is arranged on the second connection surface, the clamping thin wall (1320) is used for clamping a test tube upper cover (2) of the test tube (3), an oil inlet through hole (1210) and an oil outlet through hole (1220) are located in an area surrounded by the clamping thin wall (1320), a cap (1310) is integrally formed on the second connection surface, caps (1310) are coaxially connected to the oil inlet through hole (1210) and the oil outlet through hole (1220), the caps (1310) are of a protruding structure, the caps (1310) are in interference fit with the first puncture needle (1800) and the second puncture needle (1900), and the caps (1310) and the clamping thin wall (1320) are made of silica gel.

5. The totally enclosed and pollution-free digital nucleic acid detection chip according to claim 1, wherein the height of the tiling chamber (1150) is 0.5-2 times the diameter of the micro-droplets from the second chip (1200) to the first chip (1100);

the inlet diameter of the smooth flow passage (1130) is 1mm, the outlet diameter of the smooth flow passage (1130) is 0.3mm, the height of the inlet of the smooth flow passage (1130) is 0.3mm, and the height of the outlet of the smooth flow passage (1130) is 0.1mm.

6. The fully-enclosed pollution-free digital nucleic acid detection chip according to claim 1, further comprising an oil supply device (5) and a bottom support (4), wherein an outlet of the oil supply device (5) is connected with a rubber tube, and the rubber tube is in interference fit with an oil inlet through hole (1210);

a test tube groove (401) is formed in the bottom support (4), the test tube groove (401) is used for placing a test tube (3), and a support column (402) is integrally formed in the bottom support (4);

when the bottom support (4) is placed on the bottom of the chip body (1), the support column (402) is in contact with the bottom wall surface of the chip body (1).

7. The method for detecting a fully-enclosed, pollution-free, digitized nucleic acid chip of any one of claims 1-6, comprising the steps of:

the test tube (3) filled with micro-droplets is fixed on the chip body (1), the first puncture needle (1800) stretches into the test tube (3), the oil inlet through hole (1210) is connected with an oil phase, the oil phase enters the oil inlet groove (1110) through the oil inlet through hole (1210), the oil phase in the oil inlet groove (1110) enters the test tube (3) through the first puncture needle (1800), and the micro-droplets float at the liquid level of the oil phase;

when the oil phase is filled in the inner cavity of the test tube (3), the liquid level of the oil phase contacts the second puncture needle (1900), the oil phase continuously enters the test tube (3) from the first puncture needle (1800), micro-droplets on the liquid level of the oil phase are pushed into the second puncture needle (1900), the micro-droplets sequentially pass through the second puncture needle (1900), the liquid inlet groove (1120) and the flat chamber (1150), the micro-droplets continuously enter the flat chamber (1150), the flat is gradually completed, and after the flat is completed, the chip body (1) is moved to the optical detection platform for detection;