Quenched coating

Abstract

Described is an object surface coating comprising one or more polymers and a peptide covalently linked to at least one of said one or more polymers, said peptide comprising a) a first cleavage site, wherein said first cleavage site is cleaved by a first compound specifically provided by a microbe belonging to a first group consisting of a limited number of microbial strains, species or genera, and not cleaved by any compound provided by any microbe not belonging to said first group, b) a first fluorescent agent having an emission wavelength of 650-900 nm, c) a first non-fluorescent agent having an absorption wavelength of 650-900 nm, for quenching said emission of said first fluorescent agent, wherein cleavage of said first cleavage site results in the release of said first non-fluorescent agent from the coating, the release of said first non-fluorescent agent being indicative for the presence of a microbe belonging to said first group.

Claims

1. An object surface coating comprising one or more polymers and a peptide covalently linked to at least one of said one or more polymers, said peptide comprising: a) a cleavage site which comprises amino acids which are substrates for proteases secreted by gram negative bacteria, wherein said cleavage site is cleaved by a compound specifically provided by a microbe belonging to a group consisting of a limited number of microbial strains, species or genera, and not cleaved by any compound provided by any microbe not belonging to the group, b) a fluorescent agent having an emission wavelength of 650-900 nm, c) a non-fluorescent agent having an absorption wavelength of 650-900 nm, for quenching said emission of said fluorescent agent, wherein cleavage of said cleavage site results in the release of said non-fluorescent agent from the coating, the release of said non-fluorescent agent being indicative for the presence of a microbe belonging to the group.

2. The object surface coating according to claim 1, wherein said one or more polymers are selected from the group consisting of polyethylene glycol, polyethylene glycol diacrylate, polylactide, polyvinyl alcohol, poly DL-lactide-co-glycolide/polyethylene glycol copolymer and combinations thereof.

3. The object surface coating according to claim 1, wherein said peptide is covalently linked via the N- or C-terminus or a side chain to at least one of said one or more polymers by C4-C30 hydrocarbyl linker, preferably via the C-terminus or a side chain, most preferably via the side chain.

4. The object surface coating according to claim 4, wherein the C4-C30 linker comprises a cyclic hydrocarbyl group or a cyclic heterocyclic group, preferably said heterocyclic group is a triazole group.

5. The object surface coating according to claim 1, wherein the cleavage site is cleaved by a compound specifically provided by a microbe belonging to a group consisting of gram positive bacteria or gram negative bacteria.

6. The object surface coating according to claim 1, wherein the cleavage site is cleaved by a compound, being a protease selected from the group consisting of V8 protease, lgAl protease, and aureolysin.

7. The object surface coating according to claim 1, wherein said fluorescent agent is a cyanine having an emission wavelength of 650-900 nm.

8. The object surface coating according to claim 1, wherein said non-fluorescent agent is a cyanine dye having an absorption wavelength of 650-900 nm.

9. The object comprising a surface coating according to claim 1, said object being selected from the group consisting of medical implants, medical instruments, medical swabs, microwell plates, food preparation surfaces and furniture.

10. The object comprising a surface coating according to claim 1, said object being selected from the group consisting of needles, catheters, guide wires, screws, implant plates and implant pins.

11. A method of preparing an object surface coating comprising one or more polymers and a peptide covalently linked to at least one of said one or more polymers, said peptide comprising: a) a cleavage site which comprises amino acids which are substrates for proteases secreted by gram negative bacteria wherein said cleavage site is cleaved by a compound specifically provided by a microbe belonging to a group consisting of a limited number of microbial strains, species or genera, and not cleaved by any compound provided by any microbe not belonging to said group, b) a fluorescent agent having an emission wavelength of 650-900 nm, c) a non-fluorescent agent having an absorption wavelength of 650-900 nm, for quenching said emission of said fluorescent agent, wherein cleavage of said cleavage site results in the release of said non-fluorescent agent from the coating, the release of said non-fluorescent agent being indicative for the presence of a microbe belonging to said group, the method comprising the steps of: a) providing an object surface comprising one or more polymers, said one or more polymers further comprising a reactive group A and b) contacting the object surface according to step a) with the peptide comprising the fluorescent agent, the non-fluorescent agent, the cleavage site and a reactive group B complementary to reactive group A so that reactive group A and reactive group B form a covalent linkage to prepare said object surface coating.

12. The method according to claim 16, said reactive group A being chosen from the group consisting of alcohol (OH), thiol (SH), amine (NH2), acid (C02H), alkyne, alkene, azide,

13. The method according to claim 16, said reactive group B being chosen from the group consisting of alcohol (OH), thiol (SH), amine (NH2), acid (C02H), alkyne, alkene, azide,.

14. A method of sensing photon emission from an object comprising an object surface coating comprising one or more polymers and a peptide covalently linked to at least one of said one or more polymers, said peptide comprising: a) a cleavage site which comprises amino acids which are substrates for proteases secreted gram negative bacteria wherein said cleavage site is cleaved by a compound specifically provided by a microbe belonging to a group consisting of a limited number of microbial strains, species or genera, and not cleaved by any compound provided by any microbe not belonging to said group, b) a fluorescent agent having an emission wavelength of 650-900 nm, c) a non-fluorescent agent having an absorption wavelength of 650-900 nm, for quenching said emission of said fluorescent agent, wherein cleavage of said cleavage site results in the release of said non-fluorescent agent from the coating, the release of said non-fluorescent agent being indicative for the presence of a microbe belonging to said group, the object selected from the group consisting of medical implants, medical instruments, medical swabs, microwell plates, food preparation surfaces and furniture or selected from the group of needles, catheters, guide wires, screws, implant plates and implant pins, the method comprising the step of radiating the object with photons having a wavelength of 650-900 nm and detecting the emitted photons.

15. A method comprising: a) providing an object surface coating comprising one or more polymers and a peptide covalently linked to at least one of said one or more polymers, said peptide comprising: a cleavage site, which comprises amino acids which are substrates for proteases secreted gram negative bacteria, wherein said cleavage site is cleaved by a compound specifically provided by a microbe belonging to a group consisting of a limited number of microbial strains, species or genera, and not cleaved by any compound provided by any microbe not belonging to said group; a fluorescent agent having an emission wavelength of 650-900 nm; and a non-fluorescent agent having an absorption wavelength of 650-900 nm, for quenching said emission of said fluorescent agent, wherein cleavage of said cleavage site results in the release of said non-fluorescent agent from the coating, the release of said non-fluorescent agent being indicative for the presence of a microbe belonging to said group; and b) either dip coating an object selected from the group consisting of medical implants and medical instruments with the object surface coating from step a) or contacting an object selected from the group consisting of medical swabs, microwell plates, food preparation surfaces and furniture with the surface coating from step a).

Description

EXAMPLES

[0099] The following non-limiting examples show particular embodiments of the present invention as compared to prior art.

Peptide Synthesis

[0100] Peptides 1 and 2 were obtained from Cambridge Research Biochemicals (UK) and used without further purification.

TABLE-US-00001 Peptide1: Ac-X-[K(IRDye800RS)]-GLLEFRIVAK(IRDye800RS)-amide, whereXis-azido-norvaline Peptide2: Ac-X-GLLEFRIVAC(IRDye800CW)amide, whereXis-azido-L-norvaline,alsonamed (S)-5-azido-2-(Fmoc-amino)pentanoicacid).

V8 Protease

[0101] Bacterial protease was obtained from Sigma-Aldrich (Endoproteinase Glu-C from Staphylococcus aureus V8, P2922 SIGMA), also named protease V8 or V8 in this document.

GranzymB Protease

[0102] Human recombinant protease was obtained from MerckMillepore (368043 Granzyme B, Human, Recombinant, E. coli).

Alkyne-Functionalized Glass Slides

[0103] Alkyne-functionalized glass microscope slides (1013-1014 alkyne groups/cm2, 75 mm25 mm) were obtained from Microsurfaces, Inc. and were cut to size by a diamond glass cutter. The slides were stored in a sealed bag in the dark at 20 C. During the experiments the slides were kept in the dark in a desiccator over P.sub.2O.sub.5.

Example 1

Attachment of Peptides to Glass Slides

[0104] Peptides 1 and 2 were attached to glass surfaces.

Negative Control Experiment, no CuSO4 (no Covalent Conjugation is Possible)

[0105] 10 L of peptide stock solution of peptide 1 (10 mg/mL; in DMSO) was mixed with 26.5 L of sodium ascorbate solution (1 mg/mL; in DMSO/water 1/1) and 13.5 L of DMSO was added to increase the solubility of the peptide. The final DMSO/water ratio was 73:27. Final peptide concentration was 0.65 mM, final sodium ascorbate concentration was 2.7 mM.

Peptide 1; Conjugation with CuSO4

[0106] 10 L of peptide stock solution of peptide 3 (10 mg/mL; in DMSO) was mixed with 12.5 L of CuSO4 solution (1 mg/mL; in DMSO/water 1/1) and 14 L of sodium ascorbate solution (1 mg/mL; in DMSO/water 1/1) and 13.5 L of DMSO was added to increase the solubility of the peptide. Final DMSO/water ratio was 73:27. Final peptide concentration was 0.65 mM, final CuSO4 concentration was 1 mM, final sodium ascorbate concentration was 1.4 mM.

[0107] The resulting solution (50 L) was pipetted onto the glass slide, and the slide was kept in a humidified environment for 1H. The droplet was washed off by rinsing 3 with 1 mL DMSO:H2O 80/20; thereafter, the slide was placed in a washing solution of DMSO:H2O 80/20 and was gently agitated for 1 minute. The washing solution was rinsed off with water (51 mL) and the slide was dried by a flow of nitrogen.

[0108] The glass slides were characterized using UV-VIS spectroscopy, where only the slides that had been treated with peptide and with CuSO4 showed a residual absorption maximum of typically 0.01-0.05 in the expected wavelength region 600-800 nm.

Example 2

[0109] Monitoring digestion of peptide 1 by luminescence spectroscopy The cleavage of peptide 1 in the presence of V8 protease and GranzymB protease was monitored in solution by luminescence spectroscopy. Photo-luminescence spectra were measured on a Perkin-Elmer Luminescence Spectrometer LS50 B. Spectra were recorded from 250 to 900 nm, using an excitation wavelength of 720 nm and applying a slit width of 15 nm.

[0110] The digestion in solution was performed in a glass vial. 100 L of stock solution of peptide 1 (10 mg/mL) was diluted with 625 L H.sub.2and 250 L 100 mM phosphate buffer solution (pH=7.8). A sample of 10 L was taken at t=0. Then, 50 L of V8 or Granzyme B protease stock solution (25 units, 25 g) was added, the mixture shaken and was kept at room temperature. During the time period of the experiment the incubation solution remained clear (no precipitation of dye peptide). At various time points (30 s, 2, 5, 10, 15, 20, 25, 30, 40, 50, 60, 90, 120, 150 and 180 min., and at 18H) a 10 L incubate sample was taken and was diluted with 3 mL of water. The prepared diluted samples were measured by luminescence spectroscopy, so as to monitor the digestive cleavage of peptide 1. As blanks, cuvettes with water and the same amount of DMSO as the sample solution were used.

[0111] In the absence of V8 protease, a background fluorescence of around 1 AU was observed. After 30 s of adding V8 protease, the fluorescence intensity increases to 4 AU and within 6 minutes the fluorescence intensity is 6 AU. This shows that the peptide according to the invention is capable of detecting bacteria within minutes, which is a considerable improvement over the prior art methods, such as ATP based test for organic material (typically 4-8 hours) or bacterial cultures (24 hours).

[0112] In the presence of Granzyme B, Peptide 1 is not cut and the background fluorescence of 1 AU was maintained throughout the experiment. Granzyme B is a serine protease present at a normal concentration of 20-4-pg/ml in the blood plasma. The results show that the peptide is selective for the presence of bacterial proteases and is not cleaved in the presence of proteases released by the human body as part of autoimmune responses.

Example 3

Monitoring Digestion of Surface Bound Peptide 1 by Luminescence Spectroscopy

[0113] Li-Cor Odyssey CLX equipment was used on site at Westburg (Leusden, N L; Timo Kreike, Moniek Kors). All Odyssey CLX measurements were taken by exciting at 800 nm.

[0114] In the presence of V8 protease, the intensity increased from circa 6000 units to 25000 units, indicating that V8 triggers the release of the quencher group from peptide 1.

[0115] In the presence of V8 protease, no change in intensity was observed for peptide 2. The intensity observed remains at 8000 units, indicating that although the fluorescent group may be cleaved, the fluorescent group does not diffuse away from the surface, therefore the no decrease in signal is observed so a false negative result is obtained.

[0116] The results show that the coating according to the invention is able to detect the presence of bacteria and is not prone to false negative results.

Example 4

Catheter Coating

[0117] The object coatings are composed of a PEG polymer (MeO-PEG-alkyne, IRIS biotech) and a peptide bearing a cleavage site and first fluorescent agent and a first non-fluorescent agent. The comparative example does not contain the first non-fluorescent agent.

[0118] Peptides were synthesized by standard solid phase peptide synthesis (SPPS) using standard reagents, coupling and purification techniques.

[0119] Coupling of the peptide to the first fluorescent agent and first non-fluorescent agent was conducted post peptide purification and facilitated by maleimide coupling to cysteine or amide coupling to lysine.

[0120] Coupling of the peptide bearing a cleavage site and first fluorescent agent and a first non-fluorescent agent was conducted using standard azide alkyne Huisgen cycloaddition reaction conditions.

[0121] The catheter used was a 14F silicone Foley Catheter which was provided in 6 mm lengths and having a diameter of 4.2 mm. An aqueous slurry of each polymer coating was prepared and each piece of catheter plunged into the slurry mixture. The coated catheters were stored at 20 C. in the dark for 15 minutes prior to repeating the dipping procedure. The process was repeated four times in total. After the final coating, the coated catheters were stored at 35 C. for 48 hours to facilitate drying of the coating.

Example 5

[0122] Each coated catheter and one none coated catheter as control, were incubated in the presence of 210.sup.6 colony-forming units (CFU) ml.sup.1 of S. auereus for 4 hours. There after coated catheters were removed from the incubator and exposed to a clinical multispectral fluorescence camera (T3-imaging system, SurgOptix BV,

[0123] Groningen, The Netherlands) with an IVIS Spectrum (excitation: 710 nm, emission: 800 nm, acquisition time 5 s, binning 4, F-stop 2, FOV 21.2).

[0124] The results obtained from the IVIS Spectrum and IVIS Lumina II were analyzed with Living Image 4.2. (Caliper L S, Hopkinton Mass., USA). Optimal detection limits were set to the lowest signal at which positive signal was effortlessly discriminated from negative controls. Signal intensity was determined by drawing regions of interests (ROI's) and measuring average counts in these regions. The signal was corrected for background by subtracting the background signal from the signal of interest, referred to in the text as net counts. A strong near infrared signal was, attributed to the first fluorescent agent.

[0125] The results are shown in Table 1.

TABLE-US-00002 TABLE1 Coating response to Exam- S.aureus ple Coating Object challenge 5.1 NONE Foley ++(tested silicone by catheter microbial (14F, culture-24 4.6mm) hours) 5.2 G-(Pol)-X-LLEFRIVAC- IRDye800CW 5.3 (IRDye800RS)-KG-(Pol)- + X-LLEFRIVAK(IRDye800RS) 5.4 Ac-(IRDye800CW)CG-(Pol)- ++ X-LLEFRIVAK(IRDyeQC-1) where Pol is Meo-PEG-alkyne and X is (S)-5-Azido-2-(Fmoc-amino) pentanoic acid

[0126] The results show that the coatings according to the invention comprising a non-fluorescent agent with an absorption spectrum overlapping with the emission spectrum of the first fluorescent agent (Example 5.4) lead to very good detection of the presence of pathogenic microorganisms, for example in this case S. aureus. Quenching by the first non-fluorescent agent is strictly dependent on the distance from the first fluorescent agent thus as soon as the first non-fluorescent agent is cleaved, emitted light is detected.

[0127] Example 5.4 also shows that the same level of detection can be obtained using the coatings according to the invention as by conventional microbial culture (example 5.1) but the results are obtained much quicker, within 4 hours rather than 24-36 hours as with microbial cultures.

[0128] Example 5.3 shows a first fluorescent agent that also functions as a first non-fluorescent agent also provide suitable means to detect microorganisms. However, due to the limited diffusion of the cleaved first fluorescent agent only a weak signal is detected.

[0129] Coatings only comprising a first-fluorescent agent do not provide good detection of microorganisms (comparative example 5.2). This is attributed to the slow rate of diffusion of the first fluorescent agent away from the surface thus only a small change in intensity in emitted light is detected.