Abstract
A kit-of-parts for attaching an object on a substrate (1, 1′) having (i) at least a first solution having at least one first compound, wherein the first compound is at least one of a gelling agent, a gellable agent, and a thickening agent, and (ii) at least a first substrate (1, 1′) having a surface (2, 2′). The first solution is suitable for forming at least a first dispersion of an object in the first solution when a the object is added to the first solution. The first dispersion is suitable for attaching the object on a functionalized surface (3, 3′) of the substrate (1, 1′) when the first dispersion is added to the functionalized surface (3, 3′) of the substrate (1, 1′).
Claims
1. A kit-of-parts for attaching an object on a substrate comprising: (i) At least a first solution comprising at least one first compound, wherein the first compound is at least one of a gelling agent, a gellable agent, and a thickening agent; and (ii) At least a first substrate comprising a surface; wherein the first solution is suitable for forming at least a first dispersion of at least one object in the first solution when at least one object is added to the first solution, and wherein the first dispersion is suitable for attaching the object on the surface of the substrate when the first dispersion is added to the surface of the substrate.
2. The kit-of-parts according to claim 1, further comprising instructions for the attachment of the object, wherein the instructions comprise the step of preparing the first dispersion by dispersing the at least one object in the first solution, and wherein the instructions further comprise the step of adding the first dispersion to the optionally functionalized surface of the substrate so as to attach the object on the optionally functionalized surface of the substrate.
3. The kit-of-parts according to claim 1, wherein the first solution comprises a pH-value that is at least one of physiological, in the range of about 6 to 8, or about 7.
4. The kit-of-parts according to claim 1, wherein at least one of i) at least a first part of the surface of the substrate is at least one of physically or chemically functionalized and ii) the kit-of-parts further comprises at least one second compound being suitable for chemically functionalizing at least a first part of the surface of the substrate, and wherein the first dispersion and the functionalized surface of the substrate are suitable for attaching the object on the functionalized surface of the substrate when the first dispersion is added to the functionalized surface of the substrate.
5. The kit-of-parts according to claim 4, wherein at least one of i) the surface of the substrate is chemically functionalized, and wherein the at least one second compound is provided in at least one second solution, or ii) wherein the surface of the substrate is physically functionalized, and wherein at least one of a) at least one surface structure is generated in the surface of the substrate or b) at least one layer is generated on the surface of the substrate.
6. The kit-of-parts according to claim 5, wherein the at least one layer comprises at least one of at least one metal compound, at least one oxide compound, at least one silicon compound, at least one nitride compound, at least one sulphide compound.
7. The kit-of-parts according to claim 5, wherein the second compound is at least one of a polymer or a copolymer thereof, a polymerizable agent, a cross-linking agent, and a compound comprising at least one functional group.
8. The kit-of-parts according to claim 7, wherein at least one of i) the polymer or the copolymer thereof or ii) the polymerizable agent is at least one of a polysaccharide compound, a polyaminosaccharide compound, a polyaminoacid compound, a polydopamine compound, a glycoproteine compound, a nucleic acid compound, an epoxy resin compound, a polysilane compound, a polysiloxane compound, a polyphosphate compound, a boron nitride polymer compound, a fluoropolymer compound, a polyallylamine compound, a polysulfide compound, and a polyphenol compound.
9. The kit-of-parts according to claim 8, wherein at least one of: the polyaminosaccharide compound is chitosan, the polyaminoacid compound is polylysine, the glycoprotein compound is laminin, the nucleic acid compound is desoxy ribonucleic acid, the epoxy resin compound is at least one of a bisphenol polymer compound and polyacetylene compound, the polyphenol compound is a polyphenolic protein, or the polyallylamine compound comprises at least one of primary, secondary or tertiary polymers.
10. The kit-of-parts according to claim 7, wherein the cross-linking agent is an aldehyde-comprising cross-linking agent.
11. The kit-of-parts according to claim 7, wherein the functional group is at least one of an organosilicon compound or an organosulfur compound.
12. The kit-of-parts according to claim 5, wherein at least one of: i) the second solution comprises at least one of a protic solvent, an aprotic solvent, a nonpolar solvent, a polar solvent, an organic compound, an inorganic compound, a liquid gas, and a melt, or ii) the second solution is an aqueous solution that further comprises at least one of a polar water-soluble solvent such as an alcohol, a dissolved salt such as sodium chloride, and an acid such as acetic acid or hydrochloric acid.
13. The kit-of-parts according to claim 1, wherein the first compound is at least one of a polysaccharide, an amide-based polymer, a silicon-based polymer, and an ionomer.
14. The kit-of-parts according to claim 13, wherein at least one of: the polysaccharide is selected from agarose, agar, alginate, dextran, the amide-based polymer corresponds to polyacrylamide, the silicon-based polymer corresponds to a polymeric organosilicon compound, or the ionomer corresponds to an inorganic polymer.
15. The kit-of-parts according to claim 1, wherein at least one of: i) the first solution is an aqueous solution, or ii) the first solution is an aqueous solution that further comprises at least one of a growth medium or a preferably dissolved salt such as sodium chloride.
16. The kit-of-parts according to claim 1, wherein at least one of: i) the first compound of the first solution has a concentration in the range of between 0.0001% by weight to 10% by weight with respect to a total volume of the first solution or between 0.001% by weight to 5% by weight with respect to the total volume of the first solution or between 0.02% by weight to 1% by weight with respect to the total volume of the first solution, ii) wherein the first compound of the first solution has a concentration in the range of between 0.0001% by volume to 10% by volume with respect to a total volume of the first solution or between 0.001% by volume to 5% by volume with respect to the total volume of the first solution or between 0.02% by volume to 1% by volume with respect to the total volume of the first solution, or iii) the first compound of the first solution is added to the first solution at a temperature between −20° C. to 120° C. or between 0° C. to 100° C. or between 10° C. and 40° C.
17. The kit-of-parts according to claim 5, wherein at least one of: i) the second compound of the second solution has a concentration in the range of between 0.0001% by weight to 50% by weight with respect to a total volume of the second solution or between 0.001% by weight to 5% by weight with respect to the total volume of the second solution or between 0.01% by weight to 2% by weight with respect to the total volume of the second solution, ii) wherein the second compound of the second solution has a concentration in the range of between 0.0001% by volume to 50% by volume with respect to a total volume of the second solution or between 0.001% by volume to 5% by volume with respect to the total volume of the second solution or between 0.01% by volume to 2% by volume with respect to the total volume of the second solution, or iii) the second compound of the second solution is added to the second solution at a temperature between −100° C. to 500° C. or between 0° C. and 100° C. or between 10° C. and 40° C.
18. The kit-of-parts according to claim 1, wherein at least a second part of the surface of the substrate is functionalized, and wherein at least one of: i) the kit-of-parts comprises at least a further first solution comprising at least one further first compound that differs from the first compound of the first solution, or ii) the functionalization of said second part of the surface of the substrate differs from the functionalization of the first part of the surface of the substrate.
19. The kit-of-parts according to claim 1, wherein at least one of: i) the substrate is a flexible support or a rigid support; ii) the substrate comprises a silicone-compound such as silicone dioxide or elementary silicone, plastic, ceramic, ceramic-metallic blend, a metal, a metal oxide or sulphide, and carbon such as graphite or diamond; or iii) at least part of the surface of the substrate is coated with a coating prior to the functionalization of the surface of the substrate.
20. A method of producing a kit-of-parts for attaching an object on a substrate, the method comprising the steps of: (i) Providing at least a first solution comprising at least one first compound, wherein the first compound is at least one of a gelling agent, a gellable agent, and a thickening agent; and (ii) Providing at least a first substrate comprising a surface; wherein the first solution is suitable for forming at least a first dispersion of at least one object in the first solution when at least one object is added to the first solution, and wherein the first dispersion is suitable for attaching the object on the surface of the substrate when the first dispersion is added to the surface of the substrate.
21. The method according to claim 20, wherein at least one of i) at least a first part of the surface of the substrate is at least one of physically or chemically functionalized and ii) the method further comprises the step of providing at least one second compound being suitable for chemically functionalizing at least a first part of the surface of the substrate, and wherein the first dispersion and the functionalized surface of the substrate are suitable for attaching the object on the functionalized surface of the substrate when the first dispersion is added to the functionalized surface of the substrate.
22. (canceled)
23. (canceled)
24. (canceled)
25. (canceled)
26. (canceled)
27. (canceled)
28. (canceled)
29. (canceled)
30. (canceled)
31. (canceled)
32. (canceled)
33. (canceled)
34. A method of attaching an object on a surface of a substrate comprising the steps of: (i) Preparing at least a first dispersion of at least one object in a first solution, wherein the first solution comprises at least one first compound, wherein the first compound is at least one of a gelling agent, a gellable agent, and a thickening agent; and (ii) Adding the first dispersion to the surface of the substrate, whereby the object is attached on the surface of the substrate.
35. The method according to claim 34, further comprising the step of functionalizing at least a first part of the surface of the substrate, and wherein the first dispersion is attached to the functionalized surface of the substrate, whereby the object is attached on the functionalized surface of the substrate.
36. The method according to claim 35, wherein at least one of: i) the functionalization of the surface of the substrate corresponds to a chemical functionalization that is achieved by applying at least one second compound to the surface of the substrate, wherein the second compound is provided in at least one second solution and interacts with the surface of the substrate, or ii) the functionalization of the surface of the substrate corresponds to a physical functionalization that is achieved by at least one of a) generating at least one surface structure in the surface of the substrate or by b) generating at least one layer on the surface of the substrate.
37. The method according to claim 36, wherein the at least one layer comprises at least one of: at least one metal compound, at least one oxide compound, at least one silicon compound, at least one nitride compound, or at least one sulphide compound.
38. (canceled)
39. (canceled)
40. The method according to claim 34, wherein at least a second part of the surface of the substrate is functionalized, and wherein at least one of: i) a further first dispersion of an object in a further first solution is added to the functionalized second part of the surface, wherein the further first solution comprises at least one further first compound that differs from the first compound of the first solution that is added to the functionalized first part of the surface, or ii) the functionalization of said second part of the surface of the substrate differs from the functionalization of the first part of the surface of the substrate.
41. The method according to claim 34, wherein the object is a biological object being at least one of a cell, a virus such as a phage, and a matter of biological origin such as peptides, proteins, polysaccharides, vesicles, protein-RNA co-polymers, protein-DNA co-polymers, capsules, spores, or wherein the object is a non-biological object being at least one of a protein, a lipid, a nucleic acid such as DNA, a nanotube or nano bead, a nanodevice, a glucide, a hydrocarbon, an aliphatic or aromatic polymer such as a phenolic polymer, and the like.
42. The method according to claim 34, wherein at least one of: i) the substrate is a flexible support or a rigid support such as a glass cover slide, a ceramic tile, a rigid electrode, a dish; ii) the substrate comprises a silicone-compound such as silicone dioxide or elementary silicone, plastic, ceramic, ceramic-metallic blend, a metal, a metal oxide or sulphide, and carbon such as graphite or diamond; or iii) at least part of the surface of the substrate is coated with a coating prior to the functionalization of the surface of the substrate in step (ii).
43. (canceled)
44. A substrate comprising at least one object being attached thereon, and wherein the substrate comprises: at least one surface, and at least one first layer, wherein the first layer is arranged on at least a part of the surface and is formed from at least a first dispersion as obtained in the method according to claim 34.
45. (canceled)
46. The substrate according to claim 44, wherein at least one of: i) a thickness of the first layer is about 100 nanometer or more or about 1000 nanometer or more, or ii) a thickness of the second layer is about 10 nanometer or more, or about 100 nanometer or more.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0327] Preferred embodiments of the invention are described in the following with reference to the drawings, which are for the purpose of illustrating the present preferred embodiments of the invention and not for the purpose of limiting the same. In the drawings,
[0328] FIG. 1 shows a top view on a substrate according to a first embodiment;
[0329] FIG. 2 shows a perspective view on a substrate according to a further embodiment, wherein the substrate is attached to a mount;
[0330] FIG. 3a shows a side view of the substrate according to FIG. 2;
[0331] FIG. 3b shows a top view of the substrate according to FIG. 2;
[0332] FIG. 4 shows a top view of a substrate according to a further embodiment;
[0333] FIG. 5 shows a top view of a substrate according to a further embodiment;
[0334] FIG. 6 shows a top view of a substrate according to a further embodiment;
[0335] FIG. 7a shows a photograph of a substrate that has been treated with glutaraldehyde, and that has been subjected to E. coli B1;
[0336] FIG. 7b shows a photograph of a substrate that has been treated with glutaraldehyde, and that has been subjected to E. coli B1 submerged in an agar solution;
[0337] FIG. 8a shows a photograph of a substrate that has been treated with poly-D-lysine, and that has been subjected to E. coli ATCC 25922;
[0338] FIG. 8b shows a photograph of a substrate that has been treated with poly-D-lysine, and that has been subjected to E. coli ATCC 25922 submerged in an agar solution;
[0339] FIG. 9a shows a photograph of a substrate that has been treated with glutaraldehyde and that has been subjected to the E. coli resistant strain B1;
[0340] FIG. 9b shows a photograph of a substrate that has been treated with poly-D-lysine and that has been subjected to the E. coli resistant strain B1 submerged in an agar solution;
[0341] FIG. 9c shows a photograph of the substrate according to FIG. 9b after an incubation time of three hours;
[0342] FIG. 10 shows a photograph of an untreated substrate, and that has been subjected to E. coli ATCC 25922;
[0343] FIG. 11 shows a photograph of an untreated substrate, and that has been subjected to E. coli ATCC 25922 submerged in an agar solution;
[0344] FIG. 12 shows a photograph of a substrate that has been treated with glutaraldehyde, and that has been subjected to E. coli ATCC 25922;
[0345] FIG. 13 shows a photograph of a substrate that has been treated with glutaraldehyde, and that has been subjected to E. coli ATCC 25922, submerged in an agar solution;
[0346] FIG. 14 shows a photograph of a substrate that has been treated with poly-D-lysine, and that has been subjected to E. coli ATCC 25922;
[0347] FIG. 15 shows a photograph of a substrate that has been treated with poly-D-lysine, and that has been subjected to E. coli ATCC 25922 submerged in an agar solution;
[0348] FIG. 16 shows a photograph of an untreated substrate, and that has been subjected to E. coli ATCC 25922 submerged in a Nafion® solution;
[0349] FIG. 17 shows a photograph of a substrate that has been treated with glutaraldehyde, and that has been subjected to E. coli ATCC 25922 submerged in a Nafion® solution;
[0350] FIG. 18 shows a photograph of a substrate that has been treated with chitosan, and that has been subjected to E. coli ATCC 25922 submerged in a Nafion® solution;
[0351] FIG. 19 shows a photograph of an untreated substrate, and that has been subjected to E. coli ATCC 25922 submerged in a polyacrylamide solution,
[0352] FIG. 20 shows a photograph of a substrate that has been treated with glutaraldehyde, and that has been subjected to E. coli ATCC 25922 submerged in a polyacrylamide solution;
[0353] FIG. 21 shows a photograph of a substrate that has been treated with chitosan, and that has been subjected to E. coli ATCC 25922 submerged in a polyacrylamide solution;
[0354] FIG. 22a-22f show photographs of a substrate that has been treated with poly-D-lysine and that has been subjected to E. coli ATCC 25922 submerged in an agarose solution (a), in an agar solution (b), in an alginate solution (c), in a Nafion® solution (d), in a polydimethylsiloxane solution (e), and in a polyethylene glycol solution (f);
[0355] FIG. 23a-23g show photographs of a substrate that has been treated with laminin and that has been subjected to E. coli ATCC 25922 submerged in an agarose solution (a), in an agar solution (b), in an alginate solution (c), in a Nafion® solution (d), in a polydimethylsiloxane solution (e), and in a polyethylene glycol solution (f) as well as a photograph of a substrate that has been treated with laminin and that has been subjected to an E. coli ATCC 25922 suspension (g);
[0356] FIG. 24a-24g show photographs of a substrate that has been treated with chitosan and that has been subjected to E. coli ATCC 25922 submerged in an agarose solution (a), in an agar solution (b), in an alginate solution (c), in a Nafion® solution (d), in a polydimethylsiloxane solution (e), and in a polyethylene glycol solution (f) as well as a photograph of a substrate that has been treated with chitosan and that has been subjected to an E. coli ATCC 25922 suspension (g);
[0357] FIG. 25a-25f show photographs of a substrate that has been treated with glutaraldehyde and that has been subjected to E. coli ATCC 25922 submerged in an agarose solution (a), in an agar solution (b), in an alginate solution (c), in a Nafion® solution (d), in a polydimethylsiloxane solution (e), and in a polyethylene glycol solution (f);
[0358] FIG. 26a-26g show photographs of a substrate that has been treated with (3-aminopropyl)triethoxysilane (APTES) and that has been subjected to E. coli ATCC 25922 submerged in an agarose solution (a), in an agar solution (b), in an alginate solution (c), in a Nafion® solution (d), in a polydimethylsiloxane solution (e), and in a polyethylene glycol solution (f) as well as a photograph of a substrate that has been treated with (3-aminopropyl)triethoxysilane and that has been subjected to an E. coli ATCC 25922 suspension (g);
[0359] FIG. 27a-27f show photographs of a substrate that has been treated with poly(sodium-p-styrene sulfonate)/poly(allylamine hydrochloride) copolymer and that has been subjected to E. coli ATCC 25922 submerged in an agarose solution (a), in an agar solution (b), in an alginate solution (c), in a Nafion® solution (d), in a polydimethylsiloxane solution (e), and in a polyethylene glycol solution (f);
[0360] FIG. 28a-28g show photographs of a substrate that has been treated with MAPTrix™ and that has been subjected to E. coli ATCC 25922 submerged in an agarose solution (a), in an agar solution (b), in an alginate solution (c), in a Nafion® solution (d), in a polydimethylsiloxane solution (e), and in a polyethylene glycol solution (f) as well as a photograph of a substrate that has been treated with MAPTrix™ and that has been subjected to an E. coli ATCC 25922 suspension (g);
[0361] FIG. 29a-29g show photographs of a substrate that has been treated with 4-aminothiophenol and that has been subjected to E. coli ATCC 25922 submerged in an agarose solution (a), in an agar solution (b), in an alginate solution (c), in a Nafion® solution (d), in a polydimethylsiloxane solution (e), and in a polyethylene glycol solution (f) as well as a photograph of a substrate that has been treated with 4-aminothiophenol and that has been subjected to an E. coli ATCC 25922 suspension (g);
[0362] FIG. 30a-30g show photographs of an untreated substrate that has been has been subjected to E. coli ATCC 25922 submerged in an agarose solution (a), in an agar solution (b), in an alginate solution (c), in a Nafion® solution (d), in a polydimethylsiloxane solution (e), and in a polyethylene glycol solution (f) as well as a photograph of an untreated substrate that has been been subjected to an E. coli ATCC 25922 suspension (g);
[0363] FIG. 31a-31f show photographs of a substrate that has been treated with poly-D-lysine and that has been subjected to Mycobacterium smegmatis MC(2)155 submerged in an agar solution (a) and in an agarose solution (b), as well as a photograph of a substrate that has been treated with poly-D-lysine and that has been subjected to a Mycobacterium smegmatis MC(2)155 suspension (c), as well as photographs of an untreated substrate that has been subjected to Mycobacterium smegmatis MC(2)155 submerged in an agar solution (d) and in an agarose solution (e), as well as a photograph of an untreated substrate that has been subjected to a Mycobacterium smegmatis MC(2)155 suspension (f);
[0364] FIG. 32a-32f show photographs of a substrate that has been treated with poly-D-lysine and that has been subjected to Vero ATCC CCL-81 submerged in an agar solution (a) and in an agarose solution (b), as well as a photograph of a substrate that has been treated with poly-D-lysine and that has been subjected to a Vero ATCC CCL-81 suspension (c), as well as photographs of an untreated substrate that has been subjected to Vero ATCC CCL-81 submerged in an agar solution (d) and in an agarose solution (e), as well as a photograph of an untreated substrate that has been subjected to a Vero ATCC CCL-81 suspension (f);
[0365] FIG. 33a-33g show photographs of a substrate that has been treated with poly-D-lysine and that has been subjected to Candida albicans SC5314 submerged in an agarose solution (a), in an agar solution (b), in an alginate solution (c), in a Nafion® solution (d), in a polydimethylsiloxane solution (e), and in a polyethylene glycol solution (f) as well as a photograph of a substrate that has been treated with poly-D-lysine and that has been subjected to a Candida albicans SC5314 suspension (g);
[0366] FIG. 34a-34g show photographs of a substrate that has been treated with laminin and that has been subjected to Candida albicans SC5314 submerged in an agarose solution (a), in an agar solution (b), in an alginate solution (c), in a Nafion® solution (d), in a polydimethylsiloxane solution (e), and in a polyethylene glycol solution (f) as well as a photograph of a substrate that has been treated with laminin and that has been subjected to a Candida albicans SC5314 suspension (g);
[0367] FIG. 35a-35g show photographs of a substrate that has been treated with chitosan and that has been subjected to Candida albicans SC5314 submerged in an agarose solution (a), in an agar solution (b), in an alginate solution (c), in a Nafion® solution (d), in a polydimethylsiloxane solution (e), and in a polyethylene glycol solution (f) as well as a photograph of a substrate that has been treated with chitosan and that has been subjected to a Candida albicans SC5314 suspension (g);
[0368] FIG. 36a-36f show photographs of a substrate that has been treated with glutaraldehyde and that has been subjected to Candida albicans SC5314 submerged in an agarose solution (a), in an agar solution (b), in an alginate solution (c), in a Nafion® solution (d), in a polydimethylsiloxane solution (e), and in a polyethylene glycol solution (f);
[0369] FIG. 37a-37f show photographs of a substrate that has been treated with (3-aminopropyl)triethoxysilane and that has been subjected to Candida albicans SC5314 submerged in an agarose solution (a), in an agar solution (b), in an alginate solution (c), in a Nafion® solution (d), in a polydimethylsiloxane solution (e), and in a polyethylene glycol solution (f);
[0370] FIG. 38a-38f show photographs of a substrate that has been treated with poly(sodium-p-styrene sulfonate)/poly(allylamine hydrochloride copolymer and that has been subjected to Candida albicans SC5314 submerged in an agarose solution (a), in an agar solution (b), in an alginate solution (c), in a Nafion® solution (d), in a polydimethylsiloxane solution (e), and in a polyethylene glycol solution (f);
[0371] FIG. 39a-39f show photographs of a substrate that has been treated with MAPTrix™ and that has been subjected to Candida albicans SC5314 submerged in an agarose solution (a), in an agar solution (b), in an alginate solution (c), in a Nafion® solution (d), in a polydimethylsiloxane solution (e), and in a polyethylene glycol solution (f);
[0372] FIG. 40a-40f show photographs of a substrate that has been treated with 4-aminothiophenol and that has been subjected to Candida albicans SC5314 submerged in an agarose solution (a), in an agar solution (b), in an alginate solution (c), in a Nafion® solution (d), in a polydimethylsiloxane solution (e), and in a polyethylene glycol solution (f);
[0373] FIG. 41a-41g show photographs of an untreated substrate that has been has been subjected to Candida albicans SC5314 submerged in an agarose solution (a), in an agar solution (b), in an alginate solution (c), in a Nafion® solution (d), in a polydimethylsiloxane solution (e), and in a polyethylene glycol solution (f) as well as a photograph of an untreated substrate that has been been subjected to an Candida albicans SC5314 suspension (g);
[0374] FIG. 42a shows an image of an untreated substrate being attached to a mount recorded with an electron microscope;
[0375] FIG. 42b shows another image of the untreated substrate being attached to the mount according to FIG. 42a recorded with an electron microscope;
[0376] FIG. 43 shows an image of a substrate that has been treated with poly-D-lysine recorded with an electron microscope;
[0377] FIG. 44 shows an image of a substrate that has been treated with poly-D-lysine in a first step and that has been treated with an agarose solution in a subsequent step recorded with an electron microscope;
[0378] FIG. 45a-45f show images of a substrate that has been treated with poly-D-lysine in a first step and that has been treated with E. coli ATCC 25922 submerged in an agarose solution in a subsequent step recorded with an electron microscope.
DESCRIPTION OF PREFERRED EMBODIMENTS
[0379] FIGS. 1 to 6 depict different embodiments of a substrate 1, 1′ for attaching biological objects and/or non-biological objects for illustrative purposes.
[0380] In fact, the substrate 1′ depicted in FIG. 1 corresponds to a rigid support in the form of a glass cover slide. Four parts 3′, 3a′, 3b′, 3c′ of a top surface 2′ of the glass cover slide 1 have been functionalized so as to allow an attachment of biological objects and/or of non-biological objects at four different conditions at a time. To this end the four parts 3′, 3a′, 3b′, 3c′ of the surface 2′ of the glass cover slides 1′ have been subjected to four different second compounds. For example, said different second compounds can correspond to different concentrations of the same second compound, different mixtures of different second compounds, or different incubation times, depending on the investigator's interest. In other words, FIG. 1 illustrates different chemical functionalizations of the surface 3′, 3a′, 3b′, 3c′ of the substrate 1′.
[0381] As has already been mentioned in the introduction, the attachment of biological objects is of great interest in the field of nanomotion AST, for example. In doing so the biological objects can be attached to a flexible support such as a cantilever, and wherein the movements of the cantilever that are caused by the attached biological objects are measured. FIGS. 2 to 6 depict different embodiments of a flexible substrate 1 in the form of a cantilever according to the invention which have proven to be very suitable and effective in these types of measurements. In fact, the cantilevers 1 can simply be attached to a mount 5 and be subject to measurements as disclosed in EP 2 766 722 B1, for example. The cantilevers 1 depicted in FIGS. 3a to 6 correspond here to essentially rectangular substrates that are etched from silicone wafers. The cantilevers 1 according to FIGS. 3a and 3b have in each case a surface 2 that has been chemically functionalized, wherein one or more second compounds preferably in a second solution have been added to the surface 2 of the cantilever, and wherein said one or more second compounds interact with the surface 2 of the cantilever, whereby the chemically functionalized surface 3 is formed. This is in contrast to the physically functionalized cantilevers 1 according to FIGS. 4 to 6, wherein a surface structure 4a, 4b, 4c has been generated in the surface 2 of the cantilever 1, whereby the physically functionalized surfaces 3 are formed. In particular, the cantilever 1 according to FIG. 4 comprises a surface structure 4a in the form of a dotted pattern, wherein the dots are recesses that reach into the surface 2 of the cantilever 1. The surface structures 4b, 4c of the cantilevers 1 depicted in FIGS. 5 and 6 correspond to a striped pattern, wherein the stripes are recesses that reach into the surface 2 of the cantilever 1. The stripes 4b of the cantilever 1 according to FIG. 5 extend along a transverse direction T of the cantilever 1, and the stripes 4c of the cantilever 1 according to FIG. 6 extend along a longitudinal direction L of the cantilever 1 that runs perpendicularly to the transverse direction T. These patterns 4a, 4b, 4c confer a surface topography to the cantilever 1 and are generated here by means of a KOH etching process.
[0382] As has already been discussed in great detail above, the inventors have found out that the use of a first solution comprising at least one of a gelling agent, gellable agent, and a thickening agent in combination with a functionalized surface 3, 3′ of the substrate 1, 1′ results in an improved attachment of the biological object as compared to the attachment methods known in the state of the art. FIGS. 7a to 21 shall illustrate this effect.
[0383] Namely, FIGS. 7a and 7b depict photographs of an attached E. coli ceftriaxone resistant strain B1. The substrate 1′ in these figures corresponds in each case to a glass slide cover. The substrate 1′ depicted in FIG. 7a has been treated with a dispersion comprising a solution of 0.5% glutaraldehyde by weight per total volume of the solution and a suspension comprising the E. coli ceftriaxone resistant strain B1. The substrate 1′ depicted in FIG. 7b, however, has been functionalized with a dispersion comprising a solution of 0.5% glutaraldehyde by weight per total volume of the solution in a first step. The same surface has subsequently been treated with a solution comprising 0.04% agar by weight per total volume of the solution and the E. coli ceftriaxone resistant strain B1. As is readily evident from a comparison between FIGS. 7a and 7b, a much higher number of E. coli bacteria is attached on the substrate 1′ according to the invention and as depicted in FIG. 7b.
[0384] The same findings are found with respect to FIGS. 8a and 8b. Namely, FIG. 8a depicts a photograph of a substrate 1′ that has been functionalized with a dispersion comprising a solution of 0.1% poly-D-lysine by weight and subsequently with E. coli susceptible strain ATCC 25922. The substrate 1′ depicted in FIG. 8b, has been functionalized with a solution of 0.1% poly-D-lysine by weight. In a subsequent step, however the surface 2′ of the substrate 1′ of FIG. 8b has been with a solution comprising 0.04% agar by weight per total volume of the solution and E. coli susceptible strain ATCC 25922.
[0385] FIGS. 9a to 9c illustrate an attachment of biological objects on a substrate 1 in the form of a cantilever. The cantilever 1 of FIG. 9a has functionalized prior to the addition of the biological object with a solution comprising 0.5% glutaraldehyde by weight per total volume. However, no first compound was added to the suspension of the biological object E. coli resistant strain B1. The surface 2 of the cantilever 1 has been functionalized with a solution of 0.01% poly-D-lysine by weight prior to the addition of the biological object in the situations depicted in FIGS. 9b and 9c. Subsequently, a dispersion comprising a solution comprising 0.04% agar by weight per total volume of the solution and cells of the E. coli resistant strain B1 have been added to the functionalized surface 3 of the substrate 1 in a second step. FIG. 9c depicts a micrograph of the functionalized substrate 1 of FIG. 9b that has been recorded about 3 hours after the attachment of the cells to said substrate 1. As is readily evident from a comparison between FIGS. 9a to 9c, a much higher number of E. coli bacteria is attached on the cantilevers 1 according to the invention and as depicted in FIGS. 9b and 9c as compared to the cantilever 1 according to FIG. 9a whose surface 2 has functionalized but the cell suspension did not contain a first compound such as agar. Furthermore, FIG. 9c clearly shows that even after some time there is still a high number of attached cells present on the substrate 1. Hence, the invention allows a reliable attachment, wherein the cells remain attached to the surface during a period of time that is at least as long as a testing time.
[0386] FIGS. 10 to 21 in each case depict an attachment of the E. coli ceftriaxone susceptible strain ATCC 25922 to differently treated substrates with different first compounds in the cell suspension. In particular, FIG. 10 shows a glass substrate that is a microscopic cover slide that has not been functionalized and where there has no first compound been added to the cell suspension. As can be seen, no cells of E. coli ceftriaxone susceptible strain ATCC 25922 attached to the surface after washing with water twice. The E. coli cells were grown overnight on Columbia agar plates as described above. The OD.sub.600 of the cell suspension was 1.2 prior to the addition of the same to the surface. In FIG. 11, another untreated glass surface is shown that was treated with the same cells but this time the first compound was added to the cell suspension. The first compound was agar at a concentration 0.04% by weight. More cells adhered to the surface compared with FIG. 10. The FIGS. 12 and 13 show the same cells attached to another glass surface that has been functionalized using glutaraldehyde at a concentration of 0.5% by weight. No first compound was added to the cell suspension. FIG. 12 shows that cells adhered in small aggregates on the surface. The test was repeated with the same cells and another glass surface that was functionalized using 0.5% glutaraldehyde by weight but this time 0.04% agar by weight were added to the cell suspension. The result in FIG. 13 shows that a similar amount of cells had attached to the surface but they were evenly dispersed without cell aggregates. This test was repeated but this time, glutaraldehyde as second compound was replaced by 0.01% poly-D-lysine by weight to functionalize the surface. FIG. 14 shows that without agar more cells attached compared with FIG. 12 where the surface was functionalized using glutaraldehyde. The cells attached in larger aggregates compared with glutaraldehyde. As shown in FIG. 15, when 0.04% agar by weight was added to the surface, more cells attached compared to the test without agar. The attached cells were also more evenly distributed over the glass surface.
[0387] FIG. 16 depicts an attachment of the cells to the substrate, wherein 0.03% Nafion® as the first compound but no second compound were used. That is, the surface of the substrate was not functionalized but only a first dispersion comprising the cells and 0.03% Nafion® by weight was added to the substrate. FIG. 17 depicts an attachment of the cells to a substrate that has been treated with both, a first compound and a second compound. In particular, 0.5% glutaraldehyde as the second compound was used to functionalize the surface of the substrate in a first step. In a second step, a dispersion of the cells and 0.03% Nafion® by weight was added to the functionalized surface of the substrate. FIG. 18 depicts an attachment of the cells to the substrate that has likewise been treated with a first and a second compound. In this case, a solution of 0.1% chitosan by weight was used to functionalize the surface of the substrate. Thereafter, a dispersion comprising the cells and 0.03% Nafion® was added to the functionalized surface. A further improvement of cell attachment is apparent. FIG. 19 in turn depicts an attachment of the cells where no second compound has been used, i.e. the surface of the substrate has not been functionalized. Instead, a dispersion comprising the cells and 0.1% acrylamide was added to the unfunctionalized surface of the substrate. FIG. 20 depicts an attachment of cells to a functionalized surface. That is, the surface of the substrate has been functionalized by 0.5% glutaraldehyde, wherein subsequently the dispersion comprising the cells and 0.1% acrylamide by weight have been added to the functionalized surface. Similarly, FIG. 21 depicts an attachment of cells to a functionalized surface, wherein the surface has been functionalized with 0.1% chitosan by weight, to which a dispersion comprising the cells and 0.1% acrylamide by weight has been added. From these figures it is readily apparent that a cell attachment takes place if a first compound is added to the cell dispersion, and where no functionalization of the surface of the substrate has taken place, see FIGS. 16 and 19. Said attachment can however be further enhanced if the surface of the substrate is functionalized, see FIGS. 17 to 18 and 20 to 21.
[0388] FIGS. 22a to 41g depict different images of a substrate in the form of a glass surface to which different objects have been added along with different first compounds and/or different second compounds.
[0389] In particular, FIGS. 22a to 22f depict the attachment of Enterobacteria Escherichia coli ATCC 25922 that were grown in a subculture for 1 hour at 37° C. and provided at a cell concentration OD.sub.600=1 to hydrolytic class 1 glass, wherein the images were recorded after an attachment time of 5 minutes at 25° C. In all these figures the glass surface has been functionalized with a solution of 0.1 milligram per millilitre poly-D-lysine solution during 5 minutes at 25° C. in a first step. Thereafter, a dispersion comprising Escherichia coli ATCC 25922 and (a) 0.04% agarose by weight per total volume of the first solution (b) 0.04% agar by weight per total volume of the first solution, (c) 0.25% alginate by volume per total volume of the first solution, (d) 0.25% Nafion® by weight per total volume of the first solution, (e) 2% polydimethylsiloxane by volume per total volume of the first solution (stock solution at 20 Centistokes (cst)) resulting in 0.4 cst polydimethylsiloxane, and (f) 0.125% polyethylene glycol by weight per total volume of the first solution were added to the functionalized surface, respectively. Here and below, the expression weight per total volume of the first solution means “gram per 100 millilitre of solvent”, wherein water was used as the solvent. The expression volume per total volume of the first solution means “millilitre of a 100% stock solution per total volume of the first solution”, wherein the expression “100% stock solution” refers to a solution comprising 100% of the first compound. In other words, a “100% stock solution” is an undiluted solution of the first compound. It follows from these images that a good and homogeneous attachment of Escherichia coli ATCC 25922 was achieved.
[0390] FIGS. 23a to 23g depict the attachment of Enterobacteria Escherichia coli ATCC 25922 that were grown in a subculture for 1 hour at 37° C. and provided at a cell concentration OD.sub.600=1 to hydrolytic class 1 glass, wherein the images were recorded after an attachment time of 5 minutes at 25° C. In all these figures the glass surface has been functionalized with a solution of 5 microgram per square centimeter (μg/cm.sup.2) laminin solution during 60 minutes at 25° C. in a first step. Thereafter, a dispersion comprising Escherichia coli ATCC 25922 and (a) 0.04% agarose by weight per total volume of the first solution (b) 0.04% agar by weight per total volume of the first solution, (c) 0.25% alginate by volume per total volume of the first solution, (d) 0.25% Nafion® by weight per total volume of the first solution, (e) 0.4 cst polydimethylsiloxane, and (f) 0.125% polyethylene glycol by weight per total volume of the first solution were added to the functionalized surface, respectively. In (g) the attachment of a suspension of Escherichia coli ATCC 25922 in the absence of a first compound is depicted. It follows, that usage of a functionalized surface only resulted in only a few cells to be attached while several agglomerates are formed. The addition of a first compound to the cell suspension significantly increases the attachment quality and homogeneity, especially when Nafion® is used (FIG. 23d).
[0391] FIGS. 24a to 24g depict the attachment of Enterobacteria Escherichia coli ATCC 25922 that were grown in a subculture for 1 hour at 37° C. and provided at a cell concentration OD.sub.600=1 to hydrolytic class 1 glass, wherein the images were recorded after an attachment time of 5 minutes at 25° C. In all these figures the glass surface has been functionalized with a solution of 0.1 milligram per millilitre chitosan solution during 5 minutes at 25° C. in a first step. Thereafter, a dispersion comprising Escherichia coli ATCC 25922 and (a) 0.04% agarose by weight per total volume of the first solution (b) 0.04% agar by weight per total volume of the first solution, (c) 0.25% alginate by volume per total volume of the first solution, (d) 0.25% Nafion® by weight per total volume of the first solution, (e) 0.4 cst polydimethylsiloxane, and (f) 0.125% polyethylene glycol by weight per total volume of the first solution were added to the functionalized surface, respectively. In (g) the attachment of a suspension of Escherichia coli ATCC 25922 in the absence of a first compound is depicted. It follows, that usage of a functionalized surface only resulted in only a few cells to be attached while several agglomerates are formed. As follows from these figures, if the cell suspension is added to the functionalized surface only and in the absence of a first compound, almost no cells are attached. The addition of a first compound drastically increased the number of cells as well as the quality, i.e. homogeneity of the attachment.
[0392] FIGS. 25a to 25f depict the attachment of Enterobacteria Escherichia coli ATCC 25922 that were grown in a subculture for 1 hour at 37° C. and provided at a cell concentration OD.sub.600=1 to hydrolytic class 1 glass, wherein the images were recorded after an attachment time of 5 minutes at 25° C. In all these figures the glass surface has been functionalized with a solution of 0.5% glutaraldehyde by volume per total volume of said solution in a first step. Thereafter, a dispersion comprising Escherichia coli ATCC 25922 and (a) 0.04% agarose by weight per total volume of the first solution (b) 0.04% agar by weight per total volume of the first solution, (c) 0.25% alginate by volume per total volume of the first solution, (d) 0.25% Nafion® by weight per total volume of the first solution, (e) 0.4 cst polydimethylsiloxane, and (f) 0.125% polyethylene glycol by weight per total volume of the first solution were added to the functionalized surface, respectively. As follows from these figures the addition of a first compound to the cell suspension reduces the presence of agglomerates and increases the numbers of cells attached.
[0393] FIGS. 26a to 26g depict the attachment of Enterobacteria Escherichia coli ATCC 25922 that were grown in a subculture for 1 hour at 37° C. and provided at a cell concentration OD.sub.600=1 to hydrolytic class 1 glass, wherein the images were recorded after an attachment time of 5 minutes at 25° C. In all these figures the glass surface has been functionalized with a solution of (3-aminopropyl)triethoxysilane of 1% by volume per total volume of said solution during 5 minutes at 25° C. in a first step. Thereafter, a dispersion comprising Escherichia coli ATCC 25922 and (a) 0.04% agarose by weight per total volume of the first solution (b) 0.04% agar by weight per total volume of the first solution, (c) 0.25% alginate by volume per total volume of the first solution, (d) 0.25% Nafion® by weight per total volume of the first solution, (e) 0.4 cst polydimethylsiloxane, and (f) 0.125% polyethylene glycol by weight per total volume of the first solution were added to the functionalized surface, respectively. In (g) the attachment of a suspension of Escherichia coli ATCC 25922 in the absence of a first compound is depicted. It follows that usage of 3-aminopropyl)triethoxysilane alone results in highly agglomerated cells and an unhomogeneous attachment. The addition of a first compound to the cell suspension significantly reduces the generation of agglomerates and the number of attached cells was increased.
[0394] FIGS. 27a to 27f depict the attachment of Enterobacteria Escherichia coli ATCC 25922 that were grown in a subculture for 1 hour at 37° C. and provided at a cell concentration OD.sub.600=1 to hydrolytic class 1 glass, wherein the images were recorded after an attachment time of 5 minutes at 25° C. In all these figures the glass surface has been functionalized first with a solution comprising 1 milligram per millilitre of poly(sodium-p-styrene sulfonate) and then by a 1 milligram per millilitre solution of poly(allylamine hydrochloride to finally form a co-polymer (i.e. a PAH/PSS co-polymer) during 20 minutes at 25° C. for each solution. The PAH/PSS co-polymer is a so-called layer-by-layer polymer, wherein the co-polymer is formed by incubating one compound at a time. In the present case the PSS polymer was incubated in a first step and the PAH polymer was incubated in a subsequent second step. The formation of this layer-by-layer polymer as a surface coating of the glass was performed initially. Thereafter, a dispersion comprising Escherichia coli ATCC 25922 and (a) 0.04% agarose by weight per total volume of the first solution (b) 0.04% agar by weight per total volume of the first solution, (c) 0.25% alginate by volume per total volume of the first solution, (d) 0.25% Nafion® by weight per total volume of the first solution, (e) 0.4 cst polydimethylsiloxane, and (f) 0.125% polyethylene glycol by weight per total volume of the first solution were added to the functionalized surface, respectively. As follows from these figures the addition of a first compound to the cell suspension reduces the presence of agglomerates and increases the numbers of cells attached. Also from these figures, it follows that a high number of cells are attached homogeneously when a first compound is added to the cell suspension.
[0395] FIGS. 28a to 28g depict the attachment of Enterobacteria Escherichia coli ATCC 25922 that were grown in a subculture for 1 hour at 37° C. and provided at a cell concentration OD.sub.600=1 to hydrolytic class 1 glass, wherein the images were recorded after an attachment time of 5 minutes at 25° C. In all these figures the glass surface has been functionalized with a solution of 1 milligram per millilitre Mussel Adhesive recombinant protein (MAPTrix™) solution during 30 minutes at 25° C. in a first step. Thereafter, a dispersion comprising Escherichia coli ATCC 25922 and (a) 0.04% agarose by weight per total volume of the first solution (b) 0.04% agar by weight per total volume of the first solution, (c) 0.25% alginate y volume per total volume of the first solution, (d) 0.25% Nafion® by weight per total volume of the first solution, (e) 0.4 cst polydimethylsiloxane, and (f) 0.125% polyethylene glycol by weight per total volume of the first solution were added to the functionalized surface, respectively. In (g) the attachment of a suspension of Escherichia coli ATCC 25922 in the absence of a first compound is depicted. As can be seen from these figures MAPTrix™ alone attaches the cells with a high amount of agglomerates. The tested first compounds resulted in an increased number of cells attached in a very homogeneous manner.
[0396] FIGS. 29a to 29g depict the attachment of Enterobacteria Escherichia coli ATCC 25922 that were grown in a subculture for 1 hour at 37° C. and provided at a cell concentration OD.sub.600=1 to hydrolytic class 1 glass, wherein the images were recorded after an attachment time of 5 minutes at 25° C. In all these figures the glass surface has been functionalized with a 10 mM solution of 4-aminothiophenol during 20 minutes at 25° C. in a first step. Thereafter, a dispersion comprising Escherichia coli ATCC 25922 and (a) 0.04% agarose by weight per total volume of the first solution (b) 0.04% agar by weight per total volume of the first solution, (c) 0.25% alginate by volume per total volume of the first solution, (d) 0.25% Nafion® by weight per total volume of the first solution, (e) 0.4 cst polydimethylsiloxane, and (f) 0.125% polyethylene glycol by weight per total volume of the first solution were added to the functionalized surface, respectively. In (g) the attachment of a suspension of Escherichia coli ATCC 25922 in the absence of a first compound is depicted. As can be seen from these FIGS. 4-aminothiophenol resulted in almost no cells being attached. Usage of a first compound significantly increased the attachment quality.
[0397] FIGS. 30a to 30g depict the attachment of Enterobacteria Escherichia coli ATCC 25922 that were grown in a subculture for 1 hour at 37° C. and provided at a cell concentration OD.sub.600=1 to an unfunctionalized hydrolytic class 1 glass. In FIGS. 30a to 30f the cell dispersion comprising the bacteria and a first compound was added to the unfunctionalized substrate. The first compounds were as follows: (a) 0.04% agarose by weight per total volume of the first solution (b) 0.04% agar by weight per total volume of the first solution, (c) 0.25% alginate by volume per total volume of the first solution, (d) 0.25% Nafion® by weight per total volume of the first solution, (e) 0.4 cst polydimethylsiloxane, and (f) 0.125% polyethylene glycol by weight per total volume of the first solution. FIG. 30g depicts an image of an untreated substrate to which the said cell suspension in the absence of any first compound has been added. It follows that the absence of a functionalized surface as well as the absence of a first compound result in the formation of compact cell agglomerates. The presence of a first compound results in a homogeneous attachment of many cells.
[0398] FIGS. 31a to 31f depict the attachment of Mycobacteria Mycobacterium smegmatis MC(2)155 that were grown in a subculture for 1 hour at 37° C. and provided at a cell concentration OD.sub.600=1 to hydrolytic class 1 glass, wherein the images were recorded after an attachment time of 5 minutes at 25° C. In FIGS. 31a to 31c the glass surface has been functionalized with a solution comprising 0.1 milligram per millilitre poly-D-lysine during 5 minutes at 25° C. in a first step. Furthermore, FIGS. 31a and 31b depict the substrate after the addition of a dispersion comprising Mycobacterium smegmatis MC(2)155 and (a) 0.04% agar by weight per total volume of the first solution and (b) 0.04% agarose by weight per total volume of the first solution, respectively. FIG. 31c depicts the addition of a cell suspension in the absence of a first compound. The presence of the first compound increase the numbers of cells attached. FIGS. 31d and 31e depict an untreated substrate, wherein the cell dispersion comprising the cells and the first compound have been added to the unfunctionalized glass substrate. To this end FIG. 31d depicts the attachment of a dispersion comprising 0.04 agar by weight per total volume of the first solution and FIG. 31e depicts the attachment of a dispersion comprising 0.04% agarose by weight per total volume of the first solution. FIG. 31f depicts an untreated, i.e. unfunctionalized substrate wherein the cell suspension per se, i.e. in the absence of a first compound has been added to the substrate. It follows that the presence of a first compound decreases the presence of agglomerates and increases the number of cells being attached.
[0399] FIGS. 32a to 32f depict the attachment of Mammalian cells, here of Vero cells of the strain ATCC CCL-81. It should be noted that the cellular shape of the depicted attached cells does not correspond to the real shape of Vero cells. This is due to the trypsinization of the cells, necessary to detach them from their initial culture (Vero cells are so-called adherent cells). This process results in detachment of the cells which, without their substrate and sister cells (Vero cells naturally grow to form a confluent cell monolayer), in a loss of shape characterized by a circularization of the cells. Detached cells can be counted and diluted to the desired concentration. This step is therefore necessary to work with such cells. In the present case, as the images were recorded only a few minutes after attachment, the cells did not have the time to reacquire their natural shape. It usually takes them several hours to recover totally from trypsinization. However, despite their unnatural shape the cells still behave in their natural manner such that the present images can be seen as an adequate proof of attachment of Vero cells. To this end, FIGS. 32a to 32f depict the attachment of Vero cells of the strain ATCC CCL-81 that were grown in a subculture for 1 hour at 37° C. and provided at a cell concentration of 3.35×10.sup.3 cells per millilitre to hydrolytic class 1 glass, wherein the images were recorded after an attachment time of 5 minutes at 25° C. In FIGS. 32a and 32b the glass surface has been functionalized with a solution comprising 0.1 milligram per millilitre poly-D-lysine during 5 minutes at 25° C. in a first step. Furthermore, FIGS. 32a and 32b depict the substrate after the addition of a dispersion comprising Vero cells ATCC CCL-81 and (a) 0.04% agar by weight per total volume of the first solution and (b) 0.04% agarose by weight per total volume of the first solution, respectively. FIG. 32c depicts the addition of a cell suspension in the absence of a first compound. The presence of the first compound increases the numbers of cells attached, especially in the case of agarose. FIGS. 32d and 32e depict an untreated substrate, wherein the cell dispersion comprising the cells and the first compound have been added to the unfunctionalized glass substrate. To this end FIG. 32d depicts the attachment of a dispersion comprising 0.04 agar by weight per total volume of the first solution and FIG. 32e depicts the attachment of a dispersion comprising 0.04% agarose by weight per total volume of the first solution. FIG. 32f depicts an untreated, i.e. unfunctionalized substrate wherein the cell suspension per se, i.e. in the absence of a first compound has been added to the untreated substrate. It follows that without a second compound for the surface treatment and a first compound as additive to the cell suspension only a few cells are able to attach (Vero cells are adherent cells and therefore they have the innate capacity to attach to surfaces with time. The addition of Agarose alone resulted in slightly more cells being attached. Agar on the other hand resulted in more cells attached. It is assumed that this difference is due to the difference of the polysaccharide mesh strength (i.e. Agar is more “dense” than Agarose for a same concentration).
[0400] FIGS. 33a to 41g depict the attachment of yeast Candida albicans SC5314 that were grown in a subculture for 1 hour at 37° C. and provided at a cell concentration of OD.sub.600=1 to hydrolytic class 1 glass, wherein the images were recorded after an attachment time of 5 minutes at 25° C.
[0401] In FIGS. 33a to 33g the glass surface has been functionalized with a solution of 0.1 milligram per millilitre poly-D-lysine solution during 5 minutes at 25° C. in a first step. Thereafter, a dispersion comprising Candida albicans SC5314 and (a) 0.04% agarose by weight per total volume of the first solution (b) 0.04% agar by weight per total volume of the first solution (c) 0.25% alginate by volume per total volume of the first solution, (d) 0.25% Nafion® by weight per total volume of the first solution, (e) 0.4 cst polydimethylsiloxane, and (f) 0.125% polyethylene glycol by weight per total volume of the first solution were added to the functionalized surface, respectively. FIG. 33g depicts the attachment of a suspension of Candida albicans SC5314 in the absence of a first compound. It follows that the presence of a functionalized surface alone, i.e. an addition of a cell suspension in the absence of a first compound, results in the presence of large agglomerates. The addition of a first compounds reduces the presence of agglomerates significantly. Furthermore, the number of cells being attached is increased.
[0402] In FIGS. 34a to 34g the glass surface has been functionalized with a solution of 5 microgram per square centimeter (μg/cm.sup.2) laminin solution during 5 minutes at 25° C. in a first step. Thereafter, a dispersion comprising Candida albicans SC5314 and (a) 0.04% agarose by weight per total volume of the first solution (b) 0.04% agar by weight per total volume of the first solution, (c) 0.25% alginate by volume per total volume of the first solution, (d) 0.25% Nafion® by weight per total volume of the first solution, (e) 0.4 cst polydimethylsiloxane, and (f) 0.125% polyethylene glycol by weight per total volume of the first solution were added to the functionalized surface, respectively. In (g) the attachment of a suspension of Candida albicans SC5314 in the absence of a first compound is depicted. It follows that an attachment in the absence of a first compound in the cell suspension resulted in an attachment that is not homogeneous throughout the substrate but with some zones showing a very dense population and some zones showing only a few agglomerates. The addition of a first compound homogenizes the attachment throughout the substrate, while reducing the presence of agglomerates.
[0403] In FIGS. 35a to 35g the glass surface has been functionalized with a solution of 0.1 milligram per millilitre chitosan solution during 5 minutes at 25° C. in a first step. Thereafter, a dispersion comprising Candida albicans SC5314 and (a) 0.04% agarose by weight per total volume of the first solution (b) 0.04% agar by weight per total volume of the first solution (c) 0.25% alginate by volume per total volume of the first solution, (d) 0.25% Nafion® by weight per total volume of the first solution, (e) 0.4 cst polydimethylsiloxane, and (f) 0.125% polyethylene glycol d by weight per total volume of the first solution were added to the functionalized surface, respectively. In (g) the attachment of a suspension of Candida albicans SC5314 in the absence of a first compound is depicted. As follows from these figures the presence of chitosan alone resulted in few cells attached and regrouped in agglomerates. Usage of a first compound in the cell dispersion increased the number of cells attached and the homogeneity of the attachment.
[0404] FIGS. 36a to 36f depict a glass surface that has been functionalized with a solution of 0.5% glutaraldehyde by volume per total volume of said solution in a first step. Thereafter, a dispersion comprising Candida albicans SC5314 and (a) 0.04% agarose by weight per total volume of the first solution (b) 0.04% agar by weight per total volume of the first solution (c) 0.25% alginate by volume per total volume of the first solution, (d) 0.25% Nafion® by weight per total volume of the first solution (e) 0.4 cst polydimethylsiloxane, and (f) 0.125% polyethylene glycol by weight per total volume of the first solution were added to the functionalized surface, respectively. As follows from these figures the addition of a first compound to the cell suspension results in a homogeneous repartition of the cells.
[0405] In FIGS. 37a to 37f the glass surface has been functionalized with a solution of (3-aminopropyl)triethoxysilane of 1% by volume per total volume of said solution during 5 minutes at 25° C. in a first step. Thereafter, a dispersion comprising Candida albicans SC5314 and (a) 0.04% agarose by weight per total volume of the first solution (b) 0.04% agar by weight per total volume of the first solution, (c) 0.25% alginate by volume per total volume of the first solution, (d) 0.25% Nafion® by weight per total volume of the first solution, (e) 0.4 cst polydimethylsiloxane, and (f) 0.125% polyethylene glycol by weight per total volume of the first solution were added to the functionalized surface, respectively. The addition of these first compounds to the cell suspension resulted in an absence of any agglomerates and in a homogeneous cell dispersion instead.
[0406] In FIGS. 38a to 38f the glass surface has been functionalized with a solution comprising 1 milligram per millilitre of poly(sodium-p-styrene sulfonate) and 1 milligram per millilitre of poly(allylamine hydrochloride copolymer during 20 minutes at 25° C. in a first step as described above with reference to FIGS. 27a to 27f. Thereafter, a dispersion comprising Candida albicans SC5314 and (a) 0.04% agarose by weight per total volume of the first solution (b) 0.04% agar by weight per total volume of the first solution, (c) 0.25% alginate by volume per total volume of the first solution (d) 0.25% Nafion® by weight per total volume of the first solution, (e) 0.4 cst polydimethylsiloxane, and (f) 0.125% polyethylene glycol by weight per total volume of the first solution were added to the functionalized surface, respectively. As is readily apparent from these figures, the cell dispersion according to the invention enabled a homogeneous attachment of many cells.
[0407] In FIGS. 39a to 39f the glass surface has been functionalized with a solution of 1 milligram per millilitre Mussel Adhesive recombinant protein (MAPTrix™) solution during 30 minutes at 25° C. in a first step. Thereafter, a dispersion comprising Candida albicans SC5314 and (a) 0.04% agarose by weight per total volume of the first solution (b) 0.04% agar by weight per total volume of the first solution, (c) 0.25% alginate by volume per total volume of the first solution, (d) 0.25% Nafion® by weight per total volume of the first solution, (e) 0.4 cst polydimethylsiloxane, and (f) 0.125% polyethylene glycol by weight per total volume of the first solution were added to the functionalized surface, respectively. Also in this case it is noted that a homogeneous attachment was achieved, wherein a rather high number of attachment was achieved especially with agarose and PDMS as first compound.
[0408] In FIGS. 40a to 40f the glass surface has been functionalized with a 10 mM solution of 4-aminothiophenol during 20 minutes at 25° C. in a first step. Thereafter, a dispersion comprising Candida albicans SC5314 and (a) 0.04% agarose by weight per total volume of the first solution (b) 0.04% agar by weight per total volume of the first solution, (c) 0.25% alginate by volume per total volume of the first solution, (d) 0.25% Nafion® by weight per total volume of the first solution, (e) 0.4 cst polydimethylsiloxane, and (f) 0.125% polyethylene glycol by weight per total volume of the first solution were added to the functionalized surface, respectively. The addition of the first compound to the cell suspension increased the homogeneity of the attachment. Especially Nafion® and alginate as first compounds gave homogeneous cell attachment with almost no agglomerates.
[0409] In FIGS. 41a to 41f the cell dispersion comprising Candida albicans SC5314 and a first compound was added to the unfunctionalized substrate. The first compounds were as follows: (a) 0.04% agarose by weight per total volume of the first solution (b) 0.04% agar by weight per total volume of the first solution, (c) 0.25% alginate by volume per total volume of the first solution, (d) 0.25% Nafion® by weight per total volume of the first solution, (e) 0.4 cst polydimethylsiloxane, and (f) 0.125% polyethylene glycol by weight per total volume of the first solution. FIG. 41g depicts an image of the untreated substrate to which the said cell suspension in the absence of any first compound has been added. The addition of a first compound resulted in increased attachment quality. Especially the addition of agarose or PEG resulted in a high amount of cells being attached.
[0410] FIGS. 42a to 45f depict images of a substrate in the form of a cantilever being attached to a mount that were recorded with an electron microscope. To this end FIGS. 42a and 42b depict an untreated cantilever 1 whose surface 2 has not been functionalized yet. FIG. 43 depicts the cantilever 1 whose surface 2, 3 has been functionalized with a solution comprising 0.1 milligram per millilitre of poly-D-lysine solution for 20 minutes at 25° C. FIG. 44 depict the cantilever 1 according to FIG. 43 wherein the functionalized surface 3 has been additionally treated with a solution of 0.04% agarose by weight per total volume of said solution, incubated during 5 minutes at 25° C. FIGS. 45a to 45f depict images of the cantilever according to FIG. 43, wherein the functionalized surface has been additionally treated with a dispersion comprising E. coli ATCC 25922 submerged in an agarose solution. As just described, the functionalization of the cantilever was achieved by adding a solution comprising 0.1 milligram per millilitre of poly-D-lysine for 20 minutes at 25° C. in a first step and by incubating said functionalized cantilever with a dispersion comprising 0.04% weight by volume of agarose and Escherichia coli ATCC 25922 with an OD600=5 during 5 minutes at 25° C. in a second step. From these images it follows that the poly-D-lysine solution that is used to functionalize the surface of the cantilever forms a coating or a layer on the surface. It is said coating or layer that provides the functionalization of the cantilever. The addition of the agarose solution only (FIG. 44) as well as the addition of a cell dispersion comprising the bacteria as well as agarose (FIGS. 45a to 45f) in each case results in the formation of a further layer that is arranged on top of the layer constituting the functionalization of the cantilever. In other words, the functionalized cantilever to which the cells are attached can be seen as a layered device, wherein a first layer is arranged on top of a second layer. A thickness of the cantilever comprising the second layer only, i.e. a cantilever whose surface has been functionalized with a solution comprising the second compound according to the invention, has here a thickness in the range of about 720 to 780 nanometer. The thickness of the cantilever comprising the said second layer as well as a first layer being constituted by the agarose solution only has a thickness of about 1 micrometer to 1.5 micrometer. The thickness of a cantilever comprising the said second layer as well as a first layer being constituted by the dispersion comprising the bacteria being dispersed in the agarose solution is about 2.5 micrometer.
[0411] Regarding the attachment of other objects such as DNA the following is noted. DNA is negatively charged just as Gram-neg and Gram-pos bacteria. DNA backbone is constituted of phosphate groups which are negatively charged. In Gram-positive bacteria the reason of this negative charge is the presence of teichoic acids linked to either the peptidoglycan or to the underlying plasma membrane. These teichoic acids are negatively charged because of presence of phosphate in their structure. The Gram-negative bacteria have an outer covering of phospholipids and lipopolysaccharides. The lipopolysaccharides impart a strongly negative charge to surface of Gram-negative bacterial cells. The addition of a first compound according to the invention, i.e. the addition of a gelling agent and/or gellable agend and/or thickening agent, will help with the DNA distribution and the attachment on the substrate. Besides, it is noted that polysaccharides such as agar and agarose are commonly used in laboratories working with DNA already in these days, wherein agar and agarose are used to create hydrogels allowing DNA extraction and verification, for example.
LIST OF REFERENCE SIGNS
[0412] 1, 1′ substrate [0413] 2, 2′ surface [0414] 3, 3′, 3a′, 3b′, 3c′ functionalized surface [0415] 4a, 4b, 4c surface structure [0416] 5 mount [0417] T transverse direction [0418] L longitudinal direction
