Plasma Deposition Method For Catechol/Quinone Functionalised Layers

Abstract

The invention provides a solvent-free plasma method for depositing an adherent catechol and/or quinone functionalised layer to an inorganic or organic substrate from a precursor which comprises at least a quinone group; a protected or unprotected catechol group; a molecule substituted by a quinone group and/or a protected or unprotected catechol group; and/or a natural or synthetic derivative of a catechol group and/or a quinone group; wherein the quinone group is a 1,2-benzoquinone group and the catechol group is a 1,2-dihydroxybenzene group.

Claims

1.-16. (canceled)

17. A method for adhering a catechol and quinone functionalized layer to a substrate wherein the method comprises the steps of: a) providing a substrate; b) providing a precursor comprising at least one monomer and a molecule with an unprotected catechol group; c) applying a plasma to said precursor and said substrate in order to form a coating comprising a catechol and quinone functionalized layer on said substrate.

18. The method according to claim 17, wherein said molecule with an unprotected catechol group is ##STR00027##

19. The method according to claim 17, wherein said monomer is vinyltrimethoxysilane.

20. The method according to claim 17, wherein a step of cleaning the substrate by ultrasonic washing is carried out before step (a).

21. The method according to claim 17, wherein said substrate is an organic or inorganic substrate.

22. The method according to claim 17, wherein a polymerisation initiator agent is injected into the precursor one of before step (c) or during step (c).

23. The method according to claim 22, wherein said polymerization initiator agent is a free radical initiator.

24. The method according to claim 23, wherein said free radical initiator is one of 4,4′-azobis(4-cyanopentano acid), 2,2′-azobis[2-methyl-N-(1,1-bis-(hydroxymethyl)-hydroxyethylpropionamide],2,2′-azobis(2-methylpropionamidine)dihydrochloride, 2,2′-azobis[2-methyl-N-(2-hydroxyethyl)propionamide], 2,2′-azobis(isobutyramidine hydrochloride), 2,2′-azobis[2-methyl-N-(1,1-bis(hydroxymethyl)-2-ethyl)-propionamide], 2,2′-azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride, 2,2′-Azobis[N-(2-carboxyethyl)-2-methylpropionamidine] hydrate, 2,2′-Azobis{2-[1-(2-hydroxyethyl)-2-imidazolin-2-yl]propane}dihydrochloride, 2,2′-Azobis(1-imino-1-pyrrolidino-2 ethylpropane)dihydro chloride, 2,2′-Azobis{2-methyl-N-[1,1-bis(hydroxymethyl)-2-hydroxyethyl]propionamide},2,2′-azobis[2-methyl-N-(2-hydroxyethylpropionamide], 2,2′- azobis(isobutylamide) dihydrate, or azoinitiators having polyethylene glycol unit.

25. The method according to claim 17, wherein the plasma used in step (c) is a non-equilibrium plasma process operating at atmospheric pressure.

26. The method according to claim 17, wherein said plasma of step (c) is generated by an electrical excitation, said electrical excitation comprising an electrical signal which is delivered in a pulsed wave form.

27. The method according to 17, wherein said molecule with an unprotected catechol group is ##STR00028## ##STR00029## wherein R.sub.2 and R.sub.3 are the same or different, and independently represent a hydrogen atom, a saturated C.sub.1-4 hydrocarbon group, and wherein P.sub.1 represents separately and independently —NH.sub.2, —COOH, —OH, —SH, ##STR00030## wherein R.sub.2 and R.sub.3 are the same or different, and independently represent a hydrogen atom, a saturated C.sub.1-4 hydrocarbon group, an halogen, ##STR00031## wherein each of A.sub.1 and A.sub.2 independently represents a hydrogen atom; or ##STR00032## wherein LG represents a linking group and is chosen from oligomers having chemical structure —[C(R.sub.2)(R.sub.3)].sub.x—P.sub.2 wherein R.sub.2 and R.sub.3 are the same or different, and independently represent a hydrogen atom, a saturated C.sub.1-4 hydrocarbon group and wherein P.sub.2 represents 1'NH.sub.2, —COOH, —OH, —SH, an halogen, —NH-A.sub.5-, —C(O)A.sub.6, —CH(NHA.sub.5)-c(O)-A.sub.6 wherein A.sub.5 represents —H, —C and A.sub.6 represents —OH, —NH.sub.2.

28. The method according to claim 17, wherein said monomer is at least one of an acrylate derivative, a methacrylate derivative, a styrene derivative, a vinyl ester derivative, a vinyl amide derivative, acrylonitrile, methacrylonitrile, acrylamide, methacrylamide, an unsaturated vegetable oil, a fatty acid, an acryclic acid, a methacrylic acid, a vinyl alkoxysilanes, ethylene, hexamethyldisiloxane, hexamethyldisilazane, octamethylcyclotetrasiloxane, decamethylcylcopentasiloxane, dodecamethylcyclohexasiloxane, hexaethyldisiloxane, tetraethylorthosilicate, tetramethyldisiloxane, pentamethylcyclopentasiloxane, octamethylcyclooctasiloxane, polydimethylsiloxane and derivatives of polydimethylsiloxane.

29. A coated substrate, said substrate comprising: a catechol and quinone functionalized layer, the catechol and quinone functionalized layer adhered to the substrate by: a) providing the substrate; b) providing a precursor comprising at least one monomer and a molecule with an unprotected catechol group; and c) applying a plasma to the precursor and the substrate in order to form a coating comprising the catechol and quinone functionalized layer on the substrate.

30. A process for immobilizing at least one biomolecule on a substrate with a catechol and quinone functionalized layer, said process comprising: a) providing the substrate; b) providing a precursor comprising at least one monomer and a molecule with an unprotected catechol group; c) applying a plasma to the precursor and the substrate in order to form a coating comprising the catechol and quinone functionalized layer on the substrate; and d) immersing the coated substrate in a buffer solution comprising at least one biomolecule.

31. A process according to claim 30, wherein said at least one biomolecule is natamycin and/or an antibacterial peptide, said antibacterial peptide being preferentially nisin, lysozyme, and/or melimine peptide.

32. The method according to claims 21 wherein the inorganic substrate comprises one of a stainless steel substrate, a titanium substrate or an aluminum substrate.

Description

[0106] A first atmospheric pressure plasma deposition chamber is indicated generally at 100 on FIG. 1 of the drawings. Chamber 100 comprises a spray system indicated generally at 105, a plasma generation system indicated generally at 107, a moving table 110 and an inorganic or organic substrate 120. The spray system 105 has a precursor inlet 130 and an ultrasonic spray system 140 which generates microdroplets 150 which when directed against the substrate 120 in step 200 forms an initial coating layer 152. In some embodiments, the ultrasonic spray system 140 may be a 48 kHz ultrasonic atomising nozzle (Sono-Tek Corporation). The plasma generation system 107 has a pair of electrodes 160, a gas flux inlet 170, a high voltage circuit 180 and a gas outlet 190. The plasma generation system 107 is arranged vertically above the moving table 110 and is spaced from the substrate 120. When a plasma is generated in step 300, an adhered organic functionalised coating layer 152B is formed on the substrate 120A. The adhered organic functionalised coating layer 152B has functional catechol and/or quinone groups indicated at 154.

[0107] A second atmospheric pressure plasma deposition chamber is indicated generally at 500 on FIG. 5 of the drawings. Chamber 500 comprises a plasma generation system indicated generally at 507, a moving table 510 and an inorganic or organic substrate 520. The plasma generation system 507 has a pair of electrodes 560, a gas and precursor inlet 570, a high voltage circuit 580 and a gas and precursor outlet 590. The plasma generation system 507 is arranged vertically above the moving table 510 and is spaced from the substrate 520. The gas and precursor introduced by outlet 590 and the substrate 520 are then submitted to a plasma polymerization step 300. A substrate 520A of organic or inorganic material having an adhered coating 552B is generated. The coating 552B is an organic functionalised layer with quinone groups 554 and catechol groups 555

[0108] A low pressure plasma deposition chamber is indicated generally at 600 on FIG. 6 of the drawings. Chamber 600 comprises a deposition chamber 605, a high voltage electrode 602, a grounded electrode 601, a generated plasma 603, a plasma gas and precursor inlet 604, an outlet 606 connected to a pump, a substrate 620 and a functionalised layer 652.

[0109] A coated substrate according to a first embodiment of the invention is indicated generally at 700 in FIG. 7. The coated substrate has a substrate 704 and a solvent-free adhered coating 702. A coated substrate according to a first embodiment of the invention is indicated generally at 800 in FIG. 8. The coated substrate has a substrate 804 and solvent-free adhered coating portions 802A and 802B which partly cover substrate 804.

[0110] The present invention will be further illustrated by reference to the following non limiting examples.

PREPARATORY EXAMPLE 1

[0111] In this Preparatory Example, the synthesis of N-(3,4-Dihydroxyphenethyl)acrylamide (DOA, molecule (2)) is described.

[0112] The synthesis used a modified procedure adopted from Lee et al. [Lee, B. P.; Huang, K.; Nunalee, F. N.; Shull, K. R.; Messersmith, P. B. J. Biomater. Sci. Polym. Ed. 2004, 15, 449-464.]. A two-neck round-bottom flask was charged with 12.1 g (31.6 mmol) of Na.sub.2B.sub.4O.sub.7.10H.sub.2O and 5.0 g of Na.sub.2CO.sub.3, and 475 mL of milli-Q water (18.2 MΩ.Math.cm, Millipore). This basic aqueous solution was degassed in sonicator bath (Branson 2510, 100 W, 42 KHz) for 1 h, applying light vacuum followed by, bubbling with argon for another 2 h. 3 g (15.8 mmol) of Dopamine hydrochloride (molecule 1) was added under argon atmosphere and continued stirring for 30 minutes. The flask was then cooled at 0° C. before drop-wise addition of 5.1 mL (63.2 mmol) of acryloyl chloride with stirring. Another 9.0 g Na.sub.2CO.sub.3 was added to maintain the pH of the solution above 9 during the reaction. After stirring for 12 h at room temperature, the solution was acidified to pH 1-2 with 6N HCl and continued stirring for 1 h in an open vessel. The mixture was extracted five times with ethyl acetate, washed with 0.1 M HCl and dried over MgSO.sub.4. The solvent was removed in vacuum to yield crude greyish paste, which was further purified by flash silica gel column chromatography eluting with dichloromethane/methanol (9:1) mixture (80% yield).

##STR00025##

EXAMPLE 2

[0113] In this Example, preparation of bio-inspired antibacterial surfaces is described.

[0114] The substrates used for deposition are 1 mm thick mirror polished 304 stainless steel disks or aluminium foils. Stainless steel disks were first cleaned by successive ultrasonic washings in butanone (5 min.), acetone (1 min.) and absolute ethanol (1 min) and further dried under a nitrogen flux. Lysozyme from chicken egg, white egg (lyophilized powder, protein 90%, 40,000 units/mg protein, Aldrich) was used as received.

[0115] Before plasma deposition, the metallic substrates were plasma cleaned and activated through an Ar/O.sub.2 plasma treatment (19 slm/1 slm (standard litre per minute)) in continuous (CW) discharge mode at 1.6 W/cm.sup.2 during 30 sec.

[0116] A laboratory synthesized dopamine acrylamide (DOA, molecule (2)) prepared as described in Preparatory Example 1 was dissolved at final concentration of 0.5 mg/mL in vinyltrimethoxysilane (VTMOS, 98%, Sigma-Aldrich) for 10 hours at ambient temperature.

[0117] First, the prepared solution was sprayed by a 48 kHz ultrasonic atomising nozzle 140 (Sono-Tek Corporation) using the apparatus illustrated in FIG. 1. The created mist 150 was composed of droplets of median diameter of 40 microns and the range of size is 5 to 200 microns. 0.25 mL/min of solution was injected in the nozzle 140 by using a syringe driver. The Sonotek generator 140 was set up to 2 W to generate the mist 150, while at the output of the nozzle 140, a nitrogen flow was used in order to shape the mist 150, and entrain it on the substrate 120. Plasma curing in argon was then performed with a dielectric barrier discharge reactor 107 composed of two flat alumina covered electrodes 160 connected to high voltage 180 and ensuring an efficient plasma surface zone of 18.72 cm.sup.2. The samples were placed on the moving table 110 (i.e, grounded electrode) ensuring a dynamic deposition mode. The table 110 speed and the gap between the electrodes 160 were fixed at 100 mm.Math.s.sup.−1 and 1 mm, respectively. The plasma discharge was ignited with a sinusoidal signal at 10 kHz chopped by a 1667 Hz rectangular signal. The power density was set up to 1.6 W/cm.sup.2.

[0118] A biomolecule in the form of lysozyme (antibacterial peptide) was then immobilised on the coated surface as follows. Substrates coated with the catechol/quinone functionalized layer were immersed in a 10 mM phosphate buffer solution containing lysozyme at 5 mg/ml, at pH 11 during 1h at room temperature under gentle mechanical agitations. The samples were then washed 5 times during 5 minutes with MilliQ purified water.

[0119] Scanning Electron Microscopy (SEM) observations were performed on a Hitachi SU-70 FE-SEM after a metallization step.

[0120] FT-IR analysis was performed on a Bruker Hyperion 2000 spectrometer equipped a with MCT detector. A grazing angle or ATR objectives were used for the coated stainless steel or aluminium substrates analyses, respectively.

[0121] X-ray photon spectroscopy (XPS) was performed with a Kratos Axis-Ultra DLD instrument using a monochromatic Al Kα X-ray source (hu=1486.6 eV) at pass energy of 20 eV. PerkinElmer Lambda 950 UV-vis-NIR (InGaAs) spectrophotometer equipped with an integrating sphere was used for UV-visible spectroscopy.

[0122] The antibacterial properties of the deposited layers against Bacillus subtilis, a gram+bacteria, were investigated by using the ISO 22196 antibacterial test. A surface is considered as antibacterial if the logarithmic reduction of the survival bacteria is superior than 2. This test is largely exploited in the industry and derived from the JISZ2801 protocol.sup.27. A brief description of the method can be found in.sup.28.

[0123] The resulting coatings were well adherent to the substrate and powder-free. SEM analysis revealed that the deposits were smooth and covered homogeneously all the surface. This latter result was also confirmed by XPS analysis as no Al and/or Fe was detected.

[0124] To obtain a fully covering layer presenting functional groups, the samples are exposed less than 15 seconds under the plasma. This method is in the order of few seconds compared to all other existing methods which are in the order of about an hour. The method of the present invention is thus at least 200 times faster.

[0125] The deposition rate, measured with a contact profilometer, was estimated to be around 5 to 10 nm/s,.sup.29. Hence, catechol/quinone functionalized layers can be deposited according to the present invention with a deposition rate that is at least 200 times faster than the conventional wet chemical methods already reported.

[0126] According to FT-IR analyses reported in FIG. 2, the plasma deposited layer (spectra c) results clearly from the combination of DOA and VTMOS monomers as several characteristic peaks from dopamine acrylamide and VTMOS are detected. Hence, a broad band at 3345 cm.sup.−1 can be assigned to OH groups from catechol and NH groups from amide bond in dopamine acrylamide monomer; a very slight shoulder at 3080 cm.sup.−1 corresponding to ═C—H stretching confirms the presence of aromatic compounds. Aromatic out of plane C—H bending vibrations are also visible at 830 cm.sup.−1. A shoulder at 2920 cm.sup.−1 is ascribed to stretching of aromatic CH groups. Many peaks are overlapped between 1500 and 1600 cm.sup.−1. NH bending from amide II presents a peak at 1550 cm.sup.−1 and aromatic C═C at 1520 cm.sup.−1. A peak at 1450 cm.sup.−1 is ascribed to CN stretching. A band at 1740-1720 cm.sup.−1 might be allocated to C═O from quinones or C═O stretching. The 1650 cm.sup.−1 peak is ascribed to C═O bending from quinones groups.sup.30 and also could come from the amide bond in the dopamine acrylamide monomer. The absence of the vinylic peak 1600 cm.sup.−1 from DOA and VTMOS suggests that a plasma polymerisation through the vinyl monomer group occurred. Considering the presence of Si-O stretching, Si—OH, Si—O—CH.sub.3 peaks at 1200-1000 cm.sup.−1, 940 cm.sup.−1 and 1280 cm.sup.−1 respectively, it can be concluded that the catechol/quinone functionalized layer might be composed of a silica network resulting from the use of VTMOS. Finally, the peak at 1100 cm.sup.−1 might be related that an overlap should exist between the Si—O—Si peak and C—C/C—O peak coming from a C—C/C—O network created by the reaction between catechols and radicals as supposed in E. Faure review.sup.31.

[0127] Catechols are chromophore compounds that absorb in the UV zone. Using transmittance UV-Vis analysis, it has been shown that dopamine acrylamide in VTMOS solution shows an absorbance peak at around 290 nm (FIG. 3.a). UV-Visible analyses of plasma deposited layer (FIG. 3.c) confirm the presence of free catechol groups with a small shift of this peak toward 276 nm. The same shift was observed in the UV-analyse of a sprayed DOA/VTMOS solution onto a surface (FIG. 3b). In the plasma deposited layer, one can also notice the formation of a band at 320 nm and the emergence of a peak at 420 nm. The former peak might be due to the formation of a polymer of higher molecular mass, a dopamine derivative intermediate. The peak at 420 nm is sometimes assigned to catechol oxidation and accumulation of phenol coupling products.

[0128] By comparing the XPS analyses (Table 1) of plasma deposited layers from pure VTMOS and from a DOA/VTMOS solution in a similar process deposition condition, the successful integration of both comonomers in the layer can be pointed out. Indeed, only DOA/VTMOS plasma deposited layer contains nitrogen element up to 2 at. %.

[0129] According to XPS analyses, it can be concluded that lysozyme was successfully immobilized onto the plasma deposited catechol/quinone functionalized layer as an increased amount of nitrogen (from 2 to 13 at. %) and the presence of sulphur, a “fingerprint” of the biomolecule, were detected (Table 1.c).

TABLE-US-00001 TABLE 1 XPS atomic percentages for different plasma deposited layers. Atomic surface composition (at. %) C N O S Si a) Layer from VTMOS 38 0 45 0 17 b) Layer from 42 2 44 0 12 DOA/VTMOS c) Layer “b” after 61 13 22 0.5 3 lysozyme immobilization

[0130] JISZ2801 antimicrobial tests were performed on plasma deposited layers after the lysozyme immobilization step. Metallic grafted samples exhibited a total reduction of the bacteria population. Log reduction of the bacteria of 6.9 was achieved, which is far above the 2 log reduction efficacy limit set by the test.

EXAMPLE 3

[0131] In this Example, preparation of bio-inspired antibacterial surfaces with the use of a commercial catechol molecule as monomer is described.

[0132] The substrate used for deposition is 1 mm thick mirror polished 304 stainless steel disks. Prior to plasma deposition, stainless steel disks were cleaned by successive ultrasonic washings in butanone (5 min.), acetone (1 min.) and absolute ethanol (1min) and further dried under a nitrogen flux. Metallic substrates were then plasma activated through an Ar/O2 plasma treatment (19 slm/1 slm (standard litre per minute)) in continuous (CW) discharge mode at 1.6 W/cm2 during 30 sec.

[0133] Catechol molecule (1,2-dihydroxybenzene, Sigma-Aldrich, 99%) was dissolved at final concentration of 5 mg/mL in vinyltrimethoxysilane (VTMOS, 98%, Sigma-Aldrich) at ambient temperature.

[0134] The plasma deposition method described in Example 2 was used.

[0135] In this example, nisin (antibacterial peptide) was used as the biomolecule. The conditions for nisin antibacterial peptide immobilization were as follows. The catechol/quinone plasma functionalized layers were immersed in a 1 mL solution containing 5 mg of nisin and left to react under constant agitation during 1 hour at ambient temperature. Nisin immobilisation was performed in a 10 mM phosphate buffer solution at pH 6.8. The surfaces were then rinsed with deionized water 4 times during 5 minutes under 500 rpm stirring to remove unreacted peptides.

[0136] The antibacterial activity of the developed surfaces was investigated by carrying out the 15022196 antibacterial test.

[0137] Surfaces achieved by the plasma deposition of a functionalized catechol/quinone layer according to the present invention and further reacted with antibacterial nisine presented a logarithmic reduction of the survival bacteria up to 5.

EXAMPLE 4

[0138] In this example, preparation of bio-inspired antibiofilm surfaces is described. The materials were the same as for Example 2 except that the biomolecule used is DispersinB (antibiofilm), which has been produced according to the protocol published by Kaplan et al. (Kaplan, J. B. Ragunath, C. Ramasubbu N. Fine D. H, J. Bacteriol. 2005, 185, 4693). The plasma deposition method was the same as for Example 2.

[0139] The DispersinB (Antibio-film enzyme) immobilization conditions were as follows. Substrates coated with the catechol/quinone functionalized layer were immersed in a 10 mM phosphate buffer solution containing Dispersin B at 1 mg/ml, at pH 7 during 1 h at room temperature under gentle mechanical agitations. The samples were then washed 5 times during 5 minutes with MilliQ purified water.

[0140] Complementary data for the preparation of the Dispersin B (DspB) are as follows. pET-28a/DspB-expressing Escherichia coli was grown overnight at 37° C. with shaking in 50 ml LB medium supplemented with 50 μg/ml kanamycin. The bacterial suspension was diluted 100-fold in a total of 2 liters of LB supplemented with kanamycin (50 μg/ml), and the expression of DspB was induced with isopropyl-β-D-thiogalactopyranoside (final concentration, 0.5 mM) when the culture reached an A600 of 0.6. The induced culture was incubated for further 4 h at 37° C. with shaking. DspB was purified by nickel affinity chromatography as previously described [Faure et al., Adv. Funct. Mater. 2012, 22, 5271-5282]. Fractions were analyzed by SDS-PAGE and by the ability to hydrolyze the chromogenic substrate 4-nitrophenyl-N-acetyl-β-D-galactosaminide (Sigma Aldrich). Those fractions containing Dispersin B were pooled and dialyzed against 10 mM PO4 pH 5.9, 100 mM NaCl overnight at 4° C. Proteins were quantified using the BCA kit (Pierce).

[0141] An in-vitro anti-adhesion test was performed as follows. A preculture of biofilm forming S. epidermidis ATCC35984 was grown overnight at 37° C. in LB (3 mL) and used the next morning to seed a fresh culture in LB (50 mL). The bacterial concentration of test inoculum was adjusted to about 107 cells mL.sup.−1 in M63 medium supplemented with glucose and casamino acids. Metallic substrates coated with anti-adhesive films were placed in Petri dishes containing damp blotting paper. Test inoculum (200 μL) was pipetted onto each substrate. The Petri dishes containing the inoculated coupons were closed and incubated at 37° C. for 24 h. The treated substrates were rinsed twice with sterile deionized water (10 mL) to remove non-adherent bacteria and then placed face downward in glass jars containing 500-fold-diluted LB (20 mL) and 4-mm glass beads. The jars were shaken horizontally for 10 minutes and then their contents were sonicated in a water bath (50-60 kHz) for 2 minutes. Viable bacteria were counted by plating 10-fold dilution on LB agar. The plates were incubated at 37° C. overnight at room temperature before colony-forming units were counted.

[0142] An assessment of the bioactivity was carried out as follows. As reported in Table 2, summarizing the bioactivity results achieved with surfaces elaborated according to the present invention, it can be concluded that surfaces are elaborated through a reproducible way and present strong anti-biofouling properties with a reduction in population up to 97%.

TABLE-US-00002 TABLE 2 Antiadhesion bioactivity estimated for plasma as-deposited layer and plasma deposited layer after DispersinB immobilization. Adherent Population Samples population, % reduction, % Stainless-steel 2 .Math. 10{circumflex over ( )}6 (100%) Plasma deposited layer after DspB 6 94 immobilization 14  86 9 91

EXAMPLE 5

[0143] In this example, the preparation of bio-inspired surfaces for water depollution, hereafter for antibiotic degradation is described.

[0144] Metallic and organic synthetic substrates were used, namely, 1 mm thick mirror polished 304 stainless steel disks and nylon membranes. In this example, a becta-lactamase, an enzyme able to degrade antibiotics, was also used as the biomolecule. For the plasma deposition method, see Example 1.

[0145] The biomolecule immobilization conditions were as follows. A beta-lactamase biomolecule, was immobilized onto a plasma deposited catechol/quinone functionalized layer by using a 1 mg/ml biomolecule concentration in a 100 mM phosphate buffered solution, at pH=7.5.

[0146] Enzymatic activity was estimated through the amoxicillin degradation monitoring over time. Enzymes were incubated in a 2 mL degradation medium composed of tap water filtered through 0.22 μm, HEPES (12.5 mM), BSA (10 μg√mL.sup.−1) and amoxicillin (100 μg.Math.mL.sup.−1). Each 24 h, the degraded amoxicillin concentration was estimated by absorbance measurements at 210 nm with a 2 Synergy microplate reader (Biotek). The medium was removed every 24 hours, wells were washed 3 times with filtered tap water and a new volume of the degradation medium was put in wells.

[0147] Enzymatic activity assays for free enzymes in solution. In the wells used for the first measurements (T=24 h), enzymes were directly dissolved in a volume of 2 mL of the degradation medium containing 100 μg.Math.mL.sup.−1 of amoxicillin. For all other wells, at time T0, enzymes were dissolved in a 300 μL solution containing 20 μg.Math.mL.sup.−1 of amoxicillin. All wells were fed by this same volume every 24 hours. 24 hours prior absorbance measurement, wells were amended with 100 μg.Math.mL.sup.−1 of antibiotic and supplemented to achieve a final volume of 2 ml.

[0148] The bioactivity is assessed in the results shown in FIG. 4 which is a bar chart showing degradation of amoxicillin for free and immobilized beta-lactamase. On the y-axis, the percentage degradation of amoxicillin is shown. On the x-axis, the time in hours is measured. At the time periods of 24, 48, 72 and 96 hours, the percentage degradation of amoxicillin is shown for immobilized beta-lactamase (left hand bars) and free beta-lactamase (right hand bars). At 144, 192, 240, 288 and 312 hours, only the percentage degradation of amoxicillin is shown for immobilized beta-lactamase.

[0149] As reported in FIG. 4, one can clearly observe that immobilized enzymes allow reaching high degrading antibiotics rates during 13 days. Over 24 h, it can also be noticed that the biological performance of immobilized enzymes is clearly superior to the free enzyme one.

[0150] The resistance to erosion of the functionalized disks elaborated according to the present invention was investigated by placing the substrates in a Biofilm Reactor Annular LI 1320 (Biosurface Technologies Corporation). The coated substrates were subjected to 30 km.Math.h.sup.−1 water flows. After 48 h and 6 days, the enzymatic activity was investigated. Interestingly, the activity of immobilized enzymes after this erosion test was not altered reflecting a strong enzymes anchoring onto the functionalized surface.

EXAMPLE 6

[0151] In this example, preparation of bio-inspired antibiofilm surfaces is described based on the plasma deposition of a pre-formed polymer carrying quinone groups.

[0152] The substrates used for deposition were 1 mm thick mirror polished 304 stainless steel disks or aluminium foils. Stainless steel disks were first cleaned by successive ultrasonic washings in butanone (5 min.), acetone (1 min.) and absolute ethanol (1 min) and further dried under a nitrogen flux. Before plasma deposition, the metallic substrates were plasma cleaned and activated through an Ar/O.sub.2 plasma treatment (19 slm/1 slm (standard litre per minute)) in continuous (CW) discharge mode at 1.6 W/cm2 during 30 sec.

[0153] A homopolymer of methacrylamide bearing 3,4-dihydroxy-L-phenylalanine, noted P(mDOPA) (molecule (1)), was prepared and oxidized in basic media according to a procedure described in J. Mater. Chem. 2011, 21, 7901-7904. For that, 20 mg of P(mDOPA) was dispersed in a 10 mL distilled water solution and a NaOH solution (0.1 M) was slowly added in order to raise the pH above 10. The oxidation step lasted at least one night under air. At the end of the reaction, polymer solution presents a pink colour characteristics confirming that the oxidized Pox(mDOPA) polymer had been obtained, (molecule (2)).

##STR00026##

[0154] The plasma deposition method and conditions used were as described in Example 2. Here, a 0.5 mL/min of a Pox(mDOPA) solution was injected in the nozzle by using a syringe driver.

[0155] The antibiofilm biomolecule used was DispersinB (DspB). The production and immobilization conditions of DspB are already reported in Example 4.

[0156] An assessment of the bioactivity was carried out as follows. As reported in Table 3, summarizing the bioactivity results achieved with surfaces elaborated according to the present invention, it can be concluded that surfaces presented strong anti-biofouling properties with an average reduction in population up to 85%.

TABLE-US-00003 TABLE 3 Anti-adhesion bioactivity estimated for stainless-steel substrates and plasma deposited layers from pre-formed polymer containing quinone groups and after DispersinB immobilization. Adherent Reduction in Samples population, % population, % Stainless-steel 2 × 10.sup.6 (100%) Plasma deposited layer after DspB 10.3 89.7 immobilization 19.4 80.6 16.8 83.2