Microbial drug sensitivity detection device and method

Abstract

The invention discloses a method and device for detecting drug sensitivity of microorganisms, and relates to the field of biological detection.The device comprises two parallel chip layers, micron-sized channels and cavities are etched in each chip layer, and accurate mixing of samples and microorganisms is achieved through a sliding mechanism by means of an upper-layer chip and a lower-layer chip; when the micro flow channels on the upper-layer chip and the lower-layer chip are partially overlapped, a complete continuous fluid channel is formed, bacteria and candidate compounds are fused in the sliding process, and a multiple nanoliter-scale bacteria-compound droplet matrix is generated; wherein a microbial suspension and to-be-detected solutions with different concentrations are respectively loaded in different chambers, and the chambers are aligned through sliding operation, so that a mixed reaction of a sample and microorganisms is promoted; detection is carried out through optical signals, electrical signals and the like so as to judge the bacterial state. According to the invention, rapid drug sensitivity detection of the to-be-detected compound can be realized, relatively low sample consumption is realized, and a foundation is laid for microbial drug sensitivity detection.

Description

Technical Field

[0001] The present invention relates to the field of biological detection, and in particular to a device and method for detecting drug sensitivity of microorganisms. Background Art

[0002] Bacterial resistance to antibiotics is escalating, and there is an urgent need to find new antibiotics. In order to slow down this trend, it is necessary not only to regulate the use of antibiotics, but also to urgently explore new unexplored drug targets in bacteria to develop more effective antibacterial agents. Natural products from natural sources (including plants and microorganisms) are an ideal way to solve this problem, but natural products pose a challenge to existing screening methods due to their low content of active ingredients.

[0003] Traditional antimicrobial sensitivity screening has some significant drawbacks when applied to complex samples such as natural products. It usually takes a long time, and it may take several days from culturing bacteria to observing their response to antibiotics. Traditional screening consumes a large amount of samples to be tested, and requires a large amount of laboratory consumables and equipment, as well as professionally trained personnel to operate, resulting in relatively high costs. In addition, since traditional methods mainly rely on manual operations with poor repeatability, and it is difficult to achieve high-throughput screening of a large number of samples, the ability to simultaneously process and analyze large numbers of samples is limited.

[0004] The development of microfluidics and other technologies has made some progress in the field of antimicrobial drug screening, improving screening efficiency, reducing costs, and providing the possibility of faster and more accurate screening. Microfluidic chips can manipulate solutions with nanoliter volumes and have been shown to be very promising in antibiotic sensitivity testing. Microfluidic chips can produce a large number of microdroplets for high-throughput screening. The confinement effect formed by the small size can accelerate the reaction and obtain results faster. SlipChip is a type of microfluidic device that can manipulate droplets through the relative movement of the upper and lower chips without the need for complex fluid control systems. Therefore, we designed a set of slipchip devices for rapid microbial susceptibility testing. Summary of the invention

[0005] To achieve the above-mentioned purpose, the present invention provides a microfluidic device for microbial drug sensitivity detection, characterized in that it includes two parallel chip layers, each chip layer is etched with micron-scale channels and chambers, and the upper chip and the lower chip achieve precise mixing of samples and microorganisms through a sliding mechanism; when the microchannels on the upper chip and the lower chip partially overlap, a complete continuous fluid channel is formed, and the sliding process fuses the bacteria with the candidate compounds to generate a multi-nanoliter bacteria-compound droplet matrix.

[0006] Among them, the microbial suspension and the test solutions of different concentrations are loaded in different chambers respectively, and these chambers are aligned through sliding operations to promote the mixing reaction of samples and microorganisms; detection is carried out through optical, electrical and other signals to determine the status of bacteria.

[0007] Among them, the upper chip and the lower chip are both etched with shuttle-shaped micropores and cylindrical micropores, and the shuttle-shaped micropores and cylindrical micropores are alternately arranged in an array in the chip.

[0008] In a preferred embodiment of the present invention, a plurality of groups of loading channels are arranged on the upper chip; the loading channels include X-direction loading channels and Y-direction loading channels.

[0009] In a preferred embodiment of the present invention, the shuttle-shaped micropores on the upper chip and the micropores on the lower chip are aligned in different ways to generate fluid channels in the X-axis and Y-axis directions.

[0010] In a preferred embodiment of the present invention, after initial alignment to form an X-axis channel, candidate compounds are introduced by positive pressure; the position of the chip layer is adjusted during the sliding process so that the spindle-shaped micropores are in contact with the cylindrical micropores, thereby forming a droplet array of different candidate compounds, and then the chip layer is slid to form a complete fluid channel in the Y-axis direction and load various bacterial samples; the bacteria are fused with the candidate compounds during the sliding process to generate a bacteria-antimicrobial compound combination matrix.

[0011] The present invention also provides a method for microbial drug sensitivity analysis, comprising the following steps:

[0012] a) dispersing a sample to be evaluated and a corresponding bacterial solution into a plurality of independent liquid units; each liquid unit contains a test compound and a microorganism;

[0013] b) incubating the plurality of independent liquid units under certain conditions for a period of time;

[0014] c) imaging the individual liquid cells at the end of the incubation or during the incubation;

[0015] d) liquid units containing compounds with antibacterial activity, bacteria are killed or lysed, and the normal bacterial reproduction process is affected;

[0016] e) In the liquid unit containing the compound without antimicrobial activity, bacteria reproduce normally;

[0017] f) through testing, the number of bacteria in the liquid unit containing the compound without antibacterial activity is significantly higher than the number of bacteria in the liquid unit containing the compound with antibacterial activity;

[0018] g) by analyzing the liquid unit in which the bacteria do not significantly reproduce, the antibacterial activity of the corresponding unit compound can be obtained;

[0019] h) wherein in the above detection process, a molecule-assisted optical detection of signal changes caused by bacterial growth is used;

[0020] i) In the above detection process, molecular-assisted optical detection of signal changes caused by bacterial death or proliferation is used.

[0021] In another preferred embodiment of the present invention, the liquid unit is 1 mL to 1 pL; the number thereof is 3 to 30,000,000; and the liquid unit is a droplet, microchamber, micropore or micropit structure.

[0022] In another preferred embodiment of the present invention, the killing or lysis efficiency of phages on bacteria is analyzed by monitoring the process of cell death or proliferation in a liquid unit containing phages.

[0023] In another preferred embodiment of the present invention, the bacteria are first dispersed into a large number of independent liquid units, and then the corresponding test compound is added into the independent liquid units for detection.

[0024] In another preferred embodiment of the present invention, the independent liquid unit is generated by changing the relative physical positions of two components of the upper and lower chips.

[0025] Technical Effects

[0026] The present invention utilizes microfluidic technology to complete the sensitivity test of bacteria to antibiotics within 3 hours, which greatly shortens the detection time compared to traditional methods. This rapid detection capability is of great significance for clinical treatment decision-making and can guide the rational use of antibiotics in a timely manner. By precisely controlling the fluid and quickly generating a concentration gradient, it is possible to provide information on the sensitivity of bacteria to different antibiotics in a very short time, which is particularly important for emergency situations such as bloodstream infections because they require rapid and early determination of appropriate antimicrobial drugs. Rapid antibiotic sensitivity test results can help doctors quickly adjust treatment plans and reduce unnecessary use of antibiotics, thereby improving treatment outcomes and reducing the risk of drug resistance development. By quickly determining the sensitivity of bacteria to antibiotics, the use of antibiotics can be managed more effectively, the abuse of broad-spectrum antibiotics can be reduced, and the rational use of targeted antibiotics can be promoted. In combination with the needs of clinicians, it has the ability to develop automated equipment that can provide a variety of sensitivity information, which helps to further improve the accuracy and reliability of detection.

[0027] Low consumption: This technology significantly reduces sample and reagent consumption through nanoliter-scale droplet fusion operations. Especially when dealing with precious samples such as natural products, it can accurately control sample volume, reduce reagent consumption, improve experimental efficiency, reduce pollution risks, and meet environmental protection and sustainability requirements. Effectively reducing the consumption of detection reagents allows researchers to quickly evaluate the effectiveness of multiple conditions or multiple samples. By reducing the consumption of samples and reagents, this technology helps to reduce research costs, making more research and experiments possible, showing unique advantages and broad application prospects under limited resources.

[0028] High throughput: The microfluidic chip technology used in the present invention can achieve high-throughput screening by generating hundreds of microdroplets with positioning information on a tiny chip. Each microdroplet can contain a combination of different pathogens and antimicrobial agents, so that a large number of drug-bacteria pairs can be tested in parallel at the same time. This high-throughput screening technology significantly improves the efficiency and throughput of the experiment. Compared with the traditional single-sample testing method, it greatly increases the number of test samples per unit time. The application of this technology enables researchers to quickly evaluate the sensitivity of multiple antimicrobial agents to a series of pathogens, thereby accelerating the discovery and development process of new drugs. In the early stages of drug development, this can greatly shorten the time for drug screening, reduce research and development costs, and speed up the pace of new drugs to market. In clinical applications, this high-throughput screening capability can help doctors quickly determine the most appropriate antibiotics for specific infections, thereby improving treatment effects and reducing patient pain and medical costs. In addition, high-throughput screening technology also provides a powerful tool for studying drug resistance. By quickly identifying which antimicrobial agents are effective against specific pathogens, researchers can better understand resistance patterns and design more targeted treatment strategies. The application of this technology is not only crucial in the development of new drugs, but also has important significance for global public health security and drug resistance management.

[0029] Easy operation: The present invention realizes the generation of droplets through the shear force and surface tension changes generated by the change of physical position, and a large number of droplets can be generated without complex instruments. Simple operation reduces the requirements for operator skills, making the detection process easier to standardize and promote. The core advantage of the present invention is not only reflected in the user-friendly design, which makes it easy for non-professionals to operate, but also greatly reduces the technical threshold through simplified operation procedures and intuitive interfaces. This simplified step effectively reduces human errors in the experimental process, improves the consistency and reliability of experimental results, and speeds up sample processing and data analysis, improving work efficiency. The easy-to-standardize operation process ensures the repeatability and accuracy of the experimental results, and the simple operation characteristics also make the technology more easily accepted by different laboratories and medical institutions, promoting the widespread promotion and application of the technology. In addition, the reduced operation skill requirements reduce training time and cost, allowing more staff to quickly master this technology, and its strong adaptability also enables it to play a role in field detection in resource-limited areas or emergency situations. Simple operation also provides greater flexibility and scalability for experiments, promotes cooperation between researchers from different backgrounds, shares experimental data and results, and promotes the progress of scientific research. Ultimately, this simple operation process helps to transform laboratory technology into clinical applications and accelerate the commercialization and practical application of scientific research results. The small size of the device of the present invention makes it have great development potential and broad application prospects in biomedical research, especially in precision medicine and personalized treatment. The microfluidic technology of the present invention not only has transformation capabilities in laboratory environments, but also shows great promotion potential in clinical applications and commercialization.

[0030] Flexibility and Customizability in Production Implementation: The modular design of the present invention provides significant advantages in flexibility and customization in the production process. This design enables the production process to be quickly adjusted to accommodate different production needs from small batches to large batches. The flexibility of the present invention is particularly reflected in the ability to respond quickly to market changes, such as the ability to adjust production lines in a timely manner to meet new AST testing needs during the rapid development and launch of new drugs. In emergency public health events such as epidemics, the rapid response capability of the present invention is particularly important, and the production process can be quickly reconfigured to produce products for the detection needs of specific pathogens, effectively supporting epidemic control and response. The ability to quickly adapt to and meet changing market demands is a significant advantage in the highly competitive market for product promotion.

[0031] Mature production technology: The present invention relies on the already very mature microfluidic technology. This technical foundation not only reduces the time and cost of research and development, but also accelerates the transformation of products from concept to market. The maturity of the technology ensures the stability and reliability of the present invention in practical applications, reduces the failure rate in the production process, and improves production efficiency, which is particularly important for the medical and scientific research fields that require high precision and high reliability. In addition, the maturity of the technology also enhances the user's trust in the present invention, especially in critical applications, where users are more inclined to choose proven mature technologies. It also supports large-scale production to meet the needs of large-scale markets while maintaining cost-effectiveness, facilitating regulatory approval, and accelerating product entry into the market. The mature technology platform also provides a solid foundation for future technological innovation, enabling the present invention to expand functions and improve performance on this basis, further promoting the development and application of technology. Therefore, the maturity of the microfluidic technology of the present invention provides a strong guarantee for the rapid promotion and wide application of the present invention.

[0032] Materials have cost advantages: The present invention demonstrates significant cost-effectiveness advantages in material selection. In the design and production process, priority is given to materials that are cost-effective and easy to process. These materials are not only easy to obtain, but also have a simple processing process, thereby effectively reducing the overall production cost. Through careful selection of materials, the present invention can achieve cost control while ensuring product quality and performance, making the final product more competitive in price. This cost-effectiveness optimization not only helps to increase the market appeal of the product, but also makes the present invention easier to be widely promoted and applied. Through strategic decisions on material selection, the present invention successfully balances cost and performance, bringing users cost-effective solutions, while also creating greater market space and profit potential for enterprises.

[0033] In summary, the present invention not only has obvious advantages in terms of technology, but also shows great potential and transformation possibilities in terms of production implementation and industrial application prospects. Its rapid detection capability, low consumption, high-throughput screening and ease of operation, combined with mature production technology and cost-effective material selection, lay a solid foundation for the wide application and rapid transformation of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 It is a chip design diagram of a preferred embodiment of the present invention;

[0035] Figure 2 is a top view of an upper chip of a preferred embodiment of the present invention;

[0036] Figure 3 is a lower chip design diagram of a preferred embodiment of the present invention;

[0037] Figure 4 is a top view of a lower chip of a preferred embodiment of the present invention;

[0038] Figure 5 It is a chip combination-solution injection and liquid diffusion position design diagram of a preferred embodiment of the present invention;

[0039] Figure 6 It is a top view of a chip assembly-injection solution and liquid diffusion position of a preferred embodiment of the present invention;

[0040] Figure 7 It is a top view of a chip of a preferred embodiment of the present invention - the position of the chip when the droplet is formed;

[0041] Figure 8 It is a schematic diagram of a sample loading process of a microfluidic chip for microbial drug sensitivity detection according to a preferred embodiment of the present invention - assembling two chips (upper chip, lower chip) and injecting a solution for the first time;

[0042] Fig. 9 is a schematic diagram of the first droplet formation of a preferred embodiment of the present invention;

[0043] Fig.10 This is a schematic diagram of the second injection of solution in a preferred embodiment of the present invention;

[0044] Fig.11 is a schematic diagram of a preferred embodiment of the present invention after the second injection of solution;

[0045] Fig.12 is a schematic diagram of the formation of the final droplet matrix in a preferred embodiment of the present invention;

[0046] Fig.13 It is a design diagram of a chip of a preferred embodiment of the present invention and a schematic diagram of some detailed structures of the chip;

[0047] Fig.14 It is a schematic diagram of the principle and operation flow of a microbial drug sensitivity detection device of a preferred embodiment of the present invention;

[0048] Fig.15 The MIC measurement result of nitrofurantoin inhibiting Escherichia coli ATCC25922 in a preferred embodiment of the present invention is shown;

[0049] Fig.16 It is the growth of bacteria in different pathogens nm-SlipChip of a preferred embodiment of the present invention;

[0050] Fig.17The results of high-throughput screening of plant natural products in a preferred embodiment of the present invention and co-culture of compounds 1 and 2 with different bacterial strains. Wherein (A) Representative 30-fold magnified bright field images of Staphylococcus aureus (S. aureus), methicillin-resistant Staphylococcus aureus (MRSA), methicillin-resistant coagulase-negative Staphylococcus (MRSE), Pseudomonas aeruginosa (P. aeruginosa), Klebsiella pneumoniae (K. pneumoniae), Acinetobacter baumannii (A. baumannii), Escherichia coli (E. coli), Bacillus subtilis (B. subtilis), Salmonella typhimurium (S. typhimurium) and Shigella dysenteriae (S. dysenteriae) treated with compounds 1 and 2 and DMSO in the nano-SlipChip. (B) The results of screening 20 compounds (64 μg / mL) using nm-SlipChip. Teicoplanin, asarone and berberine were used as positive control compounds. Gray indicates an inhibitory effect on the corresponding bacteria, while white indicates no significant inhibition. (B) Results with no significant inhibitory activity are not shown. Scale bars apply to all images. (D) Analysis of bacterial pixel counts in Figures B and C. Error bars represent standard deviations, n=3. Data represent the results of three independent experiments. *p<0.05, **p<0.01 and ***p<0.001 compared to the untreated group. DETAILED DESCRIPTION

[0051] The following describes several preferred embodiments of the present invention with reference to the drawings in the specification, so that the technical content is clearer and easier to understand. The present invention can be embodied in many different forms of embodiments, and the protection scope of the present invention is not limited to the embodiments mentioned in the text.

[0052] In the drawings, components with the same structure are indicated by the same numerical reference numerals, and components with similar structures or functions are indicated by similar numerical reference numerals. The size and thickness of each component shown in the drawings are arbitrarily shown, and the present invention does not limit the size and thickness of each component. In order to make the illustration clearer, the thickness of the components is appropriately exaggerated in some places in the drawings.

[0053] like Figure 1-Figure 7 As shown, the microfluidic chip for microbial drug sensitivity detection includes two parallel chip layers, each chip layer is etched with micron-scale channels and chambers, and the upper chip and the lower chip achieve precise mixing of samples and microorganisms through a sliding mechanism; when the microchannels on the upper chip and the lower chip partially overlap, a complete continuous fluid channel is formed, and the sliding process fuses the bacteria with the candidate compounds to generate a multi-nanoliter bacteria-compound droplet matrix.

[0054] Among them, the microbial suspension and the test solutions of different concentrations are loaded in different chambers respectively, and these chambers are aligned through sliding operations to promote the mixing reaction of samples and microorganisms; detection is carried out through optical, electrical and other signals to determine the status of bacteria.

[0055] Among them, the upper chip and the lower chip are both etched with shuttle-shaped micropores and cylindrical micropores, and the shuttle-shaped micropores and cylindrical micropores are alternately arranged in an array in the chip.

[0056] In a preferred embodiment of the present invention, a plurality of groups of loading channels are arranged on the upper chip; the loading channels include X-direction loading channels and Y-direction loading channels.

[0057] In a preferred embodiment of the present invention, the shuttle-shaped micropores on the upper chip and the micropores on the lower chip are aligned in different ways to generate fluid channels in the X-axis and Y-axis directions.

[0058] In a preferred embodiment of the present invention, after initial alignment to form an X-axis channel, candidate compounds are introduced by positive pressure; the position of the chip layer is adjusted during the sliding process so that the spindle-shaped micropores are in contact with the cylindrical micropores, thereby forming a droplet array of different candidate compounds, and then the chip layer is slid to form a complete fluid channel in the Y-axis direction and load various bacterial samples; the bacteria are fused with the candidate compounds during the sliding process to generate a bacteria-antimicrobial compound combination matrix.

[0059] Implementation 1:

[0060] The microfluidic chip uses a sliding-induced self-separation mechanism to generate droplets. The device consists of two closely contacting glass chips, the top plate and the bottom plate. When the microchannels on the two plates overlap, a complete continuous fluid channel is formed ( Fig.14 A). The spindle-shaped microwells on the top plate and the microwells on the bottom plate can generate fluid channels in the X-axis and Y-axis directions through different alignment methods. After the initial alignment to form the X-axis channel, the candidate compound is introduced by positive pressure. The sliding process adjusts the position of the plate so that the spindle-shaped microwell contacts the cylindrical microwell, thereby forming a droplet array of different candidate compounds ( Fig.14 B). Then, by sliding the nm-SlipChip, the shuttle-shaped micropores formed a complete fluid channel in the Y-axis direction and loaded with various bacterial samples. The sliding process merged the bacteria with the candidate compounds, generating a multi-nanoliter bacteria-antimicrobial compound combination matrix ( Fig.14 C) Each combination contains two identical droplet groups as biological replicates ( Fig.14 D). First, combine the upper and lower chips in PMX-200 dimethyl silicone oil ( Fig.14 A). Use a pipette to load 5 nanoliters of different test compounds into the loading channel in the X direction ( Fig.14 B); then the concentration is 5-10×10 5 CFU / mL of various microorganisms to be tested are loaded into the Y direction channel by a pipette ( Fig.14 C) By simply sliding, the bacterial solution is fused with the test compound ( Fig.14 D). The chip was then placed in a 37°C incubator for three hours. Each well was photographed using a Nikon Ti2 microscope at 0 and 3 hours to count the number of bacteria and observe changes in bacterial morphology and bacterial movement ( Fig.14 E). By counting the changes in the number of bacteria in different microwells, combined with changes in bacterial morphology and bacterial movement, if a fixed concentration of ten test compounds is loaded, the sensitivity of bacteria to different components can be qualitatively analyzed, and if different concentrations of the same component are loaded, the MIC can be quantitatively analyzed ( Fig.14 F).

[0061] Implementation Method 2

[0062] The feasibility of the nm-SlipChip for high-throughput analysis of antimicrobial compounds was first evaluated using eight antibiotics, including four antibiotics (oxacillin, vancomycin, tetracycline, rifampicin) against Staphylococcus aureus ATCC25923 and four antibiotics (ceftazidime, tigecycline, nitrofurantoin, levofloxacin) against Escherichia coli ATCC25922. These MIC results were consistent with the quality control standards of the Clinical and Laboratory Standards Institute (CLSI) ( Fig.15 ). Taking furantoin as an example, the detection and analysis process is described in detail. Serial dilutions of furantoin (concentrations ranging from 0.5 to 128 μg / mL) were first loaded into the X-axis channel as the first sample. Then, the bacterial solution was introduced into the Y-axis channel and mixed with droplets containing different concentrations of furantoin by sliding. The nm-SlipChip was incubated at 37°C for 3 hours, and bright field images of each well were taken every hour. The minimum inhibitory concentration (MIC) of furantoin was determined by analyzing the occupied bacterial pixels. When the concentration of furantoin was lower than 1 μg / mL, Escherichia coli proliferated normally. However, when the concentration was between 2 and 8 μg / mL, the morphology of Escherichia coli became elongated and the proliferation rate was slower than that of the control group. At concentrations above 8 μg / mL, Escherichia coli stopped proliferating and transformed into protoplasts or spheroids ( Fig.16 A). Therefore, the MIC of nitrofurantoin determined by nm-SlipChip is 4 μg / mL ( Fig.16B, C). We also monitored the growth of ten bacterial strains, including six Gram-negative and four Gram-positive strains. All strains showed significant growth within 2-3 hours on the nm-SlipChip. Compared with traditional 96-well plate culture and optical density (OD) value measurement, all strains cultured on the nm-SlipChip reached detectable levels within 2-3 hours, showing a six-fold reduction in time, reflecting the excellent compatibility of the nm-SlipChip ( Fig.17 ).

[0063] The method of microbial drug sensitivity detection is to disperse a large volume of bacterial suspension and a solution of the compound to be tested into small volumes. Each small volume can be 1 femtoliter to 1 milliliter. The device consists of two parallel chip layers, each of which is etched with micron-scale channels and chambers. The upper chip and the lower chip achieve precise mixing of samples and microorganisms through a precision sliding mechanism. The microbial suspension and the test solution of different concentrations are loaded in different chambers respectively, and these chambers are aligned through sliding operations to promote the mixing reaction of samples and microorganisms. Due to the small volume of the microchamber and the high reaction efficiency, the test results can be obtained quickly. In no more than 3 hours, bacteria can grow and multiply in a small volume. This process can be detected by optical, electrical and other signals. Rapidly determine whether the bacterial state is inhibitory or non-inhibitory, which serves as the basis for qualitative and quantitative detection of microbial drug sensitivity.

[0064] In order to solve the problems of low content and instability of active ingredients in natural products, we have successfully developed a rapid microbial susceptibility detection device, which realizes activity-oriented separation, and specifically searches for natural product components with antibacterial activity. It can also achieve high-throughput screening with low consumption and process multiple samples at the same time. Using this device, we successfully isolated 20 single components from Callicarpa integerrima. We tested the antibacterial activity of these 20 compounds and evaluated their inhibitory activity against 10 common pathogens (Table 1).

[0065] Table 1 Bacteria used in drug sensitivity testing

[0066]

[0067] During the screening, we identified two compounds with anti-Gram-positive bacterial activity. These two compounds showed good inhibitory activity against Staphylococcus aureus, methicillin-resistant Staphylococcus aureus and methicillin-resistant Staphylococcus epidermidis ( Fig.17 ). This device provides an efficient, accurate, and low-cost solution that can significantly improve the efficiency and accuracy of drug screening and testing.

[0068] This device is a nanoliter-scale matrix slip chip (nm-SlipChip), which can construct an "N×M" matrix in nanoliter droplets through simple loading and sliding operations, containing "N" antibiotics and "M" candidate molecules. The entire operation process is precisely controlled by internal microfluidics, without relying on complex fluid control systems and operations. By phenotyping the growth of more than 100 nanoliter droplets in three hours, we can accurately evaluate the antibacterial ability of candidate molecules.

[0069] By applying this device to the antimicrobial activity-guided separation of Callicarpa plants, we discovered for the first time two plant-derived small molecule natural products with inhibitory activity against MRSA (methicillin-resistant Staphylococcus aureus), providing a new approach to addressing the problem of antibiotic resistance. As a flexible, efficient, on-demand high-throughput screening platform, nm-SlipChip has potential for application not only in the discovery of antimicrobial drugs, but also as a new tool for other broad biochemical screening analyses.

[0070] The preferred specific embodiments of the present invention are described in detail above. It should be understood that ordinary technicians in the field can make many modifications and changes based on the concept of the present invention without creative work. Therefore, all technical solutions that can be obtained by technicians in the technical field based on the concept of the present invention through logical analysis, reasoning or limited experiments on the basis of the prior art should be within the scope of protection determined by the claims.

Claims

1. A microfluidic device for microbial drug sensitivity detection, characterized in that: The invention comprises two parallel chip layers, each of which is etched with micron-scale channels and chambers. The upper chip and the lower chip achieve precise mixing of samples and microorganisms through a sliding mechanism. When the microchannels on the upper chip and the lower chip partially overlap, a complete continuous fluid channel is formed. The bacteria and the candidate compounds are merged during the sliding process to generate a matrix of multiple nanoliter bacteria-compound droplets. The microbial suspension and the test solutions of different concentrations are respectively loaded in different chambers. The chambers are aligned through sliding operations to promote the mixing reaction of samples and microorganisms. The bacteria status is determined by optical, electrical and other signals.

2. The device according to claim 1, characterized in that The upper chip and the lower chip are both etched with shuttle-shaped micropores and cylindrical micropores, and the shuttle-shaped micropores and cylindrical micropores are alternately arranged in an array in the chip.

3. The device according to claim 2, characterized in that A plurality of loading channels are arranged on the upper chip; the loading channels include an X-direction loading channel and a Y-direction loading channel.

4. The device according to claim 3, characterized in that The shuttle-shaped micropores on the upper chip and the micropores on the lower chip are aligned in different ways to generate fluid channels in the X-axis and Y-axis directions.

5. The device according to claim 4, characterized in that After initial alignment to form the X-axis channel, the candidate compound is introduced by positive pressure; The sliding process adjusts the position of the chip layer so that the spindle-shaped micropores contact the cylindrical micropores, thereby forming a droplet array of different candidate compounds. The chip layer is then slid to form a complete fluid channel in the Y-axis direction and load various bacterial samples. The sliding process fuses the bacteria with the candidate compounds to generate a bacteria-antimicrobial compound combination matrix.

6. A method for analyzing microbial drug sensitivity using the microfluidic device for detecting microbial drug sensitivity according to any one of claims 1 to 5, characterized in that: The following steps are involved: a) dispersing a sample to be evaluated and a corresponding bacterial solution into a plurality of independent liquid units; each liquid unit contains a test compound and a microorganism; b) incubating the plurality of independent liquid units under certain conditions for a period of time; c) imaging the individual liquid cells at the end of the incubation or during the incubation; d) liquid units containing compounds with antibacterial activity, bacteria are killed or lysed, and the normal bacterial reproduction process is affected; e) In the liquid unit containing the compound without antimicrobial activity, bacteria reproduce normally; f) through testing, the number of bacteria in the liquid unit containing the compound without antibacterial activity is significantly higher than the number of bacteria in the liquid unit containing the compound with antibacterial activity; g) by analyzing the liquid unit in which the bacteria do not significantly reproduce, the antibacterial activity of the corresponding unit compound can be obtained; h) wherein in the above detection process, a molecule-assisted optical detection of signal changes caused by bacterial growth is used; i) In the above detection process, molecular-assisted optical detection of signal changes caused by bacterial death or proliferation is used.

7. The method according to claim 6, characterized in that The liquid unit is 1 mL to 1 pL; the number thereof is 3 to 30,000,000; the liquid unit is a droplet, a microchamber, a micropore or a micropit structure.

8. The method according to claim 7, characterized in that By monitoring the process of cell death or proliferation in the liquid unit containing phage, the killing or lysis efficiency of phage on bacteria can be analyzed.

9. The method according to claim 8, characterized in that The bacteria are first dispersed into a large number of independent liquid units, and then the corresponding test compound is added into the independent liquid unit for detection.

10. The method according to claim 9, characterized in that Independent liquid cells are generated by changing the relative physical positions of two components on the upper and lower chips.

Images