阅读说明：本技术 一种微生物检测系统 (Microorganism detection system ) 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的制备方法,通过区别化的掩模涉及,从而在各片层上形成贯通和/或非贯通的微通道结构；然后将个片层材料组合,得的位于单独片层上的输水通道和用于容纳微液滴的容纳腔,或由多个片层组合而成的组合输送通道和组合容纳腔,本发明所制备的芯片还具有能够诱导微液滴进入容纳腔的侧流通道,所述侧流通道可以具有与容纳腔相同的深度,或仅被设在中间层,从而具有小于容纳腔的深度,通过将中间层的微通道结构设在为具有相比于其他片层的微通道结构更强的亲水特性,从而允许使用缩小的侧流通道截面积实现同等的侧向流强度。(The invention relates to a preparation method of a vertical microorganism detection chip, which forms a through and/or non-through microchannel structure on each sheet layer through differentiated mask; and then combining the materials of the sheet layers to obtain a water delivery channel and a containing cavity for containing micro liquid drops on the single sheet layer, or a combined delivery channel and a combined containing cavity formed by combining a plurality of sheet layers, wherein the chip prepared by the invention also comprises a lateral flow channel capable of inducing the micro liquid drops to enter the containing cavity, the lateral flow channel can have the same depth as the containing cavity, or is only arranged on the middle layer so as to have the depth smaller than the containing cavity, and the micro channel structure of the middle layer is arranged to have stronger hydrophilic property compared with the micro channel structure of other sheet layers, so that the equivalent lateral flow strength can be realized by using the reduced cross section area of the lateral flow channel.)

1. A preparation method of a vertical microorganism detection chip (10), wherein the chip (10) only comprises a cover sheet layer (1) and a substrate layer (2), and the cover sheet layer (1) is provided with an inlet through hole (11) and an outlet through hole (12); a non-through groove-shaped micro-channel structure is arranged on the substrate layer (2), and the micro-channel structure comprises a sample inlet groove (21), a sample outlet groove (22), a conveying channel (23) extending between the sample inlet groove and the sample outlet groove in a bending manner, an accommodating cavity (24) communicated with the horizontal section of the conveying channel and a side flow channel (25) communicated with the bottom of the accommodating cavity (24) and the horizontal section of the next stage; the method is characterized by comprising the following steps:

step 1, selecting a cover sheet layer and a substrate layer with the same shape and size;

step 2, preparing a cover plate mask and a substrate mask, wherein the cover plate mask only comprises gaps for forming an inlet through hole (11) and an outlet through hole (12); the substrate mask comprises notches for forming a sample inlet groove (21), a sample outlet groove (22), a conveying through (23), an accommodating cavity (24) and a lateral flow channel (25);

step 3, respectively spin-coating photoresist on the single sides of the cover sheet layer (1) and the substrate layer (2);

step 4, respectively measuring a cover sheet mask and a substrate mask on the gluing of the cover sheet layer (1) and the substrate layer (2);

step 5, irradiating the corresponding lamella from the mask side of each lamella by using ultraviolet light;

step 6, drying each sheet layer;

step 7, sufficiently etching the cover plate layer (1) obtained in the step 6 by using an etching solution to obtain a through inlet through hole (11) and a through outlet through hole (12); performing controlled etching on the substrate layer (2) obtained in the step 6 to obtain a non-through micro-channel structure;

and 8, removing the photoresist, and fixedly attaching any one side of the cover plate layer (1) to the micro-channel side of the substrate layer (2) to obtain the chip (10) with the double-layer structure.

2. The method for preparing a vertical microorganism detection chip (10) according to claim 1, wherein: also comprises the following steps:

step 9, preparing a coating mask only covering the containing cavities on the cover plate layer (1) and the substrate layer (2); the coating masks are respectively positioned and attached to the non-channel sides of the cover plate layer (1) and the substrate layer (2);

and step 10, respectively applying polarization coatings on the mask sides of the cover sheet layer (1) and the substrate layer (2) of the chip (10) obtained in the step 9, wherein the polarization directions of the two polarization coatings are opposite.

And 11, drying the chip (10) obtained in the step 10, and tearing off the coating mask to obtain the chip (10) with the three-layer structure of the polarization coating.

3. The method for preparing a vertical microorganism detection chip (10) according to claim 1 or 2, characterized in that: the substrate mask is provided with a notch for forming a conveying channel (23), so that the conveying channel (23) extends between a sample inlet groove (21) and a sample outlet groove (22) in a bending way, and the conveying channel (23) comprises a plurality of horizontally extending distribution channels (231) and connecting channels (232) connected to the tail end of the upper-stage distribution channel (231) and the head end of the lower-stage distribution channel (231).

4. A method for preparing a vertical microorganism detection chip (10) according to claim 3, characterized in that: the substrate mask includes a cutout for forming a receiving cavity (24) such that the receiving cavity (24) is located between adjacent ones of the distribution channels (231) and an upper half of the receiving cavity (24) is in fluid communication with a lower edge of the distribution channels (231).

5. The method for preparing a vertical microorganism detection chip (10) according to claim 4, wherein: the substrate mask comprises a notch for forming a lateral flow channel (25), so that the lateral flow channel (25) is communicated with the bottom of the containing cavity (24) and the upper edge of the next-stage distribution channel (231), and the outlet of the lateral flow channel (25) is positioned between two adjacent containing cavities (24); the accommodating cavity (24) is a circular cavity.

6. The method for preparing a vertical microorganism detection chip (10) according to claim 5, wherein: the upper half part of the accommodating cavity (24) is communicated with the lower edge of the distribution channel (231), and an eaves part (242) capable of wrapping micro liquid drops and a guide part (243) capable of guiding the micro liquid drops to enter the accommodating cavity (24) are arranged at the position close to the opening of the accommodating cavity (24); the radius of the guide portion 243 is the same as the height of the brim 242.

7. A preparation method of a vertical microorganism detection chip (10), wherein the chip (10) comprises a cover sheet layer (1), a substrate layer (2) and a structural layer (3) clamped between the cover sheet layer (1) and the substrate layer (2), and is characterized in that: 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 a through outlet through hole (12); the substrate layer (2) is provided with a corresponding non-through sample inlet groove (21) and a sample outlet groove (22); 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 micro-channel structures of the structural layer (3) are all through structures; the method comprises the following specific steps:

step 1, selecting a cover sheet layer, a substrate layer and a structural layer with the same shape and size;

step 2, respectively preparing a cover plate mask, a cover plate second mask, a substrate mask and a structural layer mask; wherein the cover sheet mask only comprises gaps for forming the inlet through holes (11) and the outlet through holes (12); the second cover sheet mask includes only notches for forming a cover sheet layer feed passage (13) and a cover sheet layer accommodating chamber (14); the substrate mask only comprises gaps for forming a sample inlet groove (21), a sample outlet groove (22), a conveying through (23) and an accommodating cavity (24); the structural layer mask comprises gaps for forming a sample inlet middle groove (31), a sample outlet middle groove (32), a middle layer conveying channel (33), a middle layer accommodating cavity (34) and a middle layer side flow channel (35); wherein the corresponding indentations on each mask have the same shape and size and are positioned such that the microstructures of each layer produced are capable of forming a complete combined microchannel;

step 3, coating glue once, namely spin-coating photoresist on the single sides of the cover layer (1) and the substrate layer (2), and spin-coating photoresist on the two sides of the structural layer (3);

step 4, respectively covering a cover plate mask and a substrate mask on the gluing sides of the cover plate layer (1) and the substrate layer (2), and respectively covering a structural layer mask and a rigid support plate on the two gluing sides of the structural layer (3);

step 5, performing primary ultraviolet irradiation, and irradiating corresponding lamella from the mask side of each lamella by using ultraviolet light;

step 6, drying each sheet layer;

step 7, etching for the first time, wherein each sheet layer obtained in the step 6 is etched by using etching liquid, and the cover sheet layer (1) is fully etched to obtain a through inlet through hole (11) and a through outlet through hole (12); the substrate layer (2) controls the etching degree to obtain a non-through micro-channel structure; fully etching the structural layer (3) to obtain a through micro-channel structure;

step 8, 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 (1);

step 10, covering a second cover plate mask on the gluing side of the cover plate layer (1) obtained in the step 9, and carrying out ultraviolet irradiation, drying, etching and glue stripping on the cover plate layer (1) according to the mode in the step 5-8; thereby obtaining a cover sheet layer (1) 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 cover plate layer (1) to one side of the structural layer (3) without the rigid support plate, removing photoresist between the structural layer (3) and the rigid support plate to separate the structural layer (3) from the rigid support plate, and fixedly attaching the micro-channel side of the substrate layer (2) to the structural layer (3) to obtain the chip (10) with a three-layer structure.

8. The method for preparing a vertical microorganism detection chip (10) according to claim 6, wherein: also comprises the following steps:

step 12, preparing a coating mask only covering the containing cavities on the cover plate layer (1) and the substrate layer (2); the coating masks are respectively positioned and attached to the non-channel sides of the cover plate layer (1) and the substrate layer (2);

and step 13, respectively applying polarization coatings on the mask sides of the cover sheet layer (1) and the substrate layer (2) of the chip (10) obtained in the step 12, wherein the polarization directions of the two polarization coatings are opposite.

And 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.

9. The method for preparing a vertical microorganism detection chip (10) according to claim 6, wherein: the side walls of the middle layer delivery channel (33) are more hydrophilic than the side walls of the delivery channels on the cover sheet layer (1) and the substrate layer (2).

Technical Field

The present invention relates to a biological detection device, and more particularly to a microorganism detection system for performing detection of a DNA specific to a microorganism such as a virus or a bacterium by a 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

To solve the above problems in the prior art, the present invention provides a microorganism detection system. The microorganism detection system can acquire clear and reliable fluorescence signal points, reduce and even eliminate background interference caused by stray light such as exciting light and the like, effectively inhibit the mixing of fluorescence signals of adjacent liquid drops and effectively improve the independence between different signal points; in addition, the microbiological detection system of the present invention enables efficient droplet filling.

In order to achieve the technical effects, the invention firstly provides a vertical microorganism detection chip, for convenience of description, a chip 10 is referred to as the vertical microorganism detection chip hereinafter, the chip 10 is vertically placed when in use and comprises a cover sheet layer 1 and a substrate layer 2, an inlet through hole 11 and an outlet through hole 12 are formed in the cover sheet layer 1, and the position of the inlet through hole 11 is higher than that of the outlet through hole 12; preferably, the inlet/outlet through hole 11 and the outlet through hole 12 are located at the upper and lower ends, for example, the left side, of the same side of the vertical central axis of the chip 10.

Micro-channel structures are arranged on the substrate layer 2 and are non-penetrating grooves, so that mutual positioning of all parts of the micro-structures is facilitated. The microchannel structure includes the kind groove 21 and the play appearance groove 22 that correspond to the import through-hole 11 and the export through-hole 12 of cover plate layer 1 respectively, communicates kind groove 21 and a play appearance groove 22 to conveying channel 23 of extending buckles between the two, conveying channel 23 includes the distribution channel 231 of a plurality of horizontal extensions and connects in the end of last level distribution channel 231 and the connecting channel 232 of the head end of next level distribution channel 231.

Except for the distribution channels 231 communicated with the sample outlet groove 22, the lower side wall of each distribution channel 231 is communicated with a plurality of accommodating cavities 24, and the accommodating cavities 24 are positioned between two adjacent distribution channels 231.

Preferably, the bottom of the receiving chamber 24 is provided with a side flow channel 25 communicated with the upper edge of the next-stage distribution channel 231; because of the channel flow resistance, the continuous phase fluid flowing in the upstream distribution channel 231 has relatively higher kinetic energy or fluid pressure, thus allowing a portion of the continuous phase fluid flowing in the upstream distribution channel 231 to flow directly into the downstream distribution channel 231 from the side flow channel 25, thereby forming a side flow at the bottom of each of the receiving chambers 24, and the continuous phase fluid and micro-droplets still flowing in the distribution channel 231 can be defined as a bulk flow; the lateral flow can induce micro-droplets in the bulk flow to enter the receiving chamber 24, and at the same time, the continuous phase fluid forming the lateral flow rejoins the bulk flow in the downstream distribution channel 231, i.e., the continuous phase fluid is not lost during the entire distribution process.

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 holding chamber 24 do not effectively close the lateral flow channel 25, and therefore, collecting or discharging the lateral flow causes a continuous decrease of continuous phase fluid in the bulk flow, resulting in a pressure loss due to fluid loss, which is detrimental to the process of dispensing microdroplets, especially for the case of a large number of microdroplets in the microdroplet array; furthermore, the continuous phase fluid acts on the one hand as a transport medium for the droplets and, on the other hand, also as a separation of adjacent droplets; therefore, the loss of continuous phase fluid also results in a smaller and smaller spacing between adjacent droplets, even the spacing becomes zero (a phenomenon that occurs when the dispensing path is long), creating a risk of droplet coalescence.

Preferably, the outlet of the lateral flow channel 25 is located between two adjacent receiving cavities 24, so that the lateral flow flowing out of the receiving cavities 24 can be prevented from impacting the micro-droplets in the receiving cavities 24. Specifically, two rows of receiving chambers 24 communicating with different distribution channels 231 may be arranged alternately, and the lateral flow channel 25 may be vertically arranged at the bottom of the receiving chamber 24; alternatively, at least the outlet portion of the side flow path 25 may be obliquely disposed in a state where the receiving chamber 24 is positioned at the same position of the distribution path 231 to prevent impact on the micro-droplets in the receiving chamber 24. The outlet portion of the side flow channel 25 is preferably inclined in a direction such that the horizontal component of the side flow is in the same direction as the bulk flow in the respective distribution channel 231, so that the vertical component of the side flow may provide additional guidance for the micro-droplets.

Preferably, the receiving cavity 24 is a circular cavity, and the depth of the cavity is equal to the diameter of the circular cavity (both in a view perpendicular to the chip), so as to allow the micro-droplets filled in the receiving cavity 24 to maintain a better sphericity. The depth of the lateral flow channel 25 is the same as that of the accommodating cavity 24, so as to facilitate the manufacture of the substrate layer 2 and ensure the uniformity of the sizes of all microstructures of the chip.

Preferably, the upper half of the receiving chamber 24 communicates with the lower edge of the distribution channel 231, forming an opening smaller than the diameter of the receiving chamber 24; therefore, on the horizontal section passing through the center of circle, two symmetrical eaves 242 capable of wrapping the partial eaves 242 of the upper half part of the micro-droplets are formed between the cavity wall 241 of the accommodating cavity 24 and the two edges of the opening, and the eaves 242 can effectively prevent the filled micro-droplets from escaping from the accommodating cavity 24 under the washing of the main body flow.

The receiving chamber 24 has a double-brim 242 structure at the opening thereof, so that the width of the opening thereof is smaller than the diameter of the receiving chamber 24, which causes the micro-droplets matching the diameter of the receiving chamber 24 to undergo a certain degree of deformation to enter the receiving chamber 24, which causes a certain difficulty in the droplet dispensing process; or the size of the micro-droplets may be further reduced to allow them to enter the receiving chamber without deformation, but too small droplets tend to cause undesirable conditions such as multiple droplet fills in the receiving chamber 24 or droplet accumulation at the entrance to the receiving chamber 24. Therefore, it is preferable that the width of the opening (referring to the dimension in the direction of the bulk flow) is not less than 3/4 of the diameter of the receiving chamber 24 to reduce the deformation requirement for the micro-droplets entering the receiving chamber 24 or to select micro-droplets with a suitably small size.

In addition, the present invention finds that the use of the single brim 242 configuration can effectively address both the deformation requirement during the droplet filling process and the risk of droplet accumulation caused by the use of small-sized droplets. Specifically, the eaves 242 are provided only on the downstream side of the opening of the accommodation chamber 24; and a guide portion 243 similar to a rounded chamfer is provided on the upstream side of the opening thereof; preferably, the radius of the guide portion 243 is the same as the height of the brim 242. The similar parcel effect to little liquid droplet of two eaves 242 constructions can be played to single eaves 242 constructions, simultaneously, because the existence of guide portion 243, the opening width who holds chamber 24 is greater than its diameter, and little liquid droplet need not to experience deformation and can get into and hold chamber 24, and then allows to choose for use and hold the little liquid droplet that the chamber diameter is more close to, reduces the liquid droplet and holds the multiple packing or the accumulational risk in the chamber.

Preferably, the chip 10 may include more layer structures, such as a cover sheet layer 1, a substrate layer 2 and a structure layer 3 sandwiched between the cover sheet layer 1 and the substrate layer 2; the structural layer 3 may be a single-layer structure or a multi-layer structure.

The cover sheet layer 1 and the substrate layer 2 have the same shape and size, and also have an overlapped (including the same channel shape, size and position relative to the layer) non-through micro-channel structure, wherein the micro-channel structure comprises a part of a conveying channel and a containing cavity; with the difference that the cover sheet layer 1 has an inlet through hole 11 and an outlet through hole 12 therethrough; the substrate layer 2 has a corresponding non-through sample inlet slot 21 and sample outlet slot 22. The structural layer 3 includes a portion overlapping with the microchannel structures on the cover sheet layer 1 and the substrate layer 2, and a sample inlet groove 31 and a sample outlet groove 32 corresponding to the inlet through hole 11 and the outlet through hole 12. Wherein, the micro-channel structures of the cover sheet layer 1 and the substrate layer 2 do not comprise a lateral flow channel, and the micro-channel structure of the structure layer 3 comprises a middle layer lateral flow channel 35; the micro-channel 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 are mutually overlapped to form a complete combined conveying channel, a sample feeding groove, a sample discharging groove and an accommodating cavity, but a side flow channel only exists in the structural layer 3.

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 lateral flow channel 25 is difficult to effectively close by the micro-droplets, so that even after the receiving chamber 24 is filled, the lateral flow channel 25 still provides an inducement to the micro-droplets in the bulk flow, which reduces the droplet dispensing rate and at the same time increases the risk of droplet build-up.

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 cover sheet layer 1 and the substrate layer 2, the mutually separated rims 37 of the structural layer 3 and the block units 36 need to be accurately positioned, which can be achieved by using rigid inert support plates during the lithography of the structural layer 3, as will be described in detail in the chip manufacturing method section below.

A chip 10 having a multi-layer (meaning three and more) structure may also have a brim 242 for enclosing the upper half of the filled droplet, thereby resisting scouring of bulk flow; in particular, a double eave 242 configuration may be used, or a single eave 242 downstream of the chamber and a guide 243 upstream of the chamber similar to those described above. The eaves 242 and the guide 243 are formed by combining the cover sheet layer 1, the substrate layer 2 and the structural layer 3.

Preferably, the side walls of the middle layer delivery channel 33 are more hydrophilic than the side walls of the water delivery channels on the cover sheet layer 1 and the substrate layer 2, so as to allow the middle layer side flow channel 35 to provide equivalent lateral flow strength with a relatively small cross-sectional area, thereby not reducing the induction of micro-droplets due to the reduction of the cross-sectional area of the middle layer side flow channel 35.

Preferably, for a chip 10 with a double-layer structure or a multi-layer structure, the non-attaching sides of the cover sheet layer 1 and the substrate layer 2 are respectively coated with polarization coatings with opposite polarization directions. This arrangement allows the excitation light source and the fluorescence signal detecting element, such as a CCD camera, to be respectively disposed on both sides of the chip 10 when the chip 10 is vertically disposed, so that after the excitation light passes through the polarized coating on the surface of the cover sheet 1, the micro-droplets are irradiated with the P-polarized excitation light transmitted through the micro-droplets, and the P-polarized excitation light transmitted through the micro-droplets continues to be directed to the substrate layer 2, while the polarized coating on the surface of the substrate layer 2 is opposite to the polarized coating on the cover sheet 1 in the polarization direction, thus allowing only the S-polarized light to be transmitted, and therefore, the excitation light in the P-polarized state will be intercepted by the substrate layer 2 and cannot continue to be directed to the CCD camera, thereby eliminating the background interference of the excitation.

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 substrate layer 2 is provided with a light-absorbing coating; the light absorbing coating does not cover the receiving cavity 24 or the combined receiving cavity. Such a light-absorbing coating can be realized by means of a mask, i.e. the receiving cavities are covered with mask parts complementary to a photolithographic mask (which can also be processed to remove masked parts of the transport and lateral flow channels while leaving only parts that can mask the receiving cavities), and then the light-absorbing coating is applied to the respective layer surface. Such an arrangement allows for the external polarizing element to be incorporated to prevent the fluorescence signals of adjacent droplets from merging with each other. Specifically, polarization elements with opposite polarization directions are respectively arranged on the light paths on the front side and the rear side of the chip 10. When the excitation light is emitted to the chip 10, the excitation light is first specially P-polarized light by the upstream polarizer, and an unexpected part of the P-polarized light passing through the accommodating cavity 24 is absorbed by the light-absorbing coating on the surface of the substrate layer 2 and cannot penetrate through the chip; the P-polarized light entering and transmitting the micro-droplets in the accommodating chamber 24 is completely intercepted by the downstream side polarization element; this works substantially the same as the previous polarization scheme, blocking the excitation light to the CCD camera. In addition, the fluorescent light emitted from the fluorescent probes in the micro-droplets is scattered and emitted, wherein the fluorescent light signal directed to the light-absorbing coating is absorbed and thus cannot be transmitted, and only the fluorescent light signal directed to the light-absorbing coating corresponding to the blank portion of the accommodating chamber 24 is transmitted, and then is converted into S-polarized fluorescent light by the downstream-side polarizing element and directed to the CCD camera, in the process, the fluorescent light portion where adjacent signals are fused due to the passage between adjacent droplets through the chamber wall is eliminated, and the propagation path of the excitation light to the CCD camera is also completely blocked, so that the obtained fluorescent light signal is clearer and easier to recognize.

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 cover sheet layer 1, and a P-polarizing coating is disposed on the backlight side; wherein the polarizing coating does not cover the receiving cavity or the combined receiving cavity.

Under the arrangement, after the excitation light passes through the P-polarization element, the excitation light is emitted to the cover sheet layer 1 of the chip 10 in a P-polarization state, wherein part of the P-polarization excitation light is blocked by the S-polarization coating layer and cannot pass through the cover sheet layer 1, and only the P-polarization light emitted to the accommodating cavity part can pass through the cover sheet layer 1 and the substrate layer 2 and then is emitted to the downstream S-polarization element, wherein the transmitted excitation light is completely blocked; the fluorescent probes in the micro-droplets are excited to emit fluorescent signals, wherein part of the fluorescent signals are changed into P-polarized fluorescent light through the P-polarized coating on the substrate layer 2, and then emitted to the S-polarized element at the downstream of the chip 10 and completely blocked at the position; part of the fluorescence signal passes through the notch part of the substrate layer 2 corresponding to the accommodating cavity and is emitted to the S-polarized element in the form of unpolarized light, and after passing through the S-polarized element, the fluorescence signal is emitted to the CCD camera in the form of S-polarized fluorescence.

The microorganism detection system is based on the chip 10, and 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 channel wall 61 disposed perpendicular to the chip 10; and a short wall 62 disposed parallel to the chip 10 and capable of abutting against the chip 10 when the chip 10 is inserted into the slot 43; the short wall 62 is positioned so as not to obscure the containing cavity on the chip 10; the channel wall 61 is provided with two sliding grooves 63 which are arranged up and down; a liquid inlet slide block 64 corresponding to the inlet through hole 11 of the chip 10 is arranged in the slide groove 63 positioned above the liquid inlet slide block in a sliding way; a liquid outlet slide block 65 corresponding to the outlet through hole 12 of the chip 10 is arranged in the lower slide groove 63 in a sliding way; the side of the liquid inlet slide block 64 opposite to the chip 10 and the side of the liquid outlet slide block 65 opposite to the chip 10 are respectively provided with a needle 66 which can be in fluid seal with the inlet through hole 11 and the outlet through hole 12 of the chip 10.

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 cover plate layer 1 and a substrate layer 2, the method comprises the following steps:

step 1, selecting a cover sheet layer and a substrate layer with the same shape and size, wherein the material of the substrate layer can adopt the conventional materials in the field, such as glass, PDMS and the like;

step 2, preparing a mask, and respectively preparing a cover plate mask and a substrate mask, wherein the cover plate mask only comprises gaps for forming an inlet through hole 11 and an outlet through hole 12; the substrate mask comprises notches for forming a sample inlet groove 21, a sample outlet groove 22, a conveying through 23, a containing cavity 24 and a lateral flow channel 25;

step 3, spin-coating photoresist, namely respectively spin-coating photoresist on the single sides of the cover plate layer 1 and the substrate layer 2;

step 4, covering masks, namely measuring the covering cover plate masks and the substrate masks respectively on the gluing of the cover plate layer 1 and the substrate layer 2;

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 cover sheet layer 1 is fully etched to obtain a through inlet through hole 11 and a through outlet through hole 12; controlling the degree of etching amount by the substrate layer to obtain a non-through micro-channel structure;

and 8, removing the photoresist, and fixedly attaching any one side of the cover layer 1 to the microchannel side of the substrate layer 2 (by adopting an attaching mode known in the art), so as to obtain the chip 10 with the double-layer structure.

When the chip 10 comprises only the cover layer 1, the substrate layer 2 and the structural layer 3, the steps are as follows:

step 1, selecting a cover sheet layer, a substrate layer and a structural layer with the same shape and size, wherein the material of the sheet layer can adopt the conventional materials in the field, such as glass, PDMS and the like;

step 2, preparing a mask, namely respectively preparing a cover plate mask, a cover plate second mask, a substrate mask and a structural layer mask; wherein the cover sheet mask includes only notches for forming the inlet through-holes 11 and the outlet through-holes 12; the cover sheet second mask includes only notches for forming the cover sheet layer feeding path 13 and the cover sheet layer accommodating chamber 14; the substrate mask only comprises gaps for forming a sample inlet groove 21, a sample outlet groove 22, a conveying through 23 and an accommodating cavity 24; the structural layer mask comprises notches for forming a sample inlet middle groove 31, a sample outlet middle groove 32, a middle layer conveying channel 33, a middle layer accommodating cavity 24 and a middle layer side flow channel 35; wherein the corresponding indentations on each mask have the same shape and size and are positioned such that the microstructures of each layer produced are capable of forming a complete combined microchannel;

step 3, coating glue once, namely spin-coating photoresist on the single sides of the cover plate layer 1 and the substrate layer 2, and spin-coating photoresist on the two sides of the structural layer 3;

step 4, covering masks, namely covering the cover plate mask and the substrate mask on the gluing sides of the cover plate layer 1 and the substrate layer 2 respectively, and covering the structural layer mask and the rigid support plate on the two gluing sides of the structural layer 3 respectively;

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 cover sheet layer 1 is fully etched to obtain a through inlet through hole 11 and a through outlet through hole 12; controlling the degree of etching amount by the substrate layer to obtain a non-through micro-channel structure; the structural layer 3 is fully etched to obtain a through micro-channel structure, at this time, the structural layer 3 is etched into a frame 37 and a block unit 36 which are separated from each other, but on the opposite side of the structural side mask, the frame 37 and the block unit 36 which are separated from each other are still fixed and positioned on the rigid supporting plate through the photoresist which is not irradiated by ultraviolet light, so that the mutual position positioning is still kept;

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 cover plate layer 1 to one side of the structural layer 3 without the rigid support plate, then taking out the photoresist between the structural layer 3 and the rigid support plate, so that the structural layer 3 is separated from the rigid support plate, and then fixedly attaching the micro-channel side of the substrate layer 2 to the structural layer 3, so as to obtain the chip 10 with a three-layer structure.

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 cover plate layer 1 and the substrate layer 2, and the coating mask can be manufactured in the form of a conventional film-coated sticker in the field in consideration of the discrete distribution of the containing cavities so as to ensure the accurate positioning of the shielding part of the mask; the coating masks are respectively positioned and attached to the non-channel sides of the cover plate layer 1 and the substrate layer 2;

and step 13, applying a polarization coating, wherein the polarization coating is respectively applied to the mask sides of the cover plate layer 1 and the substrate layer 2, and the polarization directions of the two polarization coatings are opposite.

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.