CN111621415A - Microorganism detection system - Google Patents

Abstract

The invention relates to a microorganism detection system, comprising a detection platform, a chip vertically arranged on the detection platform, the detection platform is provided with excitation light sources and fluorescence detection elements located on both sides of the chip, and further includes a chip on the detection platform. The first and second polarizing elements with opposite polarization directions are arranged on the downstream and downstream sides of the optical path of the chip, and the chip includes a conveying channel extending on the inlet and outlet parts and an accommodating cavity arranged in the horizontal part of the conveying channel, The bottom of the accommodating cavity is arranged with a side flow channel whose outlet is connected to the distribution channel arranged horizontally in the next stage, and the excitation light source and the fluorescence detection element are arranged on both sides of the chip, so that the background interference of the excitation light can be completely shielded; The inlet and outlet ends are respectively connected to the bottom of the holding chamber and the lower-level distribution channel, which can induce microdroplets without consuming the continuous phase fluid.

Description

A microbial detection system

technical field

The present invention relates to a biological detection device, in particular to a microbial detection system for performing the detection of specific DNA of microorganisms such as viruses and bacteria through PCR reaction.

Background technique

The droplet-based digital PCR chip is one of the most advanced nucleic acid quantitative detection methods. Compared with traditional PCR technology, digital PCR dilutes the solution containing target genes, primers, polymerases, etc., and divides it into dozens to dozens of Thousands of tiny, independent reactors, so that the number of nucleic acid templates in each reactor is less than or equal to 1, and traditional PCR amplification and fluorescence detection are performed on each reactor. The reactor containing the target gene was marked as 1, and the reactor without the target gene was marked as 0. According to the relative ratio and the volume of the reactor, the nucleic acid concentration of the original solution was calculated using the Poisson distribution.

Currently, the mainstream implementation of digital PCR is based on a microfluidic chip with an array of microreaction chambers; water-in-oil droplets from a droplet preparation unit are dispensed into the array of microreaction chambers, where they undergo several heating-annealing times After the amplification cycle, excitation light is introduced into the micro-reaction chamber, and the microdroplets including the target nucleic acid template emit fluorescence under the illumination of the excitation light, the secondary fluorescence is detected, and the corresponding detection results can be obtained after statistical analysis. In the main steps of the digital PCR detection process, the effective distribution of the droplets in the micro-reaction chamber and the accurate capture of the fluorescence signal greatly affect the reliability of the detection results.

In the prior art, the droplets are partially blocked by means of distribution channels and microstructures such as protrusions arranged in similar distribution channels, so as to improve the effective filling rate of the micro-reaction chamber (referring to the filling rate of a single droplet, excluding no filling rate). and multi-droplet filled reaction chamber). However, due to the resistance of the microchannel itself and the local barrier, there is a large pressure difference in the continuous phase fluid flowing between the upstream and downstream of the distribution channel, which will lead to a huge difference in the filling effect of the micro-reaction chamber upstream and downstream of the distribution channel. .

In addition, in the process of fluorescence detection of the amplified micro-reaction chamber, the reflection and refraction of the incident excitation light often cause strong background interference; The non-directional nature of the fluorescence emitted by the excited droplets in the reaction chamber makes the fluorescence signals between adjacent microwells often partially pass through the pore wall and are mixed with the fluorescence signals emitted by the adjacent droplets, making it difficult to distinguish them. The problem.

SUMMARY OF THE INVENTION

In order to solve the above problems in the prior art, the present invention provides a microorganism detection system. The microorganism detection system of the invention can obtain clear and reliable fluorescent signal points, reduce or even eliminate the background interference caused by stray light such as excitation light, and can also effectively suppress the mixing between the fluorescent signals of adjacent droplets, and effectively improve the relationship between different signal points. Independence between the two; in addition, the microorganism detection system of the present invention can achieve efficient droplet filling.

In order to achieve the above technical effect, the present invention first provides a vertical microorganism detection chip. For the convenience of description, the vertical microorganism detection chip is referred to below by the chip 10. The chip 10 is placed vertically during use, and includes The cover sheet layer 1 and the base sheet layer 2, the cover sheet layer 1 is provided with an inlet through hole 11 and an outlet through hole 12, and the position of the inlet through hole 11 is higher than the outlet through hole 12; The hole 11 and the outlet through hole 12 are located at the upper and lower ends of the same side of the vertical central axis of the chip 10 , for example, the left side.

A micro-channel structure is arranged on the substrate layer 2 , and the micro-channel structure is a non-penetrating groove, so as to facilitate the mutual positioning of various parts of the micro-structure. The microchannel structure includes a sample inlet slot 21 and a sample outlet slot 22 corresponding to the inlet through hole 11 and the outlet through hole 12 of the cover sheet layer 1 respectively, communicated with the sample inlet slot 21 and the sample outlet slot 22, and is connected between the two. The conveying channel 23 is bent and extended between them. The conveying channel 23 includes a plurality of horizontally extending distribution channels 231 and a connecting channel 232 connected to the end of the upper distribution channel 231 and the head end of the lower distribution channel 231 .

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

Preferably, the bottom of the holding chamber 24 is provided with a side flow channel 25 that communicates with the upper edge of the distribution channel 231 of the next stage; due to the flow resistance of the channel, the continuous phase fluid flowing in the upstream distribution channel 231 has relatively higher kinetic energy or fluid pressure, thereby allowing a portion of the continuous phase fluid flowing in the upstream distribution channel 231 to flow directly from the side flow channel 25 into the downstream distribution channel 231, thereby forming a lateral flow at the bottom of each of the holding chambers 24, which can The continuous phase fluid and microdroplets still flowing in the distribution channel 231 are defined as the main flow; the lateral flow can induce the microdroplets in the main flow to enter the holding chamber 24, and at the same time, the lateral flow is formed The continuous phase fluid of 100 Å is rejoined into the main flow in the downstream distribution channel 231, that is, the continuous phase fluid is not lost during the whole distribution process.

Keeping the amount of continuous phase fluid constant is important for the distribution process of microdroplets. For the treatment of continuous phase fluid in lateral flow, additional sink channels can also be set to collect or discharge it, but it should be noted that filling The microdroplets in the containment chamber 24 do not effectively close the side flow channel 25, therefore, collecting or discharging the side flow will result in a continuous reduction of the continuous phase fluid in the bulk flow, resulting in loss of fluid. This is not conducive to the distribution process of droplets, especially when the number of microdroplets in the microdroplet array is large; in addition, the role of the continuous phase fluid is on the one hand as a transport medium for droplets, on the other hand, it also It acts to separate adjacent droplets; therefore, the loss of continuous phase fluid also causes the spacing between adjacent droplets to become smaller and smaller, or even to zero (this phenomenon occurs when the distribution path is long), This creates a risk of droplet fusion.

Preferably, the outlet of the side flow channel 25 is located between two adjacent accommodating cavities 24 , so that the lateral flow flowing out therefrom can be prevented from impacting the droplets in the accommodating cavity 24 . Specifically, the two rows of accommodating cavities 24 connected to different distribution channels 231 can be arranged in a staggered manner, while the side flow channels 25 can be vertically arranged at the bottom of the accommodating cavities 24; In the case of the same position of the distribution channel 231 , at least the outlet part of the side flow channel 25 is inclined to prevent the impact on the micro droplets in the accommodating chamber 24 . The inclined direction of the outlet portion of the lateral flow channel 25 is preferably such that the horizontal component of the lateral flow is in the same direction as the bulk flow in the corresponding distribution channel 231, so that the vertical component of the lateral flow can be the droplet Provide additional guidance.

Preferably, the accommodating cavity 24 is a circular cavity, and its depth is equal to the diameter of the circular cavity (both with a viewing angle perpendicular to the chip), so as to allow the micro-droplets filled in the accommodating cavity 24 to remain relatively small. good sphericity. The depth of the side flow channel 25 is the same as the depth of the accommodating cavity 24, so as to facilitate the manufacture of the substrate layer 2 and ensure the uniformity of the size of the microstructures of each part of the chip.

Preferably, the upper half of the accommodating cavity 24 communicates with the lower edge of the distribution channel 231 to form an opening smaller than the diameter of the accommodating cavity 24; Two symmetrical eaves 242 are formed between 241 and the two edges of the opening, which can wrap the upper half of the micro-droplet, and the eaves 242 can effectively prevent the filled micro-droplets from flowing in the main body. Escape from the accommodating cavity 24 under flushing.

The above-mentioned accommodating cavity 24 has a double eaves 242 structure at its opening, so the width of its opening is smaller than the diameter of the accommodating cavity 24, which makes the microdroplets matching the diameter of the accommodating cavity 24 need to undergo a certain degree of deformation to enter the cavity. The accommodating cavity 24 can be inserted into the accommodating cavity, which will cause certain difficulties in the distribution process of the droplets; or the size of the microdroplets can be further reduced so that they can enter the accommodating cavity without being deformed, but the droplets that are too small can easily cause Undesirable situations such as multi-droplet filling in the containment chamber 24 or droplet buildup at the entrance of the containment chamber 24 . Therefore, preferably, the width of the opening (referring to the dimension along the flow direction of the main body) is not less than 3/4 of the diameter of the accommodating cavity 24, so as to reduce the requirement for the deformation of the droplets entering the accommodating cavity 24 or when selecting Microdroplets of moderately small size.

In addition, the present invention finds that the use of the single eave 242 configuration can effectively solve the two problems of 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 portion 242 is only provided on the downstream side of the opening of the accommodating cavity 24 ; and a guide portion 243 similar to an arc chamfer is provided on the upstream side of the opening thereof; preferably, the radius of the guide portion 243 is the same as that of the The heights of the eaves 242 are the same. The structure of the single eave portion 242 can play a similar role in wrapping the droplets as the structure of the double eave portion 242. At the same time, due to the existence of the guide portion 243, the opening width of the accommodating cavity 24 is larger than its diameter, and the droplets do not need to undergo deformation. The accommodating cavity 24 can be entered, thereby allowing the selection of microdroplets that are closer to the diameter of the accommodating cavity, reducing the risk of multiple filling or accumulation of droplets in the accommodating cavity.

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

The cover sheet layer 1 and the base sheet layer 2 have the same shape and size, and also have overlapping (including the same channel shape, size and position relative to the layer) non-penetrating micro-channel structure, wherein the micro-channel structure. The channel structure includes part of the delivery channel and the accommodating cavity; the difference is that the cover sheet layer 1 has through-penetrating inlet through holes 11 and outlet through-holes 12; the substrate layer 2 has corresponding non-through sample inlet grooves 21 and sample outlet holes 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 slot 31 and a sample outlet slot corresponding to the inlet through hole 11 and the outlet through hole 12. 32. Wherein, the microchannel structures of the cover sheet layer 1 and the substrate layer 2 do not include side flow channels, while the microchannel structure of the structural layer 3 includes the middle-layer side flow channels 35; the microchannel structures of the structural layer 3 are all through structures. , so that the cover sheet layer 1 , the structural layer 3 and the substrate layer 2 are superimposed to form a complete combined conveying channel, sample inlet slot, sample outlet slot and accommodating cavity, but the side flow channel only exists in the structural layer 3 .

Such an arrangement allows the side flow channel to have a square channel cross section, which can be better closed by the microdroplets filled in the combined accommodating cavity. Otherwise, the rectangular cross section of the side flow channel 25 is difficult to be effectively used by the microdroplets. so that even after the containment cavity 24 is filled, the side flow channel 25 still provides induction to the microdroplets in the bulk flow, which reduces the droplet dispensing rate and increases the risk of droplet buildup.

Since the upper microchannel structure of the structural layer 3 is a through structure, its sheet layer includes a frame 37 and a block unit 36 that are separated from each other; wherein, a middle-layer accommodating cavity 34 is formed between the two adjacent block units 36, and the middle-layer side circulates The non-edge portion of the road 35 and the middle-layer conveying channel 33; the edge portion of the middle-layer conveying channel 33 is formed between the block unit 36 and the frame 37.

In order to form an integral microchannel structure with the exact combination between the cover sheet layer 1 and the base sheet layer 2, the mutually separated frame 37 and the block unit 36 of the structure layer 3 need to be precisely positioned. The photolithography of the structural layer 3 is implemented using a rigid inert support plate, which will be described in detail in the chip fabrication method section below.

The chip 10 having a multi-layer (referring to three or more layers) structure can also have the eave portion 242 for wrapping the upper half of the filled droplet, thereby resisting the scouring of the main body flow; specifically, the double eave portion 242 structure, Or the structure of the single eave portion 242 located downstream of the accommodating cavity and the guide portion 243 located upstream of the accommodating cavity as described above. Wherein, the eaves portion 242 and the guide portion 243 are both constituted by the combination of the cover sheet layer 1 , the base sheet layer 2 and the structural layer 3 .

Preferably, the side walls of the middle layer transport channel 33 are more hydrophilic than the side walls of the water transport channels on the cover sheet layer 1 and the base sheet layer 2, so as to allow the middle layer side flow channel 35 to be In the case of having a relatively small cross-sectional area, the same lateral flow intensity is provided, so that the reduction of the cross-sectional area of the middle-layer lateral flow channel 35 will not reduce the induction effect on the microdroplets.

Preferably, for the chip 10 with a double-layer structure or more, the non-bonding sides of the cover sheet layer 1 and the base sheet layer 2 are respectively coated with polarizing coatings with opposite polarizing directions. Such an arrangement allows, when the chip 10 is vertically arranged, the excitation light source and the fluorescent signal detection element, such as a CCD camera, are respectively arranged on both sides of the chip 10, so that the excitation light passes through the polarizing coating on the surface of the cover sheet 1, and the The single polarization state (such as P-polarized light) illuminates the microdroplet, and the P-polarized excitation light transmitted through the droplet continues to be emitted to the substrate layer 2, and the polarized coating on the surface of the substrate layer 2 is the same as the one on the cover layer 1. The polarization direction of the polarizing coating is opposite, so only S-polarized light is allowed to pass through. Therefore, the excitation light of P-polarized state will be intercepted by the substrate layer 2 and cannot continue to be emitted to the CCD camera, thus eliminating the background interference of the excitation light.

The polarizing coating can also be replaced by external polarizing elements respectively disposed on the upstream and downstream optical paths of the chip 10 .

Preferably, a light-absorbing coating is provided on the non-bonding surface of the substrate layer 2; the light-absorbing coating does not cover the accommodating cavity 24 or the combined accommodating cavity. Such a light absorbing coating can be achieved with the aid of a mask, even if masked with a mask part complementary to the lithographic mask (which can also be machined to remove the masked parts of the delivery channel and the lateral flow channel, leaving only the part that can mask the containment cavity) The accommodating cavity is then implemented with the light-absorbing coating on the surface of the corresponding layer. Such an arrangement allows the use of external polarizing elements to prevent the fluorescence signals of adjacent droplets from merging with each other. Specifically, polarizing elements with opposite polarizing directions are respectively arranged on the optical paths on the front and rear sides of the chip 10 . When the excitation light is directed to the chip 10, it is firstly P-polarized light through the upstream polarizing element, and the 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 pass through the chip; The P-polarized light entering and passing through the droplets in the accommodating cavity 24 is completely intercepted by the downstream polarizing element; this has basically the same effect as the previous polarization scheme, which can block the excitation light from emitting to the CCD camera. In addition, the fluorescence emitted by the fluorescent probes in the microdroplets is scattered in all directions, in which the fluorescent signal directed to the light-absorbing coating is absorbed and thus cannot be transmitted through, and only the light-absorbing coating is directed to the corresponding cavity on the light-absorbing coating. The fluorescent signal in the blank part of 24 can pass through, and then it is converted into S-polarized fluorescence by the downstream side polarizing element and then directed to the CCD camera. During this process, adjacent droplets pass through the cavity wall, which may lead to fusion of adjacent signals. The fluorescent part of the CCD is eliminated, and the propagation path of the excitation light to the CCD camera is also completely blocked, so the obtained fluorescent signal is clearer and easier to identify.

Such an effect can also be achieved by a combination of a polarizing coating and an external polarizing element. For example, a P polarizing element is arranged on the upstream side of the optical path of the chip 10, and an S polarizing element is arranged downstream. At the same time, on the light-facing side of the chip 10, such as a cover An S polarizing coating is provided on the outer side of the sheet layer 1, and a P polarizing coating is provided on the backlight side; wherein, the polarizing coating does not cover the accommodating cavity or the combined accommodating cavity.

Under this setting, after the excitation light is transmitted through the P-polarized element, it is directed to the cover layer 1 of the chip 10 in a P-polarized state, and part of the P-polarized excitation light is blocked by the S-polarized coating and cannot pass through the cover layer 1. Only The P-polarized light directed to the cavity part can pass through the cover sheet layer 1 and the substrate layer 2, and then be directed to the downstream S-polarized element, where the transmitted excitation light is completely blocked; After the fluorescent probe is excited, it emits a fluorescent signal, wherein part of the fluorescent signal passes through the P-polarized coating on the substrate layer 2 to become P-polarized fluorescence, and then radiates to the S-polarized element downstream of the chip 10, where it is absorbed. It is completely blocked; some fluorescent signals pass through the notch on the substrate layer 2 corresponding to the accommodating cavity, and are directed to the S-polarized element in the form of unpolarized light. After passing through the S-polarized element, the S-polarized The fluorescent form is directed towards the CCD camera.

The microorganism detection system of the present invention is based on the aforementioned chip 10, and the detection system includes a detection platform 4, a chip 10 and a light shield 7; the detection platform 4 is provided with a fixing part for fixing the vertically arranged chip 10, such as the The fixing portion may be a slot 43 that allows the chip 10 to be inserted and fixed, and of course, other conventional fixing methods in the art may also be used. The slot 43 is preferably fixed perpendicular to the long axis of the detection platform 4 , and along the long axis direction of the detection platform 4 , the slots 43 are respectively arranged in a manner parallel to the chip 10 . There are a first polarizing element 42 and a second polarizing element 44 ; wherein, the polarizing directions of the first polarizing element 42 and the second polarizing element 44 are opposite. A light source frame 41 is fixedly arranged at one end of the detection platform 4, and a light source 5 for excitation light is fixed on the light source frame 41, and the light source 5 is positioned so that the excitation light emitted by the light source 5 is perpendicular to the first, A second polarizing element; a photosensitive element 45 is fixed on the opposite end of the detection platform 4 (referring to the end opposite to the position of the light source), and the photosensitive element 45 is used for receiving fluorescent signals.

On the detection platform 4, a liquid supply assembly 6 is also vertically disposed on the side corresponding to the position of the chip 10; the liquid supply assembly 6 can clamp one side of the chip 10, and can pass through the inlet of the chip 10. The through holes 11 supply it with continuous phase fluid with droplets and receive the remaining fluid after dispensing the droplets from the outlet through holes 12 of the chip 10 .

The liquid supply assembly 6 includes a channel wall 61 disposed perpendicular to the chip 10 ; and disposed parallel to the chip 10 , and can abut the chip when the chip 10 is inserted into the slot 43 . The short wall 62 of 10; the short wall 62 is positioned so as not to block the accommodating cavity on the chip 10; the channel wall 61 is provided with two upper and lower sliding grooves 63; it slides in the upper sliding groove 63 A liquid inlet slider 64 corresponding to the inlet through hole 11 of the chip 10 is provided; a liquid outlet slider 65 corresponding to the outlet through hole 12 of the chip 10 is slidably arranged in the sliding groove 63 located below; the liquid inlet slider The sides of the liquid outlet slider 64 and the liquid outlet slider 65 facing the chip 10 are respectively provided with needles 66 that can be fluidly sealed with the inlet through holes 11 and the outlet through holes 12 of the chip 10 .

A groove 46 is provided around the edge of the upper surface of the detection platform 4 , and the groove 46 is used to fit the light shield 7 . The inner surface of the light shield 7 is coated with a light absorbing material, so as to prevent the transmission of external light and absorb the scattered light emitted from the interior, thereby reducing the interference of stray light.

The present invention also provides a method for preparing the chip 10. Specifically, when the chip 10 only includes a double-layer structure of a cover sheet layer 1 and a substrate layer 2, the steps are as follows:

Step 1, select a cover sheet layer and a base sheet layer with the same shape and size, wherein the material of the sheet layer can be conventional materials in the art, such as glass, PDMS, etc.;

Step 2, prepare a mask, respectively prepare a cover sheet mask and a substrate mask, wherein the cover sheet mask only includes a notch for forming the inlet through hole 11 and the outlet through hole 12; the substrate mask Including gaps for forming the sample inlet slot 21, the sample outlet slot 22, the conveying channel 23, the accommodating cavity 24 and the side flow channel 25;

Step 3, spin-coating photoresist, spin-coating photoresist on one side of the cover sheet layer 1 and the substrate layer 2 respectively;

Step 4, cover the mask, and measure the cover sheet mask and the substrate mask by applying glue on the cover sheet layer 1 and the substrate layer 2 respectively;

Step 5, ultraviolet irradiation, using ultraviolet light to irradiate the corresponding sheet layers from the mask side of each sheet layer, and the photoresist at the mask gap undergoes a chemical reaction under the action of ultraviolet light;

Step 6, drying each sheet;

Step 7, etching, using the etching solution to etch each layer obtained in step 6, wherein, the cover layer 1 is fully etched to obtain through-hole inlet through holes 11 and outlet through holes 12; the substrate layer controls the degree of etching, to obtain a non-penetrating microchannel structure;

Step 8: Remove the photoresist, and fix any side of the cover sheet 1 with the microchannel side of the substrate layer 2 (a bonding method known in the art can be used) to obtain a chip 10 with a double-layer structure.

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

Step 1, Step 1, select 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 be conventional materials in the art, such as glass, PDMS, etc.;

Step 2, prepare a mask, respectively prepare a cover sheet mask, a second cover sheet mask, a substrate mask and a structural layer mask; wherein, the cover sheet mask only includes the through holes for forming the inlet holes 11 and the outlet holes 12; the second cover sheet mask only includes the notches for forming the cover sheet layer conveying channel 13 and the cover sheet layer accommodating cavity 14; 22. The gaps in the conveying channel 23 and the accommodating cavity 24; the structural layer mask includes a notch for forming the middle-layer groove 31, the middle-layer groove 32, the middle-layer transport channel 33, the middle-layer accommodating cavity 24 and the middle-layer side flow channel 35; Notches; wherein corresponding notches on each mask have the same shape and size and are positioned such that the resulting microstructures of the individual layers are fabricated to form a complete combined microchannel;

Step 3, applying glue once, spin-coating photoresist on one side of cover sheet layer 1 and substrate layer 2, and spin-coating photoresist on both sides of structural layer 3;

Step 4, cover the mask, cover the cover sheet mask and the substrate mask on the glue-coated sides of the cover sheet layer 1 and the substrate layer 2 respectively, and cover the structure layer mask on the two glue-applied sides of the structure layer 3 respectively. Molds and rigid support plates;

Step 5, irradiating with ultraviolet light once, using ultraviolet light to irradiate the corresponding sheet layer from the mask side of each sheet layer, and the photoresist at the gap of the mask undergoes a chemical reaction under the action of ultraviolet light;

Step 6, drying each sheet;

Step 7, one etching, using etching solution to etch each layer obtained in step 6, wherein, the cover layer 1 is fully etched to obtain through-hole inlet through holes 11 and outlet through holes 12; the substrate layer controls the degree of etching amount , to obtain a non-through microchannel structure; the structural layer 3 is fully etched to obtain a through microchannel structure. At this time, the structural layer 3 is etched into a frame 37 and a block unit 36 that are separated from each other, but the mask on the structural side is etched. On the opposite side, the frame 37 and the block unit 36 that are separated from each other are still fixed and positioned on the rigid support plate by the photoresist that is not irradiated by ultraviolet light, and thus still maintain their mutual positioning;

Step 8, remove the photoresist, and remove the photoresist on the mask side of each layer;

Step 9, secondary coating, second spin coating photoresist on either side of the cover sheet layer;

Step 10, in step 9, a second mask for coating the cover sheet layer is obtained to cover the cover sheet layer, and the cover sheet layer is subjected to ultraviolet irradiation, drying, etching and peeling according to the method in steps 5-8; thus A cover sheet layer having a through-penetrating inlet through hole 11 and an outlet through hole 12 and a non-penetrating cover sheet layer conveying channel 13 and a cover sheet layer accommodating cavity 14 is obtained;

In step 11, the microchannel side of the cover sheet layer 1 is fixedly attached to the side of the structural layer 3 without the rigid support plate, and then the photoresist between the structural layer 3 and the rigid support plate is taken out, so that the structural layer 3 It is separated from the rigid support plate, and then the microchannel side of the substrate layer 2 is fixedly attached to the structural layer 3 to obtain a chip 10 having a three-layer structure.

Preferably, it also includes the following steps:

Step 12, prepare a coating mask, the coating mask only covers the accommodating cavities on the cover sheet layer 1 and the substrate layer 2. Considering the discrete distribution of the accommodating cavities, conventional coating methods in the field can be used. The coating mask is made in the form of a film sticker to ensure the precise positioning of the mask blocking part; the coating mask is positioned and attached to the non-channel side of the cover sheet layer 1 and the substrate layer 2 respectively;

Step 13, applying a polarizing coating, respectively applying a polarizing coating on the mask side of the cover sheet layer 1 and the substrate layer 2, wherein the polarizing directions of the two polarizing coating layers are opposite.

In step 14, the chip 10 obtained in step 13 is dried, and the coating mask is torn off to obtain the chip 10 having a three-layer structure with a polarizing coating.

Compared with the prior art, the present invention can at least achieve the following beneficial effects: the vertically arranged chip allows the excitation light source and the fluorescence detection element to be arranged on both sides of the chip, so that the background interference of the excitation light can be completely shielded; A side flow channel is arranged on the channel structure, and the inlet and outlet ends of the side flow channel are respectively connected to the bottom of the holding chamber and the lower-level distribution channel, which can realize the induction of micro droplets without consuming the continuous phase fluid, and improve the droplet rate. Filling efficiency, and at the same time, it is not easy to cause droplet fusion defects; the side flow channels are separately arranged on the structural layer, so that the filled droplets can better close the corresponding side flow channels, thereby reducing or even eliminating the filled chamber pair. The induction effect of droplets improves the droplet distribution effect and reduces the risk of droplet accumulation; by applying polarizing coatings with opposite polarization directions on the outside of the chip cover layer and substrate layer, the detection of excitation light to fluorescence is realized. The optical path between the components is completely blocked. At the same time, in conjunction with the external polarizing elements arranged on both sides of the chip, it can also block the optical path between the fluorescent signal emitted through the side wall of the receiving cavity and the fluorescent detection element, avoiding adjacent fluorescent signals. Signal fusion between points.

Description of drawings

1 is a schematic diagram of a chip with a double-layer structure;

Fig. 2 is a partial enlarged view of area A in Fig. 1;

Fig. 3 is one of the side views of the microchannel of the substrate layer in Fig. 1;

Fig. 4 is the second side view of the microchannel of the substrate layer in Fig. 1;

5 is a horizontal cross-sectional view of the chip shown in FIG. 1;

FIG. 6 is the third view of the microchannel layer of the substrate layer in FIG. 1;

Fig. 7 is the three-dimensional enlarged view of area B in Fig. 6;

Fig. 8 is a partial enlarged view of region B in Fig. 6;

Figure 9 is one of the horizontal cross-sectional views of the chip shown in Figure 8;

FIG. 10 is the second horizontal cross-sectional view of the chip shown in FIG. 8 (not the area shown in FIG. 8 );

11 is a schematic diagram of a chip with a three-layer structure;

Fig. 12 is a partial enlarged view of region C in Fig. 11;

Fig. 13 is a partial enlarged view of region D in Fig. 11;

Fig. 14 is a partial enlarged view of region E in Fig. 11;

FIG. 15 is one of the combined enlarged views of the regions C, D, and E after the three-layer chips are bonded in FIG. 11;

FIG. 16 is the second enlarged view of the combination of regions C, D, and E after the three-layer chips are bonded in FIG. 11;

FIG. 17 is a horizontal cross-sectional view of the chip shown in FIG. 15;

FIG. 18 is one of the horizontal cross-sectional views of the chip shown in FIG. 16;

FIG. 19 is the second horizontal cross-sectional view of the chip shown in FIG. 16 (not the area shown in FIG. 16 );

Fig. 20 is the combined schematic diagram of detection platform and chip;

21 is a schematic view of a light hood;

Figure 22 is the first perspective of the liquid supply assembly;

Figure 23 is the second perspective of the liquid supply assembly;

In the figure: 1 is the cover sheet layer, 11 is the inlet through hole, 12 is the outlet through hole, 13 is the cover sheet layer conveying channel, 14 is the cover sheet layer accommodating cavity, 2 is the substrate layer, 21 is the injection slot, 22 23 is the conveying channel, 231 is the distribution channel, 232 is the connecting channel, 24 is the accommodating cavity, 241 is the cavity wall, 242 is the eaves, 243 is the guiding part, 25 is the side flow channel, and 3 is the structural layer , 31 is the sample inlet slot, 32 is the sample outlet slot, 33 is the middle layer conveying channel, 34 is the middle layer receiving cavity, 35 is the middle layer side flow channel, 36 is the block unit, 37 is the frame, 4 is the detection platform, 41 is the Light source frame, 42 is the first polarizing element, 43 is the slot, 44 is the second polarizing element, 45 is the photosensitive element, 46 is the groove, 5 is the light source, 6 is the liquid supply assembly, 61 is the channel wall, 62 is the short Wall, 63 is a sliding groove, 64 is a liquid inlet slider, 65 is a liquid outlet slider, 66 is a needle, 7 is a light shield, and 10 is a chip.

Detailed ways

In order to better illustrate the technical concept of the present invention, the solution of the present invention will be further described below with reference to the accompanying drawings.

Example 1

20-23, a microorganism detection system is provided, the detection system includes a detection platform 4, a chip 10 and a light shield 7; the detection platform 4 is provided with a slot 43 for fixing the vertically arranged chip 10, The slot 43 is fixed perpendicular to the long axis of the detection platform 4 , and along the long axis direction of the detection platform 4 , in the front and rear of the slot 43 are respectively arranged parallel to the chip 10 . The first polarizing element 42 and the second polarizing element 44; wherein, the polarizing directions of the first polarizing element 42 and the second polarizing element 44 are opposite. A light source frame 41 is fixedly arranged at one end of the detection platform 4, and a light source 5 for excitation light is fixed on the light source frame 41, and the light source 5 is positioned so that the excitation light emitted by the light source 5 is perpendicular to the first, A second polarizing element; a photosensitive element 45 is fixed on the opposite end of the detection platform 4 .

On the detection platform 4, a liquid supply assembly 6 is also vertically disposed on the side corresponding to the position of the chip 10; the liquid supply assembly 6 includes a channel wall 61 disposed perpendicular to the chip 10; and parallel to the The chip 10 is disposed, and when the chip 10 is inserted into the slot 43, it can abut against the short wall 62 of the chip 10; the short wall 62 is positioned so as not to block the accommodating cavity on the chip 10; The upper and lower parts of the channel wall 61 are respectively provided with a horizontal sliding groove 63; and a liquid inlet slider 64 corresponding to the inlet through hole 11 of the chip 10 is slid in the sliding groove 63 located above; the sliding groove located below A liquid outlet slider 65 corresponding to the outlet through hole 12 of the chip 10 is slidably arranged in the 63; The inlet through hole 11 and the outlet through hole 12 are fluid-tight to the needle 66 .

A groove 46 is provided around the edge of the upper surface of the detection platform 4 , and the groove 46 is used to fit the light shield 7 .

Example 2

1-2, a vertical microorganism detection chip for the detection system in Example 1 is provided, the chip 10 is placed vertically when in use, and includes a cover sheet layer 1 and a substrate layer 2, the The cover sheet layer 1 is provided with an inlet through hole 11 and an outlet through hole 12 , and the position of the inlet through hole 11 is higher than that of the outlet through hole 12 ; The upper and lower ends of the left side of the vertical axis.

A micro-channel structure is arranged on the substrate layer 2, and the micro-channel structure is a non-penetrating groove. The microchannel structure includes a sample inlet slot 21 and a sample outlet slot 22 corresponding to the inlet through hole 11 and the outlet through hole 12 of the cover sheet layer 1 respectively, communicated with the sample inlet slot 21 and the sample outlet slot 22, and is connected between the two. The conveying channel 23 is bent and extended between them. The conveying channel 23 includes a plurality of horizontally extending distribution channels 231 and a connecting channel 232 connected to the end of the upper distribution channel 231 and the head end of the lower distribution channel 231 .

Referring to FIG. 3 , except for the distribution channel 231 communicating with the sample outlet tank 22 , a plurality of accommodating cavities 24 are communicated on the lower side wall of each distribution channel 231 , and the accommodating cavities 24 are located between two adjacent distribution channels 231 . .

The bottom of the accommodating cavity 24 is provided with a side flow channel 25 that communicates with the upper edge of the distribution channel 231 of the next stage. The outlet of the side flow channel 25 is located between two adjacent accommodating cavities 24 , so that the lateral flow flowing therefrom can be prevented from impacting the droplets in the accommodating cavity 24 . Specifically, referring to FIG. 3 , the two rows of accommodating cavities 24 connected to different distribution channels 231 may be arranged in a staggered manner, while the side flow channels 25 are vertically arranged at the bottom of the accommodating cavities 24 ; alternatively, see FIG. 4 , under the condition that the accommodating cavity 24 is positioned at the same position of the distribution channel 231, at least the outlet part of the side flow channel 25 can be inclined, and the inclination direction of the outlet part of the side flow channel 25 makes the lateral flow The horizontal component is in the same direction as the bulk flow in the corresponding distribution channel 231 .

Referring to FIG. 5 , the accommodating cavity 24 is a circular cavity, and its depth is equal to the diameter of the circular cavity (both from a perspective perpendicular to the chip. The depth of the lateral flow channel 25 is the same as the depth of the accommodating cavity 24 . same.

The upper half of the accommodating cavity 24 communicates with the lower edge of the distribution channel 231 to form an opening smaller than the diameter of the accommodating cavity 24; thus, on the horizontal cross-section passing through the center of the circle, the cavity wall 241 of the accommodating cavity 24 and the Two symmetrical eaves 242 are formed between the two edges of the opening, which can wrap the upper half of the droplet.

Referring to FIG. 5 , the accommodating cavity 24 has a double eaves 242 structure at its opening, so the width of the opening is smaller than that of the accommodating cavity 24 , and the width of the opening is not less than 3/4 of the diameter of the accommodating cavity 24 .

Alternatively, referring to Figures 6-10, the receiving cavity 24 has a single eaves 242 configuration at its opening. Specifically, the eaves portion 242 is only provided on the downstream side of the opening of the accommodating cavity 24; and a guide portion 243 similar to an arc chamfer is provided on the upstream side of the opening thereof; the radius of the guide portion 243 is the same as that of the eaves. The heights of the portions 242 are the same.

Example 3

Referring to FIG. 11 , another vertical microorganism detection chip for the detection system in Example 1 is provided, the chip 10 is placed vertically when in use, and it includes a cover sheet layer 1 having the same shape and size, a base sheet layer 2 , and a structural layer 3 sandwiched between the cover sheet layer 1 and the substrate layer 2 .

Referring to FIGS. 12-14 , the cover sheet layer 1 and the substrate layer 2 have overlapping (including the same channel shape, size and position relative to the layers) non-penetrating microchannel structures; the cover sheet layer 1 There are through-penetrating inlet through holes 11 and outlet through-holes 12 ; the substrate layer 2 has corresponding non-penetrating sample inlet grooves 21 and sample outlet grooves 22 .

15-16 , 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 injection slot corresponding to the inlet through holes 11 and the outlet through holes 12 31 and the sample slot 32. Wherein, the microchannel structures of the cover sheet layer 1 and the substrate layer 2 do not include side flow channels, while the microchannel structure of the structural layer 3 includes the middle-layer side flow channels 35; the microchannel structures of the structural layer 3 are all through structures. , so that the cover sheet layer 1, the structural layer 3 and the substrate layer 2 can be superimposed to form a complete combined conveying channel, sample inlet slot, sample outlet slot and accommodating cavity, but the side flow channel only exists in the structural layer 3.

17-19 , the chip 1 has an eaves 242; specifically, it can be a double eaves 242 structure, or a single eaves 242 located downstream of the accommodating cavity and a guide part 243 located upstream of the accommodating cavity similar to those described above. structure. Wherein, the eaves portion 242 and the guide portion 243 are both constituted by the combination of the cover sheet layer 1 , the base sheet layer 2 and the structural layer 3 .

The sidewalls of the middle layer delivery channel 33 are more hydrophilic than the sidewalls 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 have a relatively small size. In the case of a cross-sectional area of 100 Å, the same lateral flow intensity is provided, so that the reduction of the cross-sectional area of the side flow channel 35 in the middle layer will not reduce the induction effect on microdroplets.

Example 4

The difference from Examples 1-3 is that the non-bonding sides of the cover layer 1 and the substrate layer 2 of the chip 10 are respectively coated with polarizing coatings with opposite polarization directions, and the polarizing coatings do not cover the accommodating cavity. 24 or combined accommodating chambers.

Example 5

The difference from Examples 1-4 is that the non-adhering 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 accommodating cavity 24 or the combined accommodating cavity.

The above are only examples of the best embodiments of the concept of the present invention, which should not be construed as limitations on all feasible embodiments of the present invention, and those of ordinary skill in the art can simply replace conventional means without creative work, etc. Appropriate technical solutions also belong to the feasible scope of the present invention. The protection scope of the present invention is defined by the claims.

Claims (10)

1. A microorganism detection system, the detection system comprising a detection platform (4), a chip (10) and a light shield (7); the chip (10) comprises an inlet through hole (11) and an outlet through hole (12) , an accommodating cavity (24) or a combined accommodating cavity; it is characterized in that: the detection platform (4) is provided with a slot (43) for fixing the vertically arranged chip (10), and the slot (43) A first polarizing element (42) and a second polarizing element (44) are respectively provided on the optical paths of the upstream and downstream excitation light parallel to the chip (10); the first polarizing element (42) and the second polarizing element (42) The polarization directions of the elements (44) are opposite; a light source frame (41) is fixedly arranged at one end of the detection platform (4), and a light source (5) for excitation light is fixed on the light source frame (41). (5) The excitation light that is positioned so that the excitation light emitted by it is perpendicular to the first and second polarizing elements; a photosensitive element (45) is fixed on the opposite end of the detection platform (4); the detection platform (4) ), a liquid supply assembly (6) is vertically provided on one side corresponding to the position of the chip (10); a groove (46) is provided around the edge of the upper surface of the detection platform (4), and the groove ( 46) For use with the hood (7). 2. The microorganism detection system according to claim 1, characterized in that: the liquid supply assembly (6) comprises a channel wall (61) arranged perpendicular to the chip (10); parallel to the chip (10) is arranged, and can abut against the short wall (62) of the chip (10) when the chip (10) is inserted into the slot (43); the short wall (62) is positioned so as not to block The accommodating cavity on the chip (10); the upper and lower parts of the channel wall (61) are respectively provided with a horizontal sliding groove (63); the sliding groove (63) located above is slidably provided with a sliding groove (63) corresponding to the chip (10). The liquid inlet slider (64) of the inlet through hole (11); the liquid outlet slider (65) corresponding to the outlet through hole (12) of the chip (10) is slidably arranged in the sliding groove (63) located below; The liquid inlet slider (64) and the liquid outlet slider (65) are respectively provided with an inlet through hole (11) and an outlet through hole (12) capable of communicating with the chip (10) on the side facing the chip (10). Fluid tight needle (66). 3. The microorganism detection system according to claim 2, characterized in that: the chip (10) comprises a cover sheet layer (1) and a substrate sheet layer (2) having the same shape and size, and the cover sheet layer ( 1) An inlet through hole (11) and an outlet through hole (12) are opened on the substrate layer (2), and the substrate layer (2) has a groove-shaped non-penetrating microchannel structure, and the microchannel structure includes corresponding to the cover layer (1). ) of the inlet through hole (11) and the outlet through hole (12) of the sample inlet slot (21) and the sample outlet slot (22), and communicated with the sample inlet slot (21) and the sample outlet slot (22), and in the A conveying channel (23) bent and extended between the two, the conveying channel (23) includes a plurality of horizontally extending distribution channels (231) and an end connected to the upper-level distribution channel (231) and the next-level distribution channel a connecting channel (232) at the head end of the channel (231); a plurality of accommodating cavities (24) communicated with the lower edge of the distribution channel (231) are provided at positions between two adjacent distribution channels (231); the The bottom of the accommodating cavity (24) is provided with a side flow channel (25) that communicates with the upper edge of the distribution channel (231) of the next stage. 4. The microorganism detection system according to claim 3, characterized in that: the accommodating cavity (24) is a circular cavity, the depth of which is equal to the diameter of the circular cavity; the depth of the side flow channel (25) The depth is the same as that of the accommodating cavity (24). 5. The microorganism detection system according to claim 4, characterized in that: the upper half of the accommodating cavity (24) communicates with the lower edge of the distribution channel (231), forming a diameter smaller than the accommodating cavity (24) Therefore, on the horizontal cross-section through the center of the accommodating cavity, between the cavity wall (241) of the accommodating cavity (24) and the edge of the opening, an eaves ( 242). 6. The microorganism detection chip according to claim 5, characterized in that: the eaves (242) are arranged on the downstream side of the opening of the accommodating cavity (24); A guide part (243); the radius of the guide part (243) is the same as the height of the eaves part (242). 7. The microorganism detection system according to claim 2, characterized in that: the chip (10) comprises a cover sheet layer (1) having the same shape and size, a substrate layer (2), and a cover sheet layer (2) sandwiched between the cover sheet Structural layer (3) between layer (1) and substrate layer (2); said cover sheet layer (1) and substrate layer (2) have overlapping non-through microchannel structures; said cover sheet layer (1) It has a through inlet through hole (11) and an outlet through hole (12), and also has a non-through cover sheet layer conveying channel (13) and a cover sheet layer accommodating cavity (14); the substrate layer (2) has The non-penetrating sample inlet slot (21), the sample outlet slot (22), the conveying channel (23) and the accommodating cavity (24); the structural layer (3) includes a cover sheet layer (1) and a substrate layer (2) ) on the overlapping part of the microchannel structure, and the inlet slot (31) and the outlet slot (32) corresponding to the inlet through hole (11) and outlet through hole (12); only the structural layer (3) ) of the microchannel structure includes a middle-layer side flow channel (35) for forming a lateral flow; the microchannel structures of the structural layer (3) are all through structures, so that the cover sheet layer (1), the structural layer (3) After being superimposed with the substrate layer (2), a complete combined conveying channel, a combined sample inlet slot, a combined sample outlet slot and a combined accommodating cavity can be formed, but the side flow channel only exists in the structural layer (3). 8. The microorganism detection system according to claim 7, characterized in that: the sidewalls of the microchannel structures on the structural layer (3) are compared with those on the cover sheet layer (1) and the substrate layer (2). The sidewalls of the microchannel structure are more hydrophilic to allow the mid-layer lateral flow channel (35) to provide the same lateral flow intensity with a relatively small cross-sectional area. 9 . The microorganism detection system according to claim 7 , wherein the non-bonding sides of the cover sheet layer ( 1 ) and the base sheet layer ( 2 ) of the chip ( 10 ) are respectively coated with opposite polarizing directions. 10 . A polarizing coating that does not cover the combined containment cavity. . 10. The vertical microorganism detection chip according to claim 7, characterized in that: the non-bonding surface of the substrate layer (2) of the chip (10) is coated with a light-absorbing coating; the light-absorbing coating is not Cover the combined housing cavity.

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