High-flux aquatic veterinary drug sensitivity testing device and method based on multi-layer micro-fluidic chip

Abstract

The invention provides a device and a method for testing the sensitivity of a veterinary drug for high-flux aquatic products based on a multi-layer micro-fluidic chip. The device comprises a pathogenic biological packaging device, a pathogenic biological culture device and a pathogenic biological detection device, the pathogenic biological packaging device sequentially comprises a mixed micro-fluidic chip, a liquid drop micro-fluidic chip and a glass substrate layer from top to bottom, the mixed micro-fluidic chip, the liquid drop micro-fluidic chip and the glass substrate layer are attached in a bonding mode, and the two chips are connected through a series of micro-channels. Conveying the fluid from the upper-layer mixed micro-fluidic chip to the lower-layer droplet micro-fluidic chip; the pathogenic organism culture device is connected with the pathogenic organism packaging device and is used for culturing the micro-droplets packaged with the pathogenic organisms in a proper environment; the pathogenic organism detection device is connected with the pathogenic organism culture device and is used for measuring the pathogenic organisms in the pathogenic organism culture device at intervals and determining the growth condition of the pathogenic organisms, so that the sensitivity analysis of the aquatic veterinary drugs is carried out.

Description

Technical Field

[0001] The present invention relates to the technical field of veterinary drug sensitivity testing for aquatic products, and in particular to a high-throughput veterinary drug sensitivity testing device and method for aquatic products based on a multilayer microfluidic chip. Background Art

[0002] Microfluidic chips are a technology platform that can precisely control the flow of fluids at the micro-nano scale. They are usually composed of a series of tiny channels and cavities. This type of chip can achieve rapid mixing, reaction and separation of liquids, and is widely used in biomedicine, chemical analysis and environmental monitoring. Among them, hybrid microfluidic chips and droplet microfluidic chips are two major application branches based on microfluidic technology. Hybrid microfluidic chips design flexible channel structures to allow multiple fluids or fluids of different concentrations to meet in the channel, diffuse and mix or react to meet the needs of different applications. Droplet microfluidic chips generate and manipulate micron-sized droplets by precisely controlling the flow parameters of the fluid to conduct high-throughput experiments. They can achieve parallel processing of multiple reactions in a small volume. The microdroplets generated based on microfluidic technology have the advantages of good monodispersity, high throughput, no cross-contamination, and high repeatability, and play an extremely important role in the application of microfluidic technology.

[0003] In recent years, with the long-term and extensive use of aquatic veterinary drugs in aquaculture, common pathogenic organisms have gradually developed resistance to commonly used drugs, which has posed a severe challenge to the sustainable development of aquaculture. In particular, as the resistance of pathogenic microorganisms continues to increase, the infection of pathogenic organisms has become more serious and difficult to control, resulting in economic losses in aquaculture and a deteriorating ecological environment. Therefore, rapid evaluation and analysis of the sensitivity of aquatic veterinary drugs to pathogenic organisms, combined with effective management measures, has become an urgent problem to be solved in the field of aquaculture.

[0004] Traditional methods for veterinary drug sensitivity analysis in aquaculture are mainly divided into two categories: methods based on liquid culture medium and methods based on solid culture medium. The former includes microdilution method, agar dilution method, etc., and the latter includes paper diffusion method, E-test method, etc. These methods usually contact pathogenic microorganisms with drug solutions of different concentrations, and analyze the sensitivity of pathogenic bacteria to specific drugs by observing and measuring the minimum antibiotic concentration that can inhibit bacterial growth, that is, the minimum inhibitory concentration (MIC). However, traditional sensitivity analysis methods have problems such as low sensitivity, lack of automation, and complex sample preparation. Therefore, various new technologies for drug sensitivity testing have emerged, among which droplet microfluidic chip technology has shown great potential. However, in the existing technology, although the throughput of microdroplets is high, due to the poor repeatability of the chip, when it is necessary to detect the sensitivity of multiple concentrations of drugs or the sensitivity of multiple drug mixtures, it is often necessary to make a large number of chips and conduct multiple repeated experiments, resulting in a long experimental cycle, causing loss of manpower and material resources, which is not conducive to the clinical analysis and diagnosis of animal diseases in aquaculture. Summary of the invention

[0005] According to the technical problems raised above, a high-throughput aquatic veterinary drug sensitivity test device and method based on a multilayer microfluidic chip is provided. The present invention can obtain multiple groups of microdroplets encapsulating different drug concentrations and ratios in a short time, which are used for drug resistance analysis of pathogenic organisms in aquaculture. It has the advantages of high throughput, fast efficiency, precise control, low cost, etc., and provides a more efficient tool for studying the rational use of drugs in aquaculture and the development of new drugs for aquaculture.

[0006] The technical means adopted by the present invention are as follows:

[0007] A high-throughput aquatic veterinary drug sensitivity testing device based on a multi-layer microfluidic chip, comprising: a pathogenic organism encapsulation device, a pathogenic organism cultivation device and a pathogenic organism detection device, wherein:

[0008] The pathogenic organism encapsulation device has a three-layer structure, which includes a hybrid microfluidic chip, a droplet microfluidic chip and a glass substrate layer from top to bottom. The three layers are bonded together by plasma cleaning bonding. The hybrid microfluidic chip and the droplet microfluidic chip are connected by a series of microchannels to transport fluid from the first layer of the hybrid microfluidic chip to the second layer of the droplet microfluidic chip. The glass substrate layer is used to support and seal the device.

[0009] The pathogenic organism cultivation device is connected to the pathogenic organism encapsulation device and is used to place the microdroplets encapsulating the pathogenic organisms in a suitable environment for cultivation;

[0010] The pathogenic organism detection device is connected to the pathogenic organism cultivation device and is used to detect the pathogenic organisms in the pathogenic organism cultivation device at regular intervals to determine the growth of the pathogenic organisms, thereby performing sensitivity analysis of aquatic veterinary drugs.

[0011] Furthermore, in the pathogenic organism encapsulation device 1:

[0012] The hybrid microfluidic chip is provided with 6 inlets and 69 outlets for aquatic veterinary drug solutions, wherein:

[0013] Among the 6 entrances:

[0014] Three inlets are used as inlets for aquatic veterinary drug solutions, and are used to inject aquatic veterinary drug solutions; three inlets are used as inlets for pathogenic biological culture solutions, and are used to inject pathogenic biological culture solutions;

[0015] Among the 69 exports of veterinary drug solutions for aquaculture:

[0016] 45 outlets lead to the next layer of droplet microfluidic chip, which is used to inject the mixed solution as the dispersed phase for generating microdroplets; 24 outlets are used to collect the mixed veterinary drug solution for aquaculture.

[0017] Further, in the pathogenic organism encapsulation device:

[0018] The mixing microfluidic chip is circular as a whole, increases the number of mixing channels, and expands the application range of the device. At the same time, a serpentine mixing channel is used to increase the channel length, thereby extending the mixing time and allowing pathogenic organisms and aquatic veterinary drug solutions to be fully mixed.

[0019] Further, in the pathogenic organism encapsulation device:

[0020] The droplet microfluidic chip is used to simultaneously generate aquatic veterinary drug microdroplets containing pathogenic organisms in different concentrations or different ratios, including 1 mineral oil inlet, 45 aquatic veterinary drug solution inlets and 45 microdroplet outlets, wherein:

[0021] The mineral oil inlet serves as the continuous phase for generating microdroplets;

[0022] 45 inlets for aquatic veterinary drug solutions are used to receive solutions from 45 outlets in the mixing microfluidic chip as dispersed phases for generating microdroplets;

[0023] The outlets of 45 micro-droplets are used to encapsulate the solutions of 45 aquatic veterinary drug solution inlets in the micro-droplets, so that each micro-droplet becomes a culture device.

[0024] Furthermore, the pathogenic organism cultivation device is provided with a micro-droplet inlet, which is connected to the micro-droplet outlet of the pathogenic organism encapsulation device for collecting micro-droplets, and the pathogenic organism cultivation device is placed in an environment of 37° C. for cultivation.

[0025] Furthermore, if 100% aquatic thiamphenicol solution, normal saline and normal saline are respectively introduced into the three inlets for aquatic veterinary drug solutions, and Aeromonas hydrophila and its culture solution are introduced into the three inlets for pathogenic organism culture solutions, the high-throughput aquatic veterinary drug sensitivity testing device can simultaneously test the sensitivity of Aeromonas hydrophila to 26 groups of 8 linear concentration gradients of thiamphenicol solutions and 19 groups of blank control groups.

[0026] Furthermore, if 100% aquatic thiamphenicol solution, 100% aquatic florfenicol solution and normal saline are respectively introduced into three inlets for aquatic veterinary drug solutions, and Aeromonas hydrophila and its culture solution are introduced into three inlets for pathogenic organism culture solutions, then the high-throughput aquatic veterinary drug sensitivity testing device can simultaneously test the sensitivity of Aeromonas hydrophila to 15 groups of 8 linear concentration gradients of thiamphenicol solutions, 15 groups of 8 linear concentration gradients of florfenicol solutions, 11 groups of sensitivity of two drug solutions mixed in 7 different proportions and 4 groups of blank control groups.

[0027] Furthermore, if the three inlets for aquatic veterinary drug solutions are respectively introduced with aquatic thiamphenicol solution, aquatic florfenicol solution and aquatic flumequine solution, and the three inlets for pathogenic organism culture fluid are introduced with Aeromonas hydrophila and its culture fluid, then the high-throughput aquatic veterinary drug sensitivity testing device can simultaneously test the sensitivity of Aeromonas hydrophila to 33 groups of three drugs mixed in two at 7 different proportions and the sensitivity of 12 groups of three drugs acting alone.

[0028] Furthermore, the inlet of the aquatic drug solution can be increased or decreased according to demand, or the number of channel layers can be changed laterally, so as to change the number of drug concentration gradients and the number of proportions.

[0029] The present invention also provides a high-throughput aquatic veterinary drug sensitivity testing method implemented by the high-throughput aquatic veterinary drug sensitivity testing device based on the multilayer microfluidic chip, comprising:

[0030] S1. Insert hoses into the inlets of the three aquatic veterinary drug solutions of the hybrid microfluidic chip, and connect them to injection pumps containing thiamphenicol solution for aquatic products, florfenicol solution for aquatic products, and flumequine solution for aquatic products, respectively;

[0031] S2, inserting hoses into the inlets of the three pathogenic biological culture solutions of the hybrid microfluidic chip, and connecting the syringe pumps containing Aeromonas hydrophila and its culture solution;

[0032] S3, insert a hose into the mineral oil inlet of the droplet microfluidic chip and connect it to a syringe pump filled with mineral oil;

[0033] S4, connecting the pathogenic organism culture device through a hose at the outlet of each micro-droplet of the droplet microfluidic chip;

[0034] S5. Inject three drug solutions, Aeromonas hydrophila culture solution and mineral oil into the chip at a slow speed. The thiamphenicol solution, florfenicol solution, flumequine solution and Aeromonas hydrophila solution are fully mixed in the first layer of the mixing microfluidic chip and flow into the second layer of the droplet microfluidic chip at the corresponding outlets. In the second layer of the droplet microfluidic chip, mineral oil is used as a continuous phase, and Aeromonas hydrophila and the drug solution are used as dispersed phases. They are encapsulated into microdroplets at the cross structure and enter the pathogenic organism culture device.

[0035] S6. When enough microdroplets are collected, the pathogenic organism culture device is placed in a pathogenic organism detection device for detection to determine the initial concentration of Aeromonas hydrophila;

[0036] S7. Place the pathogenic organism culture device in an environment of 37°C for culture, and place it in the pathogenic organism detection device for measurement every 30 minutes to determine the growth of Aeromonas hydrophila, make corresponding records and conduct drug resistance analysis.

[0037] Compared with the prior art, the present invention has the following advantages:

[0038] 1. The present invention provides a high-throughput aquatic veterinary drug sensitivity testing device based on a multi-layer microfluidic chip, which allows multiple groups of test samples to be generated in parallel on the same chip, significantly improving the throughput, shortening the test time, and thus improving the experimental efficiency.

[0039] 2. The present invention provides a high-throughput aquatic veterinary drug sensitivity testing device based on a multi-layer microfluidic chip, which can integrate more mixing channels in a limited space, organically combine the two chips, integrate drug mixing and encapsulation pathogenicity functions, and optimize the manipulation of fluids.

[0040] 3. The present invention provides a high-throughput aquatic veterinary drug sensitivity testing device based on a multi-layer microfluidic chip. Due to the micro-size characteristics of microfluidic technology, the required samples and reagents are significantly reduced, thereby reducing experimental costs and resource waste.

[0041] 4. The present invention provides a high-throughput aquatic veterinary drug sensitivity testing device based on a multi-layer microfluidic chip, which can flexibly design and adjust the chip structure according to different experimental requirements, adapt to various types of pathogenic microorganisms and drug sensitivity testing, and expand the scope of application of the device.

[0042] 5. The present invention provides a high-throughput aquatic veterinary drug sensitivity testing device based on a multi-layer microfluidic chip, which is combined with a pathogenic organism detection device to measure the concentration of pathogenic organisms. It can achieve rapid detection and obtain drug sensitivity results in a timely manner, providing strong support for aquaculture.

[0043] Based on the above reasons, the present invention can be widely promoted in the fields of research on rational use of drugs in aquaculture and development of new drugs for aquaculture. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0045] Figure 1 It is a schematic diagram of the overall structure of the device of the present invention.

[0046] Figure 2 It is a schematic diagram of the decomposition of the pathogenic organism packaging device of the present invention.

[0047] Figure 3 This is a top view of the microchannel of the pathogenic organism encapsulation device of the present invention.

[0048] Figure 4 This is a top view of the microchannel of the hybrid microfluidic chip of the present invention.

[0049] Figure 5 It is a three-dimensional diagram of the hybrid microfluidic chip of the present invention.

[0050] Figure 6 This is a top view of the microchannel of the droplet microfluidic chip of the present invention.

[0051] Figure 7 It is a three-dimensional diagram of the droplet microfluidic chip of the present invention.

[0052] Figure 8 This is an enlarged view of droplet generation in the droplet microfluidic chip of the present invention.

[0053] Fig. 9 It is a stereoscopic diagram of the pathogenic organism cultivation device of the present invention.

[0054] In the figure: 1. Pathogenic organism encapsulation device; 1-a. Hybrid microfluidic chip; 1-1. First inlet for aquatic veterinary drug solution; 1-2. First inlet for pathogenic organisms and culture solution; 1-3. Second inlet for aquatic veterinary drug solution; 1-4. Second inlet for pathogenic organisms and culture solution; 1-5. Third inlet for aquatic veterinary drug solution; 1-6. Third inlet for pathogenic organisms and culture solution; 2-1. First outlet leading to droplet microfluidic chip; 2-2. Second outlet leading to droplet microfluidic chip; 2-3. Third outlet leading to droplet microfluidic chip; 3-1. Fourth outlet leading to droplet microfluidic chip; 3-2. Fifth outlet leading to droplet microfluidic chip; 3-3. Sixth outlet leading to droplet microfluidic chip; 3-4. -4, leading to the seventh outlet of the droplet microfluidic chip; 3-5, leading to the eighth outlet of the droplet microfluidic chip; 3-6, leading to the ninth outlet of the droplet microfluidic chip; 4-1, leading to the tenth outlet of the droplet microfluidic chip; 4-2, leading to the eleventh outlet of the droplet microfluidic chip; 4-3, leading to the twelfth outlet of the droplet microfluidic chip; 4-4, leading to the thirteenth outlet of the droplet microfluidic chip; 4-5, leading to the fourteenth outlet of the droplet microfluidic chip; 4-6, leading to the fifteenth outlet of the droplet microfluidic chip; 4-7, leading to the sixteenth outlet of the droplet microfluidic chip; 4-8, leading to the seventeenth outlet of the droplet microfluidic chip; 4-9, leading to the eighteenth outlet of the droplet microfluidic chip; 4-10, leading to the droplet microfluidic chip The nineteenth outlet; 4-11, leading to the twentieth outlet of the droplet microfluidic chip; 4-12, leading to the twenty-first outlet of the droplet microfluidic chip; 5-1, leading to the twenty-second outlet of the droplet microfluidic chip; 5-2, leading to the twenty-third outlet of the droplet microfluidic chip; 5-3, leading to the twenty-fourth outlet of the droplet microfluidic chip; 5-4, leading to the twenty-fifth outlet of the droplet microfluidic chip; 5-5, leading to the twenty-sixth outlet of the droplet microfluidic chip; 5-6, leading to the twenty-seventh outlet of the droplet microfluidic chip; 5-7, leading to the twenty-eighth outlet of the droplet microfluidic chip; 5-8, leading to the twenty-ninth outlet of the droplet microfluidic chip; 5-9, leading to the thirtieth outlet of the droplet microfluidic chip; 5-10, leading to the droplet microfluidic chip 5-11, leading to the thirty-second outlet of the droplet microfluidic chip; 5-12, leading to the thirty-third outlet of the droplet microfluidic chip; 5-13, leading to the thirty-fourth outlet of the droplet microfluidic chip; 5-14, leading to the thirty-fifth outlet of the droplet microfluidic chip; 5-15, leading to the thirty-sixth outlet of the droplet microfluidic chip; 5-16, leading to the thirty-seventh outlet of the droplet microfluidic chip; 5-17, leading to the thirty-eighth outlet of the droplet microfluidic chip; 5-18, leading to the thirty-ninth outlet of the droplet microfluidic chip; 5-19, leading to the fortieth outlet of the droplet microfluidic chip; 5-20, leading to the forty-first outlet of the droplet microfluidic chip; 5-21, leading to the forty-second outlet of the droplet microfluidic chip;5-22, the forty-third outlet leading to the droplet microfluidic chip; 5-23, the forty-fourth outlet leading to the droplet microfluidic chip; 5-24, the forty-fifth outlet leading to the droplet microfluidic chip; 6-1, the first outlet for veterinary drug solution for aquatic products; 6-2, the second outlet for veterinary drug solution for aquatic products; 6-3, the third outlet for veterinary drug solution for aquatic products; 6-4, the fourth outlet for veterinary drug solution for aquatic products; 6-5, the fifth outlet for veterinary drug solution for aquatic products; 6-6, the sixth outlet for veterinary drug solution for aquatic products; 6-7, the seventh outlet for veterinary drug solution for aquatic products; 6-8, the eighth outlet for veterinary drug solution for aquatic products; 6-9, the ninth outlet for veterinary drug solution for aquatic products; 6-10, the tenth outlet for veterinary drug solution for aquatic products; 6-11, the veterinary drug solution for aquatic products 6-11th outlet of veterinary drug solution for aquatic products; 6-12th outlet of veterinary drug solution for aquatic products; 6-13th outlet of veterinary drug solution for aquatic products; 6-14th outlet of veterinary drug solution for aquatic products; 6-15th outlet of veterinary drug solution for aquatic products; 6-16th outlet of veterinary drug solution for aquatic products; 6-17th outlet of veterinary drug solution for aquatic products; 6-18th outlet of veterinary drug solution for aquatic products; 6-19th outlet of veterinary drug solution for aquatic products; 6-20th outlet of veterinary drug solution for aquatic products; 6-21th outlet of veterinary drug solution for aquatic products; 6-22th outlet of veterinary drug solution for aquatic products; 6-23th outlet of veterinary drug solution for aquatic products; 6-24th outlet of veterinary drug solution for aquatic products The twenty-fourth outlet; 1-b, droplet microfluidic chip; 7-1, mineral oil inlet; 8-1, the first inlet of aquatic veterinary drug solution; 8-2, the second inlet of aquatic veterinary drug solution; 8-3, the third inlet of aquatic veterinary drug solution; 10-1, the fourth inlet of aquatic veterinary drug solution; 10-2, the fifth inlet of aquatic veterinary drug solution; 10-3, the sixth inlet of aquatic veterinary drug solution; 10-4, the seventh inlet of aquatic veterinary drug solution; 10-5, the eighth inlet of aquatic veterinary drug solution; 10-6, the ninth inlet of aquatic veterinary drug solution; 12-1, the tenth inlet of aquatic veterinary drug solution; 12-2, the eleventh inlet of aquatic veterinary drug solution; 12-3, the twelfth inlet of aquatic veterinary drug solution; 12-4, The thirteenth inlet of veterinary drug solution for aquatic products; 12-5, the fourteenth inlet of veterinary drug solution for aquatic products; 12-6, the fifteenth inlet of veterinary drug solution for aquatic products; 12-7, the sixteenth inlet of veterinary drug solution for aquatic products; 12-8, the seventeenth inlet of veterinary drug solution for aquatic products; 12-9, the eighteenth inlet of veterinary drug solution for aquatic products; 12-10, the nineteenth inlet of veterinary drug solution for aquatic products; 12-11, the twentieth inlet of veterinary drug solution for aquatic products; 12-12, the twenty-first inlet of veterinary drug solution for aquatic products; 14-1, the twenty-second inlet of veterinary drug solution for aquatic products; 14-2, the twenty-third inlet of veterinary drug solution for aquatic products; 14-3, the twenty-fourth inlet of veterinary drug solution for aquatic products; 14-4, the twenty-fifth inlet of veterinary drug solution for aquatic products;14-5, the 26th entrance of veterinary drug solution for aquatic products; 14-6, the 27th entrance of veterinary drug solution for aquatic products; 14-7, the 28th entrance of veterinary drug solution for aquatic products; 14-8, the 29th entrance of veterinary drug solution for aquatic products; 14-9, the 30th entrance of veterinary drug solution for aquatic products; 14-10, the 31st entrance of veterinary drug solution for aquatic products; 14-11, the 32nd entrance of veterinary drug solution for aquatic products; 14-12, the 33rd entrance of veterinary drug solution for aquatic products; 14-13, the 34th entrance of veterinary drug solution for aquatic products; 14-14, the 35th entrance of veterinary drug solution for aquatic products; 14-15, the 36th entrance of veterinary drug solution for aquatic products; 14-16, the 37th entrance of veterinary drug solution for aquatic products; 14- 17. The 38th inlet of veterinary drug solution for aquatic products; 14-18. The 39th inlet of veterinary drug solution for aquatic products; 14-19. The 40th inlet of veterinary drug solution for aquatic products; 14-20. The 41st inlet of veterinary drug solution for aquatic products; 14-21. The 42nd inlet of veterinary drug solution for aquatic products; 14-22. The 43rd inlet of veterinary drug solution for aquatic products; 14-23. The 44th inlet of veterinary drug solution for aquatic products; 14-24. The 45th inlet of veterinary drug solution for aquatic products; 9-1. The first outlet of microdroplets; 9-2. The second outlet of microdroplets; 9-3. The third outlet of microdroplets; 11-1. The fourth outlet of microdroplets; 11-2. The fifth outlet of microdroplets; 11-3. The sixth outlet of microdroplets; 11-4. The seventh outlet of the microdroplet; 11-5, the eighth outlet of the microdroplet; 11-6, the ninth outlet of the microdroplet; 13-1, the tenth outlet of the microdroplet; 13-2, the eleventh outlet of the microdroplet; 13-3, the twelfth outlet of the microdroplet; 13-4, the thirteenth outlet of the microdroplet; 13-5, the fourteenth outlet of the microdroplet; 13-6, the fifteenth outlet of the microdroplet; 13-7, the sixteenth outlet of the microdroplet; 13-8, the seventeenth outlet of the microdroplet; 13-9, the eighteenth outlet of the microdroplet; 13-10, the nineteenth outlet of the microdroplet; 13-11, the twentieth outlet of the microdroplet; 13-12, the twenty-first outlet of the microdroplet; 15-1, the twenty-second outlet of the microdroplet; 15-2, the twenty-third outlet of the microdroplet outlet; 15-3, the twenty-fourth outlet of the microdroplet; 15-4, the twenty-fifth outlet of the microdroplet; 15-5, the twenty-sixth outlet of the microdroplet; 15-6, the twenty-seventh outlet of the microdroplet; 15-7, the twenty-eighth outlet of the microdroplet; 15-8, the twenty-ninth outlet of the microdroplet; 15-9, the thirtieth outlet of the microdroplet; 15-10, the thirty-first outlet of the microdroplet; 15-11, the thirty-second outlet of the microdroplet; 15-12, the thirty-third outlet of the microdroplet; 15-13, the thirty-fourth outlet of the microdroplet; 15-14, the thirty-fifth outlet of the microdroplet; 15-15, the thirty-sixth outlet of the microdroplet; 15-16, the thirty-seventh outlet of the microdroplet; 15-17, the thirty-eighth outlet of the microdroplet;15-18, the 39th outlet of the microdroplet; 15-19, the 40th outlet of the microdroplet; 15-20, the 41st outlet of the microdroplet; 15-21, the 42nd outlet of the microdroplet; 15-22, the 43rd outlet of the microdroplet; 15-23, the 44th outlet of the microdroplet; 15-24, the 45th outlet of the microdroplet; 1-c, glass substrate layer; 2, pathogenic biological culture device; 16-1, microdroplet inlet; 3, pathogenic biological detection device. ; DETAILED DESCRIPTION

[0055] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other. The present invention will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0056] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. The following description of at least one exemplary embodiment is actually only illustrative and is by no means intended to limit the present invention and its application or use. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0057] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0058] Unless otherwise specifically stated, the relative arrangement of the parts and steps described in these embodiments, the numerical expressions and numerical values ​​do not limit the scope of the present invention. At the same time, it should be clear that, for ease of description, the sizes of the various parts shown in the drawings are not drawn according to the actual proportional relationship. The technology, methods and equipment known to ordinary technicians in the relevant field may not be discussed in detail, but in appropriate cases, the technology, methods and equipment should be regarded as part of the authorization specification. In all examples shown and discussed here, any specific value should be interpreted as merely exemplary, rather than as a limitation. Therefore, other examples of exemplary embodiments may have different values. It should be noted that similar numbers and letters represent similar items in the following drawings, so once an item is defined in one drawing, it does not need to be further discussed in subsequent drawings.

[0059] In the description of the present invention, it is necessary to understand that the directions or positional relationships indicated by directional words such as "front, back, up, down, left, right", "lateral, vertical, perpendicular, horizontal" and "top, bottom" are usually based on the directions or positional relationships shown in the drawings. They are only for the convenience of describing the present invention and simplifying the description. Unless otherwise specified, these directional words do not indicate or imply that the device or element referred to must have a specific direction or be constructed and operated in a specific direction. Therefore, they cannot be understood as limiting the scope of protection of the present invention: the directional words "inside and outside" refer to the inside and outside relative to the contours of each component itself.

[0060] For ease of description, spatially relative terms such as "above", "above", "on the upper surface of", "above", etc. may be used here to describe the spatial positional relationship between a device or feature and other devices or features as shown in the figure. It should be understood that spatially relative terms are intended to include different orientations of the device in use or operation in addition to the orientation described in the figure. For example, if the device in the accompanying drawings is inverted, the device described as "above other devices or structures" or "above other devices or structures" will be positioned as "below other devices or structures" or "below their position devices or structures". Thus, the exemplary term "above" can include both "above" and "below". The device can also be positioned in other different ways (rotated 90 degrees or in other orientations), and the spatially relative descriptions used here are interpreted accordingly.

[0061] In addition, it should be noted that the use of terms such as "first" and "second" to limit components is only for the convenience of distinguishing the corresponding components. If not otherwise stated, the above terms have no special meaning and therefore cannot be understood as limiting the scope of protection of the present invention.

[0062] like Figure 1 As shown, the present invention provides a high-throughput aquatic veterinary drug sensitivity testing device based on a multi-layer microfluidic chip, comprising: a pathogenic organism encapsulation device 1, a pathogenic organism cultivation device 2 and a pathogenic organism detection device 3, wherein:

[0063] like Figure 2 , 3 As shown, the pathogenic organism encapsulation device 1 has a three-layer structure, which includes a hybrid microfluidic chip 1-a, a droplet microfluidic chip 1-b and a glass substrate layer 1-c from top to bottom. The three-layer structure is bonded by plasma cleaning. The hybrid microfluidic chip 1-a and the droplet microfluidic chip 1-b are connected by a series of microchannels to transport the fluid from the first layer of the hybrid microfluidic chip 1-a to the second layer of the droplet microfluidic chip 1-b. The glass substrate layer 1-c is used to support and close the device;

[0064] The pathogenic organism cultivation device 2 is connected to the pathogenic organism encapsulation device 1 and is used to place the micro-droplets encapsulating the pathogenic organisms in a suitable environment for cultivation;

[0065] The pathogenic organism detection device 3 is connected to the pathogenic organism cultivation device 2 and is used to detect the pathogenic organisms in the pathogenic organism cultivation device 2 at regular intervals to determine the growth of the pathogenic organisms, thereby performing sensitivity analysis of aquatic veterinary drugs.

[0066] In specific implementation, as a preferred embodiment of the present invention, in the pathogenic organism encapsulation device 1:

[0067] like Figure 4 , 5 As shown, the hybrid microfluidic chip 1-a is provided with 6 inlets and 69 outlets for aquatic veterinary drug solutions, wherein:

[0068] Among the 6 inlets: 3 inlets are used as inlets for aquatic veterinary drug solutions, for injecting aquatic veterinary drug solutions; 3 inlets are used as inlets for pathogenic organisms and culture fluids, for injecting pathogenic organism culture fluids;

[0069] Among the 69 outlets for veterinary drug solutions for aquaculture: 45 outlets lead to the droplet microfluidic chip 1-b, which is used to inject the mixed solution as the dispersed phase for generating microdroplets; 24 outlets are used to collect the mixed veterinary drug solutions for aquaculture, to be put into other applications and used for other methods for drug sensitivity analysis.

[0070] In this embodiment, the mixing microfluidic chip fully mixes aquatic veterinary drug solutions of different concentrations or types, and mixes them with the tested pathogenic organisms to generate a series of solutions of different concentrations or different ratios, which flow into the next layer of droplet microfluidic chip as the dispersed phase for generating droplets.

[0071] In specific implementation, as a preferred embodiment of the present invention, in the pathogenic organism encapsulation device 1:

[0072] The mixing microfluidic chip 1-a is circular as a whole, increasing the number of mixing channels and expanding the application range of the device. At the same time, a serpentine mixing channel is used to increase the channel length, thereby extending the mixing time and allowing pathogenic organisms and aquatic veterinary drug solutions to be fully mixed.

[0073] In specific implementation, as a preferred embodiment of the present invention, in the pathogenic organism encapsulation device 1:

[0074] The droplet microfluidic chip 1-b is used to simultaneously generate microdroplets of aquatic veterinary drugs containing pathogenic organisms in different concentrations or different ratios, thereby improving the throughput of the microdroplets and the efficiency of drug sensitivity analysis. Figure 6 , 7 As shown, it includes a mineral oil inlet 7-1, 45 inlets for aquatic veterinary drug solutions and 45 outlets for micro-droplets, wherein:

[0075] The mineral oil inlet serves as the continuous phase for generating microdroplets;

[0076] 45 inlets for aquatic veterinary drug solutions receive solutions from 45 outlets in the mixing microfluidic chip 1-a as dispersed phases for generating microdroplets;

[0077] The outlets of 45 micro-droplets encapsulate the solutions of 45 aquatic veterinary drug solution inlets in the micro-droplets, making each micro-droplet a culture device. Figure 8 Shown is an enlarged view of droplet generation in a droplet microfluidic chip.

[0078] When specifically implemented, as a preferred embodiment of the present invention, Fig. 9 As shown, the pathogenic organism cultivation device 2 is provided with a micro-droplet inlet 16-1, which is connected to the micro-droplet outlet of the pathogenic organism encapsulation device 1 to collect micro-droplets. The pathogenic organism cultivation device 2 is placed in an environment of 37°C for cultivation. The pathogenic organisms in the droplets interact with the drugs, and are measured at regular intervals by the pathogenic organism detection device to determine the growth of the pathogenic organisms and analyze their reactions to the drugs.

[0079] In specific implementation, as a preferred embodiment of the present invention, the high-throughput aquatic veterinary drug sensitivity testing device based on a multilayer microfluidic chip provided by the present invention can obtain multiple groups of aquatic veterinary drug microdroplets containing different concentrations or different ratios in a short time for drug sensitivity analysis. Its three specific application methods are as follows (taking the analysis of the sensitivity of Aeromonas hydrophila to aquatic thiamphenicol solution, aquatic florfenicol solution and aquatic flumequine solution as an example):

[0080] Application 1: If the first inlet 1-1 of the aquatic veterinary drug solution, the second inlet 1-3 of the aquatic veterinary drug solution, and the third inlet 1-5 of the aquatic veterinary drug solution are respectively introduced with a 100% aquatic thiamphenicol solution, saline and saline, and the first inlet 1-2 of the pathogenic organisms and culture fluid, the second inlet 1-4 of the pathogenic organisms and culture fluid, and the third inlet 1-6 of the pathogenic organisms and culture fluid are introduced with Aeromonas hydrophila and its culture fluid, then microdroplets of thiamphenicol solutions of different concentrations containing Aeromonas hydrophila can be obtained at the outlet. Among them, microdroplets of thiamphenicol solution with a concentration of 100% are collected at the first outlet 9-1 of the microdroplet, the fourth outlet 11-1 of the microdroplet, the tenth outlet 13-1 of the microdroplet, and the twenty-second outlet 15-1 of the microdroplet; microdroplets of thiamphenicol solution with a concentration of 87.5% are collected at the twenty-third outlet 15-2 of the microdroplet and the forty-fifth outlet 15-24 of the microdroplet; microdroplets of thiamphenicol solution with a concentration of 75% are collected at the eleventh outlet 13-2 of the microdroplet, the twenty-first outlet 13-12 of the microdroplet, the twenty-fourth outlet 15-3 of the microdroplet, and the forty-fourth outlet 15-23 of the microdroplet; microdroplets of thiamphenicol solution with a concentration of 62.5% are collected at the twenty-fifth outlet 15-4 of the microdroplet and the forty-third outlet 15-22 of the microdroplet; microdroplets of thiamphenicol solution with a concentration of 62.5% are collected at the fifth outlet 11-2 of the microdroplet, the ninth outlet 11-6 of the microdroplet, Microdroplets of thiamphenicol solution with a concentration of 50% are collected at the twelfth outlet 13-3 of the microdroplet, the twentieth outlet 13-11 of the microdroplet, the twenty-sixth outlet 15-5 of the microdroplet, and the forty-second outlet 15-21 of the microdroplet; microdroplets of thiamphenicol solution with a concentration of 37.5% are collected at the twenty-seventh outlet 15-6 of the microdroplet and the forty-first outlet 15-20 of the microdroplet; microdroplets of thiamphenicol solution with a concentration of 25% are collected at the thirteenth outlet 13-4 of the microdroplet, the nineteenth outlet 13-10 of the microdroplet, the twenty-eighth outlet 15-7 of the microdroplet, and the fortieth outlet 15-19 of the microdroplet; microdroplets of thiamphenicol solution with a concentration of 12.5% ​​are collected at the twenty-ninth outlet 15-8 of the microdroplet and the thirty-ninth outlet 15-18 of the microdroplet; and microdrops not containing thiamphenicol solution are collected at the other 19 groups of microdroplet outlets. In this application method, the device can simultaneously test the sensitivity of Aeromonas hydrophila to 26 groups of 8 linear concentration gradients of thiamphenicol solutions and 19 groups of blank control groups.

[0081] Application 2: If a 100% aquatic thiamphenicol solution, a 100% aquatic florfenicol solution and physiological saline are introduced into the first inlet 1-1 of the aquatic veterinary drug solution, the second inlet 1-3 of the aquatic veterinary drug solution and the third inlet 1-5 of the aquatic veterinary drug solution respectively, and Aeromonas hydrophila and its culture solution are introduced into the first inlet 1-2 of the pathogenic organisms and culture solution, the second inlet 1-4 of the pathogenic organisms and culture solution and the third inlet 1-6 of the pathogenic organisms and culture solution, then microdroplets of thiamphenicol solution and florfenicol solution of different concentrations containing Aeromonas hydrophila and microdroplets mixed in different proportions can be obtained at the outlet. The first outlet 9-1 of the microdroplet, the fourth outlet 11-1 of the microdroplet, the tenth outlet 13-1 of the microdroplet, and the twenty-second outlet 15-1 of the microdroplet collect microdroplets of a 100% thiamphenicol solution; the second outlet 9-2 of the microdroplet, the sixth outlet 11-3 of the microdroplet, the twelfth outlet 13-3 of the microdroplet, and the thirtieth outlet 15-9 of the microdroplet collect microdroplets of a 100% florfenicol solution; the forty-fifth outlet 15-24 of the microdroplet and the thirty-first outlet 15-10 of the microdroplet collect microdroplets of a 100% florfenicol solution; Collect 87.5% thiamphenicol solution microdroplets and 87.5% florfenicol solution microdroplets; collect 75% thiamphenicol solution microdroplets at the 21st outlet 13-12 of the microdroplets and the 44th outlet 15-23 of the microdroplets, collect 75% florfenicol solution microdroplets at the 15th outlet 13-6 of the microdroplets and the 32nd outlet 15-11 of the microdroplets; collect 62.5% florfenicol solution microdroplets at the 43rd outlet 15-22 of the microdroplets and the 33rd outlet 15-12 of the microdroplets, respectively. The invention discloses a method for collecting microdroplets of thiamphenicol solution and microdroplets of florfenicol solution with a concentration of 62.5%; microdroplets of thiamphenicol solution with a concentration of 50% are collected at the ninth outlet 11-6 of the microdroplets, the twentieth outlet 13-11 of the microdroplets, and the forty-second outlet 15-21 of the microdroplets; microdroplets of florfenicol solution with a concentration of 50% are collected at the seventh outlet 11-4 of the microdroplets, the sixteenth outlet 13-7 of the microdroplets, and the thirty-fourth outlet 15-13 of the microdroplets; and microdroplets of thiamphenicol solution with a concentration of 37.5% are collected at outlets 15-20 and 15-14, respectively. The microdroplets of thiamphenicol solution with a concentration of 25% are collected at the nineteenth outlet 13-10 of the microdroplet and the fortieth outlet 15-19 of the microdroplet, and the microdroplets of florfenicol solution with a concentration of 25% are collected at the seventeenth outlet 13-8 of the microdroplet and the thirty-sixth outlet 15-15 of the microdroplet; the microdroplets of thiamphenicol solution with a concentration of 12.5% ​​and the microdroplets with a concentration of 12.5% ​​are collected at the thirty-ninth outlet 15-18 of the microdroplet and the thirty-seventh outlet 15-16 of the microdroplet, respectively.5% florfenicol solution microdroplets; collecting drug-free microdroplets at the third outlet 9-3 of the microdroplets, the eighth outlet 11-5 of the microdroplets, the eighteenth outlet 13-9 of the microdroplets, and the thirty-eighth outlet 15-17 of the microdroplets; collecting microdroplets of a 1:1 mixture of thiamphenicol solution and florfenicol solution at the fifth outlet 11-2 of the microdroplets, the twelfth outlet 13-3 of the microdroplets, and the twenty-sixth outlet 15-5 of the microdroplets; collecting microdroplets of a 2:1 mixture of thiamphenicol solution and florfenicol solution at the eleventh outlet 13-2 of the microdroplets and the twenty-fourth outlet 15-3 of the microdroplets; collecting microdroplets of a 2:1 mixture of thiamphenicol solution and florfenicol solution at the thirteenth outlet 11-2 of the microdroplets, the twelfth outlet 13-3 of the microdroplets, and the twenty-sixth outlet 15-5 of the microdroplets. At the outlet 13-4 of the microdroplet, the microdroplets of thiamphenicol solution and florfenicol solution mixed in a ratio of 1:3 are collected at the twenty-eighth outlet 15-7 of the microdroplet; at the twenty-third outlet 15-2 of the microdroplet, the microdroplets of thiamphenicol solution and florfenicol solution mixed in a ratio of 3:1 are collected; at the twenty-ninth outlet 15-8 of the microdroplet, the microdroplets of thiamphenicol solution and florfenicol solution mixed in a ratio of 1:3 are collected; at the twenty-fifth outlet 15-4 of the microdroplet, the microdroplets of thiamphenicol solution and florfenicol solution mixed in a ratio of 3:2 are collected; at the twenty-seventh outlet 15-6 of the microdroplet, the microdroplets of thiamphenicol solution and florfenicol solution mixed in a ratio of 2:3 are collected. In this application method, the device can simultaneously test the sensitivity of Aeromonas hydrophila to 15 groups of 8 linear concentration gradients of thiamphenicol solutions, 15 groups of 8 linear concentration gradients of florfenicol solutions, 11 groups of sensitivity to two drugs mixed in 7 different proportions, and 4 groups of blank control groups. .

[0082] Application three: If the first inlet 1-1 of the veterinary drug solution for aquatic products, the second inlet 1-3 of the veterinary drug solution for aquatic products, and the third inlet 1-5 of the veterinary drug solution for aquatic products are respectively introduced into the thiamphenicol solution for aquatic products, the florfenicol solution for aquatic products, and the flumequine solution for aquatic products, and the first inlet 1-2 of the pathogenic organisms and culture fluid, the second inlet 1-4 of the pathogenic organisms and culture fluid, and the third inlet 1-6 of the pathogenic organisms and culture fluid are introduced into Aeromonas hydrophila and its culture fluid, then microdroplets containing the three drugs for Aeromonas hydrophila mixed in pairs in different proportions can be obtained at the outlet. The microdroplets containing only thiamphenicol solution are collected at the first outlet 9-1 of the microdroplets, the fourth outlet 11-1 of the microdroplets, the tenth outlet 13-1 of the microdroplets, and the twenty-second outlet 15-1 of the microdroplets; the microdroplets containing only florfenicol solution are collected at the second outlet 9-2 of the microdroplets, the sixth outlet 11-3 of the microdroplets, the fourteenth outlet 13-5 of the microdroplets, and the thirtieth outlet 15-9 of the microdroplets; and the microdroplets containing only florfenicol solution are collected at the third outlet 9-3 of the microdroplets, the eighth outlet 11-5 of the microdroplets, the eighteenth outlet 13-9 of the microdroplets, the thirtieth outlet 15-9 of the microdroplets. The eight outlets 15-17 collect microdroplets containing only flumequine solution; the fifth outlet 11-2 of the microdroplets, the twelfth outlet 13-3 of the microdroplets, and the twenty-sixth outlet 15-5 of the microdroplets collect microdroplets containing a 1:1 mixture of thiamphenicol solution and florfenicol solution; the seventh outlet 11-4 of the microdroplets, the sixteenth outlet 13-7 of the microdroplets, and the thirty-fourth outlet 15-13 of the microdroplets collect microdroplets containing a 1:1 mixture of florfenicol solution and flumequine solution; the ninth outlet 11-6 of the microdroplets, the twentieth outlet 13-11 of the microdroplets, and the , the forty-second outlet 15-21 of the microdroplet collects microdroplets of a 1:1 mixture of thiamphenicol solution and flumequine solution; the eleventh outlet 13-2 of the microdroplet and the twenty-fourth outlet 15-3 of the microdroplet collect microdroplets of a 2:1 mixture of thiamphenicol solution and florfenicol solution; the thirteenth outlet 13-4 of the microdroplet and the twenty-eighth outlet 15-7 of the microdroplet collect microdroplets of a 1:2 mixture of thiamphenicol solution and florfenicol solution; the fifteenth outlet 13-6 of the microdroplet and the thirty-second outlet 15-11 of the microdroplet collect florfenicol; The microdroplets of florfenicol solution and flumequine solution mixed in a ratio of 2:1 are collected at the seventeenth outlet 13-8 of the microdroplets and the thirty-sixth outlet 15-15 of the microdroplets, the microdroplets of florfenicol solution and flumequine solution mixed in a ratio of 1:2 are collected at the twenty-first outlet 13-12 of the microdroplets and the forty-fourth outlet 15-23 of the microdroplets, and the microdroplets of thiamphenicol solution and flumequine solution mixed in a ratio of 2:1 are collected at the nineteenth outlet 13-10 of the microdroplets and the fortieth outlet 15-19 of the microdroplets;At the twenty-third outlet 15-2 of the microdroplet, a microdroplet of a mixture of thiamphenicol solution and florfenicol solution in a ratio of 3:1 is collected; at the twenty-ninth outlet 15-8 of the microdroplet, a microdroplet of a mixture of thiamphenicol solution and florfenicol solution in a ratio of 1:3 is collected; at the thirty-first outlet 15-10 of the microdroplet, a microdroplet of a mixture of florfenicol solution and flumequine solution in a ratio of 3:1 is collected; at the thirty-seventh outlet 15-16 of the microdroplet, a microdroplet of a mixture of florfenicol solution and flumequine solution in a ratio of 1:3 is collected; at the forty-fifth outlet 15-24 of the microdroplet, a microdroplet of a mixture of thiamphenicol solution and flumequine solution in a ratio of 3:1 is collected; at the thirty-ninth outlet 15-18 of the microdroplet, a microdroplet of a mixture of thiamphenicol solution and flumequine solution in a ratio of 1:3 is collected; Microdroplets of a mixture of thiamphenicol solution and florfenicol solution in a ratio of 3:2 are collected at the twenty-fifth outlet 15-4 of the microdroplet, microdroplets of a mixture of thiamphenicol solution and florfenicol solution in a ratio of 2:3 are collected at the twenty-seventh outlet 15-6 of the microdroplet, microdroplets of a mixture of florfenicol solution and flumequine solution in a ratio of 3:2 are collected at the thirty-third outlet 15-12 of the microdroplet, microdroplets of a mixture of florfenicol solution and flumequine solution in a ratio of 2:3 are collected at the thirty-fifth outlet 15-14 of the microdroplet, microdroplets of a mixture of thiamphenicol solution and flumequine solution in a ratio of 3:2 are collected at the forty-third outlet 15-22 of the microdroplet, and microdroplets of a mixture of thiamphenicol solution and flumequine solution in a ratio of 2:3 are collected at the forty-first outlet 15-20 of the microdroplet. In this application method, the device can simultaneously test the sensitivity of Aeromonas hydrophila to 33 groups of three drugs mixed in 7 different proportions and 12 groups of three drugs acting alone.

[0083] In this embodiment, the selected pathogenic organisms include but are not limited to Vibrio alginolyticus, Vibrio harveyi, Vibrio vulnificus, Vibrio parahaemolyticus, Streptococcus (such as Streptococcus iniae), Aeromonas (such as Aeromonas hydrophila) and other pathogens, and the selected veterinary drugs for aquaculture include but are not limited to antibiotics such as thiamphenicol, florfenicol, flumequine, enrofloxacin, doxycycline hydrochloride, and neomycin sulfate.

[0084] In specific implementation, as a preferred embodiment of the present invention, the inlet of the aquatic drug solution is increased or decreased according to demand, or the number of channel layers is changed laterally, so as to change the number of drug concentration gradients and the number of ratios.

[0085] In specific implementation, as a preferred embodiment of the present invention, the hybrid microfluidic chip 1-a and the droplet microfluidic chip 1-b of the pathogenic organism encapsulation device 1 are both made of PDMS and are manufactured using photolithography technology. The specific operation steps are as follows:

[0086] Step 1, substrate pretreatment: Use a silicon wafer as a substrate, clean the silicon wafer with acetone and deionized water, and then blow dry it with nitrogen to ensure that the photoresist adheres better to the substrate surface;

[0087] Step 2, spin coating photoresist: fix the cleaned silicon wafer in the center area of ​​the spin coater, set the spin coater speed to 2500 rpm according to the chip channel height of 60 μm, so that the surface of the silicon wafer is evenly covered with photoresist;

[0088] Step 3, pre-baking: Place the spin-coated silicon wafer on a heating plate and heat it at 65°C for 2 minutes, then at 95°C for 8 minutes, and then cool it naturally to room temperature. The purpose of pre-baking is to enhance the adhesion of the photoresist and solidify it.

[0089] Step 4: Exposure: Cover the prepared mask on the silicon wafer and expose it using a UV exposure machine. Adjust the exposure time of the exposure machine to 20 seconds to provide 200mJ / cm 2 UV exposure energy;

[0090] Step 5, post-baking: The exposed silicon wafer is placed on a heating plate again for post-baking treatment, first set to 65°C for 1.5 minutes, and then set to 95°C for 7 minutes to enhance the exposure characteristics of the photoresist and make the pattern appear.

[0091] Step 6, Development: Wait for the silicon wafer to cool to room temperature and then perform the development operation. Soak the silicon wafer in the developer for about 5 minutes, then rinse the surface of the silicon wafer with clean water and observe the development. If there is excess photoresist residue, rinse it again with the developer, observing while rinsing until the desired structure is left on the silicon wafer, and finally blow it dry with nitrogen;

[0092] Step 7, hardening the film: After cleaning the developed substrate, bake it at 150°C for 1 hour to completely remove the solvent or moisture remaining in the film after development, so that the film and the silicon wafer are closely adhered to prevent the adhesive layer from falling off and enhance the corrosion resistance of the film itself;

[0093] Step 8, pouring: Mix liquid PDMS and curing agent in a ratio of 10:1, stir for 3 minutes, place in a vacuum box for 20 minutes to remove bubbles in the mixed liquid, and slowly pour the treated PDMS on the pre-made silicon wafer to ensure a moderate thickness;

[0094] Step 9, curing: Place the silicon wafer with PDMS cast on a heating plate and heat at 80°C for 1 hour to accelerate the curing speed of PDMS. After curing, peel the PDMS from the silicon wafer. At this time, the pre-designed channel shape has been etched on the PDMS chip and is wrapped with plastic wrap to prevent contamination;

[0095] Step 10, bonding: After drilling holes at the inlets and outlets of the hybrid microfluidic chip and the droplet microfluidic chip, the two and the glass sheet are placed in a vacuum plasma cleaning machine for cleaning to destroy the chemical bonds between the PDMS and the surface of the glass sheet. After taking them out, the three corresponding inlets and outlets are quickly bonded together;

[0096] Step 11, heating: Since the surfaces of the PDMS and glass sheets after plasma cleaning are hydrophilic, which is not conducive to the generation of water-in-oil microdroplets, the bonded chip is placed on a heating plate and heated at 130°C for 6 hours to restore the hydrophobicity;

[0097] The pathogenic organism encapsulation device manufactured based on the above method has good experimental results, is easy and efficient to manufacture, reduces manufacturing costs, and expands the scope of application of the device.

[0098] The present invention also provides a high-throughput aquatic veterinary drug sensitivity testing method implemented by the high-throughput aquatic veterinary drug sensitivity testing device based on the multilayer microfluidic chip (taking the above-mentioned application three as an example), comprising:

[0099] S1, insert hoses into the three inlets of aquatic veterinary drug solutions of the hybrid microfluidic chip 1-a, and connect them to injection pumps containing thiamphenicol solution for aquatic products, florfenicol solution for aquatic products, and flumequine solution for aquatic products respectively;

[0100] S2, inserting hoses into the three pathogenic biological culture fluid inlets of the hybrid microfluidic chip 1-a, and connecting the injection pumps filled with Aeromonas hydrophila and its culture fluid;

[0101] S3, insert a hose into the mineral oil inlet 7-1 of the droplet microfluidic chip 1-b, and connect it to a syringe pump filled with mineral oil;

[0102] S4, connecting the pathogenic organism culture device 2 through a hose at each micro-droplet outlet of the droplet microfluidic chip 1-b;

[0103] S5, injecting three drug solutions, Aeromonas hydrophila culture solution and mineral oil into the chip at a slow speed, the thiamphenicol solution, florfenicol solution and flumequine solution as well as the Aeromonas hydrophila solution are fully mixed in the first layer of the mixing microfluidic chip 1-a, and flow into the second layer of the droplet microfluidic chip 1-b at the corresponding outlets, in the second layer of the droplet microfluidic chip 1-b, the mineral oil is used as the continuous phase, the Aeromonas hydrophila and the drug solution are used as the dispersed phase, and are encapsulated into microdroplets at the cross structure and enter the pathogenic organism culture device 2;

[0104] S6. When enough microdroplets are collected, the pathogenic organism cultivation device 2 is placed in the pathogenic organism detection device 3 for detection to determine the initial concentration of Aeromonas hydrophila;

[0105] S7. Place the pathogenic organism culture device 2 in an environment of 37° C. for culture, and place it in the pathogenic organism detection device 3 for detection every 30 minutes to determine the growth of Aeromonas hydrophila, make corresponding records and conduct drug resistance analysis.

[0106] In summary, the veterinary drug sensitivity testing device for aquaculture provided by the present invention can obtain multiple groups of microdroplets containing different drug concentrations and ratios in a short time, which can be used for drug resistance analysis of pathogenic organisms in aquaculture. It has the advantages of high throughput, fast efficiency, precise control, and low cost. It provides a more efficient tool for studying the rational use of drugs in aquaculture and the development of new drugs for aquaculture, thereby promoting the sustainable development of aquaculture.

[0107] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A high-throughput aquatic veterinary drug sensitivity testing device based on a multilayer microfluidic chip, characterized in that: include: A pathogenic organism encapsulation device (1), a pathogenic organism cultivation device (2) and a pathogenic organism detection device (3), wherein: The pathogenic organism encapsulation device (1) has a three-layer structure, which includes, from top to bottom, a hybrid microfluidic chip (1-a), a droplet microfluidic chip (1-b) and a glass substrate layer (1-c); the three-layer structure is bonded together by plasma cleaning bonding; the hybrid microfluidic chip (1-a) and the droplet microfluidic chip (1-b) are connected by a series of microchannels to transport fluid from the first layer of the hybrid microfluidic chip (1-a) to the second layer of the droplet microfluidic chip (1-b); and the glass substrate layer (1-c) is used to support and seal the device; The pathogenic organism cultivation device (2) is connected to the pathogenic organism encapsulation device (1) and is used to place the micro-droplets encapsulating the pathogenic organisms in a suitable environment for cultivation; The pathogenic organism detection device (3) is connected to the pathogenic organism cultivation device (2) and is used to detect the pathogenic organisms in the pathogenic organism cultivation device (2) at regular intervals to determine the growth of the pathogenic organisms, thereby performing sensitivity analysis of aquatic veterinary drugs.

2. A high-throughput aquatic veterinary drug sensitivity testing device based on a multilayer microfluidic chip according to claim 1, characterized in that: In the pathogenic organism encapsulation device (1): The hybrid microfluidic chip (1-a) is provided with 6 inlets and 69 outlets for aquatic veterinary drug solutions, wherein: Among the 6 entrances: Three inlets are used as inlets for aquatic veterinary drug solutions, and are used to inject aquatic veterinary drug solutions; three inlets are used as inlets for pathogenic organisms and culture fluids, and are used to inject pathogenic organism culture fluids; Among the 69 exports of veterinary solutions for aquaculture: 45 outlets lead to the droplet microfluidic chip (1-b) for injecting the mixed solution as the dispersed phase for generating microdroplets; 24 outlets are used for collecting the mixed veterinary drug solution for aquaculture.

3. A high-throughput aquatic veterinary drug sensitivity testing device based on a multilayer microfluidic chip according to claim 2, characterized in that: In the pathogenic organism encapsulation device (1): The mixing microfluidic chip (1-a) is circular in shape as a whole, which increases the number of mixing channels and expands the application range of the device. At the same time, a serpentine mixing channel is used to increase the channel length, thereby extending the mixing time and allowing pathogenic organisms and aquatic veterinary drug solutions to be fully mixed.

4. The high-throughput aquatic veterinary drug sensitivity testing device based on a multilayer microfluidic chip according to claim 1, characterized in that: In the pathogenic organism encapsulation device 1: The droplet microfluidic chip (1-b) is used to simultaneously generate aquatic veterinary drug microdroplets containing pathogenic organisms at different concentrations or different ratios, including a mineral oil inlet (7-1), 45 aquatic veterinary drug solution inlets and 45 microdroplet outlets, wherein: The mineral oil inlet serves as the continuous phase for generating microdroplets; 45 inlets for aquatic veterinary drug solutions receive solutions from 45 outlets in the mixing microfluidic chip (1-a) as dispersed phases for generating microdroplets; The outlets of 45 micro-droplets encapsulate the solutions of 45 inlets of aquatic veterinary drug solutions in the micro-droplets, making each micro-droplet a culture device.

5. The high-throughput aquatic veterinary drug sensitivity testing device based on a multilayer microfluidic chip according to claim 1, characterized in that: The pathogenic organism cultivation device (2) is provided with a micro-droplet inlet (16-1), which is connected to the micro-droplet outlet of the pathogenic organism encapsulation device 1 to collect micro-droplets, and the pathogenic organism cultivation device (2) is placed in an environment of 37°C for cultivation.

6. The high-throughput aquatic veterinary drug sensitivity testing device based on a multilayer microfluidic chip according to claim 1, characterized in that: If 100% aquatic thiamphenicol solution, normal saline and normal saline are respectively introduced into the three inlets of aquatic veterinary drug solutions, and Aeromonas hydrophila and its culture solution are introduced into the three inlets of pathogenic organisms and culture medium, the high-throughput aquatic veterinary drug sensitivity testing device can simultaneously test the sensitivity of Aeromonas hydrophila to 26 groups of 8 linear concentration gradients of thiamphenicol solutions and 19 groups of blank control groups.

7. A high-throughput aquatic veterinary drug sensitivity testing device based on a multilayer microfluidic chip according to claim 6, characterized in that: If a 100% aquatic thiamphenicol solution, a 100% aquatic florfenicol solution and normal saline are respectively introduced into the inlets of the three aquatic veterinary drug solutions, and Aeromonas hydrophila and its culture solution are introduced into the three inlets of pathogenic organisms and culture solutions, then the high-throughput aquatic veterinary drug sensitivity testing device can simultaneously test the sensitivity of Aeromonas hydrophila to 15 groups of 8 linear concentration gradients of thiamphenicol solutions, 15 groups of 8 linear concentration gradients of florfenicol solutions, 11 groups of sensitivity of two drug solutions mixed in 7 different proportions and 4 groups of blank control groups.

8. The high-throughput aquatic veterinary drug sensitivity testing device based on a multilayer microfluidic chip according to claim 6, characterized in that: If the three inlets of aquatic veterinary drug solutions are respectively introduced into aquatic thiamphenicol solution, aquatic florfenicol solution and aquatic flumequine solution, and the three inlets of pathogenic organisms and culture medium are introduced into Aeromonas hydrophila and its culture medium, then the high-throughput aquatic veterinary drug sensitivity testing device can simultaneously test the sensitivity of Aeromonas hydrophila to 33 groups of three drugs mixed in 7 different proportions and the sensitivity of 12 groups of three drugs acting alone.

9. The high-throughput aquatic veterinary drug sensitivity testing device based on a multilayer microfluidic chip according to claim 1, characterized in that: According to the demand, the inlet of the aquatic drug solution can be increased or decreased, or the number of channel layers can be changed laterally, so as to change the number of drug concentration gradients and the number of ratios.

10. A high-throughput aquatic veterinary drug sensitivity testing method implemented by the high-throughput aquatic veterinary drug sensitivity testing device based on a multilayer microfluidic chip according to any one of claims 1 to 9, characterized in that: include: S1. Insert hoses into the three inlets of the aquatic veterinary drug solution of the hybrid microfluidic chip (1-a), and connect them to injection pumps containing thiamphenicol solution for aquatic products, florfenicol solution for aquatic products, and flumequine solution for aquatic products, respectively; S2, inserting hoses into the inlets of the three pathogenic biological culture solutions of the mixing microfluidic chip (1-a), and connecting the injection pumps containing Aeromonas hydrophila and its culture solution; S3, inserting a hose into the mineral oil inlet (7-1) of the droplet microfluidic chip (1-b), and connecting it to a syringe pump filled with mineral oil; S4, connecting each micro-droplet outlet of the droplet microfluidic chip (1-b) to the pathogenic organism culture device (2) through a hose; S5. Inject three drug solutions, Aeromonas hydrophila culture solution and mineral oil into the chip at a slow speed. The thiamphenicol solution, florfenicol solution, flumequine solution and Aeromonas hydrophila solution are fully mixed in the first layer of the mixing microfluidic chip (1-a) and flow into the second layer of the droplet microfluidic chip (1-b) at the corresponding outlets. In the second layer of the droplet microfluidic chip (1-b), mineral oil is used as a continuous phase, and Aeromonas hydrophila and the drug solution are used as a dispersed phase. They are encapsulated into microdroplets at the cross structure and enter the pathogenic organism culture device (2). S6. When enough microdroplets are collected, the pathogenic organism culture device (2) is placed in the pathogenic organism detection device (3) for detection to determine the initial concentration of Aeromonas hydrophila; S7. Place the pathogenic organism culture device (2) in an environment of 37° C. for culture, and place it in the pathogenic organism detection device (3) for detection every 30 minutes to determine the growth of Aeromonas hydrophila, make corresponding records and conduct drug resistance analysis.

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