ANTI-BACTERIAL PATTERNED SURFACES AND METHODS OF MAKING THE SAME

Abstract

The present invention relates to a substrate comprising a plurality of integrally formed surface features, said surface features being micro-sized and/or nano-sized, said surface features comprising at least one pointed terminus. As a result of this unique surface, said substrate exhibits a biocidal activity because the terminal ends of said surface feature pierce through cell membrane of any microbial cell that comes into contact with the substrate, thereby causing cell deformation and lysis. The present invention also relates to a method producing said substrate. By a simple treatment of copper or zinc foil with a reagent solution comprising an alkali and an oxidizing agent, Cu(OH)2 nanotube arrays, CuO nano-blades and ZnO nano-needles are prepared. These surfaces are proven to be very effective in killing bacterial (such as E. coli) via a physical interaction.

Claims

1. A substrate comprising a plurality of integrally formed surface features, said surface features being micro-sized and/or nano-sized, each surface feature comprising a crystalline phase and at least one pointed terminus.

2. The substrate of claim 1, wherein the substrate comprises a metal surface.

3. The substrate of claim 2, wherein the metal surface is reactive with an oxidizing agent to form an insoluble salt.

4. The substrate of claim 2 or 3, wherein said crystalline phase comprises an insoluble salt.

5. The substrate of any one of claims 2 to 4, wherein said crystalline phase comprises an oxide, or a hydroxide salt.

6. The substrate of any one of the preceding claims, wherein said crystalline phase has one of an orthorhombic crystal structure, monoclinic crystal structure, triclinic crystal structure, tetragonal crystal structure, hexagonal crystal structure, trigonal crystal structure or cubic crystal structure.

7. The substrate of claim 6, wherein said crystalline phase is selected from a hexagonal crystal structure having a wurtzite crystal structure, a crystalline phase having an X-Ray Diffraction characterization of JCPDS no. 13-0420 and a crystalline phase having an X-Ray Diffraction characterization of JCPDS no. 48-1548.

8. The substrate of any one of the preceding claims, wherein the surface feature is selected from the group consisting of tubes, blades, needles, pyramids, cones, pillars and mixtures thereof.

9. The substrate of any one of the preceding claims, wherein the integrally formed surface feature is tapered in shape, having a base end coupled to a surface of said substrate and a distal end that is smaller in dimension relative to a said base end.

10. The substrate of any one of the preceding claims, wherein a ratio of the height of said surface feature to a dimension of the terminus distal end of the surface feature is from about 10 to 200.

11. The substrate of any one of the preceding claims, wherein the surface feature comprises a height selected from about 200 nm to 10 m.

12. The substrate of any one of the claim 9, wherein the dimension is diameter or thickness.

13. The substrate of any one of the preceding claims, wherein a dimension of the terminus distal end of the surface feature is from about 1 nm to about 500 nm.

14. The substrate of any one of the preceding claims, wherein the surface features exhibits a pitch of from about 100 nm to about 2000 nm.

15. The substrate of any one of claims 2-14, wherein the substrate comprises a metal surface and wherein the metal is a transition metal selected from Group 11 or Group 12 of the Period Table of Elements,

16. The substrate of claim 15, wherein the Group 11 metal is Cu.

17. The substrate of claim 15, wherein the Group 12 metal is Zn.

18. A substrate comprising a copper surface, the copper surface comprising a plurality of surface features integrally formed thereon, said surface features being micro-sized and/or nano-sized, and wherein said surface features comprises Cu(OH).sub.2, CuO or a mixture thereof, each Cu(OH).sub.2 or CuO surface feature comprising at least one pointed terminus.

19. A substrate comprising a zinc surface, said zinc surface comprising a plurality of micro-sized and/or nano-sized ZnO surface features integrally formed thereon, said ZnO surface features comprising at least one pointed terminus.

20. A method of producing a substrate possessing antibacterial properties, the method comprising: contacting a surface of the substrate with a reagent solution to produce a plurality of integrally formed, micro-sized or nano-sized surface features on the substrate surface, each surface feature comprising a crystalline phase and at least one pointed terminus.

21. The method of claim 20, wherein the substrate comprises a metal surface, said surface being oxidisable to form insoluble salts to integrally form said surface features thereon.

22. The method of claim 20, wherein the reagent solution comprises metal ions that form insoluble salts on said substrate surface, thereby integrally forming said surface features thereon.

23. The method of claim 21 or 22, wherein said substrate comprises a transition metal surface and said surface features comprises oxide and/or hydroxide salts of said metal.

24. The method of claim 23, wherein the transition metal is selected from Group 11 or Group 12 of the periodic table.

25. The method of claim 24, wherein the Group 11 metal is Cu.

26. The method of claim 24, wherein the Group 12 metal is Zn.

27. The method of claim 21, wherein the reagent solution comprises an alkali and an oxidizing agent.

28. The method of claim 27, wherein the oxidizing agent is selected from the group consisting of persulfates, nitrates, halogen compounds, hypohalites and permanganates, and wherein the concentration of the oxidizing agent is selected from about from 0.01 M to 10 M.

29. The method of claim 28, wherein the concentration of the oxidizing agent is in a range of from about 0.01 M to about 5.0 M.

30. The method of claim any one of claims 27-29, wherein the concentration of the alkali is from about 1.0 M to about 10M.

31. The method of any one of claims 20-30, wherein the contacting step is conducted for a duration sufficient to produce the plurality of surface features.

32. The method of claim 31, wherein the contacting step is conducted for a duration of from about 10 minutes to about 1440 minutes.

33. The method of any one of claims 25-32, wherein the contacting step is conducted at room temperature or ambient temperature, or about 15 C., or about 20 C., or about 25 C., or about 30 C.

34. A method of producing a substrate possessing antibacterial properties, the method comprising: contacting a surface of the substrate with a reagent solution to produce a plurality of integrally formed, micro-sized or nano-sized surface features by precipitation on the substrate surface, each surface feature comprising a crystalline phase and at least one pointed terminus.

35. A substrate comprising a metal surface, said metal surface comprising a plurality of integrally formed, micro-sized and/or nano-sized surface features, said substrate being obtainable by a method as defined in any one of claims 20-34.

36. Use of the substrate of any one of claims 1-19 for providing antibacterial properties to an ex-vivo environment.

37. The use of claim 36 for providing bacteriostatic or bactericidal purposes to said ex-vivo environment.

38. The use of claim 36 or 37, being a non-therapeutic use.

39. The use of any one of claims 36-38, wherein the antibacterial substrate is capable of killing or inhibiting the growth of gram-negative and gram-positive bacteria.

40. The use of claim 39, wherein the gram-negative bacteria is selected from the group consisting of Escherichia, Shigella, and Salmonella.

41. The use of statement 40, wherein the gram-positive bacteria is selected from the group consisting of Staphylococcus, Enterococcus and Streptococcus.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0082] The accompanying drawings illustrate a disclosed embodiment and serves to explain the principles of the disclosed embodiment. It is to be understood, however, that the drawings are designed for purposes of illustration only, and not as a definition of the limits of the invention.

[0083] FIG. 1 contains the Scanning Electron Microscopy (SEM) images of (A) Cu foil, (B) Cu(OH).sub.2 nanotubes growing on Cu foil, (C) CuO nano-blades growing on Cu foil and the graphs of their corresponding X-Ray Diffraction (XRD) patterns (D-F), confirming their respective structures.

[0084] FIG. 2 contains the SEM images of (A) Zn foil, (B, C) ZnO nano-needles growing on Zn foil, and (D) graph of the XRD pattern of ZnO nano-needles on Zn foil.

[0085] FIG. 3 is a graph of the Colony Forming Units (CFU)/ml against the incubation time showing the killing efficacy (against E. coli) of various copper surfaces evaluated using Japanese Industrial Standard (JIS) Z 2801/ISO 22196 method.

[0086] FIG. 4 contains graphs of the CFU/ml against the incubation time demonstrating the killing efficacy (against E. coli) of various copper surfaces evaluated using JIS Z 2801/ISO 22196 method for (A) samples with Pt coating and (B) samples with Cu coating.

[0087] FIG. 5 is a graph of the CFU/ml against the incubation time showing the killing efficacy (against E. coli) of flat Zn foil and ZnO nano-needle surface evaluated using JIS Z 2801/ISO 22196 method.

[0088] FIG. 6 contains graphs of the CFU/ml against the incubation time demonstrating the killing efficacy (against S. aureus) of (A) flat Cu foil, Cu(OH).sub.2 nano-tubes, CuO nano-blades surface, and (B) flat Zn foil and ZnO nano-needle surface evaluated using JIS Z 2801/ISO 22196 method.

[0089] FIG. 7 contains graphs of the CFU/ml against the incubation time demonstrating the killing efficacy (against C. albicans) of (A) flat Cu foil, Cu(OH).sub.2 nano-tubes, CuO nano-blades surface, and (B) flat Zn foil and ZnO nano-needle surface evaluated using JIS Z 2801/ISO 22196 method.

[0090] FIG. 8 contains graphs of the CFU/ml against the incubation time demonstrating the killing profiles (against E. coli) of nano-structured surfaces (A) Cu(OH).sub.2 nanotubes surface, (B) CuO nano-blades surface, and (C) ZnO nano-needles surface in water under shaking condition. Testing conditions: 5 ml water, 37 C., shaking at 300 r/min.

EXAMPLES

[0091] Non-limiting examples of the invention and a comparative example will be further described in greater detail by reference to specific Examples, which should not be construed as in any way limiting the scope of the invention.

Example 1: Preparation of Cu(OH).SUB.2 .nanotubes and CuO nanoblades on Cu Substrate

[0092] For the growing of Cu(OH).sub.2 nanotubes, 4 ml of 1M (NH.sub.4).sub.2S.sub.2O.sub.8, 8 ml of 10M NaOH and 18 ml of water were mixed to form a solution. A Cu foil (2025 mm) was suspended in the solution for 15 min. A solid film of Cu(OH).sub.2 nanotubes was obtained on the Cu foil. The Cu foil was then washed 3 times with water and 3 times with ethanol. After washing, the foil was dried with flowing N.sub.2 and stored for future use.

[0093] For the growing of CuO nanoblades, 4 ml 1M (NH.sub.4).sub.2S.sub.2O.sub.8 solution and 8 ml 10M NaOH were mixed. A Cu foil (2025 mm) was suspended in the solution for 30 min. A black solid film of CuO nanoblades was obtained on the Cu foil. The Cu foil was then washed 3 times with water and 3 times with ethanol. After washing, the foil was dried with flowing N.sub.2 and stored for future use.

Example 2: Preparation of ZnO Nanoneedles on Zn Substrate

[0094] For the growing of ZnO nanoneedles, 10 ml of 0.5M Zn(NO.sub.3).sub.2 aqueous solution and 10 ml 4M KOH were mixed. A Zn foil (2020 mm) was suspended in the solution for 12 h at room temperature. The surface of the Zn foil was washed 3 times with water and 3 times with ethanol. Subsequently, the Zn foil was dried with flowing N.sub.2 and stored for future use.

Example 3: Characterization of Surface

[0095] The surfaces of the samples were characterized by SEM (JEOL JSM-7400E) and XRD (PANalytical X-ray diffractometer, X'pert PRO, with Cu K radiation at 1.5406 {acute over ()}). Prior to SEM, the samples were coated with thin Pt film using high resolution sputter coater (JEOL, JFC-1600 Auto Fine Coater). Coating conditions: For sample testing (20 mA, 30 s). For Pt coated sample for antibacterial testing (20 mA, 60 s).

[0096] Nano-patterns on copper substrate was prepared by treatment of copper foil in a (NH.sub.4).sub.2S.sub.2O.sub.8 and NaOH solution at room temperature (see Example 1), 2 types of nano-structures were grown on copper substrate. As shown in FIG. 1, when copper foil was treated with lower concentration of the solution for 15 min, nanotubes array was grown. The nanotube array grew upwards and covered the whole area of the copper substrate compactly. Each tube was 5-7 m in length with an open and sharp tip of 100-200 nm diameter. XRD confirmed the structure was Cu(OH).sub.2 with orthorhombic phase (JCPDS Card No. 13-0420). When the cupper foil was treated with higher concentration of the solution at ambient temperature, blade-like structure was formed on the Cu surface, with sharp edge standing upward. XRD confirmed the structure to be monoclinic symmetry of CuO on copper. (JCPDS Card No. 48-1548).

[0097] Similarly, a nano-patterned zinc surface was prepared by using a simple method (see Example 2). By treatment of a zinc foil in Zn(NO.sub.3).sub.2 and KOH solution, ZnO nano-needles array was grown on the zinc substrate as shown below in FIG. 2. After treatment in the solution for 12 hours at room temperature, highly oriented uniform nano-needles array was formed on the surface. Further study showed that the needles were typically 1-2 m in length. The diameters of the needle tips and roots are 10-40 nm and 100-200 nm, respectively. XRD analysis confirmed that the nano-needles are wurtzite ZnO structure. A strong diffraction peak at 34.4 (002) was present, indicating the highly preferential growth of ZnO nanoneedles along c-axis.

Example 4: Bacterial Growth Conditions and Sample Preparation

[0098] E. coli, S. aureus, and C. albicans were obtained from American Type Culture Collection (ATCC-8739). Prior to each bacterial experiment, bacterial cultures were refreshed on nutrient agar from stock. Fresh bacterial suspensions were grown overnight at 37 C. in 5 ml of TSB (E. coli and S. aureus) or 5 ml YM broth for C. albicans. Bacterial cells were collected at the logarithmic stage of growth and the suspensions were adjusted to OD.sub.600=0.07.

Example 5: JIS Killing Efficacy Testing

[0099] The tested bacteria were suspended in 5 mL of respective nutrient broth and adjusted to OD.sub.600=0.07. In order to cover the surface, 150 L of cell suspensions was placed on the surfaces. Experiments were carried out in triplicate at 37 C. After incubation with the surfaces, the respective cell suspensions were washed and diluted, and each dilution spread on two nutrient agar plates. Resulting colonies were then counted using standard plate counts techniques, and the number of colony forming units per mL was calculated. The number of colony forming units was assumed to be equivalent to the number of viable cells in suspension.

[0100] The antibacterial properties against E. coli were evaluated for nano-patterned Cu surfaces by using JIS Z 2801:2000 (Japanese Industrial Standard) method. As shown in FIG. 3, all the bacteria were killed after 1 h incubation on Cu(OH).sub.2 nanotubes surface. For the CuO nano-blade surface, 94.5% of E. coli bacteria were killed after 1 h incubation and all bacteria were killed after 3 hours. In relation to the control, Cu foil with a flat surface, only 28% of bacteria were killed after 1 h and there were still about 35% of E. coli surviving after 3 h.

[0101] From FIG. 3, it was observed that the E. coli killing efficacy was in the order of Cu(OH).sub.2 nanotubes>CuO nano-blades>Cu foil, which indicated that the sharper the surface, the better the killing efficacy. Considering that the chemical composition of the 3 surfaces are different (Cu(OH).sub.2, CuO, and Cu), to exclude the composition effect, three surfaces were coated with Pt and Cu, respectively, and the E. coli killing profiles were re-evaluated.

[0102] FIG. 4(A) demonstrates the killing efficacy against E. coli for the Pt coated samples. It was shown that Cu foil with Pt coating significantly changed the bacteria killing profile. Without Pt coating, flat Cu foil killed 65% of E. coli after 3 hours (FIG. 3). While after Pt coating, E. coli kept on growing instead after 3 hour incubation (FIG. 4). For Cu(OH).sub.2 nanotubes and CuO nano-blade surface, the killing profiles were almost unchanged after Pt coating as compared with the uncoated surfaces. All the bacteria were killed after 3 hour incubation, as shown in FIG. 4(A). To further confirm this result, three samples were also coated with Cu by vacuum vapour deposition method. SEM results did not show any obvious morphological change after Cu coating. After coating, all the three samples have the same chemical composition of Cu on the nano-patterned surfaces. As shown in FIG. 4(B), the killing profile of Cu-coated flat Cu foil was similar to the uncoated sample shown in FIG. 3. The killing efficacy of Cu(OH).sub.2 nanotubes surface and CuO nano-blades surface were maintained or even increased after coating with Cu. As can be seen from FIG. 4(B), all the bacteria were killed after 1 h incubation with copper coated nanotube and nano-blade surfaces. All these results indicated that the bacteria killing properties of these samples are mainly or entirely contributed by the surface nano-structures rather than chemical component.

[0103] The antibacterial activity against E. coli was also tested for zinc foil and ZnO nanoneedles. As shown in FIG. 5, all the bacteria were killed on ZnO nano-needles surface after 6 h incubation. As control, E. coli on flat Zn foil kept on growing, indicating the non-biocidal property of Zn foil. This result again demonstrated that the nano-structured zinc surface kills bacteria efficiently via physical interaction.

[0104] In addition to E. coli, which represents Gram-negative bacteria, Gram-positive bacteria were also tested. The antibacterial properties against S. aureus were also tested, as shown in FIG. 6.

[0105] As demonstrated in FIG. 6, the killing profile for S. aureus was similar to that of E. coli. The Cu(OH).sub.2 nano-tubes surface and CuO nano-blades surface killed nearly all the bacteria after 1 hour incubation, while for flat Cu foil, 23% of bacteria remained alive even after 3 hours incubation. For ZnO nano-needles surface, all the S. aureus were killed after 6 hours incubation, while 70% of S. aureus remained surviving on the flat Zn surface.

[0106] C. albicans as a sample of fungi was also tested. The killing profile for C. albicans was very different from those of E. coli and S. aureus. As shown in FIG. 7, all the tested surfaces could kill C. albicans. After 24 hours incubation, the remaining C. albicans were 2% (Cu), 4% (Cu(OH).sub.2), 0.7% (CuO), 1.3% (Zn) and 2.8% (ZnO). The nanostructured surfaces did not exhibit faster killing efficacy as compared with the flat surface. This might due to the robust cell wall of fungus as compared with other bacteria. As control, C. albicans on 6-well plate grow 25 times after 24 hours incubation, indicating the non-antibacterial of plate substrate (results not shown).

Example 6: Bacterial Killing Efficacy Under Washing Machine Condition

[0107] To simulate the washing process, E. coli was suspended in 5 ml of water and adjusted to OD.sub.600=0.07. The testing surfaces, mounted on 3.5 cm circular discs, were immersed in 5 ml of 1:10 diluted bacterial suspension for incubation intervals and shaken at a speed of 300 r/min. The cell suspensions were then sampled (100 l) at discrete time intervals, serially diluted 1:10, and each dilution spread on two nutrient agar plates. Resulting colonies were then counted, and the number of colony forming units per mL was calculated.

[0108] As an example of potential application of the nano-patterned Cu and Zn surfaces in washing machine, the bacterial killing activities of these nano-structured surfaces were tested under a simulated washing machine condition. E. coli, water and nanostructured surface were put in a bacterial culture plate, under shaking at 300 r/min. Bacteria in the solution was monitored by plate counting technique. The results show that for Cu(OH).sub.2 nanotubes and CuO nano-blades surfaces, all the bacteria in water are killed within 30 min. For ZnO nanoneedles surface, 82% of E. coli was killed after 1 h. In the control experiment, i.e. washing water without the nano-structured surface, the bacteria were still alive after 24 h. This experiment clearly demonstrated the possibility of making the inner surface of a washing machine having antibacterial surface/properties. The surface would kill bacteria during the washing session (30-60 min).

[0109] In summary, surfaces with Cu(OH).sub.2 nanotubes, CuO nano-blades and ZnO nano-needles have been prepared by simple solution treatment of respective copper or zinc foil at room temperature. All surfaces are bactericidal against E. coli. Application of these artificial surfaces are also demonstrated in washing machine condition in water, where E. coli bacteria are completely killed within 30 min by Cu(OH).sub.2 nanotubes and CuO nano-blades surfaces.

INDUSTRIAL APPLICABILITY

[0110] The nano-patterned surfaces of the present application may be useful in providing non-chemical anti-bacteria properties. Such anti-bacterial nano-patterned surfaces may be used as alternative surface materials for frequently-touched surfaces, e.g. doorknobs, handles and sanitary fittings, to provide an environment which discourages or inhibits bacteria proliferation such as in a hospital setting.

[0111] Advantageously, this would reduce the reliance on synthetic chemical disinfectants which may undesirably result in secondary contamination and may cause serious drug-resistant superbugs to develop. The disclosed patterned surfaces also lend possibility to the provision of domestic household appliances and equipment possessing such patterned metal surfaces. The anti-bacteria surface may also be used in a number of cleaning applications, e.g. to render the inner chamber surface of household or industrial scale washing machine anti-bacterial. This may advantageously reduce or completely eliminate the requirement of synthetic detergents which may be harmful to the human body. Also, the cleaning time may be reduced which results in higher cleaning efficiency of the washing machine.

[0112] It will be apparent that various other modifications and adaptations of the invention will be apparent to the person skilled in the art after reading the foregoing disclosure without departing from the spirit and scope of the invention and it is intended that all such modifications and adaptations come within the scope of the appended claims.