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- 1. Capri, Italy. September 21st to 23rd 2016 Alfredo Maria Gravagnuolo,1,* Jane Politi,2 Paola Cicatiello,1 Luca De Stefano,2 Eden Morales-Narváez,3 Arben Merkoçi,3,4 and Paola Giardina1 alfredomaria.gravagnuolo@unina.it * EUROPEAN ASSOCIATION OF MATERIAL SURFACES SCIENCE & TECHNOLOGY 1 UNIVERSITY OF NAPLES “FEDERICO II” ITALY 2 CNR, Unit of Naples Italy 3 ICN2, Barcelona Spain 4 ICREA, Barcelona Spain Self-assembled nanoscale films and complexes of the surface active protein Vmh2
- 2. BIOMEDICAL APPLICATIONS OF EMERGING (NANO)MATERIALS Kurapati et al., 2016, Adv. Mater. Capri, Italy - September 21st to 23rd 2016 Alfredo Maria Gravagnuolo - UNINA
- 3. SEM IMAGE OF A FUNGAL SPORE COATING Paris et al., 2003, Appl Environ Microbiol 100 nm 100 nm HYDROPHOBINS SILICON STEEL GLASS GOLD 2D MATERIALS Capri, Italy - September 21st to 23rd 2016 Alfredo Maria Gravagnuolo - UNINA
- 4. Bayry et al., 2012, PLoS Pathogen HYDROPHOBINS SILICON STEEL GLASS GOLD 2D MATERIALS Capri, Italy - September 21st to 23rd 2016 Alfredo Maria Gravagnuolo - UNINA
- 5. Linder, 2009, Curr. Opin. Colloid Interface Sci HYDROPHOBINS SILICON STEEL GLASS GOLD 2D MATERIALS HYDROPHOBINS: JANUS FACED PROTEINS PRODUCED BY FUNGI HIGHEST SURFACE ACTIVITY AMONG PROTEINS SELF-ASSEMBLING AT HYDROPHILIC/HYDROPHOBIC INTERFACES Capri, Italy - September 21st to 23rd 2016 Alfredo Maria Gravagnuolo - UNINA
- 6. Hydrophobic face Hydrophilic face Zampieri et al., 2010, Materials HYDROPHOBINS SILICON STEEL GLASS GOLD 2D MATERIALS Class I Highly stable layer Amyloid-like structures Class II Easily soluble layer Capri, Italy - September 21st to 23rd 2016 Alfredo Maria Gravagnuolo - UNINA ~ 3 nm ~ 2 nm
- 7. Class I Highly stable layer Amyloid-like structures Linder, 2009, Curr. Opin. Colloid Interface Sci Class II Easily soluble layer AFM images HYDROPHOBINS SILICON STEEL GLASS GOLD 2D MATERIALS Capri, Italy - September 21st to 23rd 2016 Alfredo Maria Gravagnuolo - UNINA
- 8. CLASS I HYDROPHOBINS Highly stable films Amyloid-like structures Hydrophobic face Hydrophilic face Macindoe et al., 2012, PNAS β-spine structure of rodlets monomer hexamer hexamer HYDROPHOBINS SILICON STEEL GLASS GOLD 2D MATERIALS Capri, Italy - September 21st to 23rd 2016 Alfredo Maria Gravagnuolo - UNINA
- 9. Bayry et al., 2012, PLoS Pathogen Hydrophobic surface Hydrophilic surface HYDROPHOBINS SILICON STEEL GLASS GOLD 2D MATERIALS Capri, Italy - September 21st to 23rd 2016 Alfredo Maria Gravagnuolo - UNINA
- 10. HYDROPHOBINS SILICON STEEL GLASS GOLD 2D MATERIALS Capri, Italy - September 21st to 23rd 2016 Alfredo Maria Gravagnuolo - UNINA Bayry et al., 2012, PLoS Pathogen
- 11. HYDROPHOBINS SILICON STEEL GLASS GOLD 2D MATERIALS Capri, Italy - September 21st to 23rd 2016 Alfredo Maria Gravagnuolo - UNINA Bayry et al., 2012, PLoS Pathogen
- 12. VMH2 FROM PLEUROTUS OSTREATUS MONOMER Hydrophobic Hydrophilic HYDROPHOBINS SILICON STEEL GLASS GOLD 2D MATERIALS Capri, Italy - September 21st to 23rd 2016 Alfredo Maria Gravagnuolo - UNINA Gravagnuolo et al., 2016, Biomacromolecules
- 13. Hydrophobic patch HYDROPHOBINS SILICON STEEL GLASS GOLD 2D MATERIALS Capri, Italy - September 21st to 23rd 2016 Alfredo Maria Gravagnuolo - UNINA VMH2 BY HOMOLOGY MODELING Gravagnuolo et al., 2016, Biomacromolecules
- 14. VMH2 MONOLAYER WCA = 131° WCA = 62° WCA = 6° WCA = 57° HYDROPHOBINS SILICON STEEL GLASS GOLD 2D MATERIALS Capri, Italy - September 21st to 23rd 2016 Alfredo Maria Gravagnuolo - UNINA POROUS SILICON De Stefano et al., 2008, Advanced Materials OXIDIZED POROUS SILICON INFILTRATION OF MESOPOROUS SILICON WITH VMH2 Etching by NaOH 0,1 M VMH2 MONOLAYER Thickness,nm Time, s +Vmh2 Bare surface
- 15. ≈0.2 U/chip2,6-dimethoxyphenol as substrate cSi Bare silicon Vmh2 coated silicon Laccase on chip Laccase in solution Vmh2 layer Laccase HYDROPHOBINS SILICON STEEL GLASS GOLD 2D MATERIALS Capri, Italy - September 21st to 23rd 2016 Alfredo Maria Gravagnuolo - UNINA RHODAMINE LABELLED BOVINE SERUM ALBUMIN (BSA) IS IMMOBILIZED ON VMH2 COATED CRISTALINE SILICON Activity,% Time, h IMMOBILIZATION OF LACCASE ON CRYSTALLINE SILICON De Stefano et al., 2009, The European Physical Journal E 500 ng cm-2 immobilized Laccase 20 ng cm-2 immobilized BSA
- 16. STEEL VMH2 steel 1 µL sample Salts peptides and proteins HYDROPHOBINS SILICON STEEL GLASS GOLD 2D MATERIALS Capri, Italy - September 21st to 23rd 2016 Alfredo Maria Gravagnuolo - UNINA Longobardi et al., 2014, Analytical Biochemistry WCA = 96° ± 2° WCA = 75° ± 2° Bound Vmh2 90 ng cm-2 Steel MALDI plate
- 17. MALDI TOF ANALYSIS OF OVALBUMIN TRYPTIC PEPTIDES IN 2M UREA MALDI TOF ANALYSIS OF LYSOZYME IN DENATURING BUFFER VMH2 COATED WELL HYDROPHOBINS SILICON STEEL GLASS GOLD 2D MATERIALS Capri, Italy - September 21st to 23rd 2016 Alfredo Maria Gravagnuolo - UNINA Longobardi et al., 2014, Analytical Biochemistry VMH2 COATED WELL
- 18. MALDI TOF ANALYSIS OF CRUDE HUMAN SERUM ACQUIRED IN 500-80000 M/Z RANGES HYDROPHOBINS SILICON STEEL GLASS GOLD 2D MATERIALS Capri, Italy - September 21st to 23rd 2016 Alfredo Maria Gravagnuolo - UNINA Longobardi et al., 2014, Analytical Biochemistry
- 19. five minutes on-plate reactions HYDROPHOBINS SILICON STEEL GLASS GOLD 2D MATERIALS Capri, Italy - September 21st to 23rd 2016 Alfredo Maria Gravagnuolo - UNINA Longobardi et al., 2015, Analytical Bioanalytical Chemistry Immobilization of enzymes Coating by Vmh2 MALDI TOF analysis
- 20. ANALYSIS OF A COMPLEX SAMPLE Whey milk proteins tryptic digestion (five min) Peptide, 192-198, of β-casein MALDI-TOF/TOF analysis MS2 sequencing MS spectrum 2 3 4 4 4 6 6 7 12 Lactoadherin Butyrophilin K-casein β-casein α-lactalbumin β-lactoglobulin α-S1-casein Lactotransferrin α-S2-casein Identified proteins n° of peptides HYDROPHOBINS SILICON STEEL GLASS GOLD 2D MATERIALS Capri, Italy - September 21st to 23rd 2016 Alfredo Maria Gravagnuolo - UNINA Longobardi et al., 2015, Analytical Bioanalytical Chemistry
- 21. ANALYSIS WORKFLOW Enzyme Reaction Trypsin endo-proteolysis (Arg,Lys) V8 protease endo-proteolysis (Asp,Glu) PNGaseF de-glycosylation (Asn) Alkaline phosphatase de-phosphorylation HYDROPHOBINS SILICON STEEL GLASS GOLD 2D MATERIALS Capri, Italy - September 21st to 23rd 2016 Alfredo Maria Gravagnuolo - UNINA Longobardi et al., 2015, Analytical Bioanalytical Chemistry
- 22. ANALYSIS OF A COMPLEX SAMPLE * * α-S1- casein dephosphorylated peptide Dephosphorylation (5min) m/z MS spectrum HYDROPHOBINS SILICON STEEL GLASS GOLD 2D MATERIALS Capri, Italy - September 21st to 23rd 2016 Alfredo Maria Gravagnuolo - UNINA Longobardi et al., 2015, Analytical Bioanalytical Chemistry Whey milk proteins tryptic digestion (five min) β-casein dephosphorylated peptide MS spectrum
- 23. 270 ng of Vmh2 per cm2 of glass Functionalization in four minutes net time 100 glass slides per mg of protein 20000 glass slides per Liter of fungal culture GLASS FUNCTIONALIZATION HYDROPHOBINS SILICON STEEL GLASS GOLD 2D MATERIALS Capri, Italy - September 21st to 23rd 2016 Alfredo Maria Gravagnuolo - UNINA Gravagnuolo et al., 2015. Advanced Functional Materials. 25 (28)
- 24. HYDROPHOBINS SILICON STEEL GLASS GOLD 2D MATERIALS Capri, Italy - September 21st to 23rd 2016 Alfredo Maria Gravagnuolo - UNINA Gravagnuolo et al., 2015. Advanced Functional Materials. 25 (28) Quantum dots: nanocrystals of semiconductors. Showing unique electronic properties, intermediate between those of bulk material and discrete molecules High photoluminescence High resistance to photobleaching Wide absorption spectrum Tunable emission, from UV to mid-IR in dependence of their size Graphene Oxide: C/O ratio ≈ 1. Easily processed in solution. Synthesized in large quantity. Universal long-range superquencer. Capable of direct wiring with biomolecules through its heterogeneous chemical structure.
- 25. CDTE QUANTUM DOTS FLUORESCENT BSA NANOMATERIAL AND PROTEIN MICROPATTERNING HYDROPHOBINS SILICON STEEL GLASS GOLD 2D MATERIALS Capri, Italy - September 21st to 23rd 2016 Alfredo Maria Gravagnuolo - UNINA Gravagnuolo et al., 2015. Advanced Functional Materials. 25 (28)
- 26. BIOLOGICAL IMMUNOASSAY IN MICROARRAY FORMAT HYDROPHOBINS SILICON STEEL GLASS GOLD 2D MATERIALS Capri, Italy - September 21st to 23rd 2016 Alfredo Maria Gravagnuolo - UNINA Gravagnuolo et al., 2015. Advanced Functional Materials. 25 (28)
- 27. GRAPHENE OXIDE LAYER ON VMH2 SUBSTRATE HYDROPHOBINS SILICON STEEL GLASS GOLD 2D MATERIALS Capri, Italy - September 21st to 23rd 2016 Alfredo Maria Gravagnuolo - UNINA Gravagnuolo et al., 2015. Advanced Functional Materials. 25 (28) Graphene Oxide concentration Labeled BSA
- 28. HYDROPHOBINS SILICON STEEL GLASS GOLD 2D MATERIALS Capri, Italy - September 21st to 23rd 2016 Alfredo Maria Gravagnuolo - UNINA Gravagnuolo et al., 2015. Advanced Functional Materials. 25 (28) GRAPHENE OXIDE LAYER ON VMH2 SUBSTRATE
- 29. HYDROPHOBINS SILICON STEEL GLASS GOLD 2D MATERIALS Capri, Italy - September 21st to 23rd 2016 Alfredo Maria Gravagnuolo - UNINA Della Ventura et al., 2016, Applied Surface Science SELF-ASSEMBLY OF VMH2 ON GOLD AND ITS INTERACTIONS WITH GLUCOSE QUARTZ CRYSTAL-MICROBALANCE MICROFLUIDIC SYSTEM
- 30. HYDROPHOBINS SILICON STEEL GLASS GOLD 2D MATERIALS Capri, Italy - September 21st to 23rd 2016 Alfredo Maria Gravagnuolo - UNINA Della Ventura et al., 2016, Applied Surface Science WCA = 67 ± 3° WCA = 34 ± 6°WCA = 91 ± 2° 24 nm 41 nm ANALYSIS BY WATER CONTACT ANGLE (WCA) AND SPECTROSCOPIC ELLIPSOMETRY (SE) SWELLING SELF-ASSEMBLY OF VMH2 ON GOLD AND ITS INTERACTIONS WITH GLUCOSE
- 31. Capri, Italy - September 21st to 23rd 2016 Alfredo Maria Gravagnuolo - UNINA PEG Diacid Vmh2 PEG-Vmh2-AuNPs Au2+ Politi et al., 2016, Nanotechnology HYDROPHOBINS SILICON STEEL GLASS GOLD 2D MATERIALS Au0 Sodium borohydride TEM + + Tetrachloroauric acid Vmh2-PEG hydrophobic hydrophilic
- 32. HYDROPHOBINS SILICON STEEL GLASS GOLD 2D MATERIALS Capri, Italy - September 21st to 23rd 2016 Alfredo Maria Gravagnuolo - UNINA Politi et al., 2016, Nanotechnology PEG-Vmh2-AuNPs Glucose Size = 10 ± 5 nm Size = 47 ± 7 nm
- 33. HYDROPHOBINS SILICON STEEL GLASS GOLD 2D MATERIALS Capri, Italy - September 21st to 23rd 2016 Alfredo Maria Gravagnuolo - UNINA Gravagnuolo et al., 2015, Advanced Functional Materials. 25 (18) IN SITU GENERATION OF BIOFUNCTIONALIZED GRAPHENE
- 34. HYDROPHOBINS SILICON STEEL GLASS GOLD 2D MATERIALS Capri, Italy - September 21st to 23rd 2016 Alfredo Maria Gravagnuolo - UNINA Gravagnuolo et al., 2015, Advanced Functional Materials. 25 (18) IN SITU GENERATION OF BIOFUNCTIONALIZED GRAPHENE In absence of Vmh2 SEM
- 35. QUALITY ASSESSMENT BY RAMAN SPECTROSCOPY ANALYSIS Fundamental characteristics*: N° of layers: 3.8 ± 0.4 Average lateral size: 1.0 ± 0.1 µm Oxidation degree: defect-free *According to directives from EU Graphene Flagship project HYDROPHOBINS SILICON STEEL GLASS GOLD 2D MATERIALS Capri, Italy - September 21st to 23rd 2016 Alfredo Maria Gravagnuolo - UNINA Gravagnuolo et al., 2015, Advanced Functional Materials. 25 (18)
- 36. AFM imaging AFM profile HYDROPHOBINS SILICON STEEL GLASS GOLD 2D MATERIALS Capri, Italy - September 21st to 23rd 2016 Alfredo Maria Gravagnuolo - UNINA Gravagnuolo et al., 2015, Advanced Functional Materials. 25 (18) IN SITU GENERATION OF BIOFUNCTIONALIZED GRAPHENE
- 37. HYDROPHOBINS SILICON STEEL GLASS GOLD 2D MATERIALS Capri, Italy - September 21st to 23rd 2016 Alfredo Maria Gravagnuolo - UNINA Gravagnuolo et al., 2015, Advanced Functional Materials. 25 (18) SELF ASSEMBLY OF BIO-GRAPHENE C D
- 38. APPLICATIONS OF VMH2 NANOFILMS AND NANOCOMPLEXES SILICON: Surface passivation of optical devices; Enzyme immobilization. STEEL: MALDI-MS based proteomic platform GLASS: Nanomaterial and protein patterning; Microarray platform. GOLD: Glucose interactions. 2D Materials: Syntesis of defect-free few-layer bio-2D materials HYDROPHOBINS SILICON STEEL GLASS GOLD 2D MATERIALS Capri, Italy - September 21st to 23rd 2016 Alfredo Maria Gravagnuolo - UNINA
- 39. PAOLA GIARDINA SARA LONGOBARDI PAOLA CICATIELLO DEPARTMENT OF BIOLOGY EUGENIO NOTOMISTA DEPARTMENT OF PHYSICS RAFFAELE VELOTTA CARLO ALTUCCI FELICE GESUELE JASNEET KAUR BARTOLOMEO DELLA VENTURA RICCARDO FUNARI DEPARTMENT OF CHEMICAL SCIENCES GENNARO MARINO ANGELA AMORESANO FRANCESCA PANE EUGENIO GALANO LUIGI PADUANO ALESSANDRA LUCHINI Capri, Italy - September 21st to 23rd 2016 Alfredo Maria Gravagnuolo - UNINA
- 40. ACKNOWLEDGMENTS – SCIENTIFIC COLLABORATIONS NATIONAL COUNCIL OF RESEARCH, INSTITUTE FOR MICROELECTRONICS AND MICROSYSTEMS (CNR-IMM) LUCA DE STEFANO ILARIA REA JANE POLITI CATALAN INSTITUTE OF NANOSCIENCE AND NANOTECHNOLOGY (ICN2) ARBEN MERKOÇI EDEN MORALES-NARVÁEZ CHARLENE SANTOS MATOS EVERSON THIAGO S. G. DA SILVA Capri, Italy - September 21st to 23rd 2016 Alfredo Maria Gravagnuolo - UNINA SORBONNE UNIVERSITÉS JOLANDA SPADAVECCHIA SANDRA CASALE CHRISTOPHE METHIVIER
- 41. alfredomaria.gravagnuolo@unina.it Thank you for your attention! Capri, Italy - September 21st to 23rd 2016 Alfredo Maria Gravagnuolo - UNINA
- 43. Vmh2 films were formed by repeating compression−expansion cycles at the air−water interface using the Langmuir method. Afterwards they were transferred onto a hydrophilic SiO2 substrate and analysed by AFM Vmh2 forms rod-like aggregates characteristic of class I hydrophobins HYDROPHOBINS SILICON STEEL GLASS GOLD 2D MATERIALS Capri, Italy - September 21st to 23rd 2016 Alfredo Maria Gravagnuolo - UNINA Houmadiet al., 2012, Langmuir
- 44. Vmh2–glucose complex forms a chemically stable film, obtained by drop deposition on silicon, containing 35 per cent of glucose (a) bare silicon (b) Vmh2-coated silicon (c) Vmh2-glucose coated silicon 90º 44º 17º HYDROPHOBINS SILICON STEEL GLASS GOLD 2D MATERIALS Capri, Italy - September 21st to 23rd 2016 Alfredo Maria Gravagnuolo - UNINA Rea et al., 2009, R. Soc. Interface