A microbial drug sensitivity detection device and method

Abstract

The application discloses a kind of microbial drug sensitivity detection method and device, it is related to biological detection field, including two parallelly placed chip layers, each layer chip is etched with microscale channel and chamber, and the accurate mixing of sample and microorganism is realized by sliding mechanism between upper chip and lower chip;When the micro flow channel on the upper chip and the lower chip partially overlaps, a complete continuous fluid channel is formed, the bacteria and candidate compounds are fused in the sliding process, and a multiple nanoliter bacteria-compound droplet matrix is generated;Wherein, microbial suspension and different concentrations of test solution are loaded in different chambers respectively, and the mixing reaction of sample and microorganism is promoted by aligning these chambers through sliding operation;Determine bacterial state by detecting optical, electrical and other signals.The application can realize the rapid drug sensitivity detection of test compound, realize lower sample consumption, and lay a foundation for microbial drug sensitivity detection.

Description

Technical Field

[0001] This invention relates to the field of biological detection, and more particularly to a microbial drug susceptibility testing device and method. Background Technology

[0002] Bacterial resistance to antibiotics is constantly evolving, making the search for novel antibiotics urgent. To mitigate this trend, it is necessary not only to regulate antibiotic use but also to urgently explore unexplored drug targets in bacteria to develop more effective antibacterial agents. Natural products (including plants and microorganisms) are an ideal approach to address this problem; however, the low content of active ingredients in natural products poses challenges to existing screening methods.

[0003] Traditional antimicrobial susceptibility screening has several significant drawbacks when applied to complex samples such as natural products. It typically requires a long time, potentially several days from culturing bacteria to observing their response to antibiotics. Traditional screening consumes a large amount of sample material and requires substantial laboratory supplies and equipment, as well as specially trained personnel, resulting in relatively high costs. Furthermore, because traditional methods rely heavily on manual operation, reproducibility is poor, and high-throughput screening of large numbers of samples is difficult, limiting the ability to simultaneously process and analyze large volumes of samples.

[0004] The development of microfluidics and related technologies has made progress in the field of antimicrobial drug screening, improving screening efficiency, reducing costs, and enabling faster and more accurate screening. Microfluidic chips can manipulate solutions at nanoliter levels and have proven promising for antibiotic susceptibility testing. Microfluidic chips can generate a large number of microdroplets for high-throughput screening; the confinement effect caused by their small size can accelerate the reaction and obtain results more quickly. Slip-chips are a type of microfluidic device that manipulates droplets through the relative movement of upper and lower chips without the need for a complex fluid control system. Therefore, we designed a slip-chip device for rapid microbial drug susceptibility testing. Summary of the Invention

[0005] To achieve the above objectives, the present invention provides a microfluidic device for microbial drug susceptibility testing, characterized in that it comprises two parallel chip layers, each chip layer having micron-sized channels and chambers etched on it. The upper and lower chips achieve precise mixing of samples and microorganisms through a sliding mechanism. When the microchannels on the upper and lower chips partially overlap, they form a complete continuous fluid channel. The sliding process fuses bacteria with candidate compounds, generating a multi-nanoscale bacterial-compound droplet matrix.

[0006] The microbial suspension and test solutions of different concentrations are loaded into different chambers. These chambers are aligned by a sliding operation to promote the mixing reaction of the sample and microorganisms. The bacterial status is determined by detection using optical, electrical and other signals.

[0007] Both the upper and lower chips are etched with shuttle-shaped and cylindrical micro-holes, which are arranged in an alternating array within the chip.

[0008] In a preferred embodiment of the present invention, multiple loading channels are provided on the upper-layer chip; the loading channels include an X-direction loading channel and a Y-direction loading channel.

[0009] In a preferred embodiment of the present invention, the spindle-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 and Y 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 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; then, by sliding the chip layer, the spindle-shaped micropores form a complete fluid channel in the Y-axis direction and various bacterial samples are loaded; the sliding process fuses the bacteria with the candidate compounds to generate a bacterial-antimicrobial compound combination matrix.

[0011] This invention also provides a method for microbial antimicrobial susceptibility analysis, comprising the following steps:

[0012] a) The sample to be evaluated and the corresponding bacterial solution are dispersed into a large number of independent liquid units; each liquid unit contains one test compound and one microorganism;

[0013] b) Incubate these numerous independent liquid units under certain conditions for a period of time;

[0014] c) Image individual liquid units at the end of incubation or during incubation;

[0015] d) Liquid units containing compounds with antibacterial activity can kill or lyse bacteria, thus affecting their normal reproductive processes;

[0016] e) In liquid units containing compounds that do not have antibacterial activity, bacteria multiply normally;

[0017] f) The number of bacteria in the liquid unit containing the compound that does not have antimicrobial activity was significantly higher than the number of bacteria in the liquid unit containing the compound that has antimicrobial activity.

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

[0019] h) In the above detection process, molecular-assisted optical detection is used, which involves signal changes due to bacterial reproduction;

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

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

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

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

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

[0025] Technical effect

[0026] This invention utilizes microfluidic technology to complete bacterial antibiotic susceptibility testing within 3 hours, significantly shortening the detection time compared to traditional methods. This rapid detection capability is of great significance for clinical treatment decisions, enabling timely guidance for the rational use of antibiotics. By precisely controlling the fluid and rapidly generating concentration gradients, it can provide information on bacterial susceptibility to different antibiotics in a very short time. This is particularly important in emergencies such as bloodstream infections, where rapid, early identification of appropriate antimicrobial agents is crucial. Rapid antibiotic susceptibility testing results can help physicians quickly adjust treatment plans, reduce unnecessary antibiotic use, thereby improving treatment efficacy and reducing the risk of antibiotic resistance development. By rapidly determining bacterial susceptibility to antibiotics, antibiotic use can be managed more effectively, reducing the overuse of broad-spectrum antibiotics and promoting the rational use of targeted antibiotics. Furthermore, the ability to develop automated devices that can provide a variety of susceptibility information, combined with the needs of clinicians, contributes to further improving the accuracy and reliability of the test.

[0027] Low Consumption: This technology significantly reduces sample and reagent consumption through nano-level droplet fusion, especially when processing precious samples such as natural products. It enables precise control of sample volume, reduces reagent consumption, improves experimental efficiency, lowers contamination risks, and meets environmental and sustainability requirements. Effectively reducing reagent consumption allows researchers to quickly evaluate efficacy under multiple conditions or on multiple samples. By reducing sample and reagent consumption, this technology helps lower research costs, enabling more research and experiments, demonstrating unique advantages and broad application prospects in resource-constrained situations.

[0028] High Throughput: The microfluidic chip technology employed in this invention enables high-throughput screening by generating hundreds of microdroplets with positioning information on a tiny chip. Each microdroplet can contain different combinations of pathogens and antimicrobial agents, allowing for parallel testing of a large number of drug-bacteria pairs simultaneously. This high-throughput screening technology significantly improves experimental efficiency and throughput, greatly increasing the number of samples tested per unit time compared to traditional single-sample testing methods. This technology allows researchers to rapidly assess the sensitivity of multiple antimicrobial agents to a range of pathogens, thereby accelerating the discovery and development of new drugs. In the early stages of drug development, this can significantly shorten drug screening time, reduce R&D costs, and expedite the pace of new drug launches. In clinical applications, this high-throughput screening capability helps physicians quickly identify the most suitable antibiotics for specific infections, thereby improving treatment efficacy and reducing patient suffering and medical costs. Furthermore, high-throughput screening technology provides a powerful tool for studying drug resistance. By rapidly 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 new drug development, but also of great significance for global public health security and drug resistance management.

[0029] Simple Operation: This invention generates droplets by changing shear force and surface tension resulting from changes in physical position, producing a large number of droplets without the need for complex instruments. The simplicity of operation reduces the skill requirements of operators, making the detection process easier to standardize and promote. The core advantages of this invention are not only reflected in its user-friendly design, allowing even non-professionals to operate it easily, but also in the significantly lower technical threshold achieved through simplified operating procedures and an intuitive interface. This simplified procedure effectively reduces human error during the experiment, improves the consistency and reliability of experimental results, and accelerates sample processing and data analysis, thereby increasing work efficiency. The easily standardized operating procedure ensures the reproducibility and accuracy of experimental results, while the simplicity of operation makes the technology more readily accepted by different laboratories and medical institutions, promoting its widespread adoption and application. Furthermore, the reduced skill requirements decrease training time and costs, enabling more staff to quickly master the technology, and its strong adaptability allows it to play a role in on-site testing in resource-limited areas or emergency situations. The simple operation also provides greater flexibility and scalability for experiments, promoting collaboration among researchers with different backgrounds, sharing experimental data and results, and driving scientific research progress. Ultimately, this simplified operating procedure facilitates the translation of laboratory techniques into clinical applications, accelerating the commercialization and practical application of research results. The small size of the device of this invention gives it enormous development potential and broad application prospects in biomedical research, especially in precision medicine and personalized treatment. The microfluidic technology of this invention not only has translational capabilities in a laboratory environment but also demonstrates great potential for widespread application and commercialization in clinical settings.

[0030] The invention offers flexibility and customizability in production implementation: its modular design provides significant advantages in terms of flexibility and customization within the production process. This design allows for rapid adjustments to the production process to accommodate varying production needs, from small batches to large batches. The flexibility of this invention is particularly evident in its ability to quickly respond to market changes, such as during the rapid development and launch of new drugs, enabling timely adjustments to production lines to meet new AST testing requirements. In public health emergencies such as epidemics, the rapid response capability of this invention is especially important, allowing for the rapid reconfiguration of production processes to produce products targeting specific pathogen testing needs, effectively supporting epidemic control and response. This ability to quickly adapt to and meet ever-changing market demands is a significant advantage in highly competitive markets where product promotion is crucial.

[0031] Mature Production Technology: This invention relies on the highly mature microfluidic technology. This technological foundation not only reduces R&D time and costs but also accelerates the product's transition from concept to market. The maturity of the technology ensures the stability and reliability of this invention in practical applications, reduces the failure rate during production, and improves production efficiency. This is particularly important for medical and scientific research fields that require high precision and reliability. Furthermore, the maturity of the technology enhances user trust in this invention, especially in critical applications where users tend to choose proven, mature technologies. It also supports large-scale production, meeting the needs of a large market while maintaining cost-effectiveness, facilitating regulatory approvals, and accelerating product entry into the market. The mature technology platform also provides a solid foundation for future technological innovation, enabling this invention to be expanded in function and improved in performance, further promoting technological development and application. Therefore, the maturity of the microfluidic technology in this invention provides a strong guarantee for its rapid promotion and widespread application.

[0032] Cost-effectiveness of materials: This invention demonstrates significant cost-effectiveness in material selection. During the design and production process, priority is given to cost-effective and easily processed materials. These materials are not only readily available but also simple to process, effectively reducing overall production costs. Through careful material selection, this invention achieves cost control while ensuring product quality and performance, making the final product more price-competitive. This cost-effectiveness optimization not only helps improve the product's market appeal but also makes the invention easier to widely promote and apply. Through strategic decisions in material selection, this invention successfully balances cost and performance, providing users with a cost-effective solution while creating greater market space and profit potential for enterprises.

[0033] In summary, this invention not only possesses significant advantages at the technical level, but also demonstrates enormous potential and transformative 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 widespread application and rapid transformation of this invention. Attached Figure Description

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

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

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

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

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

[0039] Figure 6 This is a top view of a chip assembly with injected solution and liquid diffusion location according to a preferred embodiment of the present invention;

[0040] Figure 7 This is a top view of a chip according to a preferred embodiment of the present invention - chip position during droplet formation;

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

[0042] Figure 9 This is a schematic diagram of the first droplet formation according to a preferred embodiment of the present invention;

[0043] Figure 10 This is a schematic diagram of the second injection of solution according to a preferred embodiment of the present invention;

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

[0045] Figure 12 This is a schematic diagram of the final droplet matrix formation according to a preferred embodiment of the present invention;

[0046] Figure 13 This is a design drawing of a chip and a schematic diagram showing some detailed structural features of the chip, representing a preferred embodiment of the present invention.

[0047] Figure 14 This is a schematic diagram illustrating the principle and operation process of a microbial antimicrobial susceptibility testing device according to a preferred embodiment of the present invention;

[0048] Figure 15 This is the MIC determination result of nitrofurantoin inhibiting Escherichia coli ATCC25922 according to a preferred embodiment of the present invention;

[0049] Figure 16 This is a preferred embodiment of the present invention showing the growth of bacteria in nm-SlipChips containing different pathogens;

[0050] Figure 17This is a preferred embodiment of the present invention showing the results of high-throughput screening of plant natural products compounds 1 and 2 with different bacterial strains. (A) Representative bright-field images at 30x magnification of Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), methicillin-resistant coagulase-negative staphylococci (MRSE), Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii, Escherichia coli, Bacillus subtilis, Salmonella typhimurium, and Shigella dysenteriae treated with compounds 1 and 2 and DMSO in a nano-SlipChip. (B) Results of screening 20 compounds (64 μg / mL) using a nm-SlipChip. Teicoplanin, asarone, and berberine were used as positive control compounds. Gray indicates inhibition of the corresponding bacteria, while white indicates no significant inhibition. (B) Results showing 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 results from three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 compared to the untreated group. Detailed Implementation

[0051] The following description, with reference to the accompanying drawings, illustrates several preferred embodiments of the present invention to make its technical content clearer and easier to understand. The present invention can be embodied in many different forms, and the scope of protection of the present invention is not limited to the embodiments mentioned herein.

[0052] In the accompanying drawings, components with the same structure are indicated by the same numerical designation, and components with similar structures or functions are indicated by similar numerical designations. The dimensions and thicknesses of each component shown in the drawings are arbitrary, and the present invention does not limit the dimensions and thicknesses of each component. To make the illustrations clearer, the thickness of some components has been appropriately exaggerated in the drawings.

[0053] like Figures 1-7 As shown, the microfluidic chip for microbial drug susceptibility testing includes two parallel chip layers, each etched with micron-sized channels and chambers. The upper and lower chips achieve precise mixing of samples and microorganisms through a sliding mechanism. When the microchannels on the upper and lower chips partially overlap, they form a complete continuous fluid channel. The sliding process fuses bacteria with candidate compounds, generating a multi-nanoscale bacterial-compound droplet matrix.

[0054] The microbial suspension and test solutions of different concentrations are loaded into different chambers. These chambers are aligned by a sliding operation to promote the mixing reaction of the sample and microorganisms. The bacterial status is determined by detection using optical, electrical and other signals.

[0055] Both the upper and lower chips are etched with shuttle-shaped and cylindrical micro-holes, which are arranged in an alternating array within the chip.

[0056] In a preferred embodiment of the present invention, multiple loading channels are provided on the upper-layer chip; the loading channels include an X-direction loading channel and a Y-direction loading channel.

[0057] In a preferred embodiment of the present invention, the spindle-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 and Y 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 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; then, by sliding the chip layer, the spindle-shaped micropores form a complete fluid channel in the Y-axis direction and various bacterial samples are loaded; the sliding process fuses the bacteria with the candidate compounds to generate a bacterial-antimicrobial compound combination matrix.

[0059] Implementation method 1:

[0060] The microfluidic chip generates droplets using a sliding-induced self-separation mechanism. The device consists of two closely contacting glass chips, a top plate and a bottom plate. When the microchannels on the two plates partially overlap, a complete continuous fluid channel is formed. Figure 14 A). The spindle-shaped micropores on the top plate and the micropores on the bottom plate, through different alignment methods, can generate fluid channels in the X and Y axes. After initial alignment to form the X-axis channel, candidate compounds are introduced via positive pressure. The sliding process adjusts the position of the plates, bringing the spindle-shaped micropores into contact with the cylindrical micropores, thereby forming an array of droplets of different candidate compounds. Figure 14 B). Next, by sliding the nm-SlipChip, a complete fluid channel was formed in the Y-axis direction using spindle-shaped micropores, and various bacterial samples were loaded. The sliding process fused the bacteria with candidate compounds, generating a multi-nano-scaled bacterial-antimicrobial compound combination matrix (B). Figure 14 C). Each combination contains two identical droplet sets as biological replicates. Figure 14 D). First, assemble the upper and lower chips together in PMX-200 dimethyl silicone oil. Figure 14 A). Using a pipette, 5 nanoliters of different test compounds were loaded into the loading channel in the X direction ( Figure 14 B); then the concentration is 5-10×10 5 Multiple analyte microorganisms at CFU / mL were loaded onto the Y-direction channel using a pipette. Figure 14 C). The bacterial solution is fused with the test compound by a simple sliding motion. Figure 14 D). The chip was then incubated at 37°C for three hours. At 0 and 3 hours, images of each well were taken using a Nikon Ti2 microscope to count the number of bacteria and observe changes in bacterial morphology and motility. Figure 14 E). By statistically analyzing the changes in bacterial number in different microwells, combined with changes in bacterial morphology and motility, if ten analyte compounds at fixed concentrations are loaded, the sensitivity of bacteria to different components can be qualitatively analyzed. If different concentrations of the same component are loaded, the MIC can be quantitatively determined. Figure 14 F).

[0061] Implementation Method 2

[0062] The feasibility of nm-SlipChip in high-throughput analysis of antimicrobial compounds was first evaluated using eight antibiotics, including four against Staphylococcus aureus ATCC25923 (oxazolidin, vancomycin, tetracycline, and rifampin) and four against Escherichia coli ATCC25922 (ceftazidime, tigecycline, nitrofurantoin, and levofloxacin). These MIC results were consistent with the quality control standards of the Clinical and Laboratory Standards Institute (CLSI). Figure 15 Taking nitrofurantoin as an example, the detection and analysis process is described in detail. A series of dilutions of nitrofurantoin (concentrations 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 nitrofurantoin by slipping. The nm-SlipChip was incubated at 37°C for 3 hours, with bright-field images of each well taken hourly. The minimum inhibitory concentration (MIC) of nitrofurantoin was determined by analyzing the occupied bacterial pixels. When the nitrofurantoin concentration was below 1 μg / mL, *E. coli* proliferated normally. However, at concentrations between 2 and 8 μg / mL, the morphology of *E. coli* was elongated, and the proliferation rate was slower than the control group. At concentrations above 8 μg / mL, *E. coli* ceased proliferation and transformed into protoplasts or spheroids (…). Figure 16 A). Therefore, the MIC of nitrofurantoin determined by nm-SlipChip was 4 μg / mL ( Figure 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 to conventional 96-well plate culture and optical density (OD) measurement, all strains cultured on the nm-SlipChip reached detectable levels within 2–3 hours, showing a six-fold time reduction, demonstrating the excellent compatibility of the nm-SlipChip. Figure 17 ).

[0063] Microbial antimicrobial susceptibility testing methods disperse large volumes of bacterial suspension and analyte solutions into small volumes. Each small volume can range from 1 feli to 1 milliliter. This device consists of two parallel chip layers, each etched with micron-sized channels and chambers. The upper and lower chips achieve precise mixing of the sample and microorganisms through a precise sliding mechanism. The microbial suspension and analyte solutions of different concentrations are loaded into different chambers, and the sliding operation aligns these chambers, promoting the mixing reaction of the sample and microorganisms. Due to the small volume of the microchambers, the reaction efficiency is high, and detection results can be obtained rapidly. Bacteria can grow and multiply within the small volume in less than 3 hours. This process can be detected by optical, electrical, and other signals. This allows for rapid determination of whether the bacteria are inhibited or not, serving as the basis for qualitative and quantitative detection of microbial antimicrobial susceptibility.

[0064] To address the challenges of low and unstable levels of active ingredients in natural products, we have successfully developed a rapid microbial susceptibility testing device. This device enables activity-guided separation, allowing for the targeted identification of natural product components with antibacterial activity. It also achieves high-throughput screening with low consumption and can process multiple samples simultaneously. Using this device, we successfully isolated 20 single components from *Callicarpa integerrima*. We then 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 antimicrobial susceptibility testing.

[0066]

[0067] In the screening, we identified two compounds with activity against Gram-positive bacteria. These two compounds showed good inhibitory activity against Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, and methicillin-resistant Staphylococcus epidermidis. Figure 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 nano-scale matrix slip chip (nm-SlipChip). This technology enables the construction of an "N×M" matrix within nano-scale droplets through simple loading and sliding operations, containing drug combinations of "N" antibiotics and "M" candidate molecules. The entire operation is precisely controlled by internal microfluidics, eliminating the need for complex fluid control systems and operations. By performing phenotypic analysis on bacterial growth in over 100 nano-scale droplets within three hours, we were able to accurately assess the antibacterial activity of candidate molecules.

[0069] By applying this device to the targeted separation of antibacterial activity in 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 antibiotic resistance. The nm-SlipChip, as a flexible, efficient, and on-demand high-throughput screening platform, not only has application potential in the field of antibacterial drug discovery but also provides a new tool for a wide range of other biochemical screening analyses.

[0070] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. A microfluidic device for microbial susceptibility testing, characterized in that, The device comprises two parallel chip layers, each etched with micron-sized channels and chambers. The upper and lower chips utilize a sliding mechanism to precisely mix samples and microorganisms. When the microchannels on the upper and lower chips partially overlap, they form a complete, continuous fluid channel. The sliding process fuses bacteria with candidate compounds, generating a multi-nanoscale bacterial-compound droplet matrix. Microbial suspensions and test solutions of varying concentrations are loaded into different chambers, which are aligned through the sliding operation to promote the mixing reaction of samples and microorganisms. The bacterial state is determined by optical and electrical signals. Both the upper and lower chips are etched with shuttle-shaped micropores and cylindrical micropores, which are arranged alternately in an array within the chip. The upper-layer chip is provided with multiple sets of loading channels; the loading channels include an X-direction loading channel and a Y-direction loading channel. The spindle-shaped micropores on the upper chip and the lower chip generate fluid channels in the X and Y axes through different alignment methods: after the initial alignment forms the X-axis channel, candidate compounds are introduced by positive pressure; the sliding process adjusts the position of the chip layer so that the spindle-shaped micropores come into contact with the cylindrical micropores, thereby forming a droplet array of different candidate compounds; then, by sliding the chip layer, the spindle-shaped micropores 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 bacterial-antimicrobial compound combination matrix.

2. A method for microbial antimicrobial susceptibility analysis using the microfluidic device for microbial antimicrobial susceptibility detection as described in claim 1: characterized in that, Includes the following steps: a) The sample to be evaluated and the corresponding bacterial solution are dispersed into a large number of independent liquid units; each liquid unit contains one test compound and one microorganism; b) Incubate these numerous independent liquid units under certain conditions for a period of time; c) Image individual liquid units at the end of incubation or during incubation; d) Liquid units containing compounds with antibacterial activity can kill or lyse bacteria, thus affecting their normal reproductive processes; e) In liquid units containing compounds that do not have antibacterial activity, bacteria multiply normally; f) The number of bacteria in the liquid unit containing the compound that does not have antimicrobial activity was significantly higher than the number of bacteria in the liquid unit containing the compound that has antimicrobial activity. g) The antibacterial activity of the corresponding unit compound can be obtained by analyzing the liquid unit in which bacteria do not reproduce significantly; h) In the above detection process, molecular-assisted optical detection is used, which involves signal changes due to bacterial reproduction; i) In the above detection process, molecular-assisted optical detection is used, which involves signal changes due to bacterial death.

3. The method as described in claim 2, characterized in that, The efficiency of bacteriophages in killing or lysing bacteria is analyzed by monitoring the process of cell death or proliferation in liquid units containing bacteriophages.

4. The method as described in claim 3, characterized in that, The bacteria are first dispersed into a large number of independent liquid units, and then the corresponding test compound is added to the independent liquid unit for detection.

5. The method as described in claim 4, characterized in that, Independent liquid cells are generated by changing the relative physical positions of two components on the upper and lower layers of the chip.

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