Polymeric film surface

Abstract

A surface on a polymeric film having an array of patterned structures, wherein the array of patterned structures influences fluid flow of the surface and causes reduced attachment of a biological material.

Claims

1. A polymeric film, comprising: a surface, wherein the surface of the polymeric film comprises an array of patterned structures, wherein topographies of the array of patterned structures are selected and arranged so that when the surface of the polymeric film is rinsed and/or dipped in a fluid, the array of patterned structures induces a turbulent fluid flow and removes biological material thereon, and wherein when a predetermined space or a predetermined width of the topographies of the polymeric film is larger than a diameter or dimension of the biological material, the rinsed and/or dipped surface of the polymeric film is characterized such that it exhibits a reduced attachment of the biological material.

2. The polymeric film according to claim 1, wherein said array of patterned structures comprises of pillars or grooves.

3. The polymeric film according to claim 2, wherein said pillars are micron-sized or nano-sized.

4. The polymeric film according to claim 2, wherein said pillars have diameters that are individually selected from the range of 10 m to 50 m.

5. The polymeric film according to claim 4, wherein said pillars have diameters that are individually selected from the group consisting of 25 nm, 100 nm, 500 um, 2 m and 10 m.

6. The polymeric film according to claim 2, wherein the spacing between a pair of pillars is the same as the spacing between another pair of pillars.

7. The polymeric film according to claim 2, wherein the spacing between a pair of pillars is different from the spacing between another pair of pillars.

8. The polymeric film according to claim 2, wherein said spacing is larger than the size of said biological material.

9. The polymeric film according to claim 1, wherein said spacing is selected from the range of 10 nm to 100 m or from 10 m to 100 m.

10. The polymeric film according to claim 2, wherein said spacing is in the nano-scale.

11. The polymeric film according to claim 2, wherein said pillars have a diameter of 10 m and a spacing in the range of 6 m to 50 m.

12. The polymeric film according to claim 2, wherein said pillars have diameters individually selected from the range of 25 nm to 10 m and spacings that are equal to the diameters of said pillars.

13. The polymeric film according to claim 2, wherein said grooves have an anisotropic cross-sectional shape.

14. The polymeric film according to claim 2, wherein grooves have a width in the range of 100 nm to 5 m.

15. The polymeric film according to claim 2, wherein said grooves have a V-shaped cross section.

16. The polymeric film according to claim 15, wherein said V-shaped groves have a width selected from 100 nm, 500 nm or 2 m, wherein said width is measured from the top of said groove.

17. The polymeric film according to claim 2, wherein said grooves have a U-shaped cross section.

18. The polymeric film according to claim 17, wherein said U-shaped groves have a width selected from 500 nm or 2 m, wherein said width is measured from the top of said groove.

19. The polymeric film according to claim 1, wherein said biological material is selected from the group consisting of proteins, eukaryotic organisms, bacteria, viruses and fungi.

20. The polymeric film according to claim 19, wherein said bacteria is selected from the group consisting of gram negative bacteria, gram positive bacteria and marine bacteria.

21. The polymeric film according to claim 20, wherein said bacteria is selected from the group consisting of E. coli ATCC 25922, S. epidermidis ATCC 12228 and P. Tunicata D2.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) 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.

(2) FIG. 1 is a schematic diagram showing the protocol used for testing the extent of microorganism adhesion on test patterned substrates.

(3) FIG. 2 is a schematic diagram showing the dip test protocol used to test the extent of microorganism adhesion on test patterned substrates after repeated washing and rinsing steps.

(4) FIG. 3a is a bar graph showing the reduced mean number of adhered E. coli ATCC 25922 per mm.sup.2 for pillars with different dimensions. FIG. 3b is a bar graph showing the reduced mean number of adhered E. coli ATCC 25922 per, mm.sup.2 for pillars with varying spacings. FIG. 3c is a bar graph showing the reduced mean number of adhered E. coli ATCC 25922 per mm.sup.2 for V grooves of different widths. FIG. 3d is a bar graph showing the reduced mean number of adhered E. coli ATCC 25922 per mm.sup.2 for U grooves of different widths.

(5) FIG. 4a is a bar graph showing the reduced mean number of adhered S. epidermidis ATCC 12228 per mm.sup.2 for pillars with varying spacings. FIG. 4b is a bar graph showing the reduced mean number of adhered S. epidermidis ATCC 12228 per mm.sup.2 for a U groove.

(6) FIG. 5a is a bar graph showing the reduced mean number of adhered P. Tunicata D2 per mm.sup.2 for pillars with varying spacings. FIG. 5b is a bar graph showing the reduced mean number of adhered P. Tunicata D2 per mm.sup.2 for V grooves of varying widths. FIG. 5c is a bar graph showing the reduced mean number of adhered P. Tunicata D2 per mm.sup.2 for U grooves of varying widths.

(7) FIG. 6 is a bar graph showing the relative number of adhered microorganism on the test patterned substrates after the dip test.

(8) FIG. 7 is a diagram showing the comparison between conventional mechanisms used to reduce biological material attachment and the disclosed polymeric film (shown as IMRE in FIG. 7).

DETAILED DESCRIPTION OF DRAWINGS

(9) Referring to FIG. 1, there is shown a schematic diagram of a protocol 100 used to test the extent of microorganism adhesion on test patterned substrates. Firstly, a shake culture 2 of a test microorganism was cultivated for a period of time in sterile culture medium. The shake culture 2 is then diluted to form a working microorganism culture 4. Following which, a certain amount of the working microorganism culture 4 is dispensed onto wells 6 containing the test patterned substrates 7. The test patterned substrates 7 are then stained with a fluorescence agent and imaged with a suitable microscope at five different spots 8 to determine the amount of adhered microorganism on the test patterned substrates 7.

(10) Referring to FIG. 2, there is shown a schematic diagram showing the dip test protocol 120 used to test the extent of microorganism adhesion on test patterned substrates after repeated washing and rinsing steps. Firstly, a shake culture 10 of a test microorganism was cultivated for a period of time in sterile culture medium until the mid-log phase. The test patterned substrates (or imprints) 12 are loaded into 6-well plate and incubated with the shake culture 10. The test patterned substrate 12 is then loaded onto a dipper 20. The test patterned substrate 12 is then dipped and rinsed successively in a number of washing media (22a, 22b, 24) following the direction of the arrow 14. During each dipping and rinsing, the horizontal and vertical directions of the test patterned substrate 12 are controlled in order to determine the directional effect of the patterned structures present on the test patterned substrate 12. After dipping and rinsing, the retained microorganism on the surface of the test patterned substrate 12 were mounted on a slide 16 and the concentration of microorganism is then determined by averaging the cell counts of five fluorescent images using a Cell Profiler 18.

(11) FIG. 7 shows the mechanism of the disclosed polymeric film (labeled as IMRE) which uses surface topography to reduce attachment of a biological material, such as bacteria. This is in comparison to conventional mechanisms of tensile force (sharklet), mechanical stiffness (Aizenberg), contact angle (Siedlecki), surface functionalization (webster) and wetting property effects (others). Hence, the conventional mechanisms are based on designs that would change the mechanical properties such as tensile strength and elasticity, or on the use of surface topography to change the wettability of the substrate and hence influence bacterial attachment. However, none of the topographies presented in the conventional mechanisms are based on topographical designs that reduce the attachment of the biological material by influencing and manipulating the fluid flow on the surface. Based on the V groove structures (having a width of 2 m and a spacing of less than 200 nm) and on E. coli as the test bacteria, up to 74.16% in reduction as compared to a control (a blank, unpatterned substrate) and up to 166.39% reduction as compared to commercial Sharklet can be achieved in the disclosed polymeric film.

EXAMPLES

(12) Non-limiting examples of the invention 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

(13) The protocol 100 of FIG. 1 was used in this example to study the attachment of micro-organism on the test patterned substrate 7. Firstly, a shake culture 2 of a test microorganism such as E. coli ATCC 25922 (a gram negative bacteria, obtained from ATCC of Virginia of the United States of America) was cultivated for 18 hours in sterile culture medium of tryptic soy broth (from BD Diagnostics, obtained from Biomed Diagnostics of Singapore) at a temperature of 35 C.2 C. E. coli ATCC 25922 is a rod-shaped bacterium with diameter of about 0.5 m and a length of about 1 to 2 m. The shake culture 2 was then diluted with sterile tryptic soy broth to form a working microorganism culture 4 such as working bacterial culture 4 with a concentration of 1.0 to 2.010.sup.8 CFU/ml. Following which, 3 ml of the working bacterial culture 4 (1-210.sup.8 CFU/ml) was dispensed onto wells 6 containing the test patterned substrates 7. The test patterned substrates 7 were imprinted with various nano and micron sized structures. The test patterned substrates 7 were incubated at 125 rpm for 1 hour at a temperature of 35 C. After incubation, the test patterned substrates 7 were rinsed in saline and stained with a fluorescence agent such as BacLight viability stains for 15 minutes. After staining, the test patterned substrates 7 were further rinsed with de-ionized water and mounted for fluorescence microscopy at five different spots 8 to determine the amount of adhered bacteria on the test patterned substrates 7. Under the fluorescence microscope, live bacteria are detected as green while dead bacteria are detected as red due to the different penetrative ability of the dyes in the viability stains. From the microscopy images, the number of pixels occupied by the bacteria were measured and the mean number of bacteria adhered on the surface after the above protocol was estimated, thus quantifying the number of bacteria adhered or retained on the surface of the test patterned substrates 7 after a series of rinsing steps.

(14) The same protocol was carried out for a gram positive bacteria such as S. epidermidis ATCC 12228 (obtained from ATCC of Virginia of the United States of America) and a marine bacteria such as P. Tunicata D2 (obtained from culture collection of the University of Gothenburg of Sweden). S. epidermidis ATCC 12228 is a spherical-shaped bacterium with a diameter of about 0.5 to 1.5 m, while P. Tunicata D2 is a rod-shaped bacterium with diameter of about 0.8 m and length of about 2 to 3 m. The results of this example are shown in Table 1 (for E. coli ATCC 25922), Table 2 (for S. epidermidis ATCC 12228) and Table 3 (for P. Tunicata D2) below. The percentage reductions are calculated as follows: each particular patterned structure is tested on 3 different days and for each particular day, 3 samples for each patterned structure were tested. The mean for each patterned structure was then calculated and the reductions compared to the controls were taken.

Table 1: Table Showing the % Reduction of E. coli ATCC 25922 for Pillars with Different Dimensions (with Spacing Equal to the Dimension), Pillars with Varying Spacing, V Grooves and U Grooves

(15) TABLE-US-00001 TABLE 1-1 (see also FIG. 3a) Patterns % reduction 10 um diameter pillars 18.53 2 um diameter pillars 49.58 500 nm diameter pillars 58.24 100 nm diameter pillars 39.45 25 nm diameter pillars 41.92

(16) TABLE-US-00002 TABLE 1-2 (see also FIG. 3b) Patterns % reduction 10 um diameter with 6 um spacing 39.99 10 um diameter with 20 um spacing 43.75 10 um diameter with 50 um spacing 57.93

(17) TABLE-US-00003 TABLE 1-3 (see also FIG. 3c) Patterns % reduction V grooves 2 um 73.5 V grooves 500 nm 26.07

(18) TABLE-US-00004 TABLE 1-4 (see also FIG. 3d) Patterns % reduction U grooves 2 um 60.45 U grooves 500 nm 33.14

(19) In Table 1-1, the spacings between the pillars were equal to the diameters of the pillars. Table 1-1 investigates the changes in diameter of the pillars while Table 1-2 investigates the effect of spacings (between the pillars) on the % bacterial reduction. The results from Table 1-1 and those of Table 1-2 were carried out in independent sets. As there is a correlation between the diameter of the pillars and the diameter of the bacteria, the most effective % reduction of the bacteria was observed from 2 um to 500 nm.

(20) As the spacing between the pillars increased while keeping the diameter of the pillars constant as seen in Table 1-2, the % reduction of the bacteria increased.

(21) With regard to Table 1-3, the diameter of the bacteria can be between 500 to 800 nm. For a 2 um groove, the bacteria will sit along the groove and thus be washed off whereas for a 500 nm groove, as this is similar to the diameter of the bacteria, the bacteria will be stuck in the grooves. Hence, it is shown that for a pattern with a larger diameter than the size of the bacteria, a higher % bacterial reduction can be obtained.

(22) With regard to Table 1-4, a U-groove pattern is more likely to trap bacteria than a V-groove as it is U-shaped. There is a higher tendency for a V-groove to trap a bacteria than a U-groove. Bacteria will thus lie in the 2 um U-groove and can be washed off leading to 60.45% reduction, whereas the 500 nm groove, due to its U shape, has a higher tendency to trap the bacteria, leading to a 33.14% reduction.

Table 2: Table Showing the % Reduction of S. epidermidis ATCC 12228 for Pillars with Different Dimensions and U Grooves

(23) TABLE-US-00005 TABLE 2-1 (see also FIG. 4a) Patterns % reduction 10 um diameter with 20 um spacing 15.09 10 um diameter with 50 um spacing 47.00

(24) TABLE-US-00006 TABLE 2-2 (see also FIG. 4b) Patterns % reduction U grooves 500 nm 18.24

(25) As shown in Table 2, as the spacing between the pillars increase, there is also a corresponding increase in the % reduction of the bacteria.

Table 3: Table Showing the % Reduction of P. Tunicata D2 for Pillars with Varying Pitch, V Grooves, U Grooves and Diamond Shapes

(26) TABLE-US-00007 TABLE 3-1 (see also FIG. 5a) Patterns % reduction 10 um diameter with 50 um pitch 29.99

(27) TABLE-US-00008 TABLE 3-2 (see also FIG. 5b and FIG. 5c) Patterns % reduction V grooves 500 nm 54.08 V grooves 100 nm 39.26 U grooves 2 um 43.56 U grooves 500 nm 65.42

(28) As observed in the above tables, it can be seen that as the spacing increases, the % reduction of E. coli ATCC 25922 and S. epidermidis ATCC 12228 improve. However, as P. Tunicata D2 is a marine bacteria, its behavior may be different to the first two bacteria. In this regard, grooves (such as V-grooves and U-grooves) may be used for P. Tunicata D2.

Example 2

(29) The protocol 120 of FIG. 2 was used to test the effect of repeated dipping and rinsing steps on the attachment of the microorganism to the test patterned substrates 12. Firstly, a shake culture 10 of a test microorganism such as E. coli ATCC 25922 was cultivated for a period of time in sterile culture medium until the mid-log phase. The test patterned substrates (or imprints) 12 are loaded into 6-well plate and incubated with the shake culture 10 (containing 1-210.sup.8 CFU/ml of bacteria) for 1 hour at a temperature of 35 C. The test patterned substrates 12 having various nano and micron sized structures used in this example were chosen based on their effective reduction in bacterial attachment based on Example 1. After incubation, the test patterned substrate 12 was then loaded onto a dipper 20 such as a robot dipper and subjected to a series of controlled rinsing and dipping steps (according to the arrow 14) whereby the direction of the test patterned substrate 12 was controlled. The dipping conditions were as follows. The first and second rinsing step were done using a speed of 20 mm per second, the test patterned substrates 12 were immersed in sterile phosphate buffer (22a,22b) for 2 seconds and then retrieved at the same speed. The third rinsing step was done in sterile de-ionized water (24) at the same conditions. After 3 rounds of rinsing, the retained bacteria on the surface of the test patterned substrate 12 were mounted on a slide 16 and the concentration of bacteria was then determined by averaging the cell counts of five fluorescent images using the software Cell Profiler 18. The results of this dipping/rinsing example is shown in FIG. 6. In FIG. 6, for samples which has 90 deg as part of its name, this means that the patterned area was tilted 90 from the normal. VG refers to V-grooves; UG refers to U-grooves; and 10P/x refers to 10 um diameter with a spacing of x where x is 6 um, 20 um or 50 um.

(30) From FIG. 6, it can be shown that most of the patterned substrates show a significant reduction in bacterial attachment, which is in agreement with the results in Example 1. It is to be noted that the data present in FIG. 6 and that from Example 1 are quantified by different methods. The data from FIG. 6 was done by controlled conditions and washing speeds were controlled with a dip coater, the samples were also washed three times in total whereas data from Example 1 were carried out by initial bacterial adhesion testing. The most prominent reduction were seen on the 2 um V and U groove structures. However, in both structures, the anticipated directional effects are absent. This can be due to 1) the low amount of adhered bacteria on these surfaces such that the washing did not have a significant effect; and 2) since the size of these groove are larger than the diameter of the rod-shape E. Coli, the bacteria that are already attached (albeit a small quantity) fit tighyly into the groove.

(31) The directional effect is pronounced in the group of groove structures with 500 nm dimension. Here, the size of the groove is smaller such that the bacteria are unable to fit well into the groove. Hence, a substantial amount of bacteria are loosely present on the surface. As a result, the washing condition significantly affects the amount of bacteria remaining on the surface.

APPLICATIONS

(32) The disclosed polymeric film having a surface with an array of patterned structures can be used in industries in which contamination with a biological material is to be reduced. Such industries can include medical (for example, medical implants, medical devices), food manufacturing (for example, food packaging, food manufacturing equipments and tools, drinking water containers or storage facilities), marine (such as marine infrastructure and seafaring vessels), water filtration industries (such as water purification systems) and construction (such as on building material surfaces, optical windows).

(33) The reduction in the biological material attachment may not require the use of chemical treatment of the surface or the adhesion of a coating on the surface. Hence, the problems of chemical contamination, environmental or health toxicity and lack of long-term durability that are commonly associated with such chemical methods are not present in the disclosed structured polymeric films. In addition, since chemical treatment is not necessary, the disclosed polymeric films can be used in a number of diverse applications that would not be otherwise possible with the conventional chemical treatments because of chemical interactions or inhibition with the targeted application.

(34) The disclosed polymeric films need not be sterilized in order to reduce the attachment of the biological material. Hence, there is no need to continuously sterilize the polymeric film to ensure minimal contamination of the biological material when the polymeric film is exposed to a non-sterile environment.

(35) 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.