A system for drug susceptibility testing

Abstract

The application provides a system for drug sensitivity detection, and belongs to the technical field of drug sensitivity detection. The system comprises a card box module, wherein a composite well plate is provided with a plurality of rows and columns of first accommodation cavities, a single-layer well plate below the composite well plate is provided with a corresponding number of second accommodation cavities, the two accommodation cavities are vertically and one-to-one corresponding, and the inner wall is a hydrophilic layer. A first sealing film is arranged between the accommodation cavities for blocking, and a second sealing film and a third sealing film are arranged at the top end and the bottom end respectively for sealing. The first accommodation cavities accommodate antimicrobial drugs and target to be detected for incubation, after incubation, the target is specifically combined with a binding agent and a target-binding photon, the sealing film is punctured to connect the two accommodation cavities, and the target is transferred to the second accommodation cavity. A dye pad is arranged in the second accommodation cavity, after screening through the dye pad and an external force field, the target is enriched at the bottom for optical measurement. The system realizes the sequential reaction of the target to be detected and reagents through a longitudinal stacking structure and a layer-to-layer sealing film, realizes the sequential reaction of the target to be detected and reagents by layer-by-layer puncturing, integrates multiple steps of operation into the card box, reduces manual pipetting, reduces operation complexity and human error, and is easier to be automated.

Description

Technical Field

[0001] This invention relates to the field of drug susceptibility testing technology, and in particular to a system for drug susceptibility testing. Background Technology

[0002] The continuous increase in drug-resistant strains has led to a growing clinical demand for rapid and reliable antimicrobial susceptibility results. Traditional drug susceptibility tests (such as the paper-diffusion method, broth dilution method, and E-test) require strain isolation, culture, and manual interpretation, typically taking 48–72 hours, which is insufficient to meet the needs of early medication decisions for infectious diseases. Furthermore, traditional drug susceptibility tests are complex to perform and require a high level of operator proficiency. To achieve rapid, high-volume testing, some companies have developed reagent kits. While these kits achieve rapid, high-volume testing, their complex structure (such as the need for microfluidic channels) results in high costs, hindering widespread adoption.

[0003] Therefore, it is necessary to optimize the existing cartridges and operating procedures, simplify the process, realize the automatic and continuous loading and detection of multiple reagents, improve detection efficiency and reduce production costs. Summary of the Invention

[0004] This invention provides a system for drug susceptibility testing, the purpose of which is to simplify the drug susceptibility testing process and realize the automatic and continuous loading and detection of multiple reagents.

[0005] To achieve the above objectives, embodiments of the present invention provide a system for drug susceptibility testing, including a cartridge module, the cartridge module comprising: A composite perforated plate having at least one row and at least one column of first receiving cavities; A single-layer perforated plate located below the composite perforated plate, the single-layer perforated plate having at least one, and the single-layer perforated plate having a second receiving cavity with the same number as the first receiving cavity; The first and second accommodating cavities are vertically aligned. The inner walls of the first and second accommodating cavities are hydrophilic layers. A first sealing membrane for blocking the first and second accommodating cavities and a second and third sealing membrane for sealing the top of the first accommodating cavity and the bottom of the second accommodating cavity are provided between the first and second accommodating cavities. The first cavity is used to contain the antimicrobial drug for incubating with the target under test, and the second cavity is used to contain the dye pad. After incubation, the target under test specifically binds to the binder and target-binding photons in the first cavity and is transferred to the second cavity when the first cavity and the second cavity are connected. After being filtered by the dye pad and the external force field, it is enriched at the bottom of the second cavity for optical measurement.

[0006] Preferably, the composite perforated plate includes an upper plate with an upper receiving cavity and a lower plate with a lower receiving cavity, wherein the upper receiving cavity and the lower receiving cavity correspond one-to-one in the vertical direction, and a fourth sealing membrane for blocking the upper receiving cavity and the lower receiving cavity is provided between the upper plate and the lower plate. The upper cavity contains an antimicrobial drug for incubating the target to be tested, and the lower cavity contains a binding agent for specifically binding to the target to be tested.

[0007] Preferably, the width-to-depth ratio of the first receiving cavity and the second receiving cavity is greater than 3.3 / 10, or... The width-to-depth ratio of the upper and second accommodating cavities is greater than 3.3 / 10.

[0008] Preferably, the third sealing film is made of a transparent material with a light transmittance of not less than 90%, and the fluorescence emission intensity generated by the third sealing film within the excitation wavelength range emitted by the optical measuring device is less than the detection limit of the optical measuring device.

[0009] Preferably, the first sealing film is a metal film or a metal composite film, and the length of the first sealing film is greater than the length of the composite orifice plate to form an upwardly bendable heating wing in the length direction of the composite orifice plate, so that the target to be tested incubated inside the composite orifice plate is heated by heat conduction through the heating wing; or, The fourth sealing membrane is a metal membrane or a metal composite membrane, and the length of the fourth sealing membrane is greater than the length of the upper plate to form an upwardly bendable heating wing in the length direction of the upper plate. The heat conduction of the heating wing heats the target to be incubated in the upper plate.

[0010] Preferably, the composite perforated plate and the single-layer perforated plate are made of rigid materials, or... The lower plate is made of elastic material. The first sealing film and the fourth sealing film are respectively sandwiched between the lower plate and the single-layer perforated plate, and between the upper plate and the lower plate. The upper plate and the single-layer perforated plate form a pressing assembly.

[0011] Preferably, the system for drug sensitivity testing further includes a first pipetting module, the bottom of which is flat and a hollow cavity is formed from the bottom upwards, the inner wall of the hollow cavity and the outer surface of the first pipetting module being a hydrophilic layer.

[0012] Preferably, the bottom side of the first pipetting module is provided with a notch, which communicates with the hollow cavity.

[0013] Preferably, the system for drug sensitivity testing further includes a second pipetting module, the outer surface of which is a hydrophilic layer.

[0014] Preferably, the contact angle of the hydrophilic layer is less than 45°.

[0015] Preferably, the system for drug susceptibility testing is operated using a first operating procedure, which includes: S100. Puncture the second sealing membrane above the first receiving cavity, add the target to be tested into the first receiving cavity, and incubate the target to be tested with the antimicrobial drug in the first receiving cavity. After incubation, the target to be tested specifically binds to the target-binding photon and the binding agent. S200. The first pipetting module is used to draw the target to be tested, which is bound to the target photon and the binder, into the hollow cavity, and after puncturing the first sealing film above the second receiving cavity, the target to be tested, which is bound to the target photon and the binder, is injected into the second receiving cavity. S300. The cartridge module is placed in the optical measurement device and an external force field is applied so that the target to be tested, which specifically binds to the target-binding photons and binder, is screened and enriched at the bottom of the second receiving cavity by the dye pad for optical measurement.

[0016] Preferably, S100 includes: S110. After a preset time has elapsed since the target to be tested was added to the first cavity, target-binding photons and a binder are added to the first cavity respectively to obtain a target to be tested that specifically binds to the target-binding photons and the binder. S120. After aspirating the target material that has specifically bound to the target-binding photons and binder using the first pipetting module, the first sealing membrane above the monolayer plate is punctured, and the target material that has specifically bound to the target-binding photons and binder is injected into the second receiving cavity; or, The composite perforated plate includes an upper plate with an upper receiving cavity and a lower plate with a lower receiving cavity. The upper receiving cavity and the lower receiving cavity correspond one-to-one in the vertical direction. A fourth sealing membrane for blocking the upper receiving cavity and the lower receiving cavity is provided between the upper plate and the lower plate. The upper cavity is used to contain the antimicrobial drug for incubating the target to be tested, and the lower cavity is used to contain the binding agent for specifically binding to the target to be tested; S100 includes: S110. Puncture the second sealing membrane above the upper receiving cavity, add the target to be tested into the upper receiving cavity, and incubate the target to be tested with the antimicrobial drug in the upper receiving cavity; S120. Target-binding photons are added into the upward-facing cavity to obtain the target to be tested that is bound to the target-binding photons; S130. The first pipetting module is used to draw in the target to be tested that is bound to the target photon, and the first pipetting module is used to puncture the fourth sealing membrane above the lower receiving cavity to inject the target to be tested that is bound to the target photon into the lower receiving cavity to bind with the binder, thereby obtaining the target to be tested that is bound to the target photon and the binder.

[0017] Preferably, the system for drug susceptibility testing is operated using a second operating procedure, which includes: S100. Puncture the second sealing membrane above the upper containment cavity and add the target to be tested into the upper containment cavity for incubation; S200. After incubation, target-binding photons are added into the upper cavity to obtain the target to be tested that is bound to the target-binding photons; S300. Place the card holder module into the centrifuge unit; S400. Using the second pipetting module, the fourth sealing membrane and the first sealing membrane are punctured in sequence, and the centrifugation unit is started to centrifuge the target to be tested, which has specifically bound to the target-binding photon and the binding agent, into the second receiving cavity; S500. The cartridge module is placed in the optical measurement device and an external force field is applied so that the target to be tested, which specifically binds to the target-binding photons and binder, is screened and enriched at the bottom of the second receiving cavity by the dye pad for optical measurement.

[0018] The above-described solution of the present invention has the following beneficial effects: In this application, by utilizing the structure of vertically stacked well plates (i.e., single-layer well plates and composite well plates) and interlayer sealing membranes, the target and different reagents can be sequentially contacted and reacted during the process of puncturing the sealing membrane layer by layer. This design integrates multi-step reactions into the cartridge structure, significantly reducing manual pipetting steps, lowering the complexity of operation and human error, and making it easier to automate the detection process.

[0019] The accommodating chambers (i.e., the first accommodating chamber and the second accommodating chamber) on each well plate form multiple independent detection channels in the longitudinal direction. This design significantly improves the throughput of a single test and supports simultaneous testing of multiple analytes, multiple antimicrobial drugs, or multiple concentration gradients, meeting the clinical demand for efficient and high-volume testing.

[0020] Different reagents are pre-loaded in layers into the corresponding well plate cavities and isolated by a sealing membrane, simplifying the reagent pre-loading process.

[0021] In the card box module, the compression assembly ensures airtightness while reducing the assembly precision requirements and production costs of multi-layer structures, making it more conducive to mass production.

[0022] The inner wall of the receiving cavity is a hydrophilic layer, which significantly reduces the residue of micro-level liquid on the cavity wall. Combined with the longitudinally stacked short flow path design and the use of dedicated pipetting modules (such as the first pipetting module and the second pipetting module), a high recovery rate of liquid is ensured during interlayer transfer, laying the foundation for the formation of sufficient and uniform test targets for subsequent optical detection, and improving the accuracy and reliability of the detection results.

[0023] Other features and advantages of the present invention will be described in detail in the following detailed description section. Attached Figure Description

[0024] Figure 1 This is an exploded view of the card module when the composite perforated plate has a single-layer structure; Figure 2 This is an exploded view of the card module when the composite perforated plate has a double-layer structure; Figure 3 yes Figure 2 A schematic diagram of part A in the middle; Figure 4a This is a schematic diagram of liquid residue in Comparative Example 1; Figure 4b This is a schematic diagram of liquid residue in Comparative Example 2; Figure 4c Comparative Example 3: Liquid Residue Diagram Figure 4d This is a schematic diagram of liquid residue in Example 1.

[0025] [Explanation of Labels in the Attached Image] 100 - Composite perforated plate, 110 - First receiving cavity, 120 - Upper plate, 121 - Upper receiving cavity, 130 - Lower plate, 131 - Lower receiving cavity 200 - Single-layer perforated plate, 210 - Second receiving cavity 300 - First sealing membrane, 310 - Heating wing, 400 - Second sealing membrane 500-Third sealing membrane, 600 - Fourth sealing membrane. Detailed Implementation

[0026] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0027] like Figures 1-3As shown, an embodiment of the present invention provides a system for drug sensitivity testing, including a cartridge module. The cartridge module includes a composite well plate 100 and at least one single-layer well plate 200. The single-layer well plate 200 is disposed below the composite well plate 100. The composite well plate 100 has at least one row and at least one column of first receiving cavities 110. The single-layer well plate 200 has the same number of second receiving cavities 210 as the first receiving cavities 110. The first and second receiving cavities 110 and 210 correspond one-to-one in the vertical direction, and the inner walls of both the first and second receiving cavities 110 and 210 are hydrophilic layers. A first sealing membrane 300 is disposed between the first and second receiving cavities 110 to prevent communication between them. A second sealing membrane 400 is disposed above the first receiving cavity 110 to seal its top end, and a third sealing membrane 500 is disposed at the bottom end of the second receiving cavity 210 to seal its bottom end.

[0028] Specifically, the first receiving cavity 110 and the second receiving cavity 210 have identical structures, both having a top opening and a bottom opening. A first sealing membrane 300 is disposed between the composite perforated plate 100 and the single-layer perforated plate 200. The first sealing membrane 300 seals the bottom opening of the first receiving cavity 110 and the top opening of the second receiving cavity 210, thereby preventing communication between the first receiving cavity 110 and the second receiving cavity 210. The inner walls of the aforementioned first receiving cavity 110 and second receiving cavity 210 are hydrophilic layers, which are formed using existing technologies, such as physical coating, chemical bonding, ultrasonic spraying, etc.

[0029] The aforementioned first receiving cavity 110 is used to contain the antimicrobial agent incubated with the test target, and the second receiving cavity 210 is used to contain the dye pad. The test target refers to the entity to be tested; for example, this invention can be used to detect cells, proteins, nucleic acids, and carbohydrates. The embodiments in this application demonstrate the detection of secreted molecules. However, this invention can be used to detect a wide range of molecules and cells, including biomarkers, hormones, cell surface proteins, intracellular proteins, nucleic acids, and intact cells. The antimicrobial agent is used to test the susceptibility of a strain to the antimicrobial agent.

[0030] During use, the target to be tested is incubated with antimicrobial drugs in the first receiving cavity 110. After a preset incubation time, a binder and target-binding photons are added to the first receiving cavity 110. The target to be tested specifically binds to the binder and the target-binding photons, forming a target to be tested that is specifically bound to the target-binding photons and the binder. When the first sealing membrane 300 between the first receiving cavity 110 and the second receiving cavity 210 loses its barrier effect, the target to be tested that is specifically bound to the target-binding photons and the binder enters the second receiving cavity 210 and is enriched at the bottom of the second receiving cavity 210 after being filtered by the dye pad and the external force field for optical measurement.

[0031] The binder is used to label the target for detection and counting. The binder is a magnetic marker, such as magnetic beads. Target-binding photons include fluorescent particles, fluorophores, chemiluminescent agents, bioluminescent agents, resonant light-scattering particles, light-absorbing or colorimetric signal transducers, and quantum dots. The dye pad is used to screen the target for specific binding to the target-binding photons and the binder, separating these targets from other components and unbound target-binding photons, and depositing them on the third sealing membrane 500 for subsequent optical measurements. The external force field attracts the target for specific binding to the target-binding photons and the binder through the dye pad. This external force field can be different depending on the binder; for example, when the binder is magnetic beads, the external force field can be a magnetic field.

[0032] In this application, multiple well plates are stacked vertically, and the cavities on each well plate are aligned vertically. When the sealing film breaks from top to bottom, the adjacent cavities are connected, forming a liquid path between them. This enables continuous and orderly contact and reaction between the target and the reagents pre-placed in the cavities. This design achieves physical separation and time-sequencing of the reaction steps: after the target enters the first cavity 110 and is incubated with the antimicrobial drug pre-placed in the first cavity 110 for a period of time, target-binding photons and binding agents are added for labeling and binding. After completion, the target bound to the target and the binding agent are obtained. After the first cavity 110 is connected to the second cavity 210, the target bound to the target and the binding agent is screened by the dye pad in the second cavity 210 and the external force field and enriched at the bottom of the second cavity 210 for optical measurement. As can be seen from the above process, this application can achieve sequential contact between the target and the reagent by opening and closing the first receiving cavity 110 and the second receiving cavity 210, which can greatly reduce the complexity of operation.

[0033] Secondly, the inner walls of both the first receiving cavity 110 and the second receiving cavity 210 in this application are hydrophilic layers, which reduces the contact angle and surface energy of the liquid, making the liquid flow more easily. Combined with the vertically stacked overall structure of this application, the liquid transfer path is significantly shortened. Compared with conventional microchannel structures, this reduces liquid loss before it flows into the second receiving cavity 210 where the dye pad is located, allowing for complete and rapid transfer of the liquid into the second receiving cavity 210. This provides sufficient test targets that bind to the target photons and binders, thereby improving the accuracy of subsequent experiments. Moreover, the fabrication process of the receiving cavities is simpler and more convenient than that of microchannels, resulting in lower manufacturing costs.

[0034] Finally, each well plate is provided with at least one row and at least one column of receiving cavities, which can effectively increase the number of detection channels, allowing multiple samples to be processed in parallel on the same cartridge module or multiple index tests to be performed on the same sample, thus achieving efficient testing.

[0035] In one embodiment of this application, the composite well plate 100 can be a single-layer structure. When the composite well plate 100 is a single-layer structure, the first receiving cavity 110 on the composite well plate 100 is used to receive antimicrobial drugs, and the second receiving cavity 210 on the single-layer well plate 200 is used to receive dye pads. Preferably, a nutrient medium may also be pre-placed in the first receiving cavity 110 to provide nutrition for the incubation of the target to be tested.

[0036] In use, the second sealing membrane 400 above the sealing composite perforated plate 100 is punctured to add the target to be tested into the first receiving cavity 110. The target reacts with the antimicrobial drug and is incubated. After incubation for a period of time, a solution containing target-binding photons and a solution containing a binding agent are added to the first receiving cavity 110 to obtain the target to be tested that binds to the target-binding photons and the binding agent. The target to be tested that binds to the target-binding photons and the binding agent is filtered by the dye pad under the action of an external force field and enriched on the third sealing membrane 500 below the second receiving cavity 210 for subsequent optical measurements.

[0037] It should be noted that the target-binding photons or binding agents should be added to the bacterial strain sample sequentially, and the order of addition can be chosen arbitrarily.

[0038] In another embodiment of this application, the composite perforated plate 100 can also be a double-layer structure. When the composite perforated plate 100 is a double-layer structure, the composite perforated plate 100 includes an upper plate 120 and a lower plate 130. The lower plate 130 is located below the upper plate 120, and the upper plate 120 has an upper receiving cavity 121 and the lower plate 130 has a lower receiving cavity 131. The upper receiving cavity 121 and the lower receiving cavity 131 penetrate the upper plate 120 and the lower plate 130 in the thickness direction corresponding to the upper plate 120 and the lower plate 130 to form a structure with an open upper end and a closed lower end. The upper receiving cavity 121 and the lower receiving cavity 131 correspond one-to-one in the vertical direction, that is, the upper receiving cavity 121, the lower receiving cavity 131 and the second receiving cavity 210 are collinear in the vertical direction. A fourth sealing membrane 600 is provided between the upper plate 120 and the lower plate 130 to block the upper receiving cavity 121 and the lower receiving cavity 131.

[0039] As described above, when the composite orifice plate 100 has a double-layer structure, the upper receiving cavity 121 and the lower receiving cavity 131 are equivalent to the first receiving cavity 110. Unlike the single-layer structure of the composite orifice plate 100, in this application, the first receiving cavity 110 is further subdivided into the upper receiving cavity 121 and the lower receiving cavity 131, and the upper receiving cavity 121 and the lower receiving cavity 131 are separated by the fourth sealing membrane 600, which fully improves the utilization rate of the first receiving cavity 110 and increases the amount of pre-placed reagents that the first receiving cavity 110 can hold.

[0040] When the composite well plate 100 has a double-layer structure, an independent containment cavity is added to the transfer path of the target to be tested, which can accommodate more reagents.

[0041] In this embodiment, the upper cavity 121 is used to contain the antimicrobial drug for incubating the target to be tested, and the lower cavity 131 is used to contain the binding agent. Preferably, the binding agent in the lower cavity 131 is in lyophilized form. In use, the second sealing membrane 400 above the upper cavity 121 is first punctured, and the target to be tested is added into the upper cavity 121. The target to be tested is incubated with the antimicrobial drug. After incubation for a period of time, target-binding photons are added into the upper cavity 121 to mark the target to be tested. After marking, the fourth sealing membrane 600 is punctured, causing the fourth sealing membrane 600 to lose its barrier function. The marked target enters the lower cavity 131 and specifically reacts with the binding agent in the lower cavity 131. The binding agent specifically binds to the marked target to obtain the target to be tested bound to the target-binding photons and the binding agent. The target to be tested, which is punctured by the first sealing membrane 300 and bound to the target photon and binder, enters the second receiving cavity 210. After being screened by the dye pad and under the action of the external force field, it is enriched at the bottom of the second receiving cavity 210.

[0042] As described above, compared to the single-layer composite well plate 100, the lower receiving cavity 131 of the double-layer composite well plate 100 contains a binder located on the transfer path. By defining the composite well plate 100 as a double-layer structure, a separate space is provided for the target to be identified and bound by the binder. This ensures sufficient and controllable space for the reaction between the target and the antimicrobial drug and the target-binding photon. Compared to the scheme of adding the target-binding photon and the binder sequentially in a single well, this structure ensures more thorough mixing of the target during the incubation, labeling, and binding stages, and ensures more complete contact between the binder and the target, greatly improving the efficiency of specific capture and reducing the loss of the target. Moreover, during the experiment, a critical and error-prone reagent addition operation is eliminated, simplifying the operation process.

[0043] In one embodiment of this application, when the composite perforated plate 100 adopts a single-layer structure, the ratio of the width to the depth of the first receiving cavity 110 and the second receiving cavity 210 is greater than 3.3 / 10.

[0044] In another embodiment of this application, when the composite perforated plate 100 adopts a double-layer structure, the width-to-depth ratio of the first receiving cavity 110 and the upper receiving cavity 121 is greater than 3.3 / 10.

[0045] In this embodiment, both the first receiving cavity 110 and the second receiving cavity 210 are square receiving cavities with rounded corners. The side length of the square receiving cavity is 3.3 mm and the hole depth is 10 mm to 20 mm. The ratio of the width to the depth of the first receiving cavity 110 and the second receiving cavity 210 is greater than 3.3 / 10, which helps to form a hydrophilic layer on the inner wall of the first receiving cavity 110 and the second receiving cavity 210, and avoids the hydrophilic layer being unable to adhere to or be implanted on the inner wall of the first receiving cavity 110 and the second receiving cavity 210.

[0046] Furthermore, when the composite perforated plate 100 has a double-layer structure, the lower receiving cavity 131 can have a shape that is larger at the top and smaller at the bottom.

[0047] The materials selected for the first sealing membrane 300, the second sealing membrane 400, the third sealing membrane 500, and the fourth sealing membrane 600 are suitable for the culture of the target to be tested, and at the same time have good flexibility and good bonding with the well plate.

[0048] The first sealing membrane 300, the second sealing membrane 400, and the fourth sealing membrane 600 can be metal membranes, metal composite membranes, or polymer membranes made of organic polymers, such as PS membranes, PMMA membranes, and COC membranes.

[0049] In the embodiments of this application, the third sealing film 500 is made of a transparent material with a light transmittance of not less than 90%. The fluorescence emission intensity generated by the third sealing film 500 within the excitation wavelength range emitted by the optical measurement device is less than the detection limit of the optical measurement device, that is, the third sealing film 500 will not generate fluorescence excitation, thus avoiding background fluorescence caused by fluorescence excitation, which would affect the accuracy of optical measurement. Preferably, an anti-scratch film is adhered to the third sealing film 500, and the anti-scratch film is located on the side of the third sealing film 500 away from the single-layer perforated plate 200. The anti-scratch film needs to be removed in advance before use.

[0050] Each sealing membrane is fixed to the corresponding perforated plate by adhesive bonding or heat sealing.

[0051] Due to the different structures of the composite perforated plate 100, the structures of the first sealing membrane 300 and the fourth sealing membrane 600 are slightly different in this application.

[0052] When the composite orifice plate 100 is a single-layer structure, the length of the first sealing film 300 can be greater than the length of the composite orifice plate 100, thereby allowing the first sealing film 300 to be bent upward in the length direction to form the heating wing 310. In this case, the first sealing film 300 is a metal film or a metal composite film.

[0053] When the composite orifice plate 100 has a double-layer structure, the length of the fourth sealing membrane 600 can be greater than the length of the composite orifice plate 100, allowing the fourth sealing membrane 600 to be bent upwards in the length direction to form a heating wing 310. In this case, the fourth sealing membrane 600 is a metal membrane or a metal composite membrane. Once the fourth sealing membrane 600 forms the heating wing 310, the first sealing membrane 300 does not form a heating wing 310.

[0054] The heating wing 310 is part of the first sealing film 300 or the fourth sealing film 600. The metal film or metal composite film has good thermal conductivity. The heat is transferred to the upper receiving cavity 121 of the upper plate 120 or the first receiving cavity 110 of the composite perforated plate 100 by contacting the heating wing 310 with an external heat source and conducting heat conduction. This heats the liquid in the receiving cavity, so that it is at a suitable temperature during the incubation process.

[0055] The upper surface of the composite perforated plate 100 is also provided with scale marks for indicating the position of the first receiving cavity 110. In this embodiment, the scale marks are respectively arranged along the row and column directions on one side of the first receiving cavities 110 arranged in a matrix. The scale marks can be sequential numbers, such as 1, 2, 3, etc., or sequential letters, such as a, b, c, etc. The first receiving cavity 110 can be quickly positioned by the scale marks in both the row and column directions, thereby ensuring accuracy.

[0056] Similarly, a scale mark for indicating the position of the upper receiving cavity 121 can also be provided on the upper end surface of the upper plate 120.

[0057] In some embodiments of this application, the composite perforated plate 100 and the single-layer perforated plate 200 are made of rigid materials, preferably rigid medical materials. The composite perforated plate 100 and the single-layer perforated plate 200 are bonded together by a sealing film.

[0058] In another embodiment of this application, the composite perforated plate 100 has a double-layer structure, wherein the lower plate 130 is made of an elastic material, preferably a medical elastic material (such as medical silicone). A first sealing film 300 is sandwiched between the lower plate 130 and the single-layer perforated plate 200, and a fourth sealing film 600 is sandwiched between the upper plate 120 and the lower plate 130, and a compression assembly is formed between the upper plate 120 and the single-layer perforated plate 200.

[0059] It should be explained that the compression assembly refers to the pre-tensioning force generated between the upper plate 120 and the single-layer perforated plate 200 by vertically limiting them. This pre-tensioning force acts on the lower plate 130, which is made of elastic material, and causes the lower plate 130 to deform, forming an assembly relationship that clamps the first sealing film 300 and the fourth sealing film 600 between them. Under the compression assembly, the first sealing film 300 is tightly attached between the lower plate 130 and the single-layer perforated plate 200, and the fourth sealing film 600 is tightly attached between the lower plate 130 and the upper plate 120, effectively preventing leakage from the upper receiving cavity 121, the lower receiving cavity 131, and the second receiving cavity 210.

[0060] The compression assembly scheme not only effectively avoids leakage in each receiving cavity, but also eliminates the process of bonding the first sealing film 300 and the fourth sealing film 600 to the lower receiving cavity 131 below and above in the production of this application, making the manufacturing and assembly process of this application simpler.

[0061] There are various ways to achieve clamping assembly. In this embodiment, a method for achieving clamping assembly is provided. Specifically, this embodiment includes an upper shell and a lower shell, which can be interlocked to prevent the upper shell and lower shell from separating from each other in the vertical direction. The upper shell has an operation window, and the lower shell has a detection window. When the composite perforated plate 100 and the single-layer perforated plate 200 are placed between the upper shell and the lower shell, the upper shell provides a downward force on the upper plate 120, and the lower shell provides an upward force on the single-layer perforated plate 200. The two forces form the preload of the clamping assembly. The first receiving cavity 110 is located inside the operation window, and the second receiving cavity 210 is located inside the detection window.

[0062] The system for drug susceptibility testing provided in this application further includes a first pipetting module or a second pipetting module. The first pipetting module has a flat bottom and a hollow cavity formed from the bottom upwards. The inner wall of the hollow cavity and the outer surface of the first pipetting module are hydrophilic layers.

[0063] The hollow cavity is used to draw liquid into the hollow cavity during pipetting, and during injection, the liquid in the hollow cavity is injected into the corresponding receiving cavity. Since the volume of liquid in the detection is relatively small, approximately tens of microliters, the outer wall of the first pipetting module and the inner wall of the hollow cavity are treated to form a hydrophilic layer. This effectively prevents liquid from adhering to the hollow cavity during the first pipetting module's ingestion and expulsion process, thus avoiding a reduction in the number of target particles bound to the target photons and binders, which would affect the accuracy and sensitivity of the detection.

[0064] Preferably, a notch is provided at the bottom side of the first pipetting module, which communicates with the hollow cavity. At the microliter scale, liquid in the narrow space of the hollow cavity is mainly governed by surface tension. Later, as the liquid exits the hollow cavity, a meniscus tends to form at the bottom, causing liquid residue to remain inside. The notch provides an additional outflow path for the liquid, disrupting the closed curved surface structure that leads to liquid retention. Together with the hydrophilic layer, it ensures complete drainage of the liquid from the hollow cavity, guaranteeing the quantitative and complete transfer at the microliter scale. Furthermore, the notch effectively prevents the freeze-dried binder from clogging the bottom of the hollow cavity.

[0065] The aforementioned second pipetting module is a solid structure, and its outer surface is a hydrophilic layer.

[0066] The contact angles of the aforementioned hydrophilic layers are all less than 45°. The contact angle refers to the angle formed by the droplet on the inner wall of the cavity.

[0067] like Figures 4a-4d As shown, experiments were conducted to investigate the transfer scenarios of different cavities: Comparative Example 1: The inner wall of the upper receiving cavity 121 was not hydrophilically treated, and the fourth sealing membrane 600 was an aluminum membrane. 50g of [amount missing] was added sequentially to each of the five upper receiving cavities 121. (Measured to be 50) The liquid is applied to the fourth sealing membrane 600 using a sharp steel needle, but without puncturing it. This causes the fourth sealing membrane 600 to form a downward-pointing conical protrusion, drawing out the liquid from each upper receiving cavity 121. The amount of liquid transferred out was measured to be 49. 49 47 43 41 .

[0068] Comparative Example 2: The inner wall of the upper receiving cavity 121 was not hydrophilically treated, and the fourth sealing membrane 600 was an aluminum membrane. 50g of [amount missing] was added sequentially to each of the five upper receiving cavities 121. (Measured to be 50) The liquid is applied to the fourth sealing membrane 600 using a blunt-tipped steel needle, without piercing it, causing the fourth sealing membrane 600 to form a downward-facing circular bulge. The liquid in each upper receiving cavity 121 is then re-extracted; the amount of liquid transferred is measured to be 44. 48 46 46 48 .

[0069] Comparative Example 3: The inner wall of the upper receiving cavity 121 was not hydrophilically treated, and the fourth sealing membrane 600 was a PS transparent membrane. 50g of [unspecified substance] was added sequentially to each of the five upper receiving cavities 121. (Measured to be 50) The liquid is not pressurized on the fourth sealing membrane 600, and the liquid in each upper receiving cavity 121 is re-drawn out. The amount of liquid transferred out is measured to be 50. 48 49 46 46 .

[0070] Example 1: The inner wall of the upper receiving cavity 121 is hydrophilically treated, and the fourth sealing membrane 600 is a PS transparent membrane. 50g of [unspecified material] is sequentially added to each of the five upper receiving cavities 121. (Measured to be 50) The liquid is not pressurized on the fourth sealing membrane 600, and the liquid in each upper receiving cavity 121 is re-drawn out. The amount of liquid transferred out is measured to be 48. 49 48 49 50 .

[0071] Considering experimental errors and other factors, the liquid transfer volume was 48. The above is considered as a complete transfer.

[0072] As can be seen from the above Comparative Examples 1-3, when the inner wall of the upper cavity 121 was not hydrophilically treated, the phenomena of two, two, and three complete transfers occurred in Comparative Examples 1-3, indicating that there is a chance of complete transfer when the liquid is pipetted without hydrophilic treatment. However, considering the specific values ​​of the transfer amount in each comparative example, the amount of liquid transferred is completely uncontrollable.

[0073] In Example 1, after hydrophilic treatment, the morphology of the inner wall of the upper cavity 121 is completely different from that of Comparative Examples 1-3, with only nano-level wetting residue remaining, and the morphology is uniform, achieving controllable transfer amount. Since this application involves layer-by-layer transfer of multiple cavities, the less the amount of target material lost in the liquid during the layer-by-layer transfer process, the more it helps to improve the detection accuracy.

[0074] Therefore, after the inner wall of the upper receiving cavity 121 is hydrophilically treated, the surface energy and wall resistance are greatly reduced, so that the liquid can be smoothly transferred under the suction of the first pipetting module or the action of centrifugal force.

[0075] The system used for drug susceptibility testing operates using the first operating procedure, which includes the following steps: S100. Puncture the second sealing membrane 400 above the first receiving cavity 110, add the target to be tested into the first receiving cavity 110, the target to be tested is incubated with the antimicrobial drug in the first receiving cavity 110, and the incubated target to be tested specifically binds to the target-binding photon and the binding agent.

[0076] The steps of S100 differ slightly depending on whether the composite perforated plate 100 adopts a single-layer structure or a double-layer structure: When the composite perforated plate 100 adopts a single-layer structure, the following steps are included: S110. After adding the target to be tested into the first receiving cavity 110 and incubating for a preset time, target-binding photons and binding agents are added into the first receiving cavity 110 in any order to obtain the target to be tested that specifically binds to the target-binding photons and binding agents. S120. The first pipetting module is used to draw the target to be tested, which is specifically bound to the target-binding photon and the binder in the first receiving cavity 110, into the hollow cavity. The first pipetting module is used to puncture the first sealing membrane 300 and connect the first receiving cavity 110 with the second receiving cavity 210. The target to be tested, which is specifically bound to the target-binding photon and the binder in the first pipetting module, is injected into the second receiving cavity 210.

[0077] When the composite well plate 100 adopts a double-layer structure, the upper cavity 121 is used to contain the antimicrobial drug incubating the target to be tested, and the lower cavity 131 is used to contain the lyophilized binding agent that specifically binds to the target to be tested, including the following steps: S110. Puncture the second sealing membrane 400 above the upper receiving cavity 121, add the target to be tested into the upper receiving cavity 121, and incubate the target to be tested with the antimicrobial drug in the upper receiving cavity 121; S120. Target-binding photons are added into the upward-facing cavity 121 to obtain the target to be tested that is bound to the target-binding photons; S130. The first pipetting module is used to draw in the target to be tested that is bound to the target photon, and the first pipetting module is used to puncture the fourth sealing membrane 600 above the lower receiving cavity 131 to inject the target to be tested that is bound to the target photon into the lower receiving cavity 131 to bind with the binder, thereby obtaining the target to be tested that is bound to the target photon and the binder.

[0078] By using a pre-filled binder, the critical but error-prone step of manually adding the binder is eliminated. This not only simplifies the operation and automates the reaction upon puncture, but also eliminates the risk of binder dosage discrepancies due to inaccurate pipetting, as well as the risk of aerosol contamination or personnel exposure that may be introduced during opening the cap.

[0079] After obtaining the target to be tested that binds to the target-binding photon and the binding agent, step S200 is performed, which includes: The first pipetting module is used to draw the target to be tested, which is bound to the target photon and the binder, into the hollow cavity. After the first pipetting module punctures the first sealing membrane 300 above the second receiving cavity 210, the target to be tested, which is bound to the target photon and the binder, is injected into the second receiving cavity 210. S300. The cartridge module is placed in the optical measurement device and an external force field is applied so that the target to be tested, which binds to the target photon and binder, is screened and enriched at the bottom of the second receiving cavity 210 for optical measurement.

[0080] In this embodiment, the external force field is a magnetic field, and the binder is a freeze-dried magnetic bead. The target, which specifically binds to the target photons and the binder, is screened by the dye pad under the control of the magnetic field and is enriched on the third sealing film 500 at the bottom of the second receiving cavity 210, which facilitates subsequent optical measurements.

[0081] The system used for drug susceptibility testing is operated using the second operating procedure, which includes the following steps: S100. Puncture the second sealing membrane 400 above the upper receiving cavity 121 and add the target to be tested into the upper receiving cavity 121 for incubation; S200. After incubation for a preset time, target-binding photons are added into the upper cavity 121 to obtain the target to be tested that is bound to the target-binding photons; S300. Place the cartridge assembly into the centrifuge unit and ensure that the axes of the upper receiving cavity 121, the lower receiving cavity 131, and the second receiving cavity 210 are parallel to the direction of centrifugal force; S400. The second pipetting module is used to puncture the fourth sealing membrane 600 and the first sealing membrane 300 in sequence, and the centrifugation unit is started to centrifuge the target to be tested, which has specifically bound to the target-binding photon and the binder, into the second receiving cavity 210.

[0082] Preferably, in this step, there are multiple second pipetting modules, and the arrangement order and number of the multiple second pipetting modules are consistent with the arrangement order and number of the first receiving cavity 110. The second pipetting module punctures the fourth sealing membrane 600 and the first sealing membrane 300 twice by translation. That is, the fourth sealing membrane 600 above the lower receiving cavity 131 and the first sealing membrane 300 above the second receiving cavity 210 each have two puncture holes. Through air pressure balance, it is ensured that the target binding photons, binding agent and the incubated target are fully centrifuged into the second receiving cavity 210 during centrifugation.

[0083] S500. The cartridge module is placed in the optical measurement device and an external force field is applied so that the target to be tested, which binds to the target photon and binder, is filtered and enriched at the bottom of the second receiving cavity 210 for optical measurement.

[0084] In the second operation process, the bottom inner wall of the lower receiving cavity 131 is bowl-shaped, thereby supporting the freeze-dried binder. The aforementioned bowl shape refers to the fact that the lower end of the wall forming the lower receiving cavity 131 is curved, and the lower end of the wall is close to the longitudinal center line of the lower receiving cavity 131 to form a bottom opening.

[0085] This application employs a rapid antimicrobial drug susceptibility analysis based on fluorescence in situ hybridization and phenotypic detection. Specifically, after co-incubating the target with an antimicrobial drug, the target is fluorescently labeled using target-binding photons. Subsequently, the target is specifically bound to the binder and transferred to the third sealing membrane 500 at the bottom of the second receiving cavity 210 for fluorescence imaging analysis. By analyzing the growth of the target bound by the target-binding photons and the binder under the action of the drug, the drug susceptibility results are obtained.

[0086] Taking the composite perforated plate 100 as a double-layer structure and the method shown in the first operation procedure as an example: Ampicillin and sulbactam are added as antimicrobial drugs in the upper cavity 121, lyophilized magnetic beads are added as binders in the lower cavity 131, and a mixture of surfactants, inorganic salts and other reagents is added as a dye pad in the second cavity 210.

[0087] In use, the second sealing membrane 400 above the upper receiving cavity 121 is punctured, and *Escherichia coli* is added to the upper receiving cavity 121. The ribosomal RNA of *E. coli* is the target for testing. The target is incubated in the upper receiving cavity 121. After incubation, a specific probe is injected into the upper receiving cavity 121 as a target-binding photon to label the ribosomal RNA in *E. coli*. After labeling, the target bound to the target-binding photon is aspirated into the first pipetting module, and the fourth sealing membrane 600 is punctured using the first pipetting module. After the fourth sealing membrane 600 is punctured, the target bound to the target photon is injected into the lower receiving cavity 131 again using the first pipetting module. The target bound to the FISH primer probe specifically binds to the reconstituted lyophilized magnetic beads pre-placed in the lower receiving cavity 131, resulting in a target bound to both the FISH primer probe and the magnetic beads. The target molecules bound to the FISH primers and magnetic beads are drawn into the first pipetting module and injected into the second receiving cavity 210 after the first sealing membrane 300 is punctured. In the second receiving cavity 210, under the magnetic force of the magnetic component arranged near the third sealing membrane 500 and the filtering effect of the dye pad, the target molecules bound to the FISH primers and magnetic beads pass through the dye pad and accumulate on the third sealing membrane 500.

[0088] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A system for drug susceptibility testing, characterized in that, Includes a card holder module, the card holder module comprising: A composite perforated plate (100) having at least one row and at least one column of first receiving cavities (110). A single-layer perforated plate (200) located below the composite perforated plate (100) is provided with at least one second receiving cavity (210) having the same number as the first receiving cavity (110). The first receiving cavity (110) and the second receiving cavity (210) correspond one-to-one in the vertical direction. The inner walls of the first receiving cavity (110) and the second receiving cavity (210) are hydrophilic layers. A first sealing membrane (300) for blocking the first receiving cavity (110) and the second receiving cavity (210) and a second sealing membrane (400) and a third sealing membrane (500) for sealing the top of the first receiving cavity (110) and the bottom of the second receiving cavity (210) are respectively provided between the first receiving cavity (110) and the second receiving cavity (210). The first cavity (110) is used to contain the antimicrobial drug for incubating with the target to be tested, and the second cavity (210) is used to contain the dye pad. After incubation, the target to be tested specifically binds to the binder and target-binding photons in the first cavity (110) and is transferred to the second cavity (210) when the first cavity (110) and the second cavity (210) are connected. After being filtered by the dye pad and the external force field, it is enriched at the bottom of the second cavity (210) for optical measurement.

2. The system for drug susceptibility testing according to claim 1, characterized in that: The composite perforated plate (100) includes an upper plate (120) having an upper receiving cavity (121) and a lower plate (130) having a lower receiving cavity (131). The upper receiving cavity (121) and the lower receiving cavity (131) correspond one-to-one in the vertical direction. A fourth sealing membrane (600) for blocking the upper receiving cavity (121) and the lower receiving cavity (131) is provided between the upper plate (120) and the lower plate (130). The upper cavity (121) contains an antimicrobial drug for incubating the target to be tested, and the lower cavity (131) contains a binding agent for specifically binding to the target to be tested.

3. The system for drug susceptibility testing according to claim 2, characterized in that: The width-to-depth ratio of the first receiving cavity (110) and the second receiving cavity (210) is greater than 3.3 / 10, or, The width-to-depth ratio of the upper cavity (121) and the second cavity (210) is greater than 3.3 / 10.

4. The system for drug susceptibility testing according to claim 1, characterized in that: The third sealing film (500) is made of transparent material with a light transmittance of not less than 90%, and the fluorescence emission intensity generated by the third sealing film (500) within the excitation wavelength range emitted by the optical measuring device is less than the detection limit of the optical measuring device.

5. The system for drug susceptibility testing according to claim 2, characterized in that: The first sealing membrane (300) is a metal membrane or a metal composite membrane, and the length of the first sealing membrane (300) is greater than the length of the composite orifice plate (100) to form an upwardly bendable heating wing (310) in the length direction of the composite orifice plate (100), so that the target to be tested incubated in the composite orifice plate (100) is heated by heat conduction through the heating wing (310); or, The fourth sealing membrane (600) is a metal membrane or a metal composite membrane, and the length of the fourth sealing membrane (600) is greater than the length of the upper plate (120) so as to form an upwardly bendable heating wing (310) in the length direction of the upper plate (120), and the target to be tested incubated in the upper plate (120) is heated by the heat conduction of the heating wing (310).

6. The system for drug susceptibility testing according to claim 2, characterized in that: The composite perforated plate (100) and the single-layer perforated plate (200) are made of rigid materials, or, The lower plate (130) is made of elastic material. The first sealing film (300) and the fourth sealing film (600) are respectively sandwiched between the lower plate (130) and the single-layer perforated plate (200) and between the upper plate (120) and the lower plate (130). The upper plate (120) and the single-layer perforated plate (200) form a pressing assembly.

7. The system for drug susceptibility testing according to claim 2, characterized in that: The system for drug sensitivity testing also includes a first pipetting module, which has a flat bottom and a hollow cavity formed from the bottom upwards. The inner wall of the hollow cavity and the outer surface of the first pipetting module are hydrophilic layers.

8. The system for drug susceptibility testing according to claim 7, characterized in that: The first pipetting module has a notch at the bottom side, and the notch communicates with the hollow cavity.

9. The system for drug susceptibility testing according to claim 2, characterized in that: The system for drug sensitivity testing also includes a second pipetting module, the outer surface of which is a hydrophilic layer.

10. The system for drug susceptibility testing according to any one of claims 1-9, characterized in that: The contact angle of the hydrophilic layer is less than 45°.

11. The system for drug susceptibility testing according to claim 7, characterized in that: The system for drug susceptibility testing operates using a first operating procedure, which includes: S100. Puncture the second sealing membrane (400) above the first receiving cavity (110), add the target to be tested into the first receiving cavity (110), the target to be tested is incubated with the antimicrobial drug in the first receiving cavity (110), and the incubated target to be tested specifically binds to the target-binding photon and the binding agent; S200. The first pipetting module is used to draw the target to be tested, which is bound to the target photon and the binder, into the hollow cavity, and after piercing the first sealing film (300) above the second receiving cavity (210), the target to be tested, which is bound to the target photon and the binder, is injected into the second receiving cavity (210). S300. The cartridge module is placed in the optical measurement device and an external force field is applied so that the target to be tested, which specifically binds to the target-binding photon and binder, is screened by the dye pad and enriched at the bottom of the second receiving cavity (210) for optical measurement.

12. The system for drug susceptibility testing according to claim 11, characterized in that: S100 includes: S110. After a preset time for adding the target to be tested into the first receiving cavity (110), target-binding photons and a binder are added into the first receiving cavity (110) respectively to obtain the target to be tested that specifically binds to the target-binding photons and the binder. S120. After aspirating the target material that has specifically bound to the target-binding photon and binder using the first pipetting module, the first sealing membrane (300) above the single-layer well plate (200) is punctured, and the target material that has specifically bound to the target-binding photon and binder is injected into the second receiving cavity (210); or, The composite perforated plate (100) includes an upper plate (120) having an upper receiving cavity (121) and a lower plate (130) having a lower receiving cavity (131). The upper receiving cavity (121) and the lower receiving cavity (131) correspond one-to-one in the vertical direction. A fourth sealing membrane (600) for blocking the upper receiving cavity (121) and the lower receiving cavity (131) is provided between the upper plate (120) and the lower plate (130). The upper cavity (121) is used to contain the antimicrobial drug for incubating the target to be tested, and the lower cavity (131) is used to contain the binding agent for specifically binding to the target to be tested; S100 includes: S110. Puncture the second sealing membrane (400) above the upper receiving cavity (121), add the target to be tested into the upper receiving cavity (121), and incubate the target to be tested with the antimicrobial drug in the upper receiving cavity (121); S120. Target-binding photons are added into the upward-facing cavity (121) to obtain the target to be tested that is bound to the target-binding photons; S130. The first pipetting module is used to draw in the target to be tested which is bound to the target photon, and the first pipetting module is used to puncture the fourth sealing membrane (600) above the lower receiving cavity (131) to inject the target to be tested which is bound to the target photon into the lower receiving cavity (131) and bind to the binder to obtain the target to be tested which is bound to the target photon and the binder.

13. The system for drug susceptibility testing according to claim 9, characterized in that: The system used for drug susceptibility testing operates using a second operating procedure, which includes: S100. Puncture the second sealing membrane (400) above the upper receiving cavity (121) and add the target to be tested into the upper receiving cavity (121) for incubation; S200. After incubation, target-binding photons are added into the upper cavity (121) to obtain the target to be tested that is bound to the target-binding photons; S300. Place the card box module into the centrifuge unit; S400. Using the second pipetting module, the fourth sealing membrane (600) and the first sealing membrane (300) are punctured in sequence, and the centrifugation unit is started to centrifuge the target to be tested, which is specifically bound to the target-binding photon and the binder, into the second receiving cavity (210); S500. The cartridge module is placed in the optical measurement device and an external force field is applied so that the target to be tested, which specifically binds to the target-binding photon and binder, is screened by the dye pad and enriched at the bottom of the second receiving cavity (210) for optical measurement.

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