Macroporous uniform azide- and alkyne-functional polymer microspheres with tuneable surface area: synthesis, in-depth characterization and click-modification

A series of uniform, macroporous poly(styrene-co-divinylbenzene) microspheres with diameters ranging from 6.6 ± 0.6 to 8.6 ± 0.2 μm was prepared in a multistep procedure involving precipitation polymerization synthesis of polystyrene seed particles, swelling of seed particles with plasticiser and porogen, and polymerization of styrene–divinylbenzene (S–DVB) inside the seed particles. Particles prepared with varying DVB feed ratios had comparable diameters (as evidenced by scanning electron microscopy) with specific surface areas increasing with DVB content from 11 to 467 m2 g−1 (measured by nitrogen adsorption). Residual double bonds were converted into azide functionality (through HBr addition and bromo-azide substitution) or alkyne functionality (Br2 addition followed by double elimination) which allowed for CuAAC-click chemistry conjugation with reagents carrying the respective complimentary alkyne or azide functional groups including the fluorescent dye derivatives 7-nitro-4-(prop-2-ynylamino)benzofuran (NBD-alkyne) and Rhodamine B hexylazide synthesised for this purpose.Efficiency of chemical transformations was determined using a combination of CHN and IC elemental analyses, solid state NMR spectroscopy, FT-IR spectroscopy, Raman spectroscopy, and confocal scanning fluorescence microscopy. Although the respective second steps in each modification route (substitution and elimination) suffered from lower yields ( 35%), porous particles with azide loadings of up to 0.71 mmol g−1 and alkyne loadings of up to 0.78 mmol g−1 were prepared. Confocal laser scanning microscopy on Rhodamine B-labelled microspheres indicated functionalization throughout the particles featuring a core–shell structure with higher functionalization in the outer layer of particles. Results are expected to contribute to the development of advanced, well-defined, macroporous particles with high, chemically accessible surface areas

Hi-tech restoration by two-steps biocleaning process of Triumph of Death fresco at the Camposanto Monumental Cemetery (Pisa, Italy)

This is the peer reviewed version of the following article: Ranalli, Giancarlo, Zanardini, E. , Andreotti, A. , Colombini, M.P. , Corti, C. , Bosch-Roig, Pilar, De Nuntiis, P., Lustrato, G. , Mandrioli, P. , Rampazzi, L. , Giantomassi, C. , Zari, D. . (2018). Hi-tech restoration by two-steps biocleaning process of Triumph of Death fresco at the Camposanto Monumental Cemetery (Pisa, Italy).Journal of Applied Microbiology, 125, 3, 800-812. DOI: 10.1111/jam.13913 , which has been published in final form at http://doi.org/10.1111/jam.13913. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.[EN] AimsIn this work, the hi-tech' complex biocleaning and restoration of the 14th-century fresco Triumph of Death (56x150m) at the Camposanto Monumental Cemetery (Pisa, Italy) is reported. Since 2000, the restoration based on the biological cleaning of noble medieval frescoes, has been successfully utilized in this site. Methods and ResultsThe novelty of this study is the two-steps biocleaning process using Pseudomonas stutzeri A29 viable cells, previously applied for recovering other valuable frescoes. In this case, after the fresco detachment from the asbestos-cement support (eternity), both the animal glue and the residues of calcium caseinate were biologically removed respectively from the front and from the back of the fresco in 3h as indicated by GC-MS and PY/GC-MS analyses. The data obtained during the monitoring of the biorestoration process confirmed that the adopted procedure does not leave residual cells on the fresco surfaces as showed by plate count method, ATP determination and also SEM observation. In addition, to avoid the risk of condensation phenomena after the relocation of the restored fresco sections onto the original walls, the use of a new support has been set up together with the design of a control system that allows a continuous monitoring of environmental parameters for prevention and conservation purposes. ConclusionsThis large-scale biorestoration work clearly shows and confirms that this biotechnology is highly efficient, safe, noninvasive, risk-free and very competitive compared to the traditional cleaning methods, offering an unusual resurrection' of the degraded artworks also in very complicated and delicate conditions such as the Triumph of Death fresco, defined for its dimension and artistic importance the Pisa's Sistina frescoes'. Significance and Impact of the StudyThese findings can be of significant importance for other future new restoration activities and they are crucial for determining preservation strategies in this field.The work was supported partially by the Opera Primaziale Pisana (OPA) Pisa, Ing. G. Bentivoglio and partially by the VAL I + d APOSTD/2013/024 project by Generalitat Valenciana, Spain. The authors wish to thank the OPA Scientific Committee, under the direction of Prof. A. Paolucci, and the OPA Restoration Laboratories for the contribution of the conservation scientists, specifically S. Lupo and C. Pucci. The authors wish to thank Termotex srl, Vicenza; Studio Progetti, Padova and De Simone, Firenze. The authors would like to devote this manuscript to the memory of Donatella Zari. Finally, the authors are grateful to S. Pool for editing the manuscript. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.Ranalli, G.; Zanardini, E.; Andreotti, A.; Colombini, M.; Corti, C.; Bosch-Roig, P.; De Nuntiis, P.... (2018). Hi-tech restoration by two-steps biocleaning process of Triumph of Death fresco at the Camposanto Monumental Cemetery (Pisa, Italy). Journal of Applied Microbiology. 125(3):800-812. https://doi.org/10.1111/jam.13913S800812125

Two in One: Use of Azide Functionality for Controlled Photo-crosslinking and Click-modification of Polymer Microspheres

Spherical, micrometer-sized, azide-functional particles were produced through dispersion copolymerization of styrene and vinylbenzyl azide (VBA, 1–100 wt-% of monomer feed) in ethanol in the presence of stabilizers. The obtained microspheres were characterized by SEM, disc centrifuge, FT-IR and NMR spectroscopy, elemental analysis, DSC, and TGA, had measured azide loadings of up to 5.58 mmol/g, and average diameters that decreased with increasing azide content from 2.8 to 0.8 μm. Microspheres were irradiated at a wavelength of 254 nm resulting in crosslinking based on azide-to-nitrene decomposition and subsequent C–H insertion and C=C addition reactions. The conversion of azide functionality was monitored by FT-IR spectroscopy, elemental analysis, and DSC and was found to roughly follow first-order kinetics with increased rates found for microspheres with lower azide contents. Photocrosslinking preserved shapes and size distributions and, above a crosslinking degree of 10%, prevented microsphere dissolution in good solvents. By controlling the irradiation time, the amount of azide consumed for photo-crosslinking could be precisely adjusted. Residual azide groups spared during the irradiation were shown to be amenable to highly efficient CuAAC click modification with a fluorescent dye, Rhodamine B propargyl ester. Given the demand for functional crosslinked microspheres and the inherent difficulties associated with common synthetic strategies in producing such materials, this methodology based on two orthogonal chemistries of the azide functionality provides simple access to well-defined microspheres with customizable degrees of crosslinking and functional group densities

Two in One: Use of Azide Functionality for Controlled Photo-crosslinking and Click-modification of Polymer Microspheres

Spherical, micrometer-sized, azide-functional particles were produced through dispersion copolymerization of styrene and vinylbenzyl azide (VBA, 1–100 wt-% of monomer feed) in ethanol in the presence of stabilizers. The obtained microspheres were characterized by SEM, disc centrifuge, FT-IR and NMR spectroscopy, elemental analysis, DSC, and TGA, had measured azide loadings of up to 5.58 mmol/g, and average diameters that decreased with increasing azide content from 2.8 to 0.8 μm. Microspheres were irradiated at a wavelength of 254 nm resulting in crosslinking based on azide-to-nitrene decomposition and subsequent C–H insertion and C=C addition reactions. The conversion of azide functionality was monitored by FT-IR spectroscopy, elemental analysis, and DSC and was found to roughly follow first-order kinetics with increased rates found for microspheres with lower azide contents. Photocrosslinking preserved shapes and size distributions and, above a crosslinking degree of 10%, prevented microsphere dissolution in good solvents. By controlling the irradiation time, the amount of azide consumed for photo-crosslinking could be precisely adjusted. Residual azide groups spared during the irradiation were shown to be amenable to highly efficient CuAAC click modification with a fluorescent dye, Rhodamine B propargyl ester. Given the demand for functional crosslinked microspheres and the inherent difficulties associated with common synthetic strategies in producing such materials, this methodology based on two orthogonal chemistries of the azide functionality provides simple access to well-defined microspheres with customizable degrees of crosslinking and functional group densities

Temperature-heavy metal- and temperature-anion/molecule-responsive systems based on PEG acrylate copolymers containing dipyridyl ligands

Thermoresponsive copolymers carrying di(2-pyridyl)methyl ligands are shown to respond sensitively and selectively to the presence of heavy metal cations, while their metal complexes respond in a likewise selective and sensitive manner to the presence of anions or molecules with higher metal affinity. A set of well-defined copolymers of poly(ethylene glycol) methyl/phenyl ether acrylate, and N-di(2-pyridyl)methylacrylamide was prepared through a combination of RAFT radical polymerization and postpolymerization modification of activated esters. Products were characterised by 1 H and 19F NMR spectroscopy, size exclusion chromatography, FT-IR spectroscopy, and turbidity measurements. Ligand–metal complexation, as observed by UV–vis spectroscopy, was found to increase lower critical solution temperature (LCST) transitions in water drastically (e.g. up to 22 C) for addition of small amounts (e.g. 0.4 mM) of Cu(II), Co(II), Fe(II) and Ag(I) salts, attributed to a tethering of charge to the polymer. Conversely, salts of Mn(II) and Gd(III) did not affect copolymer solubility. Observed LCST transitions of polymer–metal complexes decreased with the addition of anions or molecules which formed more stable complexes, poorly soluble compounds, or underwent redox reactions with the metal cation. Selectivity toward specific anion or molecule analytes could be tuned though the choice of metal. An isothermal phase separation of a polymer–Cu(II) solution (5 g/L) in response to the addition of as little as 0.4 mM sodium cyanide is demonstrated while the addition of an equal amount of sodium azide did not cause any response, signifying the potential of the proposed concept for sensing applications

Macroporous uniform azide- and alkyne-functional polymer microspheres with tuneable surface area: synthesis, in-depth characterization and click-modification

A series of uniform, macroporous poly(styrene-co-divinylbenzene) microspheres with diameters ranging from 6.6 0.6 to 8.6 0.2 mm was prepared in a multistep procedure involving precipitation polymerization synthesis of polystyrene seed particles, swelling of seed particles with plasticiser and porogen, and polymerization of styrene–divinylbenzene (S–DVB) inside the seed particles. Particles prepared with varying DVB feed ratios had comparable diameters (as evidenced by scanning electron microscopy) with specific surface areas increasing with DVB content from 11 to 467 m2 g1 (measured by nitrogen adsorption). Residual double bonds were converted into azide functionality (through HBr addition and bromo-azide substitution) or alkyne functionality (Br2 addition followed by double elimination) which allowed for CuAAC-click chemistry conjugation with reagents carrying the respective complimentary alkyne or azide functional groups including the fluorescent dye derivatives 7-nitro-4-(prop-2-ynylamino)benzofuran (NBD-alkyne) and Rhodamine B hexylazide synthesised for this purpose. Efficiency of chemical transformations was determined using a combination of CHN and IC elemental analyses, solid state NMR spectroscopy, FT-IR spectroscopy, Raman spectroscopy, and confocal scanning fluorescence microscopy. Although the respective second steps in each modification route (substitution and elimination) suffered from lower yields (35%), porous particles with azide loadings of up to 0.71 mmol g1 and alkyne loadings of up to 0.78 mmol g1 were prepared. Confocal laser scanning microscopy on Rhodamine B-labelled microspheres indicated functionalization throughout the particles featuring a core–shell structure with higher functionalization in the outer layer of particles. Results are expected to contribute to the development of advanced, well-defined, macroporous particles with high, chemically accessible surface areas

Temperature–heavy metal- and temperature–anion/molecule-responsive systems based on PEG acrylate copolymers containing dipyridyl ligands

Thermoresponsive copolymers carrying di(2-pyridyl)methyl ligands are shown to respond sensitively and selectively to the presence of heavy metal cations, while their metal complexes respond in a likewise selective and sensitive manner to the presence of anions or molecules with higher metal affinity. A set of well-defined copolymers of poly(ethylene glycol) methyl/phenyl ether acrylate, and N-di(2-pyridyl)methylacrylamide was prepared through a combination of RAFT radical polymerization and postpolymerization modification of activated esters. Products were characterised by 1 H and 19F NMR spectroscopy, size exclusion chromatography, FT-IR spectroscopy, and turbidity measurements. Ligand–metal complexation, as observed by UV–vis spectroscopy, was found to increase lower critical solution temperature (LCST) transitions in water drastically (e.g. up to 22 C) for addition of small amounts (e.g. 0.4 mM) of Cu(II), Co(II), Fe(II) and Ag(I) salts, attributed to a tethering of charge to the polymer. Conversely, salts of Mn(II) and Gd(III) did not affect copolymer solubility. Observed LCST transitions of polymer–metal complexes decreased with the addition of anions or molecules which formed more stable complexes, poorly soluble compounds, or underwent redox reactions with the metal cation. Selectivity toward specific anion or molecule analytes could be tuned though the choice of metal. An isothermal phase separation of a polymer–Cu(II) solution (5 g/L) in response to the addition of as little as 0.4 mM sodium cyanide is demonstrated while the addition of an equal amount of sodium azide did not cause any response, signifying the potential of the proposed concept for sensing applications

Synthesis and in-depth characterization of reactive, uniform, crosslinked microparticles based on free radical copolymerization of 4-vinylbenzyl azide

A direct seed-swelling copolymerization formulation affords well-defined azide-functional porous or hollow microparticles amenable to click-modification.</p

Synthesis and in-depth characterization of reactive, uniform, crosslinked microparticles based on free radical copolymerization of 4-vinylbenzyl azide

The introduction of functional groups into microparticles is commonly accomplished through, at times, low-yielding post-synthesis modification. In this detailed study, the introduction of azide functionality into uniform, crosslinked, macroporous microparticles through direct copolymerization of styrene, divinyl-benzene (DVB), and 4-vinylbenzyl azide (VBA) in varying ratios inside swollen polystyrene seed (template) particles is investigated. Formulations contained up to 40 wt% of VBA in the monomer mixture. Resulting microspheres were characterised by SEM, porosimetry, FT-IR spectroscopy, and CHN elemental analysis. Uniform spherical particles with diameters ranging from 7.3 to 10.8 μm with diameter dispersities typically below 1.01 and with tuneable azide loadings from 0.11 to 1.17 mmol g−1 were obtained. Interestingly, severe effects of VBA addition on porosity, surface smoothness, and particle shape were observed. Specific surface areas and cumulative pore volumes increased with the amount of DVB in feed, decreased with increasing VBA feed ratio, and increased drastically for the use of azide-functional template particles with measured cumulative pore volumes reaching up to 0.60 cm3 g−1. With increasing VBA feed, formation of smaller, secondary particles was observed and attributed to an incomplete swelling of VBA into seed particles, which is discussed as a main reason for lower-than-expected azide contents in product particles. For high VBA feed ratios (>25 wt%), dented, hollow, or hollow collapsed azide-functional particles were found, presumably due to immiscibility of the growing azide-functional copolymer with the polystyrene seeds. Finally, successful click-modification is demonstrated with phenyl-acetylene and an alkyne-functional Rhodamine B dye allowing for mapping of functionalization density through confocal fluorescence microscopy