• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
  • 引用文献
  • 法律状态
  • 优先权
    • 专利摘要:
    The invention relates to a pro-biofilm coating applied by polymerization of a precursor by cold atmospheric plasma on a substrate. The coating has a roughness that promotes the creation of more than 100% biofilm on the substrate, 100% biofilm being produced on the same substrate lacking this pro-biofilm coating. The invention also relates to a method of producing said pro-biofilm coating and to a substrate coated therewith. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2720026A1
    申请号:ES201930265
    申请日:2019-03-25
    公开日:2019-07-17
    发明作者:Dominguez Yolanda Saenz；Elias Fernando Alba；Martinez Maria Lopez；Garcia Elisa Sainz；Fernandez Carmen Lozano；Vidal Rodolfo Mugica；Bezares Beatriz Rojo；Marcos Ana Gonzalez；Regalado Paula Toledano；Fraguas Ignacio Muro
    申请人:Universidad de La Rioja；Fundacion Rioja Salud；
    IPC主号:
    专利说明:

    [0001] PRO-BIOFILM COATING, METHOD FOR YOUR PRODUCTION AND
    [0002]
    [0003] Field of the Invention
    [0004] The present invention relates to the field of bacterial control, more specifically the control of bacterial biofilms, and in particular to the promotion of the appearance of bacterial biofilms.
    [0005]
    [0006] Background of the invention
    [0007] Biofilm is a group of microorganisms surrounded by a matrix of extracellular polymeric substances that include water, polysaccharides, proteins, lipids, nucleic acids and other biopolymers. This extracellular matrix favors the adhesion of microorganisms to surfaces, protects them from adverse environmental conditions and antimicrobial agents, and helps them capture nutrients from the environment to facilitate microbial proliferation.
    [0008] The development of a biofilm takes place in the following stages: an initial fixation stage in which bacterial cells adhere and colonize solid surfaces. It is followed by the stages of proliferation and maturation in which, by activating intercellular communication systems, microcolonies and the extracellular matrix begin to form, reaching an optimum population density and a mature biofilm structure. Finally, the dispersion stage occurs in which bacterial cells or biofilm components can separate from it, migrate and colonize other surfaces, which is a persistent source of dissemination and contamination.
    [0009] In most cases, the appearance of a biofilm involves a series of problems and consequences. harmful Therefore, techniques focused mainly on the prevention and elimination of biofilm, such as antibiofilm coatings or procedures, are used and developed.
    [0010] The biofilm deteriorate products or surfaces on which they develop, but also, when they are formed by pathogenic microorganisms, they are a matter of great concern in the food, pharmaceutical or clinical industry since they represent a source of product contamination and a risk for public health. For example, in the case of food processing and processing equipment; as well as on the contact surfaces with them, the elimination of pathogenic biofilm plays a vital role in guaranteeing an optimal state of food microbiological quality. Biofilm acts as a reservoir that can protect or release pathogenic bacteria in the free (planktonic) state, ubiquitous organisms that are especially difficult to control, resulting in bacterial persistence in the production plant and episodes of cross-contamination of food, with obvious risks for food security.
    [0011] However, there are certain situations in which the generation of more biofilm is an advantage. An example is the case of the medical industry, for diagnostic purposes and treatment choice. When a pathogenic microorganism (which causes a disease) is studied in the laboratory (in vitro), it is desirable that this microorganism behaves similarly to what it would do inside the patient (in vivo ). Traditionally, microbiology laboratories have focused their activities on isolating and conducting sensitivity studies on bacteria in planktonic form. However, extrapolate antimicrobial sensitivity data (including biocides) to that same bacterium when it is growing inside a biofilm, leads to therapeutic failures, industrial technical problems, recurrence of chronic infections, increases the chances of contamination of food products or industrial surfaces, among many other problems.
    [0012] For in vitro studies of microorganisms it would be desirable to have a simple, manageable model that is capable of providing adequate therapy in vivo. In this way problems would be avoided by choosing the wrong antibiotic agent or improper doses. In addition, it would be desirable to have a pro-biofilm coating that would allow biofilm to be produced in said model quickly so as to accelerate the aforementioned in vitro studies.
    [0013] Other cases in which it may be desirable to encourage the emergence of a specific biofilm are the food industry and biodegradation or environmental bioremediation processes. In these cases, it would be desirable to have a pro-biofilm coating that, when applied for example to a fermentation tank and inoculated with a beneficial microorganism, allows the production of a biofilm thereof to be encouraged. Said beneficial microorganism will in turn protect the fermentation tank against colonization by other harmful microorganisms.
    [0014] JP2013173715A discloses a method of biofilm formation by plasma irradiation treatment of a surface such as a polycarbonate plate. Plasma treatment produces a chemical modification of polycarbonate that favors the adhesion of microorganisms that can form a first biofilm. This first biofilm favors the adhesion of new microorganisms that will form a new biofilm. However, the chemical modification of the surface by plasma treatment depends on the material that constitutes said surface, and therefore not all surfaces can be favorably modified by said method. In addition, the adhesive capacity of species generated on the surface of polycarbonate by plasma treatment gradually decreases over time.
    [0015] Therefore, it would be desirable to have a pro-biofilm coating that favors the production of biofilm regardless of the surface on which said coating is applied and that retains its probiofilm properties over time.
    [0016]
    [0017] Summary of the invention
    [0018] To solve the problems indicated above, the present invention discloses, in a first aspect thereof, a pro-biofilm coating applied by polymerization of a precursor by cold atmospheric plasma on a substrate. The coating according to the present invention has a roughness such that it promotes the creation of more than 100% biofilm on the substrate, with 100% biofilm being produced on the same substrate lacking said pro-biofilm coating.
    [0019] In a second aspect, the present invention discloses a method of producing a probiofilm coating as defined in the first aspect of the invention. The method comprises applying a cold atmospheric plasma flow and precursor material on a substrate surface to be coated until a roughness is obtained that promotes the creation of more than 100% biofilm on the substrate.
    [0020] The invention also relates to a substrate that is It is coated by a pro-biofilm coating according to the first aspect of the invention.
    [0021]
    [0022] Brief description of the drawings
    [0023] The present invention will be better understood with reference to the following drawings illustrating preferred embodiments of the present invention, provided by way of example, and which should not be construed as limiting the invention in any way:
    [0024] Figure 1 is a schematic cross-sectional view of a device for carrying out a pro-biofilm coating production method according to the preferred embodiment of the present invention.
    [0025] Figure 2 is a graph showing the relationship between passes, coating roughness and% of biofilm produced with respect to the control, using polystyrene as a substrate and (3-aminopropyl) triethoxysilane (APTES) as a precursor material, according to a preferred embodiment of the present invention
    [0026] Figures 3A and 3B show images of atomic force microscopy (AFM) and scanning electron microscopy (SEM), respectively, of coatings obtained according to a preferred embodiment of the present invention, in which the percentage of biofilm is also indicated. obtained with respect to control.
    [0027] Figure 4 is a graph showing the relationship between the roughness of the coating and the% of biofilm obtained with respect to the control, using polystyrene as a substrate and APTES as a precursor material, according to a preferred embodiment of the invention.
    [0028] Figure 5 is a graph showing the relationship between the roughness of the coating and the% of biofilm obtained with respect to the control according to another preferred embodiment of the invention in which a mixture of polyethylene glycol methyl ether methacrylate (PEGMA) and isopropanol (IPA) is used as the precursor material.
    [0029] Figure 6 shows SEM images of coatings obtained according to another preferred embodiment of the present invention, in which the percentage of biofilm obtained with respect to the control is also indicated.
    [0030] Figure 7 is a graph showing the ratio of the atomic percentage of carbon (C), oxygen (O), silicon (Si) and nitrogen (N) with respect to the number of passes according to an embodiment of the present invention.
    [0031] Figure 8 shows graphs depicting the deconvolution of the high resolution carbon spectrum for different numbers of passes according to a preferred embodiment of the invention.
    [0032] Figure 9 is a graph depicting the relationship between the roughness of the coating and the contact angle with the water of coatings according to a preferred embodiment of the invention.
    [0033] Figure 10 is a graph showing bacterial growth as a function of time with different coatings obtained according to a preferred embodiment of the present invention.
    [0034] Figure 11 is a graph showing the amount of biofilm produced over time with a coating according to a preferred embodiment of the present invention compared to the control.
    [0035]
    [0036] Detailed description of the preferred embodiments
    [0037] Plasma is the state that a gas reaches when it is given an amount of energy that manages to ionize its molecules and atoms. That is, the passage of matter from a gaseous state to a plasma state occurs through a dissociation of molecular bonds, accompanied by an increase or decrease in the electrons of atoms, which results in the formation of ions with positive or negative charge. Depending on whether or not there is a thermal equilibrium between the plasma particles, the thermal plasma is distinguished from the cold.
    [0038] A cold or unbalanced plasma is one in which the temperature of the electrons (105-5000 ° C) is much higher than that of the heavier particles (neutral particles and ions), which are at temperatures close to that of the ambient (25-100 ° C). In this way, the temperature of a cold plasma is generally maintained below 100 ° C, which allows its use in surface treatments on a wide variety of materials without causing their deterioration due to excessive heating.
    [0039] The cold plasma generation can be carried out at atmospheric pressure in an open environment, that is, it does not require the use of vacuum systems or chambers within which specific conditions are established. These characteristics provide cold atmospheric plasma technology with great versatility, relative simplicity and low cost. From the point of view of its industrial application, plasma has become an important tool for carrying out a multitude of surface treatments.
    [0040] One of the main modifications to which the surface of a plasma-treated substrate can be subjected is "plasma polymerization." This modification consists in the deposition of fine coatings using liquid monomers as precursors through their exposure to plasma flow.
    [0041] In a specific embodiment of the present invention, for the application of pro-biofilm coatings on Petri dishes under study, the plasma polymerization method was used using an unbalanced or cold atmospheric plasma equipment (APPJ). The selection of the different precursors to be deposited and plasma operating parameters (input variables) determine the specific characteristics of the coating obtained. This versatility is of vital importance when it is intended to modify in a specific way the physical-chemical properties of surface coatings.
    [0042] Another feature that makes this technology very attractive is that it operates at room temperature and at atmospheric pressure, which greatly facilitates its possible application in existing production lines.
    [0043] The APPJ equipment used (see Figure 1) consists of two coaxial electrodes (10, 12), among which the gas (14) circulates to generate the plasma (in this specific case, nitrogen (N 2 ) was used with a flow 80 slm). The inner electrode (10) is grounded, while the outer electrode (12) is excited with a certain frequency (high voltage current) by means of a generator (16) with a power of 300 W. Through the inner electrode ( 10) the precursor material (18) (1.5 slm of N 2 that atomizes and transports the liquid precursor material) is introduced to the plasma actuation zone (20).
    [0044] According to the preferred embodiment of the present invention, and still referring to Figure 1, the pro-biofilm coating production method first comprises the step of activating the surface of the Petri dish (22) (or other substrate to be coated ) by means of a plasma jet (for example, N 2 plasma) without polymerizable precursor material. This activation and cleaning of the surface is preferably carried out by means of 4 plasma passes. The person skilled in the art will understand that, in other embodiments According to the invention, this previous stage of surface activation can be omitted.
    [0045] Next, the actual application step of a flow (20) of cold atmospheric plasma (14) and precursor material (18) on the surface of the Petri dish (22) is performed.
    [0046] In addition, the method preferably also comprises the step of carrying out, simultaneously with the application of the plasma flow, a relative displacement between the substrate to be coated and the plasma flow to cover the entire surface of the substrate.
    [0047] The plasma flow (20) projected on the surface (which transforms, transports and projects the precursor material on the base of the Petri dish) is approximately 10 mm in diameter. Therefore, for the homogeneous application of the coating (preferred embodiment) throughout the base of the Petri dish (22), this jet must be displaced throughout the entire base (30 mm in diameter). Since the plasma flow (80 slm of N 2 ) and of the precursor (1.5 slm of N2 that atomizes and transports the precursor material) are constant, to apply a homogeneous coating, this displacement must be carried out at a constant speed.
    [0048] To do this, the Petri dish (22) rotates and moves (on an axis) simultaneously with the application of the plasma flow (20). Meanwhile, the plasma application equipment remains fixed. Obviously, according to alternative embodiments, the Petri dish can remain fixed while a plasma application nozzle is displaced. According to another additional alternative, both the Petri dish and the plasma application nozzle can move simultaneously with respect to each other.
    [0049] The linear (tangential) velocity of coating (Vt) is constant (10 mm / s). Every time a lap is covered (at a certain radius) the turning speed (W) is modified (reduced at the edge, increased in the center) so that the linear speed (Vt) always remains constant. For each complete turn made by the Petri dish (22), it travels along the axis a certain advance distance (24). Keeping the linear velocity (Vt) constant allows the applied coating to be homogeneous.
    [0050] It is defined as "pass" every time the plasma jet completely covers the base of the Petri dish. Different coatings have been obtained by applying from 2 to 96 passes, as will be described hereinafter.
    [0051] Following the method described above, various pro-biofilm coatings were made using either precursor material either (3-aminopropyl) triethoxysilane (APTES) or a mixture in various proportions of polyethylene glycol methyl ether methacrylate (PEGMA, MW: 500) and isopropanol (IPA). Likewise, Petri dishes of either 30 mm diameter polystyrene (PS) (previously treated with plasma and uncoated) or 90 mm diameter glass were used. Table 1 below details the properties of the various samples of coatings made according to the method of the present invention:
    [0052]
    [0053] Table 1
    [0054] Sample Past Precursor Substrate
    [0055] 0p 0 PS -2p 2 PS APTES '(100%)
    [0056] 4p 4 PS APTES (100%)
    [0057] 12p 12 PS APTES (100%)
    [0058] 24p 24 PS APTES (100%)
    [0059] 4 8p 48 PS APTES (100%)
    [0060] 72p 72 PS APTES (100%)
    [0061] 9 6p 96 PS APTES (100%)
    [0062] 0V 0 Glass -4 8V 48 APTES Glass (100%)
    [0063]
    [0064]
    [0065] For the quantification of the percentage of biofilm produced (with respect to that produced by an uncoated Petri dish control) generated by the Pseudomonas aeruginosa bacteria on the samples studied, a fluorescein diacetate (FDA) technique was used. This technique, according to the literature, is used both to measure microbial activity within the total biofilm (Adam and Duncan, 2001; Schnürer and Rosswall, 1982; Taylor et al., 2001), and to quantify biofilm biomass (Honraet et al., 2005; Prieto et al., 2004). The quantification of the biofilm was carried out after 24 hours of incubation at 37 ° C.
    [0066] As can be seen in Figure 2, in the case of APTES coatings applied on PS (polystyrene) there is a direct relationship between the number of passes, the roughness of the coating and the% of biofilm produced with respect to the control (substrate of PS without coating). As the number of passes increases, both the roughness and the% biofilm increase. As the passes occur, the morphology of the coating goes from being practically smooth (Ra = 6.69 nm with 2 passes) to presenting a granular structure (Ra = 105 nm with 96 passes) typical of coatings based on oxides of silicon (APTES is a siloxane that when polymerized by plasma produces silicon oxides). This granular structure It can be identified in the AFM and SEM images (see Figures 3A and 3B) and is what justifies the increase in the roughness of the coating (Figure 2).
    [0067] Figure 4 shows the relationship between the roughness of APTES-based coatings and the% of biofilm produced in each of them. Coatings that generate less biofilm than that produced in the control sample (the one that has not been coated, 0p, equivalent to 100% biofilm) are defined as anti-biofilm (<100%), while those that generate more than 100 % biofilm are defined as probiofilm (> 100%).
    [0068] Taking into account the above, it is defined as "pro-biofilm limit" that roughness from which a biofilm greater than 100% is obtained (with respect to that generated in the 0p control).
    [0069] In Figure 4 it can be identified that in coatings applied on PS and obtained by plasma polymerization based on APTES, the probiofilm limit is a minimum roughness Ra of approximately 10 nm (Ra: average arithmetic roughness).
    [0070] Then, the above method was repeated using as a substrate a glass Petri dish, instead of PS. The glass plates have a roughness very similar to those of PS (uncoated PS; 0p ^ Ra = 4.87 ± 1.13 nm and uncoated glass; 0V ^ Ra = 4.67 ± 0.98 nm).
    [0071] A glass plate was coated with 48 passes.
    [0072] Otherwise, the coating method was identical to that used with the PS Petri plates described above and APTES was used as a liquid precursor material.
    [0073] It was determined experimentally that the coating applied on the glass plate with 48 passes (48V) presented a roughness and% of biofilm with respect to the control (Ra = 33.2 ± 1.27 nm and 368 ± 18%) very similar to the sample 24p with the coating applied on a PS plate with 24 passes (Ra = 33.4 ± 1.3 nm and 300 ± 3.5%).
    [0074] According to this, it can be concluded that those APTES based coatings that have a very similar roughness, generate a similar% biofilm regardless of the substrate on which it has been applied. The only difference that has existed in this case is that, to obtain on the glass a coating with a roughness similar to that of the PS, it has been necessary to apply more passes (48 passes for the glass and 24 passes for the PS).
    [0075] According to all of the above, it can be concluded that a coating based on APTES, with a certain roughness, generates a similar amount of biofilm, regardless of the substrate (polystyrene, glass or other) on which it is applied.
    [0076] Next, the coating method described above was repeated using, instead of APTES, mixtures of PEGMA (MW500) and IPA in different concentrations.
    [0077] In this case, the number of passes was set to 2 and the weight percentage of PEGMA was varied in a dilution of PEGMA and IPA (5, 10 and 100% by weight).
    [0078] Figure 5 shows the relationship between roughness and% biofilm of coatings applied on PS using APTES (previous embodiment) and PEGMA + IPA (present embodiment) as liquid precursor materials. This figure shows that PEGMA based coatings require greater roughness to achieve the amount of biofilm generated with APTES based coatings. In this sense, although the pro-biofilm limit of APTES coatings is approximately 10 nm (roughness from which a biofilm greater than 100% with respect to the control is obtained), in the case of PEGMA based coatings the pro-biofilm limit is 160 nm (Figure 5).
    [0079] From these results, it can be concluded that it is not possible to define a common pro-biofilm limit to coatings based on different precursor materials. One of the possible reasons that justify the non-existence of a common pro-biofilm limit may be related to the fact that each precursor material produces a coating with a specific surface pattern. If one takes into account that surface morphology (roughness) has a significant relationship with the amount of biofilm generated, it is reasonable to think that different surface patterns will cause microorganisms to produce different amounts of biofilm. SEM images of the 3 samples coated with PEGMA + IPA are shown in Figure 6. In all cases, it can be seen that the morphology of the surface of the coating is substantially different from that of the coated samples using APTES (Figures 3A and 3B). Especially interesting is the case of the PG10 sample in which the morphology of its coating follows a fiber-like pattern, while that of any APTES-based sample has a particle-based pattern.
    [0080] In any case, regardless of the precursor used (APTES or PEGMA), it is found that as the roughness increases, the biofilm increases. Also, in any case there is a specific roughness from which the coating is pro-biofilm.
    [0081] Next, a chemical and wettability characterization of the pro-biofilm coatings was performed according to the embodiments of the present invention.
    [0082] The chemical characterization was carried out by means of X-ray emitted photoelectron spectroscopy analysis (XPS) and that of wettability was carried out by means of an analysis of the measure of the angle of contact with water (WCA).
    [0083] Through the XPS analysis it is possible to identify and quantify the chemical composition of the first 3 to 5 nanometers of the coating (place from which the biofilm grows). Figure 7 shows the atomic percentage of the elements present (C, O, Si and N) in each of the coatings studied based on APTES (uncoated, 0p, and coated with 2, 4, 12, 24, 48 , 72 and 96 passes). These elements (C, O, Si and N) come from the plasma polymerization of APTES (in the case of coated samples), from the surface of PS (in the case of the Petri dish without control coating) and from the air surrounding during the coating procedure.
    [0084] In the case of the uncoated sample (0p) it is possible to identify the typical percentages present in the PS (polystyrene) that are substantially different from those of the coated samples (eg high carbon percentage). This uncoated sample (0p) is the only one in which silicon is not detected as this element comes from APTES.
    [0085] From this figure 7 it can be concluded that the chemical composition of the surface of ALL coatings is very similar and independent of its roughness (or number of passes applied).
    [0086] On the other hand, by means of the XPS analysis it is also possible to identify and quantify the links of each of the previously identified elements (C, O, Si and N). Figure 8 shows the deconvolution (or decomposition) of the "total" high-resolution carbon (C1s) spectrum of samples 0p, 2p and 72p.
    [0087] Carbon deconvolution (C1s) is a common practice in the chemical characterization of surfaces. This deconvolution allows to identify the partial spectra that make up the total spectrum. Each partial spectrum corresponds to a specific bond to which the carbon is attached. The area of each partial spectrum (associated with a certain link) allows quantifying its presence on the surface with respect to the total links.
    [0088] Figure 8 shows the percentage relative to each link (inscribed in a rectangle). In the case of the deconvolution of the 0p sample (uncoated PS Petri dish) the typical bonds to which the carbon is attached in a plasma treated PS sample (uncoated, plasma only) are identified. The PS Petri plates that were used in this study had previously been treated with plasma by their supplier (Petri dishes / Nunc ™ cell cultures from Thermo Scientific ™) for surface activation. In this sample (0p) it is possible to identify the links: [A] C-C and C-H aromatic, [B] C-C and C-H aliphatic, [C] the groups C-O / C-N, [D] O-C = O and [E] n-n *. Groups [A], [B] and [E] come from the PS molecule, while groups [C] and [D] are formed during plasma treatment (performed by the supplier of PS Petri plates). When the PS Petri dishes are coated by plasma polymerization based on APTES (samples 2p and 72p, representative of all samples coated from APTES) the links [A], [D] and [E] disappear and the links appear [F] C-Si and [G] C = O from the polymerized APTES. Links [B] and [C] are common to plasma activated PS and polymerized APTES.
    [0089] From the analysis of the bonds and percentages shown in Figure 8, it can be concluded that the chemical structure of the surface of the coated samples (2p and 72p) is very different from the uncoated sample (0p), both because of the total item percentages (C, O, Si and N in Figure 7) as per the links to which the carbon is associated (Figure 8).
    [0090] In addition, the chemical structure of the surface of the coated samples (samples 2p and 72p, representative of all samples coated from APTES) is very similar regardless of the large difference in roughness between the two (Ra of 2p = 6, 7 nm and Ra of 72p = 55.1 nm).
    [0091] Finally, according to all of the above, the increase in biofilm production (at higher roughness) is not determined by the chemical nature of the coatings, since among these there are no substantial differences that justify the increase in biofilm production.
    [0092] The characterization was then carried out by means of the WCA analysis, which refers to the angle formed by the surface of a liquid when it comes into contact with a solid. The value of the contact angle depends mainly on the relationship between the adhesive forces between the liquid and the solid and the cohesive forces of the liquid. At higher adhesive forces, lower WCA.
    [0093] Figure 9 shows the relationship between roughness and WCA of the coatings studied based on APTES (uncoated, 0p, and coatings with 2, 4, 12, 24, 48, 72 and 96 passes). This figure shows that all coated samples (except 96p) have a WCA very similar to that of the uncoated sample (0p). This means that the coated surface does not have an adhesive capacity greater than that of the uncoated sample (0p). Therefore, the amount of biofilm produced in such coatings does not depend on the increase in adhesion (decrease in WCA) conferred by them.
    [0094] Therefore, in summary and with respect to PS coatings using APTES, all coatings have the same "chemistry" and "wettability", and all coatings have different "morphology" and "% biofilm".
    [0095] Therefore, the increase of the biofilm% does not depend on the "chemistry" or "wettability" (adhesion) of the coating, but depends on the modification of the "morphology" thereof (roughness).
    [0096] Next, bacterial growth (see Figure 10) was studied for 24 hours (0, 3, 6 and 24 hours) of the uncoated samples (0p) and with coatings with 2, 4, 12, 24 and 48 passes. The bacterial growth of each sample was determined using a Microplate Reader 680XR Bio-Rad spectrophotometer and measuring the absorbance at a wavelength of 620 nm.
    [0097] Figure 10 shows that only in the 12p coating more bacteria grew than in the uncoated sample (0p). That is, in most of the coated samples less bacteria grew than in the control. Therefore, the production of more biofilm by said samples (with respect to the uncoated sample, 0p), is not due to an increase in the number of microorganisms attached.
    [0098] Finally, the biofilm generation rate was determined by the coatings according to the present invention. Figure 11 shows the amount of biofilm (measured by FDA) generated in samples 0p and 48p for 24 hours (measurements taken at 6, 12 and 24 hours). In this figure it can be seen that the maximum amount of biofilm produced in the 48p sample is reached at 6 hours after inoculation, being at that time (6h) significantly greater than the biofilm produced in the uncoated sample (0p ). This speed is of vital importance when it is urgently determined which treatment is optimal for a patient in a situation determined (most effective antibiotic and dose thereof).
    [0099] Previously, preferred embodiments of the present invention have been described in detail. However, the person skilled in the art may make modifications and variations thereto without thereby departing from the scope of protection defined by the following claims. For example, specific precursor materials and substrates have been described in specific embodiments of the invention, however the person skilled in the art may apply the teachings disclosed herein to determine, by routine experimentation, other combinations of precursor materials. , substrates and numbers of plasma passes to provide probiofilm coatings that exhibit sufficient roughness to encourage the creation of more than 100% biofilm, compared to the biofilm produced on the same substrate lacking pro-biofilm coating, according to the present invention.
    权利要求:
    Claims (15)
    [1]
    1 Pro-biofilm coating applied by polymerization of a cold atmospheric plasma precursor on a substrate, the coating presenting a roughness that promotes the creation of more than 100% biofilm on the substrate, with 100% of biofilm being produced on the substrate. same substrate lacking said pro-biofilm coating.
    [2]
    2 Pro-biofilm coating according to claim 1, characterized in that the precursor material constituting the coating is selected from the group consisting of (3-aminopropyl) triethoxysilane (APTES) and a mixture of polyethylene glycol methyl ether methacrylate (PEGMA, MW: 500) and isopropanol (IPA).
    [3]
    3 Pro-biofilm coating according to claim 2, characterized in that the precursor material constituting the coating is APTES and the coating has a minimum arithmetic average roughness (Ra) of more than 10 nm.
    [4]
    4 Pro-biofilm coating according to claim 2, characterized in that the precursor material constituting the coating is a mixture of PEGMA and IPA and the coating has a minimum arithmetic average roughness (Ra) of more than 160 nm.
    [5]
    Pro-biofilm coating according to any of the preceding claims, characterized in that the substrate is selected from the group consisting of polystyrene and glass.
    [6]
    Method of producing a pro-biofilm coating according to any one of claims 1 to 5, characterized in that it comprises applying a cold atmospheric plasma flow and coating precursor material on a substrate surface a coating until a roughness is obtained that promotes the creation of more than 100% biofilm on the substrate, with 100% biofilm being produced on the same substrate lacking said probiofilm coating.
    [7]
    Method according to claim 6, characterized in that it further comprises the step of carrying out, simultaneously with the application of the plasma flow, a relative displacement between the substrate to be coated and the plasma flow to cover the entire surface of the substrate.
    [8]
    8. Method according to any of claims 6 and 7, characterized in that it further comprises the previous step of activating the surface of the substrate by plasma without precursor material.
    [9]
    9. Method according to any of claims 6 and 8, characterized in that the plasma applied is N2 plasma.
    [10]
    10. Method according to claim 9, characterized in that the plasma flow consists of 80 slm of N 2 .
    [11]
    Method according to any one of claims 6 to 10, characterized in that the applied precursor material is selected from the group consisting of APTES and a mixture of PEGMA and IPA.
    [12]
    12. Method according to any of claims 6 to 11, characterized in that the precursor material is applied in liquid phase, transported by a flow of 1.5 slm of N 2 .
    [13]
    13. Method according to any of claims 6 to 12, characterized in that the substrate is selected from the group consisting of polystyrene and glass.
    [14]
    14. Method according to any of claims 6 to 13, characterized in that the application of the coating It is done evenly over the entire surface of the substrate.
    [15]
    15. Substrate coated by a pro-biofilm coating according to any one of claims 1 to 5 applied on at least one surface thereof.
    类似技术:
    公开号 | 公开日 | 专利标题
    Kelleher et al.2016|Cicada wing surface topography: an investigation into the bactericidal properties of nanostructural features
    Di et al.2017|A transparent wound dressing based on bacterial cellulose whisker and poly |
    Ploux et al.2009|Opposite responses of cells and bacteria to micro/nanopatterned surfaces prepared by pulsed plasma polymerization and UV-irradiation
    Zhang et al.2008|Ag and Ag/N2 plasma modification of polyethylene for the enhancement of antibacterial properties and cell growth/proliferation
    US20060051522A1|2006-03-09|Method of pulsed laser assisted surface modification
    US8992590B2|2015-03-31|Biocompatibility layer and coated objects
    Noorisafa et al.2016|Surface modification of polyurethane via creating a biocompatible superhydrophilic nanostructured layer: role of surface chemistry and structure
    Favia2012|Plasma deposited coatings for biomedical materials and devices: Fluorocarbon and PEO-like coatings
    Gittard et al.2009|Assessing the antimicrobial activity of zinc oxide thin films using disk diffusion and biofilm reactor
    Şen et al.2012|Modification of food-contacting surfaces by plasma polymerization technique: reducing the biofouling of microorganisms on stainless steel surface
    Ploux et al.2007|Quantitative and morphological analysis of biofilm formation on self-assembled monolayers
    Pandiyaraj et al.2014|Influence of operating parameters on surface properties of RF glow discharge oxygen plasma treated TiO2/PET film for biomedical application
    Sedlarik2013|Antimicrobial modifications of polymers
    JP2018511377A|2018-04-26|Biodegradable free-standing film based on hydrophobized hyaluronic acid, its preparation and use
    Griffin et al.2018|Evaluation of sterilisation techniques for regenerative medicine scaffolds fabricated with polyurethane nonbiodegradable and bioabsorbable nanocomposite materials
    CN104311866A|2015-01-28|Surface-modified vulcanized silicone rubber material and preparation method thereof
    Maenosono et al.2014|A transparent polyimide film as a biological cell culture sheet with microstructures
    Vargas-Alfredo et al.2018|Fabrication of biocompatible and efficient antimicrobial porous polymer surfaces by the Breath Figures approach
    ES2720026B2|2020-07-03|PRO-BIOFILM COATING, METHOD FOR ITS PRODUCTION AND SUBSTRATE COVERED BY THE SAME
    Manju et al.2010|Layer-by-Layer modification of poly | intra ocular lens: drug delivery applications
    Soffe et al.2019|Comparison of replica leaf surface materials for phyllosphere microbiology
    El-Sayed et al.2017|Surface analysis of nitrogen plasma-treated C60/PS nanocomposite films for antibacterial activity
    Peršin et al.2014|Ammonia plasma treatment as a method promoting simultaneous hydrophilicity and antimicrobial activity of viscose wound dressings
    Levchenko et al.2016|Novel biomaterials: plasma-enabled nanostructures and functions
    Morisaki et al.2009|Bacterial attachment over a wide range of ionic strengths
    同族专利:
    公开号 | 公开日
    EP3951005A1|2022-02-09|
    WO2020193830A1|2020-10-01|
    ES2720026B2|2020-07-03|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    JP2013173715A|2012-02-27|2013-09-05|恒二 ▲浜▼崎|Method of forming biofilm and biofilm fixed material|
    法律状态:
    2019-07-17| BA2A| Patent application published|Ref document number: 2720026 Country of ref document: ES Kind code of ref document: A1 Effective date: 20190717 |
    2020-07-03| FG2A| Definitive protection|Ref document number: 2720026 Country of ref document: ES Kind code of ref document: B2 Effective date: 20200703 |
    优先权:
    申请号 | 申请日 | 专利标题
    ES201930265A|ES2720026B2|2019-03-25|2019-03-25|PRO-BIOFILM COATING, METHOD FOR ITS PRODUCTION AND SUBSTRATE COVERED BY THE SAME|ES201930265A| ES2720026B2|2019-03-25|2019-03-25|PRO-BIOFILM COATING, METHOD FOR ITS PRODUCTION AND SUBSTRATE COVERED BY THE SAME|
    PCT/ES2020/070195| WO2020193830A1|2019-03-25|2020-03-20|Pro-biofilm coating, production method thereof, and substrate coated with same|
    EP20724541.6A| EP3951005A1|2019-03-25|2020-03-20|Pro-biofilm coating, production method thereof, and substrate coated with same|
    [返回顶部]