Device and method for detecting antibacterial adhesion of coating based on micrometer hole sensor

Abstract

The invention discloses a coating antibacterial adhesion detection device and detection method based on a micrometer hole sensor, the coating antibacterial adhesion detection device comprises a pool body, a to-be-detected film, a first electrode, a second electrode and a power supply, the to-be-detected film is vertically arranged on the cross section of the pool body, and the pool body is divided into a first liquid pool and a second liquid pool; the first liquid pool and the second liquid pool are respectively used for containing buffer solutions containing bacteria; the first electrode and the second electrode are respectively inserted into the first liquid pool and the second liquid pool and are respectively connected with positive and negative electrodes of a power supply; array micron holes are distributed in the to-be-detected film, and zwitterionic polymer brush coatings are attached to the inner walls of the micron holes. The current change is recorded by adopting an electrochemical measurement method (resistance pulse detection), when bacteria pass through holes, a hole passing signal is generated, and the current generated by micron holes modified with coatings and the current generated by micron holes unmodified with coatings are different, so that the cell adhesion condition can be represented.

Description

Technical Field

[0001] The present invention belongs to the technical field of detecting the antibacterial adhesion of coatings, and particularly relates to a device and method for detecting the antibacterial adhesion of coatings based on a micro-pore sensor. By means of micro-pores with different diameters, this method obtains the current signals when biological particles such as bacteria, cells, and exosomes pass through the pores through electrochemical measurement, so as to characterize the anti-adhesion of the coating, and can realize high-throughput, rapid, and miniaturized and convenient detection of anti-adhesion performance. Background Art

[0002] The statements here only provide background art related to the present invention and do not necessarily constitute prior art.

[0003] Medical catheters are extremely vulnerable to the adhesion of bacteria and cells during clinical applications, leading to the formation of biofilms, infections, and related complications (such as catheter-related urinary tract infections, bloodstream infections, etc.). To reduce the risk of bacterial adhesion, surface modification technologies have been widely studied, and among them, zwitterionic polymer brush coatings have attracted attention due to their excellent anti-fouling performance and biocompatibility. Such coatings form a physical barrier through strong hydration, which can significantly reduce the adsorption of proteins and non-specific cells, thereby inhibiting bacterial adhesion.

[0004] Currently, evaluating the amount of bacteria adhered to the surface of medical catheters usually relies on microscopic observation, plate colony counting method, or fluorescence labeling method. Among them, fluorescence staining method: bacteria are labeled with fluorescent dyes such as SYTO 9 and DAPI, and a fluorescence microscope or confocal laser scanning microscope (CLSM) is used to quantitatively analyze the number of adhered bacteria. Scanning electron microscope (SEM): directly observes the morphology and distribution of bacteria on the material surface, but requires sample fixation, drying, and coating, which may damage the biofilm structure. Plate colony counting method (CFU method): by eluting the adhered bacteria and culturing and counting them on an agar plate, but the operation is cumbersome, and the elution efficiency is greatly affected by the hydrophilicity of the coating.

[0005] However, these methods have the following key problems: strong equipment dependence: microscopes and fluorescence detection require expensive instruments, and sample preparation is complex, such as fluorescence labeling may interfere with bacterial activity; contradiction between sensitivity and throughput: high-sensitivity methods (such as QCM, CLSM) are costly and have low throughput, while high-throughput methods (such as turbidimetry) are difficult to detect low bacterial loads.

[0006] In addition, for the particularity of zwitterionic polymer brush coatings, traditional methods face additional challenges: for example, hydrophilic coatings interfere with detection: the high hydration of polymer brushes may lead to incomplete elution of bacteria, affecting the accuracy of CFU counting. Summary of the Invention

[0007] In view of the technical problems existing in the above-mentioned prior art, the present invention provides a device and method for detecting the antibacterial adhesion of a coating based on a micropore sensor. The method combines resistance pulse detection technology to measure the change in the current signal of an array of micropores when biological particles such as bacteria / cells / exosomes pass through the pores, so as to characterize the anti-adhesion of the coating.

[0008] To achieve the above object, the present invention is realized through the following technical solutions:

[0009] In a first aspect, the present invention provides a device for detecting the antibacterial adhesion of a coating based on a micropore sensor, which includes a cell body, a thin film to be detected, a first electrode, a second electrode and a power supply. The thin film to be detected is vertically arranged on the cross-section of the cell body, dividing the cell body into a first liquid cell and a second liquid cell;

[0010] The first liquid cell and the second liquid cell are respectively used to hold a buffer solution containing bacteria;

[0011] The first electrode and the second electrode are respectively inserted into the first liquid cell and the second liquid cell, and are respectively connected to the positive and negative electrodes of the power supply;

[0012] An array of micropores is distributed on the thin film to be detected, and an amphoteric ion polymer brush coating is attached to the inner wall of the micropores.

[0013] In some embodiments, the micropores include a small pore end and a large pore end. The diameter of the small pore end is 1-10 μm, and the diameter of the large pore end is 10-50 μm.

[0014] Preferably, the number of the micropores is 2-100.

[0015] In some embodiments, the interval between the micropores is 100-1000 μm, preferably 300-700 μm.

[0016] In some embodiments, it further includes a second cell body, a second thin film to be detected, a third electrode, a fourth electrode and a second power supply. The second thin film to be detected is vertically arranged on the cross-section of the second cell body, dividing the second cell body into a third liquid cell and a fourth liquid cell;

[0017] The third liquid cell and the fourth liquid cell are respectively used to hold a buffer solution containing bacteria;

[0018] The third electrode and the fourth electrode are respectively inserted into the third liquid cell and the fourth liquid cell, and are respectively connected to the positive and negative electrodes of the second power supply;

[0019] An array of micropores is distributed on the second thin film to be detected.

[0020] The current change is recorded by an electrochemical measurement method (resistance pulse detection). When bacteria pass through the pores under the drive of an external pressure, a pore passing signal will appear, and the currents generated by the micron pores modified with a coating and the micron pores without a modified coating are different, whereby the bacterial adhesion situation can be characterized. When bacteria pass through the micron pores modified with a coating, the measured current will decrease. For a coating with excellent anti-adhesion performance, the bacteria can smoothly pass through the pores, and at this time the current quickly returns to the original current value, as Figure 2 shown; when bacteria pass through the micron pores without a modified coating, the bacteria entering the pores first cause the current to decrease. And because it is easy to adhere to the inner wall surface of the micron pores and cannot smoothly pass through the pores. It is difficult for the current to return to the original value after the decrease, from which it can be judged that the bacteria have adhered to the pore wall.

[0021] In some embodiments, the material of the film to be detected or the second film to be detected is polyurethane PU, polyvinyl chloride PVC, or polyethylene terephthalate PET.

[0022] Preferably, the thickness of the film to be detected or the second film to be detected is 30-100 μm.

[0023] In a second aspect, the present invention provides a method for detecting antibacterial adhesion based on a coating-modified micron pore, comprising the following steps:

[0024] Prepare a conical array of micron pores on a polymer film;

[0025] Modify the inner wall of the micron pores with an anti-adhesive zwitterionic polymer brush coating to obtain a film to be detected;

[0026] Vertically fix the film to be detected on the cross-section of the cell body, divide the cell body into two liquid pools, and the two liquid pools are connected through the micron pores;

[0027] Inject buffer solutions containing bacteria into the two liquid pools, and the buffer solutions cover the micron pores;

[0028] Insert two electrodes into the two liquid pools respectively, apply a voltage to allow the bacteria to pass through the micron pores, and record the current when the bacteria pass through the pores;

[0029] At the same time, use a polymer film whose inner wall surface of the micron pores is not modified with a zwitterionic polymer brush coating, and use the same detection method to detect the current when the bacteria pass through the pores;

[0030] Characterize the cell adhesion situation according to the difference in the current when the bacteria pass through the pores of the two films.

[0031] The cell body is made of an inert non-conductive material, such as polychlorotrifluoroethylene (PCTFE), etc.

[0032] In some embodiments, the method for modifying the inner wall of the micron pores with an anti-adhesive zwitterionic polymer brush coating is:

[0033] First, the polymer film is pretreated. First, it is ultrasonically cleaned with deionized water, and then its surface is made to carry active groups through plasma treatment;

[0034] The polydopamine layer is formed using the dopamine-assisted method: Add a dopamine solution (1 - 5 mg / mL) to Tris buffer (pH 8.5), soak the film at room temperature for 10 - 14 hours to form a polydopamine layer, which provides a basis for subsequent grafting of the zwitterionic polymer brush coating;

[0035] Then, the film with micron pores is soaked in a solution containing the zwitterionic polymer brush. After 10 - 30 h, the film is taken out, rinsed, and dried in vacuo to obtain the film to be tested grafted with the zwitterionic polymer brush.

[0036] In some embodiments, the diameter of the micron pores modified with the zwitterionic polymer brush coating is substantially the same as that of the micron pores without the zwitterionic polymer brush coating, and the coating thickness is very small compared to the pore diameter.

[0037] In some embodiments, in the buffer solution containing bacteria, the concentration of bacteria is 1×10^5 - 9×10^9 CFU / mL.

[0038] In some embodiments, the applied voltage is -5V to 5V.

[0039] In some embodiments, the zwitterionic polymer brush coating is a poly(sulfobetaine) coating, a poly(sulfobetaine) coating, or a poly(phosphorylcholine) coating.

[0040] In some embodiments, the electrodes are all Ag / AgCl electrodes.

[0041] In some embodiments, the bacteria are Escherichia coli, Staphylococcus aureus, or Fusobacterium nucleatum.

[0042] The beneficial effects obtained from one or more of the above embodiments of the present invention are as follows:

[0043] The principle of the measurement method of the present invention is as follows: The measurement principle of the present invention is based on the resistive pulse technique, which converts the complex cell adhesion situation into a more easily measurable current signal for characterization. The anti-adhesion property of the coating can be directly characterized by current detection, without the need for large and complex devices such as microscopes and fluorescence tests. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] The accompanying drawings forming a part of this specification are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention.

[0045] Figure 1Schematic diagram of the current measurement system structure of the array of micro - pores according to the embodiment of the present invention;

[0046] Figure 2 Schematic diagram of the current signal generated when bacteria pass through the micro - pores in the embodiment of the present invention.

[0047] Wherein: 1 - electrode, 2 - micro - pore array, 3 - thin film, 4 - liquid cell. Detailed implementation manners

[0048] It should be noted that the following detailed descriptions are all illustrative and are intended to provide further explanations of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0049] The present invention will be further described below in conjunction with embodiments.

[0050] Embodiment

[0051] As Figure 1 shown, a coating antibacterial adhesion detection device based on a micro - pore sensor includes a cell body, a thin film to be detected, a first electrode, a second electrode, and a power supply. The thin film to be detected is vertically arranged on the cross - section of the cell body, dividing the cell body into a first liquid cell and a second liquid cell;

[0052] The first liquid cell and the second liquid cell are respectively used to hold buffer solutions containing bacteria;

[0053] The first electrode and the second electrode are respectively inserted into the first liquid cell and the second liquid cell, and are respectively connected to the positive and negative electrodes of the power supply;

[0054] An array of micro - pores is distributed on the thin film to be detected, and an amphoteric ion polymer brush coating is attached to the inner wall of the micro - pores.

[0055] The micro - pores include a small - hole end and a large - hole end. The diameter of the small - hole end is 6μm, and the diameter of the large - hole end is 10μm.

[0056] The number of the micro - pores is 6, and the interval between the micro - pores is 500μm;

[0057] It further includes a second cell body, a second thin film to be detected, a third electrode, a fourth electrode, and a second power supply. The second thin film to be detected is vertically arranged on the cross - section of the second cell body, dividing the second cell body into a third liquid cell and a fourth liquid cell;

[0058] The third liquid cell and the fourth liquid cell are respectively used to hold buffer solutions containing bacteria;

[0059] The third electrode and the fourth electrode are respectively inserted into the third liquid cell and the fourth liquid cell, and are respectively connected to the positive and negative electrodes of the second power supply;

[0060] The second thin film to be detected is distributed with an array of micron holes.

[0061] The materials of the thin film to be detected and the second thin film to be detected are polyurethane (PU), and the thickness is 50 μm.

[0062] The method for detecting the anti-bacterial adhesion of the coating based on the micron hole sensor using the device in Example 1 is as follows: Prepare a conical array of micron holes on the polymer thin film;

[0063] Modify the inner wall of the micron holes with an anti-adhesion zwitterionic polymer brush coating (poly-sulfobetaine coating) to obtain the thin film to be detected;

[0064] First, pre-treat the polymer thin film. First, ultrasonically clean it with deionized water, and then make its surface carry active groups through plasma treatment;

[0065] Use the dopamine-assisted method to form a polydopamine layer: Add a 2 mg / mL dopamine solution to Tris buffer (pH 8.5), soak the thin film at room temperature for 12 hours to form a polydopamine layer, which provides a basis for subsequent grafting of the zwitterionic polymer brush coating;

[0066] Then soak the thin film with micron holes in a solution containing 0.1 g / ml sulfobetaine. After 24 hours, take out the thin film, rinse it and dry it in vacuum to obtain the thin film to be tested grafted with the zwitterionic polymer brush.

[0067] Vertically fix the thin film to be detected on the cross-section of the cell body, divide the cell body into two liquid pools, and the two liquid pools are connected through the micron holes;

[0068] Inject buffer solutions containing bacteria (Escherichia coli) into the two liquid pools respectively, and the buffer solutions cover the micron holes;

[0069] Insert two electrodes into the two liquid pools respectively, apply a voltage of 2 V to allow the bacteria to pass through the micron holes, and record the current when the bacteria pass through the holes;

[0070] At the same time, use a polymer thin film whose inner wall surface of the micron holes is not modified with a zwitterionic polymer brush coating, and use the same detection method to detect the current when the bacteria pass through the holes;

[0071] The cell body is made of an inert non-conductive material, such as polychlorotrifluoroethylene (PCTFE), etc.

[0072] The diameter of the micron holes modified with the zwitterionic polymer brush coating is basically the same as that of the micron holes not modified with the zwitterionic polymer brush coating, and the coating thickness is very small compared with the pore diameter.

[0073] In the buffer solution containing bacteria (Escherichia coli), the concentration of Escherichia coli is 10^9 CFU / mL.

[0074] All the electrodes are Ag / AgCl electrodes.

[0075] The method of electrochemical measurement (resistance pulse detection) is used to record the current change. When bacteria pass through the pores, a pore passing signal will appear, and the pore passing times through the micron pores modified with a coating and the micron pores without a modified coating are different. Thus, the cell adhesion situation can be characterized. The longer the pore passing time, the stronger the adhesion of the coating, that is, the worse the anti-adhesion property.

[0076] When bacteria pass through the micron pores modified with a coating, it will cause the measured current to decrease. This is because in the confined space of the micron pores, when bacteria pass through, the pore channels will be partially blocked. This blockage reduces the effective cross-sectional area of fluid flow, resulting in an instantaneous increase in the resistance inside the micron pores. According to Ohm's law V = IR, at a constant voltage, the increase in resistance R will directly cause a decrease in current I. This principle is similar to the Coulter principle, that is, the characteristics of particles are analyzed by detecting the resistance change when particles pass through small pores.

[0077] For a coating with excellent anti-adhesion performance, bacteria smoothly pass through the pores after a time of Δt. At this time, the interference with ion transport in the pores disappears, and the current quickly returns to the original current value. Its I-t curve is as Figure 2 shown in a. The longer the pore passing time Δt, the longer the time for bacteria to pass through the pores, and the stronger the adhesion of the coating. When bacteria pass through the micron pores without a modified coating, the bacteria entering the pores first cause the current to decrease. Since it easily adheres to the inner wall surface of the micron pores and cannot smoothly pass through the pores, the bacteria continuously block the pores, affecting the ion transport in the pores, and it is difficult for the current to return to the original value after the decrease. Its I-t curve is as Figure 2 shown in b. From this, it can be judged that the bacteria have adhered to the pore wall.

[0078] A bacterial pore passing experiment is carried out to detect the anti-adhesion performance of the coating according to the above electrical signal characteristics after bacteria pass through the pores. Each group of experiments measures the current 20 times repeatedly to avoid the influence of experimental contingency on the measurement results. When bacteria pass through the pores, the current decreases by ΔI and returns to its original value after a time of Δt. The measured current-time (I-t) curve is as Figure 2 shown in a, then it is determined that the anti-adhesion performance of the coating in this experiment is "effective"; when the total current decreases by ΔI, and the current does not return to the original value due to bacteria adhering to the pore wall, its I-t curve is as Figure 2 shown in b, then it is considered that the coating cannot effectively resist adhesion and is determined to be "invalid".

[0079] Statistically analyze the proportion of "effective" among 20 current measurements. When it reaches 90%, it is determined that the coating has excellent anti-adhesion. Coatings with the proportion of "effective" reaching 75% but not exceeding 90% are determined to have general anti-adhesion. Coatings with the proportion of "effective" at 75% or below are determined to have poor anti-adhesion.

[0080] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A coating anti-bacterial adhesion detection device based on a micropore sensor, characterized in that: It includes a cell body, a film to be detected, a first electrode, a second electrode and a power supply, wherein the film to be detected is vertically arranged on the cross section of the cell body to separate the cell body into a first liquid pool and a second liquid pool; The first liquid pool and the second liquid pool are respectively used to hold a buffer solution containing bacteria; The first electrode and the second electrode are respectively inserted into the first liquid pool and the second liquid pool, and are respectively connected to the positive and negative electrodes of the power supply; An array of micrometer holes is distributed on the film to be detected, and a zwitterionic polymer brush coating is attached to the inner wall of the micrometer hole.

2. The device for detecting antibacterial adhesion of coatings based on micropore sensors according to claim 1, characterized in that: The micron pore includes a small pore end and a large pore end, the diameter of the small pore end is 1-10 μm, and the diameter of the large pore end is 10-50 μm; Preferably, the number of the micropores is 2-100; Preferably, the interval between the micropores is 100-1000 μm, preferably 300-700 μm.

3. The device for detecting antibacterial adhesion of coatings based on micropore sensors according to claim 1, characterized in that: It also includes a second cell body, a second film to be detected, a third electrode, a fourth electrode and a second power supply, wherein the second film to be detected is vertically arranged on the cross section of the second cell body to separate the second cell body into a third liquid pool and a fourth liquid pool; The third liquid tank and the fourth liquid tank are respectively used for holding buffer solutions containing bacteria; The third electrode and the fourth electrode are respectively inserted into the third liquid tank and the fourth liquid tank, and are respectively connected to the positive and negative electrodes of the second power supply; The second film to be detected is provided with array micrometer holes.

4. The device for detecting antibacterial adhesion of coatings based on micropore sensors according to claim 1, characterized in that: The material of the film to be detected or the second film to be detected is polyurethane PU, polyvinyl chloride PVC, or polyethylene terephthalate PET; Preferably, the thickness of the film to be detected or the second film to be detected is 30-100 μm.

5. A method for detecting the anti-bacterial adhesion of a coating based on a micropore sensor, characterized in that: The steps include: Fabrication of conical array micrometer holes on polymer films; The inner wall of the micropore is modified with a zwitterionic polymer brush coating having anti-adhesion properties to obtain a film to be detected; The film to be tested is fixed vertically on the cross section of the cell body, and the cell body is divided into two liquid pools, which are connected by micrometer holes; Buffer containing bacteria is injected into the two liquid pools respectively, and the buffer does not cover the micron hole; Two electrodes are inserted into two liquid pools respectively, and voltage is applied to make bacteria pass through the micron hole, and the current when the bacteria pass through the hole is recorded; At the same time, a polymer film without modified zwitterionic polymer brush coating on the inner wall surface of the micropore was used to detect the current when bacteria passed through the pore using the same detection method; The cell adhesion is characterized by the difference in current when bacteria pass through the two films.

6. The method for detecting anti-bacterial adhesion of coatings based on micropore sensors according to claim 5, characterized in that: The method for modifying the inner wall of the micropore with a zwitterionic polymer brush coating having anti-adhesion properties is as follows: First, the polymer film is pretreated by ultrasonic cleaning with deionized water and then treated with plasma to provide active groups on its surface; Formation of polydopamine layer using dopamine-assisted method: Add dopamine solution to Tris buffer and soak the film at room temperature for 10-14 hours to form a polydopamine layer; Then, the film with micron pores is immersed in a solution containing zwitterionic polymer brushes, and after 10-30 hours, the film is taken out, rinsed and vacuum dried to obtain a film to be tested grafted with zwitterionic polymer brushes; Alternatively, the diameter of the micropores modified with the zwitterionic polymer brush coating is the same as the diameter of the micropores not modified with the zwitterionic polymer brush coating.

7. The method for detecting anti-bacterial adhesion of coatings based on micropore sensor according to claim 5, characterized in that: In the bacteria-containing buffer, the concentration of bacteria is 1×10^5-9×10^9 CFU / mL.

8. The method for detecting anti-bacterial adhesion of coatings based on micropore sensors according to claim 5, characterized in that: The applied voltage is -5V to 5V.

9. The method for detecting anti-bacterial adhesion of coatings based on micropore sensor according to claim 5, characterized in that: The zwitterionic polymer brush coating is a polysulfobetaine coating, a polysulfobetaine coating or a polyphosphorylcholine coating; Alternatively, the electrodes are all Ag / AgCl electrodes.

10. The method for detecting anti-bacterial adhesion of coatings based on micropore sensor according to claim 5, characterized in that: The bacteria are Escherichia coli, Staphylococcus aureus or Bacillus nucleatus.

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