CN113846002A - Totally-enclosed pollution-free digital nucleic acid detection chip and detection method - Google Patents
Totally-enclosed pollution-free digital nucleic acid detection chip and detection method Download PDFInfo
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Abstract
本发明公开了一种全封闭无污染的数字化核酸检测芯片，包括芯片本体、第一穿刺针和第二穿刺针，所述芯片本体为透明材质，所述芯片本体上设有若干个进油通孔，进油通孔的出口连接进油槽，所述进油槽上连接有第一穿刺针的一端开口，第一穿刺针的另一端开口伸入试管中，所述试管用于接收进油通孔输送的油相，第二穿刺针的一端开口连接在芯片本体内腔中开设的进液槽内，所述进液槽的出口连接有平铺腔室，所述平铺腔室的底部平面处于水平方向，所述第二穿刺针的另一端开口伸入试管中；第一穿刺针的另一端开口始终伸入试管中的油相内，在油相充满试管时，第二穿刺针的另一端开口接触油相的顶部液面。本发明避免了交叉污染风险，操作简单，耗时短。
The invention discloses a fully enclosed and pollution-free digital nucleic acid detection chip, comprising a chip body, a first puncture needle and a second puncture needle, the chip body is made of transparent material, and a plurality of oil inlet ports are arranged on the chip body. The outlet of the oil inlet through hole is connected to the oil inlet groove, the oil inlet groove is connected with an opening of one end of the first puncture needle, and the other end opening of the first puncture needle extends into the test tube, and the test tube is used to receive the oil inlet through hole. For the oil phase to be transported, one end of the second puncture needle is opened in the liquid inlet groove opened in the inner cavity of the chip body, the outlet of the liquid inlet groove is connected with a tiling chamber, and the bottom plane of the tiling chamber is at In the horizontal direction, the opening of the other end of the second puncture needle extends into the test tube; the opening of the other end of the first puncture needle always extends into the oil phase in the test tube. When the oil phase fills the test tube, the other end of the second puncture needle extends into the test tube. The opening contacts the top surface of the oil phase. The invention avoids the risk of cross-contamination, has simple operation and short time consumption.
Description
Technical Field
The invention belongs to the technical field of microfluidic droplet digital polymerase chain reaction, and particularly belongs to a totally-enclosed pollution-free digital nucleic acid detection chip and a detection method.
Background
With the shift of medical treatment modes and the continuous development of individual medicine, the medical inspection world needs a fast and accurate detection means urgently, and the molecular detection exerts unique advantages. At present, the molecular detection technology mainly comprises nucleic acid molecular hybridization, Polymerase Chain Reaction (PCR), a biochip technology and the like, and molecular detection products are mainly applied to detection of clinical departments such as tumors, infections, heredity, prenatal screening and the like, aspects such as a physical examination center, a technical service center, a third-party detection mechanism, a microorganism rapid detection market and the like, and play an important role in detection of novel coronaviruses. The Digital PCR (dPCR) technique is a technique for nucleic acid quantification based on a single-molecule PCR method, and is a technique for nucleic acid molecular amplification and absolute quantification. The digital PCR of the liquid drop based on the liquid drop microfluidic technology comprises the following components: the advantages of easy providing of micro-reactor with small volume and high flux, etc. are widely paid attention to.
The working principle of the micro-droplet digital PCR system is as follows: firstly, a sample to be tested is evenly distributed into a plurality of nano-liter (diameter is several microns to hundreds of microns) "water-in-oil" micro-droplets by a special micro-droplet generator, and the number of the micro-droplets is in the million level. Because the micro-droplets are enough in quantity and are mutually isolated by the oil layer, each micro-droplet is equivalent to a micro-reactor, and the micro-droplets only contain DNA single molecules of a sample to be detected; then, PCR amplification reactions are performed on the droplets, and the droplets are individually detected for their fluorescent signals by a droplet analyzer, and droplets with a fluorescent signal are interpreted as 1, and droplets without a fluorescent signal are interpreted as 0. And finally, obtaining the number of target DNA molecules of the sample to be detected according to the Poisson distribution principle and the number and proportion of the positive microdroplets, and realizing absolute quantification of the nucleic acid sample. The judgment of the fluorescence signal of the micro-droplet sample depends on a core technology: the design and processing of the micro-droplet fluorescence detection device are characterized in that negative micro-droplets and positive micro-droplets are distinguished by using the fluorescence signal of a product in the laser-excited micro-droplets.
Currently available instruments such as BIO-RAD QX200TMCombining water-in-oil emulsification microdroplet technology with microfluidics, 20000 uniform nanoliter-scale microdroplets can be generated for each sample by means of a microdroplet generator, wherein the target fragments and background sequences are randomly distributed in the microdroplets, and each microdroplet is an independent reactor. After PCR using a thermal cycler, the QX200 droplet analyzer will analyze the droplets in each sample individually. After the droplets are inhaled, the droplets are sequentially passed 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 an absolute quantitative analysis of the target DNA in a digital format.
For the micro-droplet observation of the digital PCR detector, a droplet array micro-imaging mode is adopted, the forms of all droplets can be clearly obtained, interference factors such as non-specific amplification and the like are reduced, and the reliability of a detection result is improved.
Patent document CN109536380A (a droplet microfluidic chip for highly sensitive detection of nucleic acid and its using method) discloses a droplet microfluidic chip for highly sensitive detection of nucleic acid and its using method, which separates functional areas, and completes three links of digital PCR, droplet preparation, PCR amplification and tiling statistics technology in independent areas. Because the liquid drop preparation, the PCR amplification and the tiled statistical area are in the same plane, the area of the chip is larger, and the detection flux is greatly reduced; the preparation process of the liquid drop is difficult to realize in practice, and the size of the generated liquid drop is difficult to control; the structure of the amplification and transfer part is complex, and strict sealing is difficult to achieve; the requirement for the oil phase is high, two oil phases which are not soluble with each other and do not influence the subsequent reaction and observation results need to be used, so that the experiment difficulty is increased, and the experiment cost is increased.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a totally-enclosed pollution-free digital nucleic acid detection chip and a detection method, which solve the problems that the prior liquid drop pipetting operation is complex, the time is consumed and the cross contamination is easy to generate.
In order to achieve the purpose, the invention provides the following technical scheme: a totally-enclosed pollution-free digital nucleic acid detection chip 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, an outlet of each oil inlet through hole is connected with an oil inlet tank, one end opening of the first puncture needle is connected onto each oil inlet tank, the other end opening of the first puncture needle extends into a test tube, the test tube is used for receiving oil phase conveyed by the oil inlet through holes, one end opening of the second puncture needle is connected into the oil inlet tank formed in an inner cavity of the chip body, an outlet of each oil inlet tank is connected with a tiled cavity, the bottom plane of the tiled cavity is in the horizontal direction, and the other end opening of the second puncture needle extends into the test tube;
the opening at the other end of the first puncture needle always extends into the oil phase in the test tube, and when the oil phase is filled in the test tube, the opening at the other end of the second puncture needle contacts 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, the first connecting surface on the first chip is used for being connected with 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 with the side wall of the 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; a lateral wall intercommunication in feed liquor groove has smooth runner, smooth runner's export intercommunication is to the tiling cavity, smooth runner's entry diameter is reduced gradually by little liquid drop diameter 3 ~ 5 times to the exit diameter, the coaxial intercommunication in feed liquor groove has the feed liquor through-hole, the feed liquor through-hole link up second chip and third chip, feed liquor through-hole and second pjncture needle interference fit, the area of tiling cavity is 1.2 times of total little liquid drop individual layer tiling area in the test tube.
Further, exhaust through holes are further penetrated through the first chip, the second chip and the third chip, the tail end of the tiled cavity is communicated to the side wall of the exhaust through holes, and the diameter of the tail end of the tiled cavity is smaller than that of the micro liquid drops;
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 on the third chip is connected the face and 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, the oil feed through-hole is located the region that the screens thin wall encloses and closes with the through-hole that produces oil, integrated into one piece has the cap on the second connection face, equal coaxial coupling has the cap on oil feed through-hole and the through-hole that produces oil, the cap is protruding structure, the cap is interference fit with first pjncture needle and second pjncture needle, cap and screens thin wall are the silica gel material.
Furthermore, a tiled cavity supporting column is integrated on the first connecting surface and is in contact with the second chip.
Furthermore, in the direction from the second chip to the first chip, the height of the tiled cavity is 0.5-2 times of the diameter of the micro liquid drop;
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 0.1 mm.
Furthermore, a plurality of positioning marks are integrated on the first chip, the positioning marks are located between adjacent tiled chambers, and the shapes of the adjacent positioning marks between the same tiled chambers are inconsistent.
Furthermore, 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 needle to the opening of the test tube is greater than the distance from the opening of the short puncture needle to the opening of the test tube;
the one end opening of long pjncture needle and short pjncture needle all with first connecting face parallel and level, the homogeneous body shaping has the boss on the one end opening of long pjncture needle and short pjncture needle, and the boss is all pushed up and is pressed on the wall of first chip.
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;
a test tube groove is formed in the bottom support and used for placing a test tube, and a support column is integrally formed on the bottom support;
when the bottom support is placed at the bottom of the chip body, the support column is contacted 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 filled with micro-droplets is fixed on the chip body, the first puncture needle extends into the test tube, the oil inlet through hole is connected with an oil phase, the oil phase enters the oil inlet tank through the oil inlet through hole, the oil phase in the oil inlet tank enters the test tube through the first puncture needle, and the micro-droplets float on the liquid level of the oil phase;
when the inner cavity of the test tube is filled with the oil phase, the liquid surface of the oil phase contacts the second puncture needle, the oil phase continuously enters the test tube through the first puncture needle, micro liquid drops on the liquid surface of the oil phase are pushed into the second puncture needle, the micro liquid drops sequentially pass through the second puncture needle, the liquid inlet groove and the flat paving cavity, the micro liquid drops continuously enter the flat paving cavity to gradually finish the flat paving, and after the flat paving is finished, 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 a plurality of images to obtain the copy number and confidence interval of the original nucleic acid, 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, which takes a transparent material as a chip body to facilitate real-time observation, and realizes that an oil phase enters a test tube with micro-droplets through arranging an oil inlet through hole, an oil inlet groove and a first puncture needle on the chip body, wherein the density of the micro-droplets is less than that of the oil phase, and the micro-droplets are always positioned at the liquid level of the oil phase, so that the oil phase is continuously filled into the test tube when the test tube is filled with the oil phase, the micro-droplets on the liquid level of the oil phase are passively pushed into a second puncture needle and enter a tiling cavity through the liquid outlet groove, and are tiled in the tiling cavity, and then fluorescence information of the amplified droplets can be obtained by carrying out optical detection operation The method has the advantages of effectively promoting the liquid drops to be tiled, avoiding two oil phases, having less chip material consumption and lower cost, particularly avoiding the cross contamination risk possibly brought by the uncovering operation of the integrated chip in the liquid transferring process, having simple operation and short time consumption, being capable of observing the micro-liquid drop state compared with the flow detection, improving the detection success rate and timeliness, being also applied to the protein or nucleic acid, such as PCR \ LAMP \ RPA reaction and other technologies, and meeting the analysis requirements of different application occasions.
Furthermore, the diameter from the inlet to the outlet of the smooth flow channel is uniformly reduced, so that the liquid inlet groove and the flat paving chamber can be smoothly connected, the liquid drops can smoothly enter the flow channel, the liquid drops are prevented from being fused due to extrusion and collision 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.
Furthermore, the exhaust through holes penetrating through the first chip, the second chip and the third chip facilitate gas exhaust and are beneficial to the spreading process of micro-droplets.
Furthermore, the screens thin wall can be more zonulae occludens test tube, and the cap can be more zonulae occludens first pjncture needle and second pjncture needle, plays screens and sealed effect.
Further, the tiled cavity supporting column can support the tiled cavity, so that collapse of hot-press bonding is avoided, and the tiled effect of liquid drops is guaranteed.
Furthermore, the diameter and height design of the smooth flow channel can ensure that liquid drops with specific volumes can be generated, such as liquid drops with the diameter of 20-200 um, the volume of the micro liquid drops is controllable, and the tiling quality of the micro liquid drops can be ensured when the height of the tiling chamber is 0.5-2 times of the diameter of the micro liquid drops.
Furthermore, after image processing is completed, each picture can be correspondingly spliced through the positioning mark of each tiled chamber, and the tiled state of the micro-droplets can be conveniently observed.
Furthermore, the bosses of the long puncture needle and the short puncture needle are beneficial to the oil phase and the micro liquid drops to enter the cavity along the pipeline, the structure is simple, and the complex structure and the complex manufacturing process are avoided.
Furthermore, the pillar can support the chip body, guarantee the level of chip body, ensure to move the quality of liquid back micro-droplet.
The invention also provides a detection method of the totally-enclosed pollution-free digital nucleic acid chip, which adopts an integrated design to realize the functions of micro-droplet tiling and imaging analysis. After PCR thermal cycle amplification of micro-droplets in a test tube is completed, the micro-droplets are assembled with the chip set for use, an 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 and enables the micro-droplets in the test tube to float upwards under the action of buoyancy, the micro-droplets enter the tiled cavity of the chip through the second puncture needle and then flow into the tiled cavity to realize single-layer tiling of the droplets, and then optical detection operation is carried out to obtain fluorescence information of the amplified droplets, so that cross contamination risk possibly brought by uncapping operation is avoided, and the operation is simple.
Drawings
FIG. 1 is a schematic overall flow diagram of the present invention;
FIG. 2 is a schematic view of the overall structure of the present invention;
FIG. 3 is a schematic structural diagram of a chip body;
FIG. 4 is a bottom view of the first chip;
FIG. 5 is a schematic cross-sectional view of C-C of FIG. 4;
FIG. 6 is an enlarged partial view of H of FIG. 5;
FIG. 7 is an axial view of the second chip;
FIG. 8 is a schematic view of the first and second needles;
FIG. 9 is a bottom view of the third chip;
FIG. 10 is a schematic cross-sectional view of B-B of FIG. 9;
FIG. 11 is a schematic view of a four-row test tube structure;
FIG. 12 is a schematic view of the bottom holder and test tube in mating configuration;
FIG. 13 is a schematic cross-sectional view of D-D of FIG. 12;
FIG. 14 is a diagram showing a process of filling an oil phase into a test tube, FIG. 14a is a diagram showing an initial state of the test tube, FIG. 14b is a diagram showing a state where the oil phase in the test tube is filled, FIG. 14c is a diagram showing an initial state of the test tube in another embodiment, and FIG. 14d is a diagram showing a state where the oil phase in the test tube is filled;
FIG. 15 is a schematic view of the tiling of microdroplets under a 10 objective lens;
fig. 16 is a schematic diagram of the tiling of microdroplets under a 5 x objective lens.
In the drawings: 1-chip body, 1100-first chip, 1110-oil inlet groove, 1120-liquid inlet groove, 1130-smooth flow channel, 1140-smooth flow channel outlet, 1150-tiled chamber, 1160-positioning mark, 1170-tiled 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 chamber, 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 and 5-oil supply device.
Detailed Description
The invention is further described with reference to the following figures and detailed description.
As shown in figures 2 and 3, the invention provides a totally-enclosed pollution-free digital nucleic acid detection chip, which separates PCR amplification from subsequent detection, reduces the complexity of the chip, avoids the restriction of a detection link on reaction flux relieved by the PCR amplification, can quickly and effectively promote the spreading of liquid drops, does not need two oil phases, has less chip material consumption and lower cost, particularly avoids the cross contamination risk possibly brought by uncovering operation in the liquid transfer process of an integrated chip, can observe the state of micro-liquid drops compared with flow detection, improves the detection success rate and timeliness, can be applied to protein or nucleic acid, such as PCR \ LAMP \ RPA reaction and other technologies, and meets the analysis requirements of different application occasions.
In this embodiment, a totally-enclosed pollution-free digital nucleic acid detection chip comprises an oil supply device 5, a chip body 1 for spreading micro-droplets and detecting micro-flow control, 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 1210, an oil inlet 1110, an oil outlet 1220, an oil inlet 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 laying cavity 1150, a flat laying cavity supporting column 1170, an exhaust 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 arranged on the chip body 1, the outlets of the oil inlet through holes 1210 are connected with an oil inlet groove 1110, one end opening of a first puncture needle 1800 is connected onto the oil inlet groove 1110, the other end opening of the first puncture needle 1800 extends into a test tube 3, the test tube 3 is used for receiving oil phase conveyed by the oil inlet through holes 1210, one end opening of a second puncture needle 1900 is connected into a liquid inlet groove 1120 formed in the inner cavity of the chip body 1, the outlet of the liquid inlet groove 1120 is connected with a tiled cavity 1150, the bottom plane of the tiled cavity 1150 is in the horizontal direction, and the other end opening of the second puncture needle 1900 extends into the 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 fills the test tube 3, the other end opening of the second puncture needle 1900 contacts the top liquid level of the oil phase.
Specifically, the transparent material of the chip body 1 is transparent glass, and may also be made of polymer materials 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 stacked, the first chip 1100 and the second chip 1200 are formed by hot-pressing and bonding polymer materials after injection molding or are made by etching transparent glass, and a high-precision cavity and a matching relation are obtained.
As shown in fig. 4, 5 and 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 sidewall of the oil inlet groove 1110 is communicated to a sidewall of the oil inlet through hole 1210, wherein the oil inlet through hole 1210 is communicated with 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 is communicated with 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; one side wall of the liquid inlet groove 1120 is communicated with a smooth flow passage 1130, an outlet of the smooth flow passage 1130 is communicated to the tiled cavity 1150, the diameter of the inlet of the smooth flow passage 1130 to the diameter of the outlet 1140 of the smooth flow passage is uniformly reduced by 3-5 times of the diameter of the micro-droplets, 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 and the second puncture needle 1900 are in interference fit, and the area of the tiled cavity 1150 is 1.2 times of the total micro-droplet single-layer tiled area in the test tube 3. The volumes of the oil inlet 1110 and the liquid inlet 1120 can be adjusted according to the size of the tiled 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 cavity of 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 cavity of 3mm in diameter and 0.3mm in height, the liquid inlet groove 1120 passes through the smooth flow passage 1130, the height of the smooth flow passage 1130 is gradually reduced to 0.1mm from 0.3mm, and the width is also gradually reduced to 0.3mm from 1 mm. The liquid inlet channel 1120 is smoothly connected with the smooth flow channel 1130 entering the tiled chamber 1150, the height of the smooth flow channel 1130 is smoothly reduced, so that micro droplets can smoothly enter the smooth flow channel 1130, the droplet fusion caused by extrusion collision of the droplets in the smooth flow channel 1130 is avoided, the micro droplets can be prevented from being broken or seriously deformed, and the unique design of the smooth flow channel 1130 enables the invention to generate micro droplets with specific volume, such as micro droplets with the diameter of 20-200 um.
Further, the height of the tiled chamber 1150 is 0.5-2 times of the diameter of the micro-droplets, preferably, the height is 1.25 times of the diameter of the droplets, wherein the area of the tiled chamber 1150 is greater than about 20% of the total area of the single-layer micro-droplets.
In this embodiment, the end of the tiling chamber 1150 is connected to the sidewall of the exhaust through hole 1400, 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, thereby facilitating the tiling process of micro-droplets. The width of the end flow channel of the tiling chamber 1150 is smaller than the diameter of the micro-droplets, and the end of the flow channel is connected with the exhaust through hole 1400 for exhausting, so that the tiling process can be smoothly performed. The entry of exhaust through-hole 1400 is connected with waste liquid chamber 1330 upper end, and wherein, waste liquid chamber 1330 is arranged in the inner chamber of third chip 1300, and the lower surface fretwork of third chip 1300, waste liquid chamber 1330 are used for collecting the waste liquid, avoid the polluted environment, and the purpose is in order to release the pressure that continuously flows in liquid and produce in collecting tiling cavity 1150, reduces cross contamination simultaneously.
As shown in fig. 7, in the present embodiment, the second chip 1200 is further provided with a positioning pin through hole 1500, the positioning pin through hole 1500 penetrates through the first chip 1100, the second chip 1200 and the third chip 1300, and the positioning pin through hole 1500 is used for precise matching when the chip body 1 is manufactured.
Specifically, a tiled cavity support column 1170 is further integrated on the first connection surface, the tiled cavity support column 1170 is in contact with the second chip 1200, and the tiled cavity support column 1170 plays a role in supporting the tiled cavity 1150, so that collapse in hot-press bonding is avoided, and a liquid drop tiling effect is ensured.
In this embodiment, positioning marks are disposed between adjacent tiled chambers 1150, the positioning marks 1160 and the first chip 1100 are integrally formed, and the shapes of the adjacent positioning marks 1160 between the same tiled chambers 1150 are not consistent, such as rectangular, triangular, circular or square, when obtaining an image of each tiled chamber 1150, the images are spliced by aligning each positioning mark 1160, so that the splicing operation is performed during image processing.
As shown in fig. 9 and 10, in detail, the third chip 1300 is provided with a second connecting surface, the second connecting surface is a surface of the third chip 1300 close to the test tube, a clamping thin wall 1320 is provided on the second connecting surface, the clamping thin wall 1320 is used for clamping the upper cover 2 of the test tube to perform a strict clamping and sealing function, the oil inlet through hole 1210 and the oil outlet through hole 1220 are located in an area enclosed by the clamping thin wall 1320, a cap 1310 is integrally formed on the second connecting surface, the cap 1310 is coaxially connected to the oil inlet through hole 1210 and the oil outlet through hole 1220, the cap 1310 is 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 upper cover 2 of the test tube to enter the test tube 3, the second puncture needle 1900 penetrates through the oil outlet through hole and the cap 1310 and then penetrates through the upper cover 2 of the test tube to enter the test tube 3, wherein the first puncture needle 1800 and the second puncture needle 1900 are fixed by the cap 1310, the cap 1310 and the first puncture needle 1800 are in interference fit with the first puncture needle 1800 and the second puncture needle 1220, the stability of the connection of the puncture needle and the chip body 1 is improved, the cap 1310 and the clamping thin wall 1320 are made of silica gel, and the interference fit effect is achieved by utilizing the deformation of the silica gel, so that the compression sealing is realized.
As shown in fig. 8, specifically, the first puncture needle 1800 and the second puncture needle 1900 are a long puncture needle 1700 or a short puncture needle 1600, and the distance from the opening of the long puncture needle 1700 to the opening of the test tube 3 is greater than the distance from the opening of the short puncture needle 1600 to the opening of the test tube 3; preferably, the openings of the one ends of the long puncture needle 1700 and the short puncture needle 1600 are flush with the first connection surface, the bosses are integrally formed on the openings of the one ends of the long puncture needle 1700 and the short puncture needle 1600, and the bosses are all pressed against the wall surface of the first chip 1100, so that the oil phase and the micro-droplets can conveniently enter the cavity along the pipeline, the structure is simple, and the complex structure and the 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 to the liquid inlet 1120, the stability of the matching is increased by the cap 1310 structure at the lower end of the third chip 1300, and 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 should be above the liquid level of the emulsion in the initial state, so as to ensure that the 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 0.2mL PCR eight-way tube to reduce the cost of consumables, the test tube 3 is fixed by the bottom support 4, wherein the test tube groove 401 is arranged on the test tube 3, the bottom support 4 is further integrally formed with a support column 402, the circular support column 402 is for supporting the chip body 1, so as to prevent the observation caused by the inclination of the chip, and when the bottom support 4 is placed at the bottom of the chip body 1, the support column 402 is in contact with the bottom wall surface of the chip body 1.
In this embodiment, still include oil supply unit 5, oil supply unit 5's exit linkage has the pipe that produces oil, and in this embodiment, the pipe that produces oil adopts the rubber tube, and rubber tube and oil feed through-hole 1210 interference fit prevent that the oil phase from revealing.
In this embodiment, the joints of the puncture needle, the oil outlet conduit, the chip body 1, the oil supply device 5 and the test tube 3 can be sealed by the silica gel plug with the cross-shaped notch or the cross-shaped notch, so that cross contamination can be effectively avoided.
The invention adopts an integrated design to realize the functions of micro-droplet tiling and imaging analysis. After the micro-droplets finish PCR thermal cycle amplification in a PCR tube, the micro-droplets are assembled with the chip set for use, an oil phase is pressed into a chip oil inlet 1210, the oil phase enters the PCR tube through a long puncture steel needle 1700, the micro-droplets in the PCR tube float upwards under the action of buoyancy, the micro-droplets enter a tiled cavity of the chip through a short puncture steel needle 1600, then flow into the tiled cavity to realize single-layer tiling 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 and contamination-free digital nucleic acid chip is provided, which comprises the following steps:
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 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 on the liquid level of the oil phase;
when the inner cavity of the test tube 3 is filled with the oil phase, the liquid surface of the oil phase contacts the second puncture needle 1900, the oil phase continuously enters the test tube 3 through the first puncture needle 1800, micro droplets on the liquid surface 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 tiling chamber 1150, the micro droplets continuously enter the tiling chamber 1150, tiling is gradually completed, and after the tiling is completed, the chip body 1 is moved to an optical detection platform for detection;
obtaining a detection image through an optical detection platform, splicing and enhancing a plurality of images, obtaining the total number of liquid drops by processing bright field images, obtaining the number of positive liquid drops by processing fluorescent field images, identifying the fusion and the breakage of the liquid drops, eliminating false positive and false negative liquid drops, further deducing the original nucleic acid copy number and a confidence interval through a Poisson probability model, and completing the detection of the digital nucleic acid chip.
As shown in fig. 14a and 14b of fig. 1 and 14, the first puncture needle is a long puncture needle, and the second puncture needle is a short puncture needle, specifically, the first step: the PCR test tube with micro-droplets is combined and fixed with the clamping thin wall of the chip body through the upper cover of the PCR test tube, and the long and short puncture steel needles enter the interior of the PCR tube.
The second step is that: the oil supply device injects 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.
The third step: because the density of the micro-droplets is less 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.
The fourth step: the micro-droplets enter the flat laying cavity along the short puncture steel needle, the liquid inlet groove and the smooth flow channel, and the gas is discharged from the vent hole at the tail end.
The fifth step: and the micro liquid drops continuously enter the tiling cavity to gradually complete tiling, and after the tiling process is completed, the chip is moved to an optical detection platform for detection.
And a sixth step: and carrying out processing such as splicing, enhancing and the like on the image to obtain an analysis result.
As shown in fig. 14c and 14d of fig. 14, the first puncture needle is a short puncture needle, and the second puncture needle is a long puncture needle, specifically, the first step: the PCR test tube with micro-droplets is combined and fixed with the clamping thin wall of the chip body through the upper cover of the PCR test tube, and the long and short puncture steel needles enter the interior of the PCR tube.
The second step is that: the oil supply device injects 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.
The third step: because the density of the micro-droplets is less 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.
The fourth step: the micro-droplets enter the tiled cavity along the long puncture needle, the liquid inlet groove and the smooth flow channel and along the flow channel, and the gas is discharged from the vent hole at the tail end.
The fifth step: and the micro liquid drops continuously enter the tiling cavity to gradually complete tiling, and after the tiling process is completed, the chip is moved to an optical detection platform for detection.
And a sixth step: and carrying out processing such as splicing, enhancing and the like on the image to obtain an analysis result.
As shown in fig. 15 and 16, it is demonstrated that uniform micro-droplets having a good morphology can be produced by the apparatus for producing droplets in a tube of the present invention.
Finally, it should be noted that: the above-mentioned embodiments are only specific embodiments of the present invention, which are used for illustrating the technical solutions of the present invention and not for limiting the same, and the protection scope of the present invention is not limited thereto, although the present invention is described in detail with reference to the foregoing embodiments, those skilled in the art should understand that: any person skilled in the art can modify or easily conceive the technical solutions described in the foregoing embodiments or equivalent substitutes for some technical features within the technical scope of the present disclosure; such modifications, changes or substitutions do not depart from the spirit and scope of the embodiments of the present invention, and they should be construed as being included therein. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims (10)
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