Chip for drug sensitivity analysis and related equipment

Abstract

The invention provides a chip for drug sensitivity analysis and related equipment. A chip platform comprises a plurality of detection units; the detection unit comprises a plurality of reagent chambers, a plurality of reaction units and independent culture units; the reagent chamber is connected with the reaction unit and is used for storing a reagent; the reaction unit is used for generating reagents with different concentrations according to the reagents in the reagent chamber so as to observe the growth states of bacteria under different reagent concentrations; and the independent culture unit is connected with the reaction unit and is used for forming a control group. Reagents with different concentrations are added into a plurality of reaction units, the growth states of bacteria under various concentrations are measured at a time, the bacterial activity is calculated by adopting a micro-triangular bacterial enrichment area, and more precise drug resistance analysis is realized by outputting multi-dimensional parameters, so that the experimental process of drug sensitivity test is simplified, the cost of drug sensitivity test is reduced, and the test efficiency is improved. And the group detection capability is improved.

Description

Technical Field

[0001] This invention relates to the technical field of microfluidic rapid drug sensitivity analysis, specifically to a chip and related equipment for drug sensitivity analysis. Background Technology

[0002] As bacterial resistance mechanisms continue to evolve, antibiotics classified as sensitive in clinical susceptibility testing often lead to treatment failure because current MIC-based susceptibility testing methods generally neglect the classification and analysis of subdrug-resistant bacteria (persistent resistance, tolerance). A common characteristic of subdrug-resistant bacteria is their ability to tolerate high doses of antibiotics and regain activity after antibiotic removal, leading to recurrent illness and increased risk of death. More worryingly, continuous antibiotic stimulation may encourage them to evolve into fully resistant strains, further accelerating the global antibiotic resistance (AMR) crisis. Currently, rapid susceptibility testing methods for subdrug-resistant bacteria are lacking.

[0003] Currently, real-time drug susceptibility testing technologies mainly include two categories: genotyping and phenotypic analysis. Genotyping methods (such as rapid detection of drug resistance genes) can complete initial screening within hours, but they have three limitations: 1. Mechanism blind spots: they cannot identify non-genotypic drug resistance (such as changes in membrane permeability); 2. Missed detection of complex mutations: it is difficult to simultaneously analyze drug resistance phenotypes generated by multiple gene co-variations; 3. Unknown gene limitations: they can only cover known drug resistance gene libraries. Phenotypic analysis, as the gold standard, directly observes the inhibitory effect of antibiotics on pathogen growth. Some studies have achieved rapid drug susceptibility results output within 2 hours using portable fingertip spinner combined with metabolic chromogenic technology, but it cannot obtain the minimum inhibitory concentration (MIC) necessary for clinical guidance of drug use.

[0004] Microfluidics, with its advantages of automation and integration, has become a key direction for overcoming this bottleneck and has been widely used in high-throughput drug susceptibility analysis and precise MIC detection. However, existing colorimetric methods have two major limitations: reagents may interfere with bacterial / antimicrobial agent action; and binary qualitative analysis is difficult to identify sub-drug-resistant strains. Although new technologies such as droplet microfluidics and sliding arrays are constantly emerging, they are still constrained by bottlenecks such as complex operation, high cost, poor culture compatibility, or insufficient population detection capabilities. Summary of the Invention

[0005] The main objective of this application is to provide a chip and related equipment for drug susceptibility analysis, which aims to simplify the experimental process of drug susceptibility testing, reduce the cost of drug susceptibility testing, improve the population detection capability, and achieve more accurate and rapid bacterial resistance classification by outputting multi-dimensional parameters for comprehensive analysis at one time through the chip. To achieve the above objectives, one aspect of this application provides a chip for drug sensitivity analysis, comprising multiple detection units; The detection unit includes: multiple reagent chambers, multiple reaction units, and independent culture units; The reagent chamber, connected to the reaction unit, is used to store reagents; The reaction unit is used to generate reagents of different concentrations according to the reagents in the reagent chamber in order to observe the growth status of bacteria under different reagent concentrations. The independent culture unit is connected to the reaction unit and is used to form a control group.

[0006] In some embodiments, the reaction unit includes: a first culture chamber, a first detection zone, a pressure-balanced microchannel, a drainage channel, and a plurality of first quantitative dispensing structures; The drainage channel is connected to the reagent chamber and the first quantitative dispensing structure, and is used to introduce reagents or bacterial suspensions into the first quantitative dispensing structure. The pressure balancing microchannel is connected to the metering chamber and the first culture chamber and is used to balance the pressure inside the chamber. The first culture chamber is connected to the first quantitative dispensing structure and is used for culturing bacteria; The first detection area, connected to the first culture chamber, is used to detect bacterial activity; The first quantitative dispensing structure is used to dispense a plurality of reagents at a first concentration.

[0007] In some embodiments, the independent culture unit includes: a second quantitative distribution structure, a waterproof and breathable channel, a capillary microchannel, pores, a second culture chamber, and a second detection area; The second quantitative dispensing structure, connected to the waterproof and breathable channel, is used to dispense a reagent of a second concentration; The waterproof and breathable channel is connected to the capillary microchannel and is used to transfer a reagent of a fixed concentration to the capillary microchannel. The capillary microchannel is connected to the second culture chamber and is used to control the flow direction of the reagent; The pores are connected to the culture chamber and are used for gas exchange; The second culture chamber is connected to the second detection area and is used to deliver cultured bacteria into the second detection area for detection.

[0008] In some embodiments, the first quantitative dispensing structure is shaped like a steep slope, allowing reagents to flow into the corresponding culture chambers at different doses; The first quantitative distribution structure includes: a metering chamber, a capillary burst valve, and a pressure balance channel; The metering chamber is connected to the drainage channel and the culture chamber, and is used to generate reagents of different concentrations; The capillary burst valve is connected to the capillary microchannel and is used to control the flow direction of the reagent; The air pressure balancing channel is connected to the capillary microchannel and is used to balance air pressure.

[0009] In some embodiments, the drug sensitivity analysis chip includes a bottom cover, a microchannel layer, a chamber layer, and a top cover distributed from bottom to top.

[0010] In some embodiments, the connection area between the first detection area and the first culture chamber is sloped. The first detection area is triangular in shape.

[0011] To achieve the above objectives, another aspect of this application proposes a drug sensitivity analysis method, applied to a chip used for drug sensitivity analysis, comprising: Reagents are added quantitatively to the reagent chamber using high-speed centrifugation. A steep-slope quantitative distribution structure was used to add bacterial suspension; Bacteria and reagents were transferred to each culture chamber of the independent culture unit and reaction unit for culture using low-speed centrifugation. High-speed centrifugation was used to enrich bacteria in the culture chamber into the detection area of ​​the reaction unit; The activity parameters of bacteria in the detection area were calculated based on the bacterial microtriangle enrichment area in the presence of reagents and the bacterial microtriangle enrichment area in the absence of reagents. A time- and concentration-based antimicrobial susceptibility evaluation model was used to perform bacterial antimicrobial susceptibility analysis based on bacterial activity parameters.

[0012] In some embodiments, the use of a time- and concentration-based drug susceptibility evaluation model to perform bacterial drug susceptibility analysis based on bacterial activity parameters includes: Based on the maximum growth rate of bacteria under drug-free conditions, the drug concentration at which the bacterial growth percentage is inhibited to 60% and the steepness of the concentration-response curve, a time- and concentration-based drug susceptibility evaluation model is constructed. Based on the time- and concentration-based drug susceptibility evaluation model, the minimum inhibitory concentration, the specific growth activity of pathogenic microorganisms under high drug concentration conditions, and the specific drug concentration at which the bacterial growth inhibition rate reaches 50% are calculated.

[0013] To achieve the above objectives, another aspect of this application provides a drug sensitivity analysis device, comprising: The reagent addition module is used to quantitatively add reagents to the reagent chamber using high-speed centrifugation. A bacterial addition module for adding bacterial suspension using a steep-slope quantitative dispensing structure; The bacterial culture module is used to transfer bacteria and reagents to each culture chamber of the independent culture unit and reaction unit for culture using low-speed centrifugation. The bacterial isolation module is used to enrich bacteria in the culture chamber to the detection area of ​​the reaction unit using high-speed centrifugation. The bacterial processing module is used to calculate the bacterial activity parameters in the detection area based on the bacterial microtriangle enrichment area under reagent-present conditions and the bacterial microtriangle enrichment area under reagent-free conditions. The antimicrobial susceptibility analysis module is used to perform bacterial antimicrobial susceptibility analysis based on bacterial activity parameters using a time- and concentration-based antimicrobial susceptibility evaluation model.

[0014] To achieve the above objectives, another aspect of the embodiments of this application proposes a drug sensitivity analysis chip platform, including a chip for drug sensitivity analysis and a drug sensitivity analysis device.

[0015] The advantages of the chip of the present invention are as follows: The present invention sets up multiple reaction units, and the reaction reagents are added to the reagent chamber. The reaction reagents flow from the reagent chamber into different reaction units, so that different reaction units generate reagents of different concentrations, thereby enabling the testing of the growth status (i.e., activity) of bacteria under different concentrations of reagents. The whole process is completed on a chip platform, which simplifies the detection process and shortens the detection time.

[0016] The beneficial effects of the method of the present invention are as follows: The present invention establishes a brand-new drug sensitivity evaluation system based on a drug sensitivity evaluation model of time and concentration, which comprehensively considers the influence of factors such as time and concentration on the drug sensitivity evaluation results. Combined with a chip for drug sensitivity analysis, it achieves more accurate drug sensitivity evaluation. Attached Figure Description

[0017] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention. To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Figure 1 A chip structure diagram for drug sensitivity analysis provided in an embodiment of this application; Figure 2 A flowchart of a drug sensitivity analysis method provided in this application embodiment; Figure 3 A diagram showing the connection between the reagent chamber and the drainage channel provided in an embodiment of this application; Figure 4 This is a schematic diagram of the reaction unit structure provided in an embodiment of this application; Figure 5 A schematic diagram of a chip application for drug sensitivity analysis provided in an embodiment of this application; Figure 6 A schematic diagram comparing the traditional drug susceptibility evaluation system and the drug susceptibility evaluation system of the present invention provided in the embodiments of this application; Figure 7 This is a schematic diagram of a steep slope quantitative distribution structure provided in an embodiment of this application; Figure 8 A schematic diagram of quantitative structural simulation analysis under different pressure conditions provided for embodiments of this application; Figure 9 This is a schematic diagram illustrating the pigment verification principle provided in an embodiment of this application. Figure 10 This is a schematic diagram of the microbial vitality calculation structure provided in the embodiments of this application; Figure 11 This is a schematic diagram illustrating the verification of the drug sensitivity analysis chip provided in an embodiment of this application; Figure 12 The drug susceptibility test data diagram provided in the embodiments of this application; Figure 13 CCD images of kanamycin sulfate antibiotic concentration and bacterial microenrichment phenotype in each culture chamber after centrifugation at 3500 RPM for 8 minutes at different culture times, provided for embodiments of this application. Figure 14 Images taken with a mobile phone at different culture times (centrifuged at 3500 RPM for 8 minutes) for different culture chambers, showing the concentration of kanamycin sulfate antibiotic and the bacterial microenrichment phenotype, as provided in the embodiments of this application.

[0018] Explanation of reference numerals in the attached figures: 100, Detection unit 1; 200, Detection unit 2; 220, Independent culture unit; 210, Reaction unit; 221, Second quantitative distribution structure; 222, Waterproof and breathable channel; 223, Capillary microchannel; 224, Vent; 225, Second culture chamber; 226, Second detection area; 211, Connector vent; 212, Siphon valve; 213, Drainage channel 1; 214, Drainage channel 2; 215, Drainage channel 3; 216, Reagent chamber 2; 217, Reagent chamber 1; 410, First quantitative distribution structure; 411, Metering chamber; 412, Pressure balance microchannel; 1101, Waste liquid chamber; 1102, Control well; 1103, Positioning chamber; 1104, Sacrificial chamber; 711, Capillary burst valve. Detailed Implementation

[0019] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0020] It should be noted that all directional indications (such as up, down, left, right, front, back, etc.) in the embodiments of the present invention are only used to explain the relative positional relationship and movement of each component in a certain specific posture (as shown in the figure). If the specific posture changes, the directional indication will also change accordingly.

[0021] Furthermore, the use of terms such as "first" and "second" in this invention is for descriptive purposes only and should not be construed as indicating or implying their relative importance or implicitly specifying the number of technical features indicated. Therefore, features defined with "first" and "second" may explicitly or implicitly include at least one of those features. Additionally, the technical solutions of the various embodiments can be combined with each other, but only on the basis of being achievable by those skilled in the art. When the combination of technical solutions is contradictory or impossible to implement, such a combination of technical solutions should be considered non-existent and not within the scope of protection claimed by this invention.

[0022] Microfluidics, with its advantages of automation and integration, has become a key direction for overcoming this bottleneck and has been widely used in high-throughput drug susceptibility analysis and precise MIC detection. However, existing colorimetric methods have two major limitations: reagents may interfere with bacterial / antimicrobial agent action; and binary qualitative analysis is difficult to identify sub-drug-resistant strains. Although new technologies such as droplet microfluidics and sliding arrays are constantly emerging, they are still constrained by bottlenecks such as complex operation, high cost, poor culture compatibility, or insufficient population detection capabilities.

[0023] Based on this, the main objective of the embodiments of this application is to provide a chip and related equipment for drug susceptibility analysis, which aims to simplify the experimental process of drug susceptibility testing, reduce the cost of drug susceptibility testing, improve the population detection capability, and achieve more accurate drug resistance classification and rapid identification and screening of sub-drug resistant strains.

[0024] This application provides a chip and related equipment for drug sensitivity analysis, which will be described in detail through the following embodiments. First, a chip for drug sensitivity analysis is described in this application embodiment.

[0025] Figure 1 This is a chip structure diagram for drug sensitivity analysis provided in an embodiment of this application. Please refer to [link / reference]. Figure 1 The chip for drug sensitivity analysis provided in this application embodiment may include, but is not limited to, multiple detection units; The detection unit includes: multiple reagent chambers, multiple reaction units, and an independent culture unit 220. For example, the drug sensitivity analysis chip platform is disk-shaped, and one drug sensitivity analysis chip platform has two detection units. (Reference) Figure 1In one detection unit, there are two reagent chambers, namely reagent chamber 1217 and reagent chamber 2216. The number of reaction units can be determined according to specific experimental requirements, and an independent culture unit 220 is set up to form a control group. In some embodiments, the reaction units can meet the reaction requirements of different concentrations of reagents and form a control with the independent culture unit 220, making the entire experimental process simpler and improving experimental efficiency.

[0026] The reagent chamber, connected to the reaction unit, is used to store reagents.

[0027] In some embodiments, the reagent chamber of the drug susceptibility analysis chip platform is a core functional unit integrated into the chip substrate and featuring a micro-nano-scale structured design. It is connected to the sample loading area, reaction detection area, and waste liquid chamber 1101 via precise microfluidic channels, enabling the partitioned storage, precise distribution, and controllable reaction of various reagents. Specifically, the reagent chamber is typically fabricated using biocompatible materials and incorporates an array of micro-trapways, micropillars, or microchannel structures. This allows for rapid mixing and uniform incubation of the test bacterial solution with antimicrobial reagents of varying concentrations. Furthermore, the hydrophilic / hydrophobic modification or functionalized coating on the chamber surface reduces non-specific adsorption, ensuring the stability of the reaction system. Simultaneously, the miniaturized design of the reagent chamber significantly reduces the amount of sample and reagents required. Combined with the chip's high-throughput array layout, it enables simultaneous detection of the susceptibility of multiple bacterial strains to multiple antimicrobial drugs on a single chip, significantly improving the efficiency and throughput of drug susceptibility analysis. This provides a crucial miniaturized and integrated solution for rapid clinical drug susceptibility diagnosis and antimicrobial drug screening.

[0028] The reaction unit is used to automatically generate reagents of different concentrations according to the reagents in the reagent chamber in order to observe the growth status of bacteria under different reagent concentrations.

[0029] Optionally, the reaction unit is the core functional module that carries out the incubation reaction between bacteria and antimicrobial reagents and enables the visualization of growth status. This unit is precisely connected to the reagent chamber and sample channel of the chip and can receive a mixed system of quantitatively dispensed gradient concentration antimicrobial reagents and test bacterial solutions. As a further optional implementation, the reaction unit typically integrates an array of micro-observation wells or a microfluidic reaction chamber. The inner wall of the chamber is biocompatible to avoid non-specific bacterial adsorption, and it is equipped with a light-transmitting substrate to adapt to optical detection equipment. During isothermal incubation, external equipment such as microscopes and spectrometers can be used to observe bacterial colony formation, cell proliferation density, and morphological changes under different reagent concentration gradients in real time or at the endpoint. This allows for the differentiation of the degree of bacterial growth inhibition at different drug concentrations, providing direct visual data support for drug sensitivity determination.

[0030] The independent culture unit 220 is connected to the reaction unit and is used to form a control group.

[0031] It should be noted that the independent culture unit 220 is a functional module specifically designed to construct an experimental control group. It uses the same microstructure design and biocompatible materials as the reaction unit of the chip, and is independent of the reaction chamber array containing antibacterial reagents. It only introduces an equal volume of the test bacterial solution and a blank culture medium without antibacterial activity. Furthermore, by maintaining the same constant temperature incubation conditions, fluid control parameters, and optical observation environment as the reaction unit, the independent culture unit 220 can eliminate interference from irrelevant variables such as differences in chip structure and fluctuations in the incubation environment. This allows for the observation of the natural growth state of bacteria without drug stress, thereby obtaining baseline data on normal bacterial proliferation. This provides a standardized reference for comparing the growth inhibition rate of bacteria under different concentrations of antibacterial reagents and determining the minimum inhibitory concentration, ensuring the accuracy and reliability of the drug sensitivity analysis results.

[0032] Further, refer to Figure 4 The reaction unit includes: a first culture chamber 218, a first detection zone, a pressure balance microchannel, a drainage channel, and multiple first quantitative distribution structures 410; The drainage channel is connected to the reagent chamber and the first quantitative dispensing structure 410, and is used to introduce reagent from the reagent chamber to the first quantitative dispensing structure 410. The pressure balancing microchannel 412 is connected to the metering chamber 411 and the first culture chamber 218, and is used to balance the pressure inside the chamber. The culture chamber 218 is connected to the first quantitative distribution structure 410 and is used to culture bacteria; The first detection area, connected to the first culture chamber 218, is used to detect bacterial activity; The first quantitative dispensing structure 410 is used to dispense a plurality of reagents of a first concentration.

[0033] As a further optional implementation, the present invention uses a larger chamber for bacterial community culture, and finally centrifuges the culture at high speed (including but not limited to 3000 rpm-6000 rpm), see reference. Figure 5 , Figure 5In this invention, Q1 and Q2 represent fluid flow rates, and A1, A2, A3, and A4 represent four different antibiotic solutions or culture media. The invention achieves bacterial enrichment in a micro-triangular sensor at the end of the chamber, using the area of ​​micro-enriched bacteria to reflect the bacterial growth status within the chamber. Secondly, precise quantitative distribution is achieved using a centrifugal microfluidic chip, automatically generating antibiotic concentration gradients based on the three-dimensional depth of the metering chamber (preset concentration points are 90% MIC, 80% MIC, 70% MIC, 60% MIC, 50% MIC, 40% MIC, 30% MIC, 20% MIC, and 10% MIC; here, MIC values ​​include, but are not limited to, the breakpoint concentration values ​​for drugs identified as sensitive by CLSI). Finally, the microfluidic chip is designed to contain high antibiotic concentrations (10 times the MIC (MIC is the minimum inhibitory concentration), C... 10倍MIC ) and the second culture chamber 225 with no antibiotic concentration control.

[0034] Therefore, by comparing the microenrichment area of ​​bacteria cultured under different antibiotic concentrations with the control culture chamber 218 (0 antibiotic concentration), the specific growth activity of bacteria in each chamber can be obtained. This chip can acquire four key parameters in a single measurement: MIC (minimum inhibitory concentration), IC50 (antibiotic concentration at which bacterial growth percentage is below 50%), concentration-growth percentage effect curve (h), and the specific growth activity of pathogenic microorganisms under high antibiotic concentrations (Amax).

[0035] A mature mathematical prediction model for the survivability of antimicrobial drugs is introduced: The survival rate formula is expressed as:

[0036] Wherein, S(c,t) represents the microbial survival rate at drug concentration c and time t; It is the killing rate of a drug, which is the ability of a drug to kill microorganisms per unit time.

[0037] The formula for the kill rate is expressed as:

[0038] in, is the maximum killing rate, which is the maximum killing effect achieved by the drug at a high concentration (far higher than MIC); C is the actual concentration of the drug; MIC is the minimum inhibitory concentration; k is the Hill coefficient, which indicates the strength (steepness) of the effect of drug concentration on the killing rate. This Hill function describes how the killing rate increases with increasing drug concentration.

[0039] This model not only describes the inhibitory effect of drug concentration (MIC) on microorganisms, but also the killing effect of drug exposure time (MDK99) on microorganisms; through the Hill coefficient, it describes how the killing rate changes with the increase of drug concentration, enabling more refined drug sensitivity classification.

[0040] Further, refer to Figure 7 The first quantitative distribution structure 410 has a steep slope shape, which allows the reagents to flow into the corresponding culture chambers at different concentrations. The first quantitative distribution structure 410 includes: a metering chamber 411, a capillary burst valve 711, and a pressure balance channel 412; The metering chamber 411 is connected to the drainage channel and is used to generate reagents of different concentrations; The capillary burst valve 711 is connected to the capillary microchannel 223 and is used to control the reagent flow direction; The air pressure balancing channel 412 is connected to the capillary microchannel 223 and is used to balance air pressure.

[0041] It is easy to understand that, reference Figure 8 and Figure 9 A ramp-type quantitative dispensing unit structure was designed to achieve precise measurement through the size of the quantitative chamber, further forming a concentration gradient. In some embodiments, exemplarily, refer to Figure 9 , Figure 9 Part A represents the quantification results without using a steep ramp design: (I-II) The solution has moved to the next microdose unit before the current chamber completes quantification; (III-V) The sector self-connection design makes accurate quantification impossible.

[0042] Figure 9 Part B represents the quantitative results using the steep slope design: precise quantitative procedures for the solution in the central region of main channel 1 (I-Ⅳ); precise quantitative procedures for the solution in main channel 2 (V-Ⅷ); and precise quantitative procedures for the solution in the distal region of main channel 3 (Ⅸ-XII).

[0043] This invention achieves fluid flow direction control by varying the resistance of microchannels. Figure 9In this diagram, R2 represents the resistance of the capillary burst valve, and R1 represents the resistance of the microchannel. For example, if R1 is smaller than R2, the resistance of R2 (the capillary burst valve) can directly block the liquid from entering the microchannel, while R1 can temporarily impede fluid movement. According to Poiseuille's law of fluid flow in pipes, the longer and narrower the channel, the greater the resistance. Since the metering chamber initially has virtually no resistance, the fluid preferentially enters the metering chamber due to the fluid disproportionation effect within the microfluidic chip. The forward thrust decreases sharply, even falling below the microchannel resistance, thus preventing the solution from continuing to move forward (simulation results demonstrate this process). Once the metering chamber is filled with liquid, the resistance of the capillary burst valve increases, making R1 greater than R2. Under the fluid disproportionation effect, the fluid's driving force continues to concentrate in the main channel, overcoming the resistance of R1 and continuing forward. Simulation data shows that this structure can adapt to large pressure changes and stabilize, and is compatible with manual thrust to achieve concentration gradient formation. This has great potential for simplifying operation, improving portability, and enhancing compatibility. Furthermore, this structural design utilizes… Figure 9 The pigment experiments have been confirmed, with experimental theoretical models and experimental data to support them.

[0044] Further, refer to Figure 10 The shape of the connection area between the first detection area and the first culture chamber 218 is sloped.

[0045] Figure 10 Part A of the diagram is the force analysis of the first culture chamber 218 of the disk-type chip. Figure 10 Part B in the design refers to the first culture chamber 218 and the detection area. Figure 10 Part C in the diagram is the analysis of the forces acting on the bacteria. Figure 10 Part D in the diagram represents the shape difference in the detection area caused by processing accuracy errors under the same culture conditions, but it does not affect the final enrichment area size. Figure 10 Part E in the text describes the bacterial viability calculation method and viability level classification.

[0046] The first detection area is triangular in shape.

[0047] It will be understood by those skilled in the art that reference Figure 10 This invention connects a larger culture chamber 218 to a micro-triangular sensing structure via a ramp at the end furthest from the heart. Through high-speed centrifugation (3000 rpm-6000 rpm), bacteria in the first culture chamber 218 are concentrated in the micro-triangular sensing structure under centrifugal force. Because the centrifugal radii of the chambers are uniform, the force applied is consistent, causing the bacteria to aggregate at the tip of the micro-triangular sensing chamber. Figure 10(In the AC section), experimental verification shows that the shape change of the triangle due to differences in batch processing precision does not affect the final detection result. This effectively solves the long-standing problem of poor detection stability and unreliable results caused by chip processing precision deviations, greatly reducing processing precision requirements and significantly improving feasibility for chip processing cost reduction and mass production. Figure 10 (Part D). Furthermore, this invention can obtain a precise percentage of bacterial growth viability by comparing the size of the micro-area enrichment with that of the control well 1102. Combined with the ingenious micro-triangular sensing structure design, this invention provides a new solution for similar drug screening and microbial growth activity assessment. Figure 10 (Part E). The reasoning process for the specific activity calculation formula is as follows: Bacterial counts typically do not directly reflect whether an antimicrobial agent inhibits bacterial growth activity, as this value is also affected by culture time and initial bacterial concentration. This invention assesses the inhibitory effect of the antimicrobial agent by comparing the bacterial counts in the antimicrobial-treated group and the control group (culture group without added drug) to calculate the percentage of bacterial growth. The percentage of bacterial growth is expressed as follows:

[0048] in, Indicates the percentage of bacterial growth. This represents the final concentration at which bacteria achieve optimal growth without the addition of any medication. This refers to the concentration of bacteria after culturing at a specific concentration of antibacterial drug. Clearly, The lower the value, the worse the inhibitory effect of the antibacterial drug; the higher the value, the more significant the inhibitory effect. When the value exceeds 100%, it indicates that the antibacterial drug promotes bacterial growth.

[0049] The relationship between bacterial count and enrichment region can be expressed as:

[0050] in, Indicates the number of bacteria. The correlation coefficient between the microtriangular enrichment area of ​​bacteria and the number of bacteria is given. The size, number, and concentration of bacterial microregions are represented as follows:

[0051]

[0052] in, Indicates bacterial concentration. The volume of the solution in the microchamber is given. By converting the bacterial microtriangular enrichment area into a concentration formula, the percentage of bacterial growth inhibition used to assess effectiveness can be converted into the following formula:

[0053] in, This indicates the area of ​​bacterial microtriangle accumulation in the presence of antibacterial agents. This represents the bacterial microtriangular enrichment area under antibacterial agent-free conditions. Since the chips have the same radius and control unit, this chip structure can directly calculate the antibacterial agent inhibition rate according to the above formula. By ranking the inhibitory effects of drugs, this system provides clinical pharmacists with more intuitive and effective medication guidance. Figure 10 Part E of the middle section).

[0054] Further, refer to Figure 1 The independent culture unit 220 includes: a second quantitative distribution structure 221, a waterproof and breathable channel 222, a capillary microchannel 223, pores 224, a second culture chamber 225, and a second detection area 226; The second quantitative dispensing structure 221 is connected to the waterproof and breathable channel 222 and is used to dispense reagents; The waterproof and breathable channel 222 is connected to the capillary microchannel 223 and is used to transfer a fixed concentration of reagent to the capillary microchannel 223. The capillary microchannel 223 is connected to the second culture chamber 225 and is used to control the flow of reagents; The vent is connected to the second culture chamber 225 and is used for gas exchange. The second culture chamber 225 is connected to the second detection area 226 and is used to send the cultured bacteria into the second detection area 226 for detection.

[0055] Furthermore, the independent culture unit 220 integrates six functional components: a second quantitative distribution structure 221, a waterproof and breathable channel 222, a capillary microchannel 223, an air pore 224, a second culture chamber 225, and a second detection area 226. Each component is precisely connected in sequence according to the fluid transmission logic. As an optional implementation, the second quantitative distribution structure 221 is connected to the waterproof and breathable channel 222, which undertakes the quantitative distribution and initial transfer function of the reagents required for the experiment; the waterproof and breathable channel 222 is connected to the capillary microchannel 223, which can realize the stable transfer of reagents of fixed concentration and avoid fluid leakage; the capillary microchannel 223 is connected to the second culture chamber 225, and the flow direction and flow rate of the reagent are precisely controlled through the microscale flow channel structure to ensure that the reagent enters the second culture chamber 225 evenly; the pores 224 are directly connected to the second culture chamber 225 for gas exchange during the culture process to maintain the aerobic or anaerobic environment required for bacterial growth; the second culture chamber 225 is finally connected to the second detection area 226, which can smoothly deliver the cultured bacterial samples into the detection area to carry out the observation and analysis of bacterial growth status, and provide a complete and controllable culture and detection link for the control group experiment.

[0056] Further, refer to Figure 11 The drug sensitivity analysis chip platform includes a bottom cover, a microchannel layer, a chamber layer, and a top cover distributed from bottom to top.

[0057] It should be noted that, combining the hand-operated quantitative distribution structure and the triangular detection zone, based on the characteristics of drug sensitivity analysis, this invention can rapidly obtain the minimum inhibitory concentration (MIC) cutoff point, and the feasibility of generating a drug concentration gradient has been demonstrated through soluble pigments. Further, as an optional implementation method, refer to... Figure 11 , Figure 11 Part A in the middle is a top-down view of the chip. Figure 11 Part B in the diagram illustrates the chip's layering. Figure 11 Part C describes the manual addition of the first antibiotic, 1. Figure 11 Part D involves manually adding either the first antibiotic or the culture medium. Figure 11 Part E is for manually adding bacterial suspension. Figure 11 The middle F section is a manually operated control chamber. Figure 11 The middle G section involves low-speed centrifugation to generate a precise concentration gradient. Figure 11 The middle H section is a control chamber unit structure design. Figure 11 The middle I part is a linear gradient of pigment RGB values. Figure 11 The J section represents the difference in chamber volume, and CH1-7 corresponds to chambers 1-7, which are chamber numbers 1-7.

[0058] The chip structure is designed as a four-layer structure, fabricated using CNC technology. The disc-shaped chip has two detection units and seven culture chambers (218), enabling efficacy evaluation at seven concentration points. Through a hand-driven quantitative dispensing structure with three drainage channels, it achieves quantitative dispensing of three layers of different reagents or bacterial solutions. This hand-driven approach effectively avoids the shortcomings of traditional microfluidic quantitative dispensing structures that rely on low-speed centrifugation (1000 RPM to 1500 RPM) to achieve concentration distribution, which can lead to variations in bacterial content in each chamber due to centrifugal force and affect the final structure. The ability of this chip structure to generate precise concentration gradients (R0.05) is demonstrated through the use of solutions of different colors. 2 =0.9908). Therefore, in a specific embodiment, by replacing the pigment solution in drainage channels 1213 and 2214 with the corresponding drug concentration, and adding a specific bacterial concentration to drainage channel 3215, the drug concentration can be generated manually for efficacy evaluation and bacterial activity analysis.

[0059] Figure 1 This is a schematic diagram of a chip structure for drug sensitivity analysis according to the present invention. Figure 2 This is a flowchart of a drug sensitivity analysis method according to the present invention. Figure 12 The following is a graph of the drug sensitivity test data of the present invention, in conjunction with... Figure 1 , Figure 2 and Figure 12 The present invention provides a detailed description and explanation of the solutions in specific drug sensitivity analysis scenarios.

[0060] A chip for drug sensitivity analysis includes the following structure: refer to Figure 1Each detection chip has two identical detection units, each containing two reagent chambers and three drainage channels. Reagent chamber 1217 is connected to drainage channel 1213 via siphon valve 212, and reagent chamber 2216 is connected to drainage channel 2214 via siphon valve 212. Reagent chambers 1217 and 2216 are connected to connecting vent 211 via airflow microchannels. Drainage channel 3215 is connected to the first culture chamber 218 via capillary microchannel 223. Simultaneously, drainage channels 1213, 2214, and 3215... 215 are directly connected to the metering chamber. The metering chamber of the drainage channel 3215 is connected to the first culture chamber 218, which is further away from the center, through the capillary microchannel 223. The first culture chamber 218 is additionally connected to the drainage channel 3215 through the air pressure balance microchannel. The metering chamber connected by the drainage channel 1213 is connected to the drainage channel 2214 through the siphon valve 212, and is directly connected to the metering chamber in the corresponding drainage channel 2214. The metering chamber in the drainage channel 2214 is directly connected to the culture chamber 218 through the microchannel of the siphon valve 212. Therefore, when releasing the pressure of the connecting vent 211, high-throughput quantitative distribution of the two reagents can be achieved by high-speed centrifugation (3000RPM to 6000RPM), and the microbial suspension can be added by manual operation. Finally, the reagents 1 and 2 and the microbial suspension in the corresponding metering chamber can be collected into the culture chamber 218 for culture by centrifugation at a speed of 1000RPM to 1500RPM.

[0061] In a specific embodiment, the drainage channel 1213 is connected to the reagent chamber 1217, from which reagents are introduced. The drainage channel 2214 is connected to the reagent chamber 2216, from which reagents are introduced. The reagents introduced by the drainage channels 1213 and 2214 flow into the first culture chamber 218 through two first quantitative distribution structures 410. Then, bacterial suspension is added manually. The bacterial suspension is finally introduced into the first culture chamber 218 through the drainage channel 3215 and the first quantitative distribution structure 410. During this process, the first quantitative distribution structure 410 connected to the drainage channels 1213 and 2214 can change the concentration of the reagent, so that the concentration of the reagent in each culture chamber is different, while the concentration of the bacterial suspension added to each culture chamber is the same.

[0062] Each detection unit also includes two independent culture units 220. Each independent culture unit 220 is designed with a quantitative distribution structure, a waterproof and breathable membrane channel, capillary microchannels 223, pores, a culture chamber 218, and a detection area. A fixed concentration of drug can be added to the culture chamber 218, or no drug can be added to form a control culture chamber 218. The specific process is as follows: microbial suspension is added manually, precise measurement is achieved through the quantitative distribution structure, and finally, a fixed concentration of microorganisms is cultured by low-speed centrifugation.

[0063] This specific embodiment applies the chip for drug susceptibility analysis of the present invention to the drug susceptibility analysis of an engineered drug-resistant bacterium. This strain exhibits different drug resistance characteristics and expresses GFP fluorescent protein (GFP-E. coli BL21(pLXRN-EGFP)) (resistant to ampicillin and sensitive to kanamycin sulfate). Based on the quantified chamber size, an automatically generated concentration gradient percentage was designed (Ch1-7): 54%, 47%, 40%, 34%, 27%, 20%, and 15%. Therefore, the automatically generated concentration gradient by adding 35 μg / mL kanamycin sulfate is a concentration gradient containing MIC breakpoints: Ch1: 18.9 μg / mL, Ch2: 16.45 μg / mL, Ch3: 14 μg / mL, Ch4: 11.9 μg / mL, Ch5: 9.45 μg / mL, Ch6: 7 μg / mL, Ch7: 5.25 μg / mL. Therefore, the antibiotic gradient for 50 μg / mL ampicillin was: Ch1: 27 μg / mL, Ch2: 23.5 μg / mL, Ch3: 20 μg / mL, Ch4: 17 μg / mL, Ch5: 13.5 μg / mL, Ch6: 10 μg / mL, and Ch7: 7.5 μg / mL. The results showed that, based on the 60% growth percentage standard, the bacteria were sensitive to kanamycin (MIC value 14 μg / mL), while the MIC value for ampicillin was higher than 27 μg / mL. This is consistent with the drug resistance characteristics of the engineered bacteria, demonstrating that the drug susceptibility analysis platform can obtain reliable drug susceptibility analysis results within three hours. Figure 12 ,exist Figure 12 middle, Figure 12 Parts A and B in the figure show a linear curve comparison of bacterial growth percentage versus fluorescence intensity after culturing for 3 hours under a gradient of specific concentrations of kanamycin sulfate (A) or ampicillin (B). Figure 12 The CD section is a linear curve comparing the percentage of bacterial growth with fluorescence intensity after culturing for 6 hours under a gradient of antibiotics such as kanamycin sulfate (C) or ampicillin (D) at specific concentrations. Figure 12The middle EF section is a comparison chart of growth percentage at 3 hours and 6 hours: 35 μg / mL kanamycin sulfate antibiotic gradient (E) and 50 μg / mL ampicillin antibiotic gradient (F). Figure 12 The GH section shows a comparison of bright-field and fluorescence fields of engineered drug-resistant bacteria at 3 hours (G) or 6 hours (H) under kanamycin sulfate or ampicillin concentration gradients. Scale bar (black): 0.5 mm; Scale bar (red): 0.5 mm.

[0064] refer to Figure 13 and Figure 14 In this specific embodiment, the chip for drug susceptibility analysis of the present invention is applied to verify the drug susceptibility analysis of Escherichia coli strain K-12 to kanamycin sulfate. Different culture times are used to verify the time point for determining the drug susceptibility results. Simultaneously, signal acquisition is performed synchronously on a smartphone using a portable micro-lens, verifying the portability of the signal acquisition method. The results show that a drug susceptibility breakpoint of 14 μg / mL is obtained after 3 hours, consistent with the previous verification results. Although the two strains are different, both are sensitive to kanamycin sulfate. Furthermore, the image results captured by the portable smartphone are consistent with those obtained by a professional camera, indicating that the detection method of the present invention has high portability and compatibility.

[0065] Based on the aforementioned methods for determining drug susceptibility activity, a bacterial drug susceptibility analysis chip capable of obtaining multi-dimensional parameters was further designed to achieve more comprehensive bacterial resistance analysis and more efficient screening of sub-drug-resistant bacteria. Furthermore, in the specific drug susceptibility analysis design, it was found that by combining the micro-concentration gradient spacing generated by the chip with bacterial growth percentage activity, an effect curve of bacteria to micro-concentration changes can be obtained. This effect curve can further differentiate bacterial resistance. Further integration with the multi-dimensional parameter design within the chip allows for more accurate drug susceptibility classification. Therefore, the chip structure design proposed in this invention can simultaneously obtain MIC and IC data. 50 h, A max Multidimensional parameters were used to evaluate drug efficacy and analyze bacterial resistance characteristics.

[0066] The beneficial effects of this invention include: Based on Poiseuille's channel flow law, this invention designs a novel hand-driven quantitative dispensing structure that achieves precise concentration gradient generation. It provides a portable, accurate, and automated quantitative dispensing design for various analytical reactions that require concentration optimization and drug evaluation.

[0067] This invention addresses the shortcomings of current drug susceptibility analysis, which relies on chromogenic reagents, requires high chip fabrication precision, and necessitates population culture. It designs a micro-enrichment area sensing structure to determine bacterial activity. This micro-enrichment area evaluation system eliminates the need for indicator reagents, avoiding their influence on microorganisms and different drugs. Furthermore, experiments demonstrate that the triangular micro-enrichment area can negate deviations caused by fabrication precision issues, providing a solid foundation for batch processing of product structures and obtaining reliable results. This solves the long-standing bottleneck of high cost and high batch-to-batch variability in microfluidic chips, hindering their industrialization.

[0068] Preliminary experiments have confirmed that this signal sensing technology can be used to detect and analyze signals using a smartphone combined with a portable macro probe. By combining AI deep learning algorithms to build a model, the drug sensitivity analysis time is less than 3 hours. This invention proposes a drug sensitivity analysis mode and chip structure design, which have significant application value in the future economic, health and other fields.

[0069] Figure 2 Please refer to the flowchart of a drug susceptibility analysis method provided in the embodiments of this application. Figure 2 This application also provides a drug sensitivity analysis method, applied to the above-mentioned chip for drug sensitivity analysis. The drug sensitivity analysis method includes, but is not limited to, steps S100 to S600.

[0070] Step S100: Add reagents quantitatively to the reagent chamber using high-speed centrifugation.

[0071] It's easy to understand that in the operation of the drug sensitivity analysis chip platform, adding reagents to the reagent chambers requires relying on the chip's preset microfluidic interface or independent injection port. Furthermore, during operation, a micropipette or automated fluid manipulation device can be used to precisely inject a quantitative amount of antibacterial reagent or blank culture medium into the corresponding reagent chamber. During injection, the reagent will spread evenly along the preset flow guide structure inside the chamber, and the injection port can be sealed by the chip's matching sealing component to prevent reagent evaporation or the entry of external contaminants. For the addition of reagents with multiple concentration gradients, the chip's array-style reagent chamber layout allows for the sequential injection of reagents of corresponding concentrations into different chambers, ensuring consistent reagent volume and uniform distribution in each chamber, providing a stable reagent supply basis for subsequent mixing and incubation with bacterial solutions and drug sensitivity reactions.

[0072] Step S200: Add bacterial suspension using a steep-slope quantitative distribution structure; In some embodiments, during the experimental operation of the drug sensitivity analysis chip platform, the addition of bacterial suspension must be performed through the chip's dedicated sample injection port. Furthermore, a micropipette or automated sample loading device can be used to precisely inject the concentration-calibrated bacterial suspension into the chip's sample distribution area. The injected bacterial suspension flows along preset microfluidic channels into the reaction unit containing gradient concentrations of antibacterial reagents and the independent culture unit 220 used for control experiments, respectively. The chip's quantitative distribution structure ensures consistent volumes of bacterial suspension in each unit. After injection, the injection port must be sealed to prevent evaporation or contamination of the suspension, while ensuring thorough contact and mixing between the bacterial suspension and the reagents within the chamber, laying a uniform reaction system foundation for subsequent isothermal incubation and growth status observation.

[0073] In step S300, bacteria and reagents are transferred to each culture chamber of the independent culture unit and reaction unit for culture using low-speed centrifugation.

[0074] Optionally, during the incubation preparation stage of the drug sensitivity analysis chip platform, after the antibacterial reagent and bacterial suspension are injected into their respective chambers, the chip is placed in a suitable high-speed centrifuge. By setting the centrifugation speed and duration to match the chip's microstructure tolerance, the centrifugal force drives the mixture to overcome the surface tension and fluid resistance within the microchannels, precisely and synchronously transferring it to each of the arrayed culture chambers 218. Furthermore, this process ensures that the bacterial-reagent mixture in each culture chamber 218 has a uniform volume and distribution, while the brief force generated by high-speed centrifugation does not significantly affect bacterial activity. After transfer, the chip is sealed and placed in a constant-temperature incubation environment for static bacterial culture, providing a stable and consistent reaction system for subsequent drug sensitivity observation.

[0075] Step S400: High-speed centrifugation is used to enrich the bacteria in the culture chamber into the detection area of ​​the reaction unit.

[0076] As is easily understood, in the detection preparation stage of the drug sensitivity analysis chip platform, after the bacteria have completed the predetermined incubation time in the culture chamber 218, the chip is placed in a suitable high-speed centrifuge. Based on the chip's microchannel dimensions and the connection structure between the culture chamber 218 and the detection area, a reasonable centrifugation speed and duration are set. Utilizing the directional driving effect of centrifugal force, the bacterial cells in the culture chamber 218 overcome the fluid resistance and surface tension within the microchannels, achieving rapid and efficient enrichment and directional migration to the connected detection area. Furthermore, this process can increase the bacterial concentration in the detection area without damaging the bacterial morphology and activity, enhancing the signal intensity of subsequent optical observations or biosensor detections, and providing a high signal-to-noise ratio detection sample for accurately determining the bacterial growth status and drug sensitivity results.

[0077] Step S500: Calculate the bacterial activity parameters in the detection area based on the bacterial microtriangle enrichment area under reagent presence and reagent-free conditions.

[0078] Furthermore, in the detection and analysis stage of the drug sensitivity analysis chip platform, based on the bacterial samples enriched within the detection area, combined with images or spectral data acquired by optical detection equipment, bacterial activity parameters are calculated using image analysis algorithms and biosensor signal analysis models. As an optional implementation, image recognition technology is used to statistically analyze the number of colonies, cell density, and morphological characteristics in the detection area. Fluorescent staining is used to quantify the ratio of live to dead bacteria. Simultaneously, the intensity of characteristic absorption peaks of bacterial metabolites is obtained through spectral detection. These multi-dimensional data are then substituted into a preset activity evaluation formula to calculate core parameters such as bacterial proliferation rate and activity inhibition rate. Finally, combined with control group data, the quantitative determination of the antimicrobial reagent's drug sensitivity effect is completed.

[0079] Step S600: A time- and concentration-based drug susceptibility evaluation model is used to perform bacterial drug susceptibility analysis based on bacterial activity parameters.

[0080] Specifically, a time- and concentration-based drug susceptibility evaluation model is used for bacterial drug susceptibility analysis. The core of this model is to quantify the correlation between drug concentration and duration of action on bacterial growth inhibition or killing effects by dynamically monitoring bacterial activity parameters at different drug concentrations and time points. This model overcomes the limitations of traditional methods that rely solely on minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) as single criteria. It can not only intuitively present the time-dependent bactericidal characteristics of drugs at subinhibitory concentrations, but also clearly define the optimal concentration range for concentration-dependent drugs. In some embodiments, by combining the changing trends of bacterial activity parameters, key indicators such as the drug's post-efficacy effect (PAE) are further analyzed, ultimately achieving precise evaluation and optimization of drug antibacterial activity, mechanism of action, and clinical medication regimens.

[0081] Furthermore, step S600 includes, but is not limited to, steps S610 to S620.

[0082] Step S510: Based on the maximum bacterial growth rate under drug-free conditions, the drug concentration at which the bacterial growth percentage is inhibited to 60%, and the steepness of the concentration-effect curve, construct a time- and concentration-based drug susceptibility evaluation model.

[0083] Optionally, the microenrichment area of ​​bacteria cultured under different antibiotic concentrations can be compared with that of the control culture chamber 218 (0 antibiotic concentration) to obtain the specific growth activity of bacteria in each chamber. Furthermore, the chip platform of this invention can acquire four key parameters in a single measurement: MIC (minimum inhibitory concentration), IC50 (antibiotic concentration at which bacterial growth percentage is below 50%), concentration-growth percentage effect curve (h), and the specific growth activity of pathogenic microorganisms under high antibiotic concentrations (Amax). These parameters are consistent with the key parameters of the prediction model. Therefore, this invention further combines the unique design of two structures to construct a novel drug susceptibility analysis chip that can simultaneously obtain multidimensional drug susceptibility information (MIC, IC50 ... 50 h, A max This invention establishes a novel drug susceptibility evaluation system based on a mathematical prediction model of microbial viability, achieving more accurate drug susceptibility assessment. This more refined assessment also provides new research subjects and directions for subsequent research on drug resistance mechanisms. Based on chip structure design, this invention proposes a novel drug susceptibility evaluation model that integrates drug susceptibility information from both time and concentration dimensions, expressed as:

[0084] It is the percentage of bacterial growth at drug concentration c; It is the maximum growth rate under conditions without drugs (0 concentration); It is the drug concentration at which bacterial growth is inhibited by 60%; It is the steepness of the concentration-effect curve (similar to the Hill coefficient).

[0085] Step S520: Based on the drug sensitivity evaluation model of time and concentration, calculate the minimum inhibitory concentration, the specific growth activity of pathogenic microorganisms under high drug concentration conditions, and the specific drug concentration when the bacterial growth inhibition rate reaches 50%.

[0086] refer to Figure 6 The novel drug susceptibility evaluation system proposed in this invention includes four key parameters: MIC, IC50, h, and Amax. In principle, it integrates time dimension and concentration gradient response information, and further combines artificial intelligence deep learning algorithm to conduct comprehensive evaluation of multi-dimensional drug susceptibility characteristics. It can realize rapid classification and identification of sub-drug resistant bacteria, and provide a new paradigm for accurate drug susceptibility classification.

[0087] Another aspect of this application provides a drug sensitivity analysis device, comprising: The reagent addition module is used to quantitatively add reagents to the reagent chamber using high-speed centrifugation. A bacterial addition module for adding bacterial suspension using a steep-slope quantitative dispensing structure; The bacterial culture module is used to transfer bacteria and reagents to each culture chamber of the independent culture unit and reaction unit for culture using low-speed centrifugation. The bacterial isolation module is used to enrich bacteria in the culture chamber to the detection area of ​​the reaction unit using high-speed centrifugation. The bacterial processing module is used to calculate the bacterial activity parameters in the detection area based on the bacterial microtriangle enrichment area under reagent-present conditions and the bacterial microtriangle enrichment area under reagent-free conditions. The antimicrobial susceptibility analysis module is used to perform bacterial antimicrobial susceptibility analysis based on bacterial activity parameters using a time- and concentration-based antimicrobial susceptibility evaluation model.

[0088] It is understood that the content of the above method embodiments is applicable to the present device embodiments. The specific functions implemented by the present device embodiments are the same as those of the above method embodiments, and the beneficial effects achieved are also the same as those achieved by the above method embodiments.

[0089] To achieve the above objectives, another aspect of the embodiments of this application proposes a drug sensitivity analysis chip platform, including a chip for drug sensitivity analysis and a drug sensitivity analysis device.

[0090] It will be understood by those skilled in the art that all or some of the steps and systems in the methods disclosed above can be implemented as software, firmware, hardware, and suitable combinations thereof. Some or all of the physical components can be implemented as software executed by a processor, such as a central processing unit, digital signal processor, or microprocessor, or as hardware, or as an integrated circuit, such as an application-specific integrated circuit. Such software can be distributed on a computer-readable medium, which can include computer storage media (or non-transitory media) and communication media (or transient media). As is known to those skilled in the art, the term computer storage media includes volatile and non-volatile, removable and non-removable media implemented in any method or technology for storing information (such as computer-readable instructions, data structures, program modules, or other data). Computer storage media includes, but is not limited to, RAM, ROM, EEPROM, flash memory or other memory technologies, CD-ROM, digital versatile disc (DVD) or other optical disc storage, magnetic cartridges, magnetic tape, disk storage or other magnetic storage devices, or any other medium that can be used to store desired information and is accessible to a computer. Furthermore, as is known to those skilled in the art, communication media typically include computer-readable instructions, data structures, program modules, or other data in modulated data signals such as carrier waves or other transmission mechanisms, and may include any information delivery medium.

[0091] The device embodiments described above are merely illustrative. The units described as separate components may or may not be physically separate; that is, they may be located in one place or distributed across multiple network units. Some or all of the modules can be selected to achieve the purpose of this embodiment according to actual needs.

Claims

1. A chip for drug sensitivity analysis, characterized in that, Includes multiple detection units; The detection unit includes: multiple reagent chambers, multiple reaction units, and independent culture units; The reagent chamber, connected to the reaction unit, is used to store reagents; The reaction unit is used to generate reagents of different concentrations according to the reagents in the reagent chamber in order to observe the growth status of bacteria under different reagent concentrations. The independent culture unit is connected to the reaction unit and is used to form a control group.

2. The chip for drug sensitivity analysis according to claim 1, characterized in that, The reaction unit includes: a first culture chamber, a first detection zone, a pressure-balanced microchannel, a drainage channel, and multiple first quantitative distribution structures; The drainage channel is connected to the reagent chamber and the first quantitative dispensing structure, and is used to introduce reagents or bacterial suspensions into the first quantitative dispensing structure. The pressure balancing microchannel is connected to the metering chamber and the first culture chamber and is used to balance the pressure inside the chamber. The first culture chamber is connected to the first quantitative dispensing structure and is used for culturing bacteria; The first detection area, connected to the first culture chamber, is used to detect bacterial activity; The first quantitative dispensing structure is used to dispense a plurality of reagents at a first concentration.

3. The chip for drug sensitivity analysis according to claim 1, characterized in that, The independent culture unit includes: a second quantitative distribution structure, a waterproof and breathable channel, a capillary microchannel, pores, a second culture chamber, and a second detection area; The second quantitative dispensing structure, connected to the waterproof and breathable channel, is used to dispense a reagent of a second concentration; The waterproof and breathable channel is connected to the capillary microchannel and is used to transfer a reagent of a fixed concentration to the capillary microchannel. The capillary microchannel is connected to the second culture chamber and is used to control the flow direction of the reagent; The pores are connected to the second culture chamber and are used for gas exchange; The second culture chamber is connected to the second detection area and is used to deliver cultured bacteria into the second detection area for detection.

4. A chip for drug sensitivity analysis according to claim 2, characterized in that, The first quantitative distribution structure is shaped like a steep slope, allowing reagents to flow into the corresponding culture chambers at different doses; The first quantitative distribution structure includes: a metering chamber, a capillary burst valve, and a pressure balance channel; The metering chamber is connected to the drainage channel and the culture chamber, and is used to generate reagents of different concentrations; The capillary burst valve is connected to the capillary microchannel and is used to control the flow direction of the reagent; The air pressure balancing channel is connected to the capillary microchannel and is used to balance air pressure.

5. A chip for drug sensitivity analysis according to claim 1, characterized in that, The drug sensitivity analysis chip includes a bottom cover, a microchannel layer, a chamber layer, and a top cover, distributed from bottom to top.

6. A chip for drug sensitivity analysis according to claim 2, characterized in that, The connection area between the first detection area and the first culture chamber is sloped in shape. The first detection area is triangular in shape.

7. A drug sensitivity analysis method, applied to a chip for drug sensitivity analysis as described in any one of claims 1-6, characterized in that, include: Reagents are added quantitatively to the reagent chamber using high-speed centrifugation. A steep-slope quantitative distribution structure was used to add bacterial suspension; Bacteria and reagents were transferred to each culture chamber of the independent culture unit and reaction unit for culture using low-speed centrifugation. High-speed centrifugation was used to enrich bacteria in the culture chamber into the detection area of ​​the reaction unit; The activity parameters of bacteria in the detection area were calculated based on the bacterial microtriangle enrichment area in the presence of reagents and the bacterial microtriangle enrichment area in the absence of reagents. A time- and concentration-based antimicrobial susceptibility evaluation model was used to perform bacterial antimicrobial susceptibility analysis based on bacterial activity parameters.

8. The drug sensitivity analysis method according to claim 7, characterized in that, The aforementioned time- and concentration-based antimicrobial susceptibility evaluation model for bacterial antimicrobial susceptibility analysis based on bacterial activity parameters includes: Based on the maximum growth rate of bacteria under drug-free conditions, the drug concentration at which the bacterial growth percentage is inhibited to 60% and the steepness of the concentration-response curve, a time- and concentration-based drug susceptibility evaluation model is constructed. Based on the time- and concentration-based drug susceptibility evaluation model, the minimum inhibitory concentration, the specific growth activity of pathogenic microorganisms under high drug concentration conditions, and the specific drug concentration at which the bacterial growth inhibition rate reaches 50% are calculated.

9. A drug sensitivity analysis device, characterized in that, include: The reagent addition module is used to quantitatively add reagents to the reagent chamber using high-speed centrifugation. A bacterial addition module for adding bacterial suspension using a steep-slope quantitative dispensing structure; The bacterial culture module is used to transfer bacteria and reagents to each culture chamber of the independent culture unit and reaction unit for culture using low-speed centrifugation. The bacterial isolation module is used to enrich bacteria in the culture chamber to the detection area of ​​the reaction unit using high-speed centrifugation. The bacterial processing module is used to calculate the bacterial activity parameters in the detection area based on the bacterial microtriangle enrichment area under reagent-present conditions and the bacterial microtriangle enrichment area under reagent-free conditions. The antimicrobial susceptibility analysis module is used to perform bacterial antimicrobial susceptibility analysis based on bacterial activity parameters using a time- and concentration-based antimicrobial susceptibility evaluation model.

10. A drug sensitivity analysis chip platform, characterized in that, Includes the chip for drug sensitivity analysis as described in any one of claims 1-6 and the drug sensitivity analysis device as described in claim 9.

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