Vertical microorganism detection chip
阅读说明:本技术 一种立式微生物检测芯片 (Vertical microorganism detection chip ) 是由 郑同玉 于 2020-05-14 设计创作,主要内容包括:本发明涉及一种立式微生物检测芯片,包括在芯片进口和出口之间弯折延伸的输送通道和设置在所述输送通道水平部分的容纳腔,容纳腔的底部布置有出口连通至下一级水平设置的分配通道的侧流通道,竖直设置的芯片允许将激励光光源和荧光检测元件设置在芯片两侧,从而可以完全屏蔽激励光的背景干扰;侧流通道的进出口端分别连通于容纳腔底部和下级分配通道,能够在不消耗连续相流体的情况下实现对微液滴的诱导;侧流通道可以单独设置在结构层,从而减小甚至消除已被填充的容纳腔对液滴的诱导效应;通过在芯片盖片层和基片层的外侧涂覆偏振方向相反的偏振涂层,从而实现对激励光到荧光检测元件间的光路的完全阻断,同时,配合在芯片两侧设置的外部偏振元件,还可以阻断透过容纳腔侧壁射出的荧光信号到荧光检测元件之间的光路,避免相邻荧光信号点之间的信号融合。(The invention relates to a vertical microorganism detection chip, which comprises a conveying channel extending between an inlet and an outlet of the chip in a bending way and an accommodating cavity arranged at the horizontal part of the conveying channel, wherein the bottom of the accommodating cavity is provided with a side flow channel of which the outlet is communicated to a next-stage horizontally arranged distribution channel; the inlet and outlet ends of the lateral flow channel are respectively communicated with the bottom of the containing cavity and the lower-stage distribution channel, so that micro liquid drops can be induced under the condition of not consuming continuous phase fluid; the lateral flow channel can be arranged on the structural layer separately, thereby reducing or even eliminating the induction effect of the filled accommodating cavity on liquid drops; through the polarization coating that the direction of polarization is opposite at the outside coating of chip cover plate layer and substrate layer to the realization is to the complete blocking of the light path between fluorescence detection component of exciting light, simultaneously, the cooperation is at the outside polarization component that the chip both sides set up, can also block to see through the fluorescence signal that holds the chamber lateral wall and jet out to the light path between the fluorescence detection component, avoids the signal fusion between the adjacent fluorescence signal point.)
1. A vertical microorganism detection chip (10), characterized in that: the chip (10) is vertically placed when in use and comprises a cover sheet layer (1) and a substrate layer (2), wherein the cover sheet layer (1) is provided with an inlet through hole (11) and an outlet through hole (12), and the inlet through hole (11) is higher than the outlet through hole (12); one side of the substrate layer (2) is provided with a non-through groove-shaped microchannel, the microchannel comprises a sample inlet groove (21) and a sample outlet groove (22) which correspond to the inlet through hole (11) and the outlet through hole (12) of the cover plate layer (1), a conveying channel (23) which is communicated with the sample inlet groove (21) and the sample outlet groove (22) and is bent and extended between the sample inlet groove and the sample outlet groove, the conveying channel (23) comprises a plurality of horizontally extending distribution channels (231) and a connecting channel (232) which is connected with the tail end of the upper-stage distribution channel (231) and the head end of the lower-stage distribution channel (231); a plurality of accommodating cavities (24) communicated with the lower edges of the distribution channels (231) are arranged between every two adjacent distribution channels (231); the bottom of the containing cavity (24) is provided with a side flow channel (25) communicated with the upper edge of the next-stage distribution channel (231).
2. The microorganism detection chip according to claim 1, wherein: the outlet of the side flow channel (25) is positioned between two adjacent containing cavities (24), so that the side flow flowing out of the side flow channel can be prevented from impacting micro liquid drops in the containing cavities (24).
3. The microorganism detection chip according to claim 1, wherein: the containing cavity (24) is a circular cavity, and the depth of the containing cavity is equal to the diameter of the circular cavity; the lateral flow channel (25) has the same depth as the receiving chamber (24).
4. The microorganism detection chip according to claim 1, wherein: the upper half of the containing cavity (24) is communicated with the lower edge of the distribution channel (231) to form an opening smaller than the diameter of the containing cavity (24); so that, in a horizontal section passing through the center of the containing cavity, an eaves (242) capable of wrapping the upper half of the micro-droplet is formed between the cavity wall (241) of the containing cavity (24) and the edge of the opening.
5. The microorganism detection chip according to claim 4, wherein: the eaves (242) are arranged on the downstream side of the opening of the accommodating cavity (24); and a guide part (243) with a circular arc chamfer is arranged at the upstream side of the opening; the radius of the guide portion 243 is the same as the height of the brim 242.
6. A vertical microorganism detection chip (10), characterized in that: the chip (10) is vertically placed in use and comprises a cover sheet layer (1) with the same shape and size, a substrate layer (2) and a structural layer (3) clamped between the cover sheet layer (1) and the substrate layer (2); the cover sheet layer (1) and the substrate layer (2) are provided with overlapped non-through micro-channel structures; the cover plate layer (1) is provided with a through inlet through hole (11) and an outlet through hole (12), and is also provided with a non-through cover plate layer conveying channel (13) and a cover plate layer accommodating cavity (14); the substrate layer (2) is provided with a non-through sample inlet groove (21), a sample outlet groove (22), a conveying channel (23) and an accommodating cavity (24); the structural layer (3) comprises a part overlapped with the micro-channel structures on the cover plate layer (1) and the substrate layer (2), and a sample inlet middle groove (31) and a sample outlet middle groove (32) corresponding to the inlet through hole (11) and the outlet through hole (12); only the microchannel structure of the structural layer (3) comprises a middle-layer side-flow channel (35) for forming a lateral flow; the microchannel structure of the structural layer (3) is a through structure, so that the cover sheet layer (1), the structural layer (3) and the substrate layer (2) can form a complete combined conveying channel, a combined sample feeding groove, a combined sample outlet groove and a combined accommodating cavity after being mutually overlapped, but a side flow channel only exists in the structural layer (3).
7. The vertical microorganism detection chip according to claim 6, wherein: the side walls of the microchannel structure on the structural layer (3) are more hydrophilic than the side walls of the microchannel structure on the cover sheet layer (1) and the substrate layer (2) to allow the middle layer side flow channel (35) to provide equivalent lateral flow strength with a relatively small cross-sectional area.
8. The vertical microorganism detection chip according to claim 6, wherein: the non-attached sides of the cover plate layer (1) and the substrate layer (2) of the chip (10) are respectively coated with polarization coatings with opposite polarization directions, and the polarization coatings completely cover the non-attached sides of the cover plate layer (1) and the substrate layer (2).
9. The vertical microorganism detection chip according to claim 6, wherein: the non-attaching sides of the cover plate layer (1) and the substrate layer (2) of the chip (10) are respectively coated with polarization coatings with opposite polarization directions, and the polarization coatings do not cover the combined accommodating cavity.
10. The vertical microorganism detection chip according to claim 6, wherein: the non-contact surface of the substrate layer (2) of the chip (10) is coated with a light-absorbing coating; the light absorbing coating does not cover the combining cavity.
Technical Field
The invention relates to a biological detection device, in particular to a vertical microorganism detection chip for detecting specific DNA of microorganisms such as viruses and bacteria through PCR reaction.
Background
Compared with the traditional PCR technology, the digital PCR is divided into dozens of to hundreds of thousands of tiny independent reactors after diluting a solution containing a target gene, a primer, polymerase and the like, so that the number of nucleic acid templates in each reactor is less than or equal to 1, and each reactor is subjected to traditional PCR amplification and fluorescence detection. The reactor containing the target gene is labeled as 1, and the reactor containing no target gene is labeled as 0, and the nucleic acid concentration of the original solution is calculated from the relative proportion and the volume of the reactor, using the poisson distribution.
At present, the mainstream implementation of digital PCR is based on microfluidic chips with micro-reaction chamber arrays; the water-in-oil droplets from the droplet preparation unit are distributed into the micro-reaction cavity array, excitation light is introduced into the micro-reaction cavity after a plurality of heating-annealing amplification cycles, the micro-droplets including the target nucleic acid template emit fluorescence under the irradiation of the excitation light, the secondary fluorescence is detected, and a corresponding detection result can be obtained after statistical analysis. In the main steps of the digital PCR detection process, the effective distribution of micro-droplets in a micro-reaction cavity and the accurate capture of fluorescence signals influence the reliability of detection results to a great extent.
Disclosure of Invention
In order to solve the above problems in the prior art, the present invention provides a vertical microorganism detection chip. Compared with the prior art, the vertical microorganism detection chip provided by the invention can provide a higher effective filling rate of liquid drops, reduce and even eliminate background interference caused by stray light such as exciting light and the like, effectively inhibit mixing of fluorescence signals of adjacent liquid drops and effectively improve independence between different signal points.
In order to achieve the technical effects, the invention specifically provides the following scheme:
for convenience of description, a chip 10 is referred to as a vertical microorganism detection chip, and the chip 10 is vertically placed in use and comprises a
Micro-channel structures are arranged on the
Except for the
Preferably, the bottom of the
Maintaining a constant volume of continuous phase fluid is important for the process of dispensing microdroplets, and for the treatment of continuous phase fluid in the lateral flow, an additional collecting channel may be provided to collect or discharge it, but it should be noted that the microdroplets filled in the
Preferably, the outlet of the
Preferably, the
Preferably, the upper half of the
The
In addition, the present invention finds that the use of the
Preferably, the chip 10 may include more layer structures, such as a
The
Such an arrangement allows the lateral flow channel to have a square channel cross-section and thus better closure by the micro-droplets filled in the combined receiving chamber, otherwise the rectangular cross-section of the
Because the upper micro-channel structures of the structural layer 3 are all through structures, the sheet layers of the structural layer comprise a frame 37 and a block unit 36 which are separated from each other; wherein, the non-edge portions of the middle layer accommodating chamber 34, the middle layer side flow path 35 and the middle layer conveying path 33 are formed between the adjacent two block units 36; the block unit 36 and the frame 37 form an edge portion of the middle layer conveyance path 33 therebetween.
In order to form a monolithic microchannel structure with an accurate combination between the
A chip 10 having a multi-layer (meaning three and more) structure may also have a
Preferably, the side walls of the microchannel structure on the structural layer 3 are more hydrophilic than the side walls of the microchannel structures on the
Preferably, for a chip 10 with a double-layer structure or a multi-layer structure, the non-attaching sides of the
The polarizing coating may be replaced by an external polarizing element disposed on the upstream and downstream optical paths of the chip 10.
Preferably, the non-attaching surface of the
Such an effect can also be achieved by means of a combination of a polarizing coating and an external polarizing element, for example, a P-polarizing element is disposed on the upstream side of the optical path of the chip 10, and an S-polarizing element is disposed downstream, while an S-polarizing coating is disposed on the light-facing side of the chip 10, for example, the outer side of the
Under the arrangement, after the excitation light passes through the P-polarization element, the excitation light is emitted to the
The invention also provides a microorganism detection system based on the chip 10, which comprises a detection platform 4, the chip 10 and a light shield 7; the detection platform 4 is provided with a fixing portion for fixing the vertically arranged chip 10, for example, the fixing portion may be a slot 43 allowing the chip 10 to be inserted and fixed, and of course, other fixing manners conventional in the art may also be adopted. The slot 43 is preferably fixed perpendicular to the long axis of the detection platform 4, and a first polarization element 42 and a second polarization element 44 are respectively arranged in front of and behind the slot 43 and parallel to the chip 10 along the long axis direction of the detection platform 4; wherein the polarization directions of the first polarization element 42 and the second polarization element 44 are opposite. A light source frame 41 is fixedly arranged at one end of the detection platform 4, a light source 5 of excitation light is fixedly arranged on the light source frame 41, and the light source 5 is positioned such that the excitation light emitted by the light source 5 vertically irradiates the first and second polarizing elements; a light sensing element 45 is fixedly arranged at the opposite end (the end opposite to the light source position) of the detection platform 4, and the light sensing element 45 is used for receiving the fluorescence signal.
A liquid supply assembly 6 is also vertically arranged on one side of the detection platform 4 corresponding to the position of the chip 10; the liquid supply assembly 6 may hold one side of a chip 10 and may supply a continuous phase fluid with droplets thereto through an inlet through hole 11 of the chip 10 and receive a remaining fluid after dispensing droplets from an outlet through hole 12 of the chip 10.
The liquid supply assembly 6 includes a
A groove 46 is formed in the periphery of the upper surface of the detection platform 4, and the groove 46 is used for being matched with the light shield 7. The inner surface of the light shield 7 is coated with a light absorption material, so that external light can be prevented from penetrating through the light shield, scattered light emitted to the light shield from the inside can be absorbed, and stray light interference is reduced.
The invention also provides a method for preparing the chip 10, and specifically, when the chip 10 only comprises a
step 3, spin-coating photoresist, namely respectively spin-coating photoresist on the single sides of the
step 4, covering masks, namely measuring the covering cover plate masks and the substrate masks respectively on the gluing of the
step 5, ultraviolet irradiation, namely irradiating the corresponding sheet layer from the mask side of each sheet layer by using ultraviolet light, and carrying out chemical reaction on the photoresist at the mask gap under the action of the ultraviolet light;
step 6, drying each sheet layer;
step 7, etching, namely etching each sheet layer obtained in the step 6 by using etching liquid, wherein the
and 8, removing the photoresist, and fixedly attaching any one side of the
When the chip 10 comprises only the
step 3, coating glue once, namely spin-coating photoresist on the single sides of the
step 4, covering masks, namely covering the cover plate mask and the substrate mask on the gluing sides of the
step 5, primary ultraviolet irradiation is carried out, ultraviolet light is used for irradiating corresponding sheet layers from the mask side of each sheet layer, and the photoresist at the mask gap is subjected to chemical reaction under the action of the ultraviolet light;
step 6, drying each sheet layer;
step 7, etching for the first time, namely etching each sheet layer obtained in the step 6 by using etching liquid, wherein the
step 8, removing the photoresist, and removing the photoresist on the mask side of each lamella;
step 9, secondary coating glue, namely, secondary spin coating of photoresist on any side of the cover plate layer;
step 10, obtaining a second mask of the gluing measurement covering cover sheet layer of the cover sheet layer in the step 9, and carrying out ultraviolet irradiation, drying, etching and glue stripping on the cover sheet layer according to the mode in the step 5-8; thereby obtaining the cover sheet layer with a through inlet through hole 11 and an outlet through hole 12, a non-through cover sheet layer conveying channel 13 and a cover sheet layer accommodating cavity 14;
and 11, fixedly attaching the micro-channel side of the
Preferably, the method further comprises the following steps:
step 12, preparing a coating mask, wherein the coating mask only covers the containing cavities on the
and step 13, applying a polarization coating, wherein the polarization coating is respectively applied to the mask sides of the
And step 14, drying the chip 10 obtained in the step 13, and tearing off the coating mask to obtain the chip 10 with the three-layer structure of the polarization coating.
Compared with the prior art, the invention can at least obtain the following beneficial effects: the vertically arranged chip allows the excitation light source and the fluorescence detection element to be arranged on two sides of the chip, so that the background interference of the excitation light can be completely shielded; a side flow channel is arranged on a micro-channel structure of the chip, and the inlet and outlet ends of the side flow channel are respectively communicated with the bottom of the accommodating cavity and the lower-level distribution channel, so that micro liquid drops can be induced without consuming continuous phase fluid, the liquid drop filling efficiency is improved, and the defect of liquid drop fusion is not easy to occur; the lateral flow channels are independently arranged on the structural layer, so that the filled liquid drops are allowed to better close the corresponding lateral flow channels, the induction effect of the filled accommodating cavities on the liquid drops is reduced or even eliminated, the liquid drop distribution effect is improved, and the risk of liquid drop accumulation is reduced; through the polarization coating that the direction of polarization is opposite at the outside coating of chip cover plate layer and substrate layer to the realization is to the complete blocking of the light path between fluorescence detection component of exciting light, simultaneously, the cooperation is at the outside polarization component that the chip both sides set up, can also block to see through the fluorescence signal that holds the chamber lateral wall and jet out to the light path between the fluorescence detection component, avoids the signal fusion between the adjacent fluorescence signal point.
Drawings
FIG. 1 is a schematic diagram of a chip having a two-layer structure;
FIG. 2 is an enlarged view of a portion of region A of FIG. 1;
FIG. 3 is one of the microchannel side views of the substrate layer of FIG. 1;
FIG. 4 is a second side view of a microchannel in the substrate layer of FIG. 1;
FIG. 5 is a horizontal cross-sectional view of the chip shown in FIG. 1;
FIG. 6 is a third view of the microchannel layer of the substrate layer of FIG. 1;
FIG. 7 is an enlarged perspective view of area B of FIG. 6;
FIG. 8 is an enlarged view of a portion of area B of FIG. 6;
FIG. 9 is a horizontal cross-sectional view of one of the chips shown in FIG. 8;
FIG. 10 is a second horizontal cross-sectional view of the chip shown in FIG. 8 (excluding the area shown in FIG. 8);
FIG. 11 is a schematic diagram of a chip having a three-layer structure;
FIG. 12 is an enlarged view of a portion of region C of FIG. 11;
FIG. 13 is an enlarged view of a portion of region D of FIG. 11;
FIG. 14 is an enlarged view of a portion of area E of FIG. 11;
fig. 15 is one of the enlarged views of the three-layer die attach area C, D, E of fig. 11;
fig. 16 is a second enlarged view of the three-layer die attach area C, D, E of fig. 11;
FIG. 17 is a horizontal cross-sectional view of the chip of FIG. 15;
FIG. 18 is a horizontal cross-sectional view of one of the chips shown in FIG. 16;
FIG. 19 is a second horizontal cross-sectional view of the chip of FIG. 16 (excluding the area shown in FIG. 16);
FIG. 20 is a schematic view of the combination of the detection platform and the chip;
FIG. 21 is a schematic view of a light shield;
FIG. 22 is a first view of the liquid supply assembly;
FIG. 23 is a second view of the liquid supply assembly;
in the figure: 1 is a cover sheet layer, 11 is an inlet through hole, 12 is an outlet through hole, 13 is a cover sheet layer conveying channel, 14 is a cover sheet layer accommodating cavity, 2 is a substrate layer, 21 is a sample introduction groove, 22 is a sample outlet groove, 23 is a conveying channel, 231 is a distribution channel, 232 is a connecting channel, 24 is an accommodating cavity, 241 is a cavity wall, 242 is an eave part, 243 is a guide part, 25 is a lateral flow channel, 3 is a structural layer, 31 is a sample inlet groove, 32 is a sample outlet groove, 33 is a middle layer conveying channel, 34 is a middle layer accommodating cavity, 35 is a middle layer lateral flow channel, 36 is a block unit, 37 is a frame, 4 is a detection platform, 41 is a light source frame, 42 is a first polarizing element, 43 is an insertion groove, 44 is a second polarizing element, 45 is a photosensitive element, 46 is a groove, 5 is a light source, 6 is a liquid supply assembly, 61 is a channel wall, 62 is a short wall, 63 is a sliding groove, 64 is a liquid inlet sliding block, 65 is a liquid, 7 is a light shield, 10 is a chip.
Detailed Description
In order to better illustrate the technical idea of the present invention, the following further describes the solution of the present invention with reference to the accompanying drawings.
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