CN110819507B - Microdroplet preparation chip for intestinal microbial detection - Google Patents

Abstract

The invention relates to a liquid drop preparation chip for intestinal microorganism detection, which can accurately control and regulate the size of multiple packaged liquid drops by arranging a regulating channel; according to the change of the preparation requirements of the liquid drops, multiple wrapped liquid drops with different sizes can be obtained by adjusting the change of the dynamic parameters of the channels; meanwhile, the adjustment of the invention is not limited to the overall size of the multi-wrapped liquid drops, and the inner core size, the thickness of the wrapping layer and the like of the multi-wrapped liquid drops can be independently adjusted; the liquid drop preparation chip provided by the invention allows the preparation of the uniform double emulsion liquid drops with different specifications on the same chip, has universality within a certain liquid drop size range, and can be used as an independent liquid drop preparation unit to be combined with liquid drop using units with various specifications, so that the detection cost is greatly reduced.

Description

Microdroplet preparation chip for intestinal microbial detection

Technical field

The present invention relates to the field of microfluidic technology, and specifically to a microdroplet preparation chip for intestinal microbial detection.

Background technique

Intestinal microorganisms refer to the general term for the microbial community living in the human intestine, including bacteria, archaea, and single-cell eukaryotes, etc., which together with the intestinal environment constitute a huge and complex ecosystem. The human intestine is a nutrient-rich microenvironment, carrying up to 100 trillion bacteria. The number of genes of human intestinal microorganisms reaches 5 million, which is 150 times the number of human genes. Intestinal microorganisms are related to metabolic diseases, cardiovascular diseases, digestive system diseases, cancer, immune system diseases and central nervous system diseases. Research on their structure, function and pathogenic mechanism will be beneficial to the treatment of diseases. and develop new treatments.

With the development of microfluidic technology, its importance in the field of microbial/nucleic acid detection has gradually become prominent. Among them, the digital PCR method is currently the mainstream method for microorganism/nucleic acid detection based on microfluidic means. It has extremely high sensitivity and accuracy and is suitable for accurate quantitative analysis of specific nucleic acids in trace samples. Digital PCR technology is a detection method based on statistical principles. Its main method is to distribute the sample solution or its dilution into a large number of micro-droplets, use the droplets as micro-reactors to perform amplification reactions, and then analyze the reaction results. Fluorescence detection is used to determine the initial copy number of the target DNA in the sample based on the Poisson distribution principle.

At present, the microdroplets used for digital PCR detection are usually water-in-oil single-wrapped droplet systems. Chinese patent (CN109746061A) introduces the controlled preparation method of such droplets in its disclosure. It introduces that for three common droplet generation modes, namely coaxial flow, cross flow and flow focusing mode, all can be Adjusting the flow rate of the continuous phase fluid controls the size of the generated droplets, where the droplets have larger sizes in the squeeze mode with lower flow rates and the smaller droplets in the jet mode with higher flow rates. .

The Chinese patent (CN107429426A) proposed in its disclosure that since the emulsion carrier phase of the single-wrapped droplet system for digital PCR detection is an inert oil phase, this will prevent the droplet reactor from being used with available methods (such as with the oil carrier phase). Incompatible fluorescence-activated cell sorting) for detection, quantification, and sorting. The patent also proposes to overcome the above problems by using double emulsion for digital PCR detection.

However, the preparation of multiple emulsions, especially the preparation of multiple emulsions for digital PCR detection purposes, faces challenges in terms of uniformity in dimensions such as droplet size, droplet multiplicity, and core droplet number.

For single-wrapped droplets, the size of the droplets can be controlled by adjusting the flow rate of the continuous phase; however, in the preparation process of multiple emulsions, there are multiple continuous phases, and usually the injection speed of the last continuous phase does not allow arbitrary Change, for example, for the preparation of double emulsion, the first continuous phase (oil) shears the aqueous core fluid to generate water-in-oil droplets and moves forward along the microchannel, and the second continuous phase (water phase) shears Cut the first continuous phase containing water-in-oil droplets to generate water-in-oil droplets. In this process, due to the spacing between the water-in-oil droplets in the first continuous phase, the second continuous phase The injection speed is essentially controlled so that the first-level continuous phase breaks exactly in the middle of the two water-in-oil droplets under the shear of the second-level continuous phase; otherwise, due to the continuation of the process, the first-level continuous phase breaks Under the shear of the second continuous phase, the fracture position will be variable and uncontrollable. The result is that the generated double droplets are uncontrollable. For example, under the shear of the second continuous phase, it may be Double droplets of water-in-oil-in-water may be generated, or single-wrapped droplets of oil-in-water may be generated, mixed with water-in-oil droplets generated during the shearing process of the first-stage continuous phase; thus causing chaos in the droplet system.

Therefore, current multiple emulsion preparation chips are usually dedicated chips. Multiple emulsion preparation chips often require different hydrophilic and hydrophobic modifications on different parts of the microchannel, which makes the manufacturing cost and difficulty of multiple emulsion preparation chips very high. If the multiple emulsion preparation chips can be made capable of preparing droplets of different sizes, The universal properties will reduce the cost of droplet preparation to a great extent.

In other words, the resulting water-in-oil-in-water droplets are essentially segmented

Generally speaking, it is difficult to produce droplets that are uniform in all dimensions. As mentioned above, when preparing droplets of different sizes, there are differences in the flow rate of the continuous phase fluid, which causes the distance between two adjacent droplets in the continuous phase to change due to size changes (for example, when preparing different batches of liquids). when the droplets are dropped); this may result in two types of water-in-oil-in-water double droplets and oil-in-water single-wrapped droplets when the droplets are re-wrapped. The single-wrapped liquid that appears during the secondary wrapping process The reason for the droplets is that the distance between the two adjacent droplets generated in the previous stage is too large. As a result, during the second wrapping, the partially wrapped oil continuous phase does not contain aqueous droplets. Although such a problem can be solved by adjusting the flow rate of the continuous phase during secondary wrapping so that the speed of generated droplets exactly matches the droplet spacing of the previous stage to avoid the occurrence of single-wrapped droplets during secondary wrapping, but in this way The adjustment of PCR testing is unfavorable.

Contents of the invention

In order to solve the above-mentioned problems in the prior art, the present invention provides a micro-droplet preparation chip for intestinal microbial detection. The micro-droplet preparation chip of the present invention allows precise control and adjustment of the size of double emulsion droplets, and can prepare homogenized double emulsion droplets of different specifications on the same chip, with a certain droplet size range. Due to its versatility, it can be used as an independent droplet preparation unit and combined with various specifications of droplet usage units (such as digital PCR chips), thereby greatly reducing detection costs.

Although the size of single-wrapped droplets can be flexibly controlled by adjusting the flow rate of the continuous phase, the final size of multi-wrapped droplets is limited by many factors and usually cannot be adjusted. Taking the preparation of double emulsion as an example, see Figure 1-3. The single-wrapped droplets generated after the inner core fluid (generally the water phase) is sheared by the first continuous phase (generally the oil phase) are arranged at a substantially constant distance. In the microchannel, the first continuous phase entraining the single-wrapped droplets is then sheared by the second continuous phase (usually the water phase) as a new core fluid, thereby forming double emulsion droplets of water-in-oil-in-water.

Theoretically, the sum of the volumes of each single-wrapped droplet in the microchannel and the first continuous phase distributed by the single-wrapped droplet in the microchannel (V1 as shown in Figures 1-3 and 5-7 and V1' in Figure 5-7. The V1' is the adjusted V1. The continuous phase volume between two adjacent micro droplets is equally divided, and the sum of the continuous phase volumes obtained by distributing on both sides of the micro droplet is In addition, the volume of the micro-droplet itself is V1 or V1' (hereinafter referred to as the distribution volume), and the volume of the final prepared double-wrapped droplet (V2 in Figure 1-3) should be equal to ensure continuous Stable generation of uniform double-wrapped droplets.

Figure 1 shows the double emulsion preparation process in V1=V2 and extrusion mode. In the extrusion mode, the first continuous phase shears the inner core fluid at a lower flow rate to form single-wrapped droplets with larger particle sizes, and due to the lower flow rate of the first continuous phase, the gap between two adjacent droplets The distance is small and the distribution volume V1 is small. Since V2=V1, the shear force provided by the second continuous phase is predetermined, corresponding to its predetermined flow rate.

Figure 2 shows the double emulsion preparation process in V1=V2 and jet mode. In the jet mode, the first continuous phase shears the core fluid at a higher flow rate to form a single-wrapped emulsion with a smaller particle size. However, due to the higher flow rate of the first continuous phase, the distance between two adjacent droplets decreases. Large, the distribution volume V1 is large. Since V2=V1, the shear force provided by the second continuous phase is also predetermined, corresponding to its predetermined flow rate.

The two situations shown in Figures 1 and 2 show that although smaller-sized single-wrapped emulsions can be prepared by increasing the flow rate of the first continuous phase, the increase in flow rate will introduce more first continuous phase fluid, resulting in a single-wrapped emulsion. The distance between the droplets increases, so that the volume of the double-wrapped droplets obtained when the first continuous phase is in the flow mode (Figure 2) is larger than the volume of the double-wrapped droplets obtained when the first continuous phase is in the extrusion mode. (figure 1). This makes it very difficult to prepare smaller-sized double-wrapped droplets.

Figure 3 shows the double emulsion preparation process in V1≠V2 and jet mode. The meaning of V1≠V2 is that the flow rate of the second continuous phase is adjusted arbitrarily without considering other factors. In this case, although the size of the droplets obtained by shearing of the second continuous phase can be reduced, this shearing mode cannot guarantee that the first continuous phase containing a single wrapped droplet will be exactly at the size of the droplet sheared by the second continuous phase. The two single-wrapped droplets break at the middle position. Therefore, the resulting droplet is a chaotic system including: double-wrapped droplets of water-in-oil-in-water, single-wrapped droplets of oil-in-water, and single-wrapped droplets of water-in-oil. . Such droplet systems cannot meet the needs of downstream applications and should be avoided.

Based on such understanding and analysis, the present invention provides a micro-droplet preparation chip for intestinal microbial detection. The droplet preparation chip is used to prepare multiple emulsion droplets wrapped in at least two layers with controllable particle size. Including the core fluid channel, the first continuous phase channel, the single-wrapped droplet channel, the second continuous phase channel, the double-wrapped droplet channel..., and so on, according to the number of packages of the required prepared droplets, there can be an N+th 1-fold continuous phase channel and N-fold wrapped droplet channel; the inner core fluid channel intersects with the first continuous-phase channel, and the downstream fluid of the intersection is connected to the single-wrapped droplet channel; similarly, the N-fold wrapped droplet channel is connected to the Nth +1 continuous phase channels intersect, and the downstream fluid connection of the intersection is N+1 heavily wrapped droplet channels; the micro droplet preparation chip also includes an adjustment channel, and the adjustment channel N multiple wrapped channels are fluidly connected, thus having a series, Such as the Nth level adjustment channel, which allows adjusting the size of the distribution volume V1 of the N-fold wrapped droplets in the corresponding droplet channel.

Preferably, the regulating channel only exists between two adjacent continuous phase channels.

Preferably, the adjustment channel is used to extract the continuous phase fluid from the droplet channel, thereby reducing the distribution volume V1; the adjustment channel is connected to an independent power unit.

Preferably, the micro-droplet preparation chip includes a cover layer and a micro-channel layer, the adjustment channel, the core fluid channel, the first continuous phase channel, the single-wrapped droplet channel, the second continuous phase channel and the double-wrapped droplet. The channels are all located on the same side of the microchannel layer. All microchannels on the microchannel layer preferably have the same depth to allow processing of the microchannel layer in a single photolithography process.

Preferably, the adjustment channels are symmetrically arranged on both sides of the droplet channel, and the size of the adjustment channel (including width, height and diagonal size) is smaller than the diameter of the droplets in the corresponding droplet channel; more preferably is smaller than the radius of the droplet.

Preferably, the adjustment channel on each side is composed of several sub-channels arranged side by side, and the several sub-channels disperse the connection openings between the adjustment channel and the droplet channel over a larger length to allow Each sub-channel uses a smaller connection opening to connect to the droplet channel and achieve the same or even greater adjustment capability. The smaller connection opening size allows for a better formation of droplets in the droplet channel during the adjustment process. Small disturbance helps small-sized droplets maintain their position and shape within the droplet channel.

Preferably, the adjustment channel includes a collection channel that gradually expands along the direction of liquid flow and a drainage grid arranged at the connection between the collection channel and the droplet channel. The drainage grid is composed of several micro-pillars arranged at intervals. The gap between two adjacent micro-columns forms a flow path of the continuous phase between the droplet channel and the collection channel; the collection channel is connected to a liquid collection channel at the largest expansion along the liquid flow direction; the micro-column It can be a body with any cross-sectional shape, preferably with a square or circular cross-section.

The function of the drainage grid is the same as that of several sub-channels arranged side by side, both of which can reduce the size of a single opening connected to the droplet channel and reduce the interference to the droplets; and the several gaps of the drainage grid pass through the collection The channels finally converge into the liquid collection channel. Therefore, the liquid collection channel can be allowed to have a size exceeding that of the droplet, thereby enabling the distribution volume V1 to be adjusted at a faster speed without causing significant changes to the position and shape of the droplet in the channel. Interference; and, because the collection channel has a gradually expanding structure along the direction of liquid flow, when the continuous phase of entrained droplets flows through the drainage grid, as the expansion of the collection channel increases, the continuous phase will increase at an increasing speed. Pass through the gap and enter the collection channel; thereby further improving the adjustment speed of the distribution volume V1, which is conducive to speeding up the overall droplet preparation process, and thus can better meet the needs of digital PCR detection and other needs to quickly prepare a large amount in a short time Requirements for uniform droplets.

In the foregoing scheme, all microchannels preferably have the same depth. In other words, the microchannels in the foregoing scheme are planar channel structures; for example, they can be formed by partial photolithography or mechanical processing of one side of the microchannel layer. The non-penetrating microchannel formed may also be a microchannel structure that penetrates the microchannel layer and is then closed by the cover sheet layer and the base sheet layer.

When the micro-droplet preparation chip is used to prepare double-wrapped droplets, it may also have a three-dimensional microchannel structure. The three-dimensional microchannel structure has the following characteristics: the upstream side of the core fluid channel, the first continuous phase channel, and the single-wrapped droplet channel are non-penetrating equal-depth microchannels, and they are arranged on the microchannel layer. side; the downstream side of the single-wrapped droplet channel, the second continuous phase channel, and the double-wrapped droplet channel are also non-penetrating equal-depth microchannels, but they are arranged on the lower side of the microchannel layer; the single-wrapped droplet channel The middle part of the droplet channel penetrates the microchannel layer, and is fluidly connected to the upstream and downstream sides of the single-wrapped droplet channel; the first and last ends of the middle part of the single-wrapped droplet channel are respectively provided with non-penetrating adjustment channels, wherein, The adjustment channel at the head end is arranged on the lower side of the microchannel layer, and the adjustment channel at the tail end is arranged on the upper side of the microchannel layer; the depth of the adjustment channel is not greater than the radius of a single wrapped droplet.

Single-wrapped droplets flow in such a microchannel structure. When moving from the upstream side of the upper single-wrapped droplet channel to the middle of the penetrating single-wrapped droplet channel, since the specific gravity of the water-based droplets is greater than that of the oily continuous Phase, the droplets will sink in a curved path in the through-channel, and then enter the downstream side of the single-wrapped droplet channel; the adjustment channels at the head and tail ends can respectively suck the bottom of the channel near the head end where the droplets have not completely sunk. The continuous phase, and the continuous phase at the top of the suction channel near the tail end where the droplet has basically sunk to the bottom, thereby adjusting the distribution volume V1.

Preferably, the adjustment channel is a flat channel with a depth no greater than 1/4 of the diameter of a single wrapped droplet.

Preferably, the width of the adjustment channel is half of the length of the middle part of the single-wrapped droplet channel, so that when the single-wrapped droplet moves in the middle part of the penetrating single-wrapped droplet channel, the adjustment channel is caused by the suction of the continuous phase. The vertical force on a single-wrapped droplet is weak and basically stable along the entire path, thus reducing the interference to the movement process and shape of a single-wrapped droplet.

Preferably, the two adjustment channels located on the upper and lower layers of the microchannel layer can be connected through a through hole, thereby allowing the same power unit to provide the same power to the adjustment channels at different locations.

Preferably, the intersection of the core fluid channel and the first continuous phase channel is in fluid communication with the single-wrapped droplet channel through a reduced diameter section.

In the foregoing solution, the preparation process of double-wrapped droplets is mainly used as an example, but the device of the present invention can also be applied to the controllable preparation of droplets with more wrapping numbers. When it is necessary to prepare more droplets with a larger number of packages, an adjustment channel can be set on one side or both sides of the droplet channel downstream of each stage of the continuous phase channel except for the last stage of the continuous phase channel. It is used to adjust the distribution volume in the corresponding droplet channel, thereby achieving controllable preparation of multiple wrapped droplets.

The chip of the present invention can use existing methods to modify various parts of the microchannel to be hydrophilic and hydrophobic, wherein the surface properties of the adjustment channel are the same as those of the droplet channels connected to them.

The invention also provides a droplet preparation method. The droplet preparation method is based on the micro-droplet preparation chip provided by the invention. The distribution volume of the droplets in the corresponding droplet channel is adjusted through the adjustment channel, so that the following The droplet volume obtained by primary packaging is controllable.

Compared with the prior art, the present invention can at least achieve the following beneficial effects: from the analysis of the formation mechanism of multi-wrapped droplets, the present invention finds the key factors affecting the size of multi-wrapped droplets, and accordingly designs a device that can adjust the size of the droplets. The distribution volume in the channel further enables the droplet preparation chip and method to control the final volume of the multiple wrapped droplets; the droplet preparation chip of the present invention can allow precise control and adjustment of the size of the multiple wrapped droplets; according to the droplet With changes in preparation requirements, multiple wrapped droplets of different sizes can be obtained by changing the dynamic parameters of the adjustment channel; at the same time, the adjustment of the present invention is not limited to the overall size of the multiple wrapped droplets, but also to the core size, The thickness of the wrapping layer, etc. can be adjusted independently; the droplet preparation chip of the present invention allows the preparation of uniform double emulsion droplets of different specifications on the same chip, has versatility within a certain droplet size range, and can be used as an independent The droplet preparation unit is combined with droplet usage units of various specifications to greatly reduce detection costs.

Description of the drawings

Figure 1 is a schematic diagram of the preparation process of double-wrapped droplets with V1=V2 in the extrusion mode;

Figure 2 is a schematic diagram of the preparation process of double-wrapped droplets with V1=V2 in jet mode;

Figure 3 is a schematic diagram of the droplet preparation process for V1≠V2 in jet mode;

Figure 4 is a top view of the chip structure of the present invention;

Figure 5 is a schematic diagram of the adjustment channel. The adjustment channel adjusts the distribution volume V1 to V1’;

Figure 6 is a schematic diagram of an adjustment channel with sub-channels;

Figure 7 is a schematic diagram of an adjustment channel with a drainage grille;

Figure 8 is a partially enlarged schematic diagram of an adjustment channel with a drainage grille;

Figure 9 is a cross-sectional view of a chip with three-dimensional microchannels;

Figure 10 is a schematic diagram of the three-dimensional structure of a chip with three-dimensional microchannels;

Figure 11 is a top view of the chip in Figure 10;

Figure 12 is a schematic diagram of another three-dimensional structure of the chip in Figure 10;

Figure 13 is another top view of a chip with three-dimensional microchannels.

In the figure: 1 is the core fluid channel, 2 is the first continuous phase channel, 3 is the single-wrapped droplet channel, 4 is the reduced diameter section, 5 is the second continuous phase channel, 6 is the double-wrapped droplet channel, and 7 is the adjustment channel. , 71 is a sub-channel, 72 is a collection channel, 73 is a drainage grid, 731 is a micro-column, 732 is a micro-column gap, 74 is a liquid collection channel, and 75 is a through hole.

Detailed ways

In order to further clarify the concept of the present invention, the present invention provides the following specific embodiments.

Example 1

As shown in Figure 4-5, a micro-droplet preparation chip for intestinal microbial detection is provided. The micro-droplet preparation chip is used to prepare double-wrapped droplets, including a cover layer and a microchannel layer; A non-penetrating microchannel structure is formed on the upper surface of the microchannel layer. The cover layer covers the upper surface of the microchannel layer, and four through holes are provided on one side of the cover layer in fluid communication with the microchannel layer. hole; the microchannel structure includes an inner core fluid channel 1, a first continuous phase channel 2, a single wrapped droplet channel 3, a second continuous phase channel 5, and a double wrapped droplet channel 6; the inner core fluid channel 1 and the The first continuous phase channel 2 intersects at a cross, and downstream of its intersection is fluidly connected to the single-wrapped droplet channel 3 through a reduced diameter section 4. The single-wrapped droplet channel 3 intersects with the second continuous phase channel 5, and its intersection is downstream The double-wrapped droplet channel 6 is fluidly connected; the downstream section of the single-wrapped droplet channel 3 is symmetrically provided with two adjustment channels 7 on both sides, and the two symmetrically arranged adjustment channels 7 are in fluid communication with the single-wrapped droplet channel 3 , and is connected to the same syringe pump, thereby allowing the first continuous phase to be sucked from the single-package droplet channel 3 through the adjustment channel 7 or to replenish the first continuous phase into the single-package droplet channel 3, thereby converting the single-package droplet into the single-package droplet channel 3. The distribution volume of the droplets is adjusted from V1 to V1'. The width of the adjustment channel 7 is smaller than the diameter of a single-wrapped droplet, and it has the same surface properties as the single-wrapped droplet channel 3 .

Example 2

As shown in Figures 4 and 7, a micro-droplet preparation chip for intestinal microbial detection is provided, which has a similar main structure to the micro-droplet preparation chip described in Example 1. The difference is that it is located in a single coating liquid The adjustment channel 7 on each side of the dripping channel 3 is composed of four sub-channels 71 arranged in parallel, and the distance between any two adjacent sub-channels 71 is 0.5-3 times the width of the sub-channel 71 itself.

Example 3

As shown in Figures 4 and 7-8, a micro-droplet preparation chip for intestinal microbial detection is provided. The difference from Examples 1 and 2 is that the adjustment located on each side of the single-wrapped droplet channel 3 The channels 7 each include a collection channel 72 that gradually expands along the liquid flow direction and a drainage grille 73 arranged at the connection between the collection channel and the droplet channel. The connection line formed by the inner edges of the plurality of drainage grilles 73 and the The side wall extension lines of the single-wrapped droplet channel 3 coincide with or are located outside the extension line, so that the single-wrapped droplet will not be hindered during the flow in the single-wrapped droplet channel 3; the drainage grids 73 are arranged at intervals. It is composed of several micro-columns 731, and the gap 732 between two adjacent micro-columns 731 forms the flow path of the first continuous phase between the single-wrapped droplet channel 3 and the collection channel 72; the collection channel 72 is along the liquid flow. The largest expansion in the direction is connected to the liquid collection channel 74; the micro-column 731 has a square cross-section.

Example 4

The scheme of this embodiment is not shown in the drawings, but the difference from Examples 1-3 is that the micro-droplet preparation chip is used to prepare triple-wrapped droplets. The microchannel layer also includes a third continuous phase channel that intersects with the double-wrapped liquid channel 6, and a triple-wrapped droplet channel fluidly connected at the intersection; the downstream sides of the double-wrapped droplet channel 6 are symmetrical. A second adjustment channel is provided, and the second adjustment channels located on both sides are jointly connected to another syringe pump for adjusting the distribution volume of the double-wrapped droplets in the second continuous phase channel, and the second adjustment channel has a function similar to that of the second continuous phase channel. The double-wrapped droplet channel 6 has the same surface properties.

Example 5

As shown in Figures 4 and 9-11, a micro-droplet preparation chip for intestinal microbial detection is provided. The difference from Embodiment 1-3 is that the micro-droplet preparation chip also includes a substrate layer, and The microchannel layer has a three-dimensional microchannel structure. Specifically, the three-dimensional microchannel structure has the following characteristics: the upstream sides of the core fluid channel 1, the first continuous phase channel 2, and the single-wrapped droplet channel 3 are non-penetrating. equal-depth microchannels, which are arranged on the upper side of the microchannel layer; the downstream side of the single-wrapped droplet channel 3, the second continuous phase channel 5, and the double-wrapped droplet channel 6 are also non-penetrating equal-depth microchannels. channel, which is arranged on the lower side of the microchannel layer; the middle part of the single-wrapped droplet channel 3 penetrates the microchannel layer, and is fluidly connected to the upstream and downstream sides of the single-wrapped droplet channel 3 respectively; the single-wrapped liquid droplet channel 3 The middle part of the dripping channel 3 is provided with non-penetrating adjustment channels 7 at both ends. The adjustment channel 7 at the head end is arranged on the lower side of the microchannel layer, and the adjustment channel 7 at the tail end is arranged on the bottom of the microchannel layer. Upper side; the depth of the adjustment channel 7 is not greater than the radius of a single wrapped droplet. The adjustment channel 7 is a flat channel, and its width is half of the length of the central portion of the single-wrapped droplet channel 3 . The adjustment channels 7 located on the upper and lower sides of the microchannel layer are connected to different syringe pumps.

Example 6

As shown in Figures 4, 9, and 12-13, a microdroplet preparation chip for intestinal microbial detection is provided. What is different from Embodiment 5 is that the two adjustment channels 7 on the same side of the single-wrapped droplet channel 3 and located on the upper and lower surfaces of the microchannel layer are connected through a through hole 75 respectively; the two through holes 75 are connected to each other. The holes 75 are jointly connected to the same syringe pump.

Example 7

A method for preparing multiple wrapped droplets is provided. The method is based on the microdroplet chip described in any one of the previous embodiments. The size of the N-thick wrapped droplets is controlled by adjusting the flow rate of the Nth continuous phase, and then connected to The adjustment channel 7 on the N-heavy-wrapped droplet channel adjusts the distribution volume of the N-heavy-wrapped droplet in the corresponding droplet channel, so that the flow rate of the N+1 continuous phase can change with the change of the distribution volume, thereby adjusting the next level of wrapping thickness. The N is a positive integer.

The above embodiments are only examples of the preferred embodiments of the present invention, and should not be understood as limiting all possible implementations of the present invention. Those skilled in the art can make routine decisions without creative work based on the technical concepts of the present invention. The implementations obtained by substitution or improvement shall be regarded as feasible solutions of the present invention. The actual protection scope of the present invention shall be determined by the claims.

Claims (7)

1. A microdroplet preparation chip for intestinal microbial detection, which is used to prepare at least two layers of microdroplets with controllable particle size, including a cover layer and a microchannel layer, characterized in that: The microchannel layer includes an inner core fluid channel, a first continuous phase channel, an N-fold wrapped droplet channel, an N+1 continuous phase channel, and an N+1-fold wrapped droplet channel; the inner core fluid channel intersects with the first continuous phase channel. , the downstream fluid of its intersection is connected to the single-wrapped droplet channel; the N-fold wrapped droplet channel intersects with the N+1th continuous phase channel, and the downstream fluid of its intersection is connected to the N+1-fold wrapped droplet channel; the micro-liquid The droplet preparation chip also includes an Nth-level adjustment channel, which is fluidly connected to the N-fold wrapped droplet channel, and each level of adjustment channel is connected to an independent power unit, where N is a positive integer; the Nth-level adjustment channel The adjustment channels are symmetrically arranged on both sides of the N-fold wrapped droplet channel. The adjustment channel is used to extract the continuous phase fluid from the droplet channel and thereby reduce the distribution volume V1; the Nth-level adjustment channel is composed of several It consists of sub-channels arranged side by side, and the spacing between the several sub-channels is 0.5-3 times its own width; the Nth-level adjustment channel includes a collection channel that gradually expands along the direction of the liquid flow and is arranged between the collection channel and N A drainage grid at the junction of the re-wrapped droplet channel. The drainage grid is composed of a number of micro-pillars arranged at intervals. The gap between two adjacent micro-pillars forms the Nth continuous phase between the N re-wrapped droplet channel and The flow path between the collection channels; the collection channel is connected to a liquid collection channel at the maximum expansion along the direction of liquid flow; the micro droplet preparation chip is used to prepare double-wrapped droplets, and also includes a substrate layer; The upstream side of the core fluid channel, the first continuous phase channel, and the single-wrapped droplet channel are non-penetrating equal-depth microchannels, and they are arranged on the upper side of the microchannel layer; the downstream side of the single-wrapped droplet channel , the second continuous phase channel and the double-wrapped droplet channel are also non-penetrating equal-depth microchannels, which are arranged on the lower side of the microchannel layer; the middle part of the single-wrapped droplet channel penetrates the microchannel layer, and The upstream and downstream sides of the single-wrapped droplet channel are fluidly connected respectively; non-penetrating adjustment channels are respectively provided at the beginning and the end of the middle part of the single-wrapped droplet channel. 2. The micro-droplet preparation chip for intestinal microbial detection according to claim 1, characterized in that: the N-th level adjustment channel has the same surface properties as the N-heavy wrapped droplet channel. 3. The micro-droplet preparation chip for intestinal microbial detection according to claim 1, characterized in that the micro-column has a square or circular cross-section. 4. The micro-droplet preparation chip for intestinal microbial detection according to claim 1, characterized in that: the adjustment channel located at the head end of the middle part of the single-wrapped droplet channel is arranged on the lower side of the micro-channel layer, located at The adjustment channel at the tail end is arranged on the upper side of the microchannel layer; the depth of the adjustment channel is no greater than the radius of a single wrapped droplet. 5. The micro-droplet preparation chip for intestinal microbial detection according to claim 4, characterized in that: the adjustment channel is a flat channel, and its depth is not greater than 1/4 of the diameter of a single wrapped droplet; and The width is half the length of the middle of the single-wrapped droplet channel. 6. The micro-droplet preparation chip for intestinal microbial detection according to any one of claims 1 to 5, characterized in that: the two adjustment channels on the same side of the single-wrapped droplet channel pass through one The through holes are connected; the two through holes are jointly connected to the same syringe pump. 7. A method for preparing multiple coated droplets, characterized in that, based on the microdroplet preparation chip according to any one of the preceding claims 1-6, the N-fold coated liquid is controlled by adjusting the flow rate of the Nth continuous phase. The size of the droplet is then adjusted through the adjustment channel connected to the N-heavy wrapped droplet channel in the corresponding droplet channel, so that the flow rate of the N+1th continuous phase can change with the distribution volume. And change to adjust the thickness of the next level of wrapping layer.