Separating device of micro-pore plate for anti-biofilm dressing research and micro-pore plate

Abstract

The utility model discloses a separation device of a micro-pore plate for anti-biofilm dressing research and the micro-pore plate, which comprises a separation structure capable of being placed in a standard micro-pore plate, and the separation structure divides the internal space of the standard micro-pore plate into an upper layer and a lower layer, the separation structure does not influence the normal formation of bacteria / biological membranes at the bottom of the standard trace pore plate or at the liquid-gas junction; the separation structure is provided with a fixed position, and the fixed position is set to ensure that dressings or medicine carrying materials placed on the separation structure are not in direct contact with bacteria / biological membranes at the bottom or the liquid-gas junction of the standard micro-pore plate, and meanwhile, the dressings or the medicine carrying materials are placed in the middle of the culture solution. According to the utility model, the research on the inhibition effect of the dressing on the biological membrane is not interfered by external factors, and the accuracy and the reliability of an experimental result are improved.

Description

Technical Field

[0001] This invention relates to the field of biomedical research. More specifically, this invention relates to a separator and a microwell plate for use in research on anti-biofilm dressings. Background Technology

[0002] Chronic wound infection is a common problem in clinical medicine, and its treatment is challenging, often accompanied by the formation of bacterial biofilms. According to the Centers for Disease Control and Prevention (CDC), 60% of chronic wounds involve bacterial infection in the form of a biofilm. Bacterial biofilms are the primary form of bacteria in wound infections and a major factor contributing to the difficulty in healing chronic skin wounds. Furthermore, the formation of bacterial biofilms makes infection difficult to control effectively using conventional methods, thus increasing the complexity of treatment and patient suffering.

[0003] In terms of diagnosis, although images of bacterial biofilm morphology obtained through scanning electron microscopy and laser scanning confocal microscopy are considered the gold standard for diagnosing bacterial biofilms, this method has not yet been widely used in clinical practice due to its high level of expertise and complex operation.

[0004] In terms of treatment, early debridement and wound closure are effective methods to combat bacterial biofilms. However, due to a shortage of skin resources, patient intolerance to surgery, or other reasons, wound repair is sometimes not timely and effective. Once biofilm-associated wound infection occurs, traditional medications and dressings are often ineffective or have minimal effect on bacterial biofilm infection. Although some biofilm can be removed through debridement and / or washing, there is currently no method that can completely remove biofilm. Therefore, residual bacteria / biofilm have the potential to regenerate and form a mature biofilm over a period of time.

[0005] To address this problem, researchers have begun exploring new strategies to combat bacterial biofilm infections, one of which is the development and application of antibiofilm dressings. Antibiofilm dressings can prevent new bacteria from entering the wound and effectively kill bacteria in the wound bed and disrupt bacterial biofilms. However, research on antibiofilm dressings is still relatively limited, and most methods used are quite complex. These include isothermal calorimetry, colony-forming flow reactor (CDFR) and standard trickle flow reactor (DFR) culture, animal experiments, and case reports. Furthermore, there are currently no clinically available dressing products specifically designed to combat bacterial biofilms.

[0006] In in vitro studies of antibiofilm dressings, the conventional microplate method is limited because it is not suitable for research on antibiofilm dressings. Since the dressing sinks under gravity, it interferes with biofilm formation, increasing the unreliability of experimental results. Therefore, developing a microplate method suitable for antibiofilm dressing research has become an urgent problem to be solved.

[0007] In summary, to address the challenge of controlling bacterial biofilms in chronic wound infections and to advance the research and development of anti-biofilm dressings, a highly efficient research method for anti-biofilm dressings is needed. The numerous problems and challenges existing in current technologies are precisely the key issues that this invention aims to solve. Utility Model Content

[0008] One object of this invention is to solve at least the problems described above and to provide at least the advantages that will be explained later.

[0009] The purpose of this invention is to provide a separator for microplates used in research on anti-biofilm dressings. This device can be conveniently placed in a fixed position within a standard microplate for studying the inhibitory effect of dressings or drug-free materials on biofilms. Simultaneously, it avoids direct contact between the dressing and the mature biofilm at the bottom of the standard microplate or the liquid-gas interface of the standard microplate, ensuring that the dressing is placed in the middle of the culture medium and does not affect bacterial / biofilm formation. Furthermore, this invention also provides a method for conducting high-throughput anti-biofilm dressing research using this separator and microplate.

[0010] To achieve these objectives and other advantages according to the present invention, a separation device for a microporous plate for research on anti-biofilm dressings is provided, comprising:

[0011] A partition structure that can be placed inside a standard microplate, the partition structure dividing the internal space of the standard microplate into upper and lower layers;

[0012] The partition structure has a fixed position, which is configured to ensure that the dressing or drug-loaded material placed on the partition structure does not come into direct contact with the bacteria / biofilm at the bottom of the standard microplate or at the liquid-gas interface.

[0013] The partition structure includes:

[0014] A main structure, wherein the main structure is configured as a mesh structure at a predetermined height from the bottom of the standard micro-well plate;

[0015] At least three connecting posts extend outward along the edge of the mesh structure and overlap the top surface of the standard micro-perforation plate;

[0016] The mesh structure is configured to allow the placement of dressings or drug-loaded materials.

[0017] Preferably, the partition structure is detachable.

[0018] Preferably, the mesh structure consists of loosely spaced intersecting strips or grids.

[0019] Preferably, the height and position of the connecting column are configured such that when the separator is placed inside the standard microplate, the mesh structure is maintained at a predetermined height, ensuring that it does not directly contact the bacteria / biofilm at the bottom of the standard microplate or at the liquid-gas interface, while allowing the dressing or drug-loaded material to be placed on the separator and located in the middle of the culture medium.

[0020] Preferably, the separating device is one or multiple devices arranged in a row or in a display.

[0021] Preferably, the material of the separating device is a biocompatible material.

[0022] Preferably, the material of the separating device is a transparent material.

[0023] Preferably, one end of the connecting column extending outward is fixed to the same frame, which overlaps the top surface of the standard micro-orifice plate;

[0024] It also includes multiple pads, which are selectively placed between the frame and the top surface of the standard micro-orifice plate.

[0025] This invention provides a microplate for research on anti-biofilm dressings, comprising:

[0026] A partition device for the study of anti-biofilm dressings, which may be arranged in rows or in displays;

[0027] Standard microplate.

[0028] This utility model has at least the following beneficial effects:

[0029] First, this invention provides a new, high-throughput research tool for studying the inhibitory effect of dressings on biofilms by designing a simple microplate separation device. This tool not only simplifies experimental procedures but also improves experimental efficiency, providing strong technical support for the development of anti-biofilm dressings.

[0030] Secondly, this invention allows the dressing and drug-loaded material to be placed in the middle of the culture medium, thereby ensuring that the study of the inhibitory effect of the dressing on the biofilm is not affected by external factors, and improving the accuracy and reliability of the experimental results.

[0031] Third, the equipment and materials used in this invention are relatively simple, such as microdilution plates, crystal violet staining, XTT method, and detection equipment such as inverted fluorescence microscopes, live / dead bacteria staining-confocal microscopes, and electron microscopes, all of which can be found or purchased in conventional laboratories. This greatly reduces research costs, enabling more research institutions and individuals to participate in the research and development of anti-biofilm dressings.

[0032] Fourth, this separation device allows for the simultaneous processing of multiple samples, significantly improving screening efficiency. This is particularly important for the initial screening stage of anti-biofilm dressings, enabling the identification of dressings with potential anti-biofilm activity in a short time, providing strong support for subsequent in-depth research.

[0033] Fifth, this invention is not only applicable to studying the inhibitory effect of dressings on bacterial biofilms, but also to studying the cytotoxicity of dressings on adherent cells. Furthermore, this separating device can be customized according to experimental needs to adapt to different types of biofilm models and experimental conditions. Therefore, this invention has broad application prospects and potential market value.

[0034] Other advantages, objectives and features of this invention will be partly apparent from the following description, and partly understood by those skilled in the art through study and practice of this invention. Attached Figure Description

[0035] Figure 1 This is a three-dimensional structural diagram of the separating device placed on a standard micro-well plate, according to one of the technical solutions of this utility model.

[0036] Figure 2 This is a three-dimensional structural diagram of an array of multiple separating devices placed on a standard micro-well plate, one of the technical solutions of this utility model.

[0037] Figure 3 An example of a biofilm formed by Candida albicans in a 24-well plate without a separator;

[0038] Figure 4 An example of a biofilm formed by Candida albicans in a 24-well plate using a separator.

[0039] Explanation of the reference numerals in the accompanying drawings: 1. Mesh structure; 2. Connecting column; 3. Frame; 4. Standard micro-perforated plate. Detailed Implementation

[0040] The present invention will now be described in further detail with reference to the accompanying drawings, so that those skilled in the art can implement it based on the description.

[0041] It should be noted that, unless otherwise specified, the experimental methods described in the following embodiments are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified. In the description of this utility model, the orientation or positional relationship indicated by the terms is based on the orientation or positional relationship shown in the accompanying drawings, and is only for the convenience of describing this utility model and simplifying the description. It does not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of this utility model.

[0042] like Figure 1 and 2 As shown, this utility model provides a separating device for a microplate used in research on anti-biofilm dressings, comprising:

[0043] A partition structure that can be placed inside a standard microplate 4, the partition structure dividing the internal space of the standard microplate 4 into upper and lower layers. After the culture medium is added to the standard microplate 4, the partition structure is located in the culture medium. The partition structure does not affect the normal formation of bacteria / biofilm at the bottom of the standard microplate 4 or at the liquid-gas interface.

[0044] The partition structure has a fixed position, which is configured to ensure that the dressing or drug-loaded material placed on the partition structure does not directly contact the bacteria / biofilm at the bottom of the standard microplate 4 or at the liquid-gas interface, while placing the dressing or drug-loaded material in the middle part of the culture medium to avoid the influence of gravity on bacterial growth. Furthermore, the partition structure allows for sample addition, fixation, and staining operations, thereby fixing the sample and reducing the amount of biofilm formation damaged by human factors.

[0045] In the above technical solution, the dressing and drug-loaded material are placed on a separator, thus positioned in the middle of the culture medium, ensuring that the study of the dressing's inhibitory effect on biofilm is not affected by external factors. Simultaneously, this separator reduces human interference with biofilm formation, improving the accuracy and reliability of experimental results. The separator is also suitable for high-throughput screening of anti-biofilm dressings, enabling simultaneous testing of the anti-biofilm effects of multiple dressings or drug-loaded materials.

[0046] In another technical solution, the partition structure is detachable, making it easy to replace and clean.

[0047] In another technical solution, the separating structure includes:

[0048] A main structure is provided, wherein the main structure is configured as a mesh structure 1 at a certain height from the bottom of the standard microplate 4; wherein the mesh structure 1 is configured to allow dressings or drug-loaded materials to be placed therein, and the mesh structure 1 has no effect on the bacterial biofilm on the bottom of the standard microplate 4.

[0049] At least three connecting posts 2 extend outward along the edge of the mesh structure 1 and overlap the top surface of the standard micro-orifice plate 4 to enhance the stability of the mesh structure 1 within the standard micro-orifice plate 4. Preferably, there are three or four connecting posts 2. Preferably, one end of the outward-extending connecting post 2 is fixed to the same frame 3, which overlaps the top surface of the standard micro-orifice plate 4. Preferably, the frame 3 is a rectangular plate with a circular through hole in the middle, the diameter of which is larger than the diameter of the standard micro-orifice plate 4, and the upper end of the connecting post 2 is fixed to the side wall of the circular through hole; preferably, the frame 3, connecting posts 2, and mesh structure 1 are integrally formed. Preferably, multiple frames 3 are connected together in an array and are adapted to the standard micro-orifice plate 4.

[0050] In another technical solution, the mesh structure 1 consists of loosely spaced intersecting strips or grids to provide sufficient space for placing and manipulating dressings.

[0051] In another technical solution, the height and position of the connecting column 2 are configured such that when the separator is placed inside the standard microplate 4, the mesh structure 1 is kept at a predetermined height, ensuring that it does not directly contact the bacteria / biofilm at the bottom of the standard microplate 4 or at the liquid-gas interface, while allowing the dressing or drug-loaded material to be placed on the separator, thereby being located in the middle part of the culture medium.

[0052] In another technical solution, the separating device is one or multiple devices arranged in rows or arrays to accommodate different numbers of samples and facilitate batch processing of samples.

[0053] In another technical solution, one end of the connecting column 2 extending outward is fixed to the same frame 3, which overlaps the top surface of the standard micro-orifice plate 4.

[0054] It also includes multiple pads, which are selectively placed between the frame 3 and the top surface of the standard micro-orifice plate 4. Preferably, the pads are also made of transparent material.

[0055] In the above technical solution, the height of the mesh structure can be adjusted by changing the number of pads (0 to n, where n is a positive integer) to adapt to the growth characteristics of different bacteria in the culture medium. For example, some bacteria grow at the bottom of the microplate, while others grow at the gas-liquid interface, or the culture medium volume (the height formed by different volumes of culture medium in a standard microplate 4) varies.

[0056] In another technical solution, the separating device is made of a biocompatible material that will not adversely affect the growth or analysis of the bacterial biofilm. Biocompatible materials are selected from polyurethane, polylactic acid (PLA), polycaprolactone (PCL), polyglycolic acid (PGA), etc., and possess excellent mechanical strength, high elasticity, wear resistance, lubricity, fatigue resistance, biocompatibility, and processability. Preferably, the separating device is made of a transparent material to facilitate observation of biofilm formation.

[0057] This invention also provides a microplate for research on anti-biofilm dressings, comprising:

[0058] A partition device for the study of anti-biofilm dressings, which may be arranged in rows or in displays;

[0059] Standard microplate 4. Specifically, standard microplate 4 is selected from 24-well plates, 48-well plates, 96-well plates, etc. There are various models of standard microplates, and the selection should be based on a comprehensive consideration of factors such as experimental requirements, sample size, and experimental conditions. Furthermore, when using standard microplates, it is essential to follow correct operating procedures and precautions to ensure the accuracy and reliability of experimental results.

[0060] In the above technical solution, depending on the experimental needs, the separating device can be used alone, in rows, or in a display to work with the standard microplate 4 to process different numbers of samples.

[0061] In another technical solution, the mesh structure 1 adapted to the 24-well plate has a total area of ​​1.5 cm². 2 It contains 30 evenly distributed square holes, each with a side length of 0.2cm, and the overall thickness of the mesh structure is uniformly 0.5mm, aiming to optimize gas exchange efficiency and drug delivery of the dressing;

[0062] Mesh Structure 1 for 48-well plates: The total area of ​​the mesh structure is reduced to 0.71 cm². 2 To accommodate a denser perforated plate layout, it contains 10 evenly distributed square holes, each with a side length of 0.2cm. The overall thickness of the mesh structure is uniformly 0.5mm, maintaining the thinness of the structural design.

[0063] Mesh Structure 1 for 96-well plates: The total area of ​​the mesh structure is further reduced to 0.24 cm². 2 To meet the needs of higher-density cell culture, it is designed with 5 evenly distributed square wells, each with a side length of 0.2cm, to maintain the necessary cell survival environment. The thickness of the mesh structure is also set at 0.5mm to ensure a balance between the strength and functionality of the mesh structure.

[0064] The design of the aforementioned mesh structure aims to optimize gas exchange, nutrient delivery, and dressing drug delivery in cell culture plates with different well numbers by precisely controlling the number, size, and total area of ​​the pores.

[0065] In another technical solution, for a 24-well plate: the length of the connecting column 2 is set to 1.5 cm to optimize the fit to the conventional sample loading range of 0.5 to 1 mL for each well, ensuring that the sample liquid has sufficient surface area to contact the mesh structure 1;

[0066] For 48-well plates: the length of the connecting column 2 is adjusted to 1.44 cm to accommodate the typical sample volume range of 0.2 to 0.3 mL per well. Through precise length design, it is ensured that the sample liquid has sufficient surface area to contact the mesh structure 1.

[0067] For 96-well plates: the length of connecting column 2 is further shortened to 0.47 cm to match the standard sample loading volume of 100 μL per well;

[0068] The length of the connecting column 2 is designed based on the conventional sample loading volume of cell culture plates with different numbers of wells. It aims to precisely control the contact area between the liquid and the surface area of ​​the mesh structure 1, facilitating gas exchange, nutrient delivery, and drug delivery. Furthermore, the connecting column is adjustable, allowing its length to be adjusted according to specific experimental needs or the specifications of the cell culture plate, thus adapting to a wider range of applications.

[0069] This invention provides a method for using a separation device with a micro-orifice plate of this invention, comprising the following steps:

[0070] Step 1: Place the separator at the predetermined position within the standard micro-well plate 4;

[0071] Step 2: Place the dressing in the mesh structure 1 of the separating device;

[0072] Step 3: Perform sample addition, fixation, and staining operations within the separation device;

[0073] Step 4: The separation device maintains sample stability during operation and reduces human factors that could damage the biofilm formation. Depending on experimental needs, the separation device can be used individually, in rows, or in a display to process different numbers of samples.

[0074] In the above method, the dressing and drug-loaded material are placed within a separator, thus positioning them in the middle of the culture medium. This ensures that the study of the inhibitory effect of the dressing on the biofilm is not affected by external factors. Simultaneously, this separator reduces the impact of human factors on biofilm formation, improving the accuracy and reliability of the experimental results.

[0075] like Figure 3 and 4As shown, one embodiment of this utility model is illustrated using a 24-well plate Candida albicans biofilm as an example.

[0076] Experimental preparation: Prepare 24-well plates, the separator provided by this invention, Candida albicans culture, culture medium, and necessary staining agents and reagents.

[0077] Group setup: The 24-well plates were divided into two groups: a control group without separators and an experimental group using separators. Multiple replicate wells were set in each group to ensure the reliability of the experimental results.

[0078] Experimental procedure:

[0079] Control group without separators: Culture medium containing Candida albicans was added directly to the 24-well plates without any separator treatment.

[0080] Experimental group using the separator: The separator provided by the utility model was placed in a 24-well plate, and gauze was placed in the mesh structure 1 as a drug-carrying material. Then, culture medium containing Candida albicans bacterial solution was added to the 24-well plate.

[0081] The culture medium for Candida albicans suspension was SDB medium, i.e., Sabouraud dextrose liquid medium, in which the concentration of Candida albicans suspension was 1×10⁻⁶. 6 CFU / mL.

[0082] Culture and observation: After two groups of 24-well plates were incubated at 37℃ for 24 hours, the biofilm was stained with crystal violet and the formation of the biofilm was observed under a microscope.

[0083] Data analysis: Compare the biomass differences between the two groups of biofilms. If there is no significant difference in biomass between the two groups, it indicates that the separation device does not affect the formation of the biofilm, thus verifying the effectiveness of the separation device of this invention.

[0084] Precautions:

[0085] For different biofilm models, it is necessary to examine factors such as bacterial concentration, culture medium type, and culture time based on experimental research to ensure the stability of the biofilm model construction.

[0086] When conducting high-throughput anti-biofilm dressing research, the number and arrangement of the separators can be adjusted according to experimental needs to accommodate different sample processing requirements.

[0087] like Figure 3 and Figure 4As shown, there was no significant difference in biomass between the two groups of biofilms. The separator of this invention can also be used for the formation of biofilms from Pseudomonas aeruginosa, Burkholderia, Staphylococcus aureus, Propionibacterium acnes, etc., which can then be detected and quantified using crystal violet staining.

[0088] In summary, the microwell plate separation device and its application method in high-throughput anti-biofilm dressing research provided by this utility model are characterized by simple operation, high efficiency and high throughput, providing a powerful tool for the development of new antibacterial drugs and dressings.

[0089] Although the embodiments of this utility model have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for this utility model. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, this utility model is not limited to the specific details and the illustrations shown and described herein.

Claims

1. A partition device for micro-well plates used in anti-biofilm dressing research, characterized in that, The application relates to a device for separating a sample in a standard microplate, comprising: a separation structure which can be placed in a standard microplate, the separation structure separating the interior space of the standard microplate into two layers; the separation structure has a fixed position which is arranged to ensure that the dressing or drug-loaded material placed in the separation structure does not directly contact bacteria / biofilm at the bottom of the standard microplate or the liquid-gas interface; wherein the separation structure comprises: a main structure which is arranged as a mesh structure at a predetermined height from the bottom of the standard microplate; at least three connecting columns which extend outward along the edges of the mesh structure and are lapped on the top surface of the standard microplate; wherein the mesh structure is arranged to allow the dressing or drug-loaded material to be placed therein.

2. The divider for micro-well plates for anti-biofilm dressing research of claim 1, wherein, The separation structure is detachable.

3. The divider for micro-well plates for anti-biofilm dressing research of claim 1, wherein, The mesh structure is composed of sparse cross strips or grids.

4. The divider for micro-well plates for anti-biofilm dressing research of claim 1, wherein, The height and position of the connecting columns are configured to maintain the mesh structure at a predetermined height when the separation structure is placed in the standard microplate, to ensure that the mesh structure does not directly contact bacteria / biofilm at the bottom of the standard microplate or the liquid-gas interface, and to place the dressing or drug-loaded material on the separation structure and in the middle part of the culture solution.

5. The divider for micro-well plates for anti-biofilm dressing research of claim 1, wherein, The separation device is one or a plurality arranged in rows or columns.

6. The divider for micro-well plates for anti-biofilm dressing research of claim 1, wherein, The material of the separation device is biocompatible.

7. The divider for micro-well plates for anti-biofilm dressing research of claim 6, wherein, The material of the separation device is transparent.

8. The divider for micro-well plates for anti-biofilm dressing research of claim 1, wherein, The outwardly extending end of the connecting column is fixed to the same frame which is lapped on the top surface of the standard microplate; a plurality of pads are selectively arranged between the frame and the top surface of the standard microplate.

9. Micro-well plate for anti-biofilm dressing research, characterized in that, The application relates to a device for separating a sample in a standard microplate, comprising: one or a plurality arranged in rows or columns of the separation device according to any one of claims 1 to 8; a standard microplate.

Images