The Galapagos Chip Platform for High-Throughput Screening of Cell Adhesive Chemical Micropatterns.

In vivo cells reside in a complex extracellular matrix (ECM) that presents spatially distributed biochemical and -physical cues at the nano- to micrometer scales. Chemical micropatterning is successfully used to generate adhesive islands to control where and how cells attach and restore cues of the ECM in vitro. Although chemical micropatterning has become a powerful tool to study cell-material interactions, only a fraction of the possible micropattern designs was covered so far, leaving many other possible designs still unexplored. Here, a high-throughput screening platform called "Galapagos chip" is developed. It contains a library of 2176 distinct subcellular chemical patterns created using mathematical algorithms and a straightforward UV-induced two-step surface modification. This approach enables the immobilization of ligands in geometrically defined regions onto cell culture substrates. To validate the system, binary RGD/polyethylene glycol patterns are prepared on which human mesenchymal stem cells are cultured, and the authors observe how different patterns affect cell and organelle morphology. As proof of concept, the cells are stained for the mechanosensitive YAP protein, and, using a machine-learning algorithm, it is demonstrated that cell shape and YAP nuclear translocation correlate. It is concluded that the Galapagos chip is a versatile platform to screen geometrical aspects of cell-ECM interaction.

Reversible Diels-Alder and Michael Addition Reactions Enable the Facile Postsynthetic Modification of Metal-Organic Frameworks.

Functionalization of metal-organic frameworks (MOFs) is critical in exploring their structural and chemical diversity for numerous potential applications. Herein, we report multiple approaches for the tandem postsynthetic modification (PSM) of various MOFs derived from Zr(IV), Al(III), and Zn(II). Our current work is based on our efforts to develop a wide range of MOF platforms with a dynamic functional nature that can be chemically switched via thermally triggered reversible Diels-Alder (DA) and hetero-Diels-Alder (HDA) ligations. Furan-tagged MOFs (furan-UiO-66-Zr) were conjugated with maleimide groups bearing dienophiles to prepare MOFs with a chemically switchable nature. As HDA pairs, phosphoryl dithioester-based moieties and cyclopentadiene (Cp)-grafted MOF (Cp-MIL-53-Al) were utilized to demonstrate the cleavage and rebonding of the linkages as a function of temperature. In addition to these strategies, the Michael addition reaction was also applied for the tandem PSM of IRMOF-3-Zn. Maleimide groups were postsynthetically introduced in the MOF lattice, which were further ligated with cysteine-based biomolecules via the thiol-maleimide Michael addition reaction. On the basis of the versatility of the herein presented chemistry, we expect that these approaches will help in designing a variety of sophisticated functional MOF materials addressing diverse applications.

Thioacetate-Based Initiators for the Synthesis of Thiol-End-Functionalized Poly(2-oxazoline)s.

New functional initiators for the cationic ring-opening polymerization of 2-alkyl-2-oxazolines are described to introduce a thiol moiety at the α terminus. Both tosylate and nosylate initiators carrying a thioacetate group are obtained in multigram scale, from commercial reagents in two steps, including a phototriggered thiol-ene radical addition. The nosylate derivative gives access to a satisfying control over the cationic ring-opening polymerization of 2-ethyl-2-oxazoline, with dispersity values lower than 1.1 during the entire course of the polymerization, until full conversion. Cleavage of the thioacetate end group is rapidly achieved using triazabicyclodecene, thereby leading to a mercapto terminus. The latter gives access to a new subgeneration of α-functional poly(2-oxazoline)s (butyl ester, N-hydroxysuccinimidyl ester, furan) by Michael addition with commercial (meth)acrylates. The amenability of the mercapto-poly(2-ethyl-2-oxazoline) for covalent surface patterning onto acrylated surfaces is demonstrated in a microchannel cantilever spotting (µCS) experiment, characterized by X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary-ion mass spectrometry (ToF-SIMS).

In Operando Synchrotron Diffraction and in Operando X-ray Absorption Spectroscopy Investigations of Orthorhombic V2O5 Nanowires as Cathode Materials for Mg-Ion Batteries.

Orthorhombic V2O5 nanowires were successfully synthesized via a hydrothermal method. A cell-configuration system was built utilizing V2O5 as the cathode and 1 M Mg(ClO4)2 electrolyte within acetonitrile, together with Mg xMo6S8 ( x ≈ 2) as the anode to investigate the structural evolution and oxidation state and local structural changes of V2O5. The V2O5 nanowires deliver an initial discharge/charge capacity of 103 mAh g-1/110 mAh g-1 and the highest discharge capacity of 130 mAh g-1 in the sixth cycle at C/20 rate in the cell-configuration system. In operando synchrotron diffraction and in operando X-ray absorption spectroscopy together with ex situ Raman and X-ray photoelectron spectroscopy reveal the reversibility of magnesium insertion/extraction and provide information on the crystal structure evolution and changes of the oxidation states during cycling.

The Multisensor Array Based on Grown-On-Chip Zinc Oxide Nanorod Network for Selective Discrimination of Alcohol Vapors at Sub-ppm Range.

We discuss the fabrication of gas-analytical multisensor arrays based on ZnO nanorods grown via a hydrothermal route directly on a multielectrode chip. The protocol to deposit the nanorods over the chip includes the primary formation of ZnO nano-clusters over the surface and secondly the oxide hydrothermal growth in a solution that facilitates the appearance of ZnO nanorods in the high aspect ratio which comprise a network. We have tested the proof-of-concept prototype of the ZnO nanorod network-based chip heated up to 400 °C versus three alcohol vapors, ethanol, isopropanol and butanol, at approx. 0.2-5 ppm concentrations when mixed with dry air. The results indicate that the developed chip is highly sensitive to these analytes with a detection limit down to the sub-ppm range. Due to the pristine differences in ZnO nanorod network density the chip yields a vector signal which enables the discrimination of various alcohols at a reasonable degree via processing by linear discriminant analysis even at a sub-ppm concentration range suitable for practical applications.

Site-Specific Surface Functionalization via Microchannel Cantilever Spotting (µCS): Comparison between Azide-Alkyne and Thiol-Alkyne Click Chemistry Reactions.

Different types of click chemistry reactions are proposed and used for the functionalization of surfaces and materials, and covalent attachment of organic molecules. In the present work, two different catalyst-free click approaches, namely azide-alkyne and thiol-alkyne click chemistry are studied and compared for the immobilization of microarrays of azide or thiol inks on functionalized glass surfaces. For this purpose, the surface of glass is first functionalized with dibenzocyclooctyne-acid (DBCO-acid), a cyclooctyne with a carboxyl group. Then, the DBCO-terminated surfaces are functionalized via microchannel cantilever spotting with different fluorescent and nonfluorescent azide and thiol inks. Although both routes work reliably for surface functionalization, the protein binding experiments reveal that using a thiol-alkyne route will obtain the highest surface density of molecular immobilization in such spotting approaches. The obtained achievements and results from this work can be used for design and manufacturing of microscale patterns suitable for biomedical and biological applications.

Spatially-Resolved Multiple Metallopolymer Surfaces by Photolithography.

A tetrazole-based photoligation protocol for the spatially-resolved encoding of various defined metallopolymers onto solid surfaces is introduced. By using this approach, fabrication of bi- and trifunctional metallopolymer surfaces with different metal combinations were achieved. Specifically, α-ω-functional copolymers containing bipyridine as well as triphenylphosphine ligands were synthesized through reversible addition-fragmentation chain transfer (RAFT) polymerization, and subsequently metal loaded to afford metallopolymers of the widely-used metals gold, palladium, and platinum. Spatially-resolved surface attachment was achieved by means of a nitrile imine-mediated tetrazole-ene cycloaddition (NITEC) based photoligation protocol, exploiting tethered tetrazoles and metallopolymers equipped with a maleimide chain terminus. Metallopolymer coated surfaces with three different metals were prepared and characterized by time-of-flight secondary ion mass spectrometry (ToF-SIMS) and spatially-resolved X-ray photoelectron spectroscopy (XPS) mapping, supporting the preserved chemical composition of the surface-bound metallopolymers. The established photochemical technology platform for arbitrary spatially-resolved metallopolymer surface designs enables the patterning of multiple metallopolymers onto solid substrates. This allows for the assembly of designer metallopolymer substrates.

Dynamic Nitroxide Functional Materials.

A substrate-independent and versatile coating platform for (spatially resolved) surface functionalization, based on nitroxide radical coupling (NRC) reactions and the formation of thermo-labile alkoxyamine functional groups, was introduced. Nitroxide-decorated poly(glycidyl methacrylate) (PGMA) microspheres, obtained through bioinspired copolymer surface deposition using dopamine and a nitroxide functional dopamine derivative as monomers, were conjugated with small functional groups in a rewritable process. Reversible coding of the nitroxide functional microspheres by NRC and decoding through thermal alkoxyamine fission were monitored and characterized by electron paramagnetic resonance (EPR) spectroscopy and X-ray photoelectron spectroscopy (XPS). In addition, this nitroxide coating system was exploited in "grafting-to" polymer surface ligations of poly(methyl methacrylate) (PMMA) and poly(2,2,2-trifluoroethyl methacrylate) (PTFEMA) in spatially confined areas. Polymer strands terminated with an Irgacure 2959 (2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone) photoinitiator were obtained through chain-transfer polymerization, and subsequently coupled to nitroxide-immobilized poly(dopamine) (PDA)-coated silicon substrates by using rapid photoclick NRC reactions. Light-driven polymer surface coding was visualized by time-of-flight secondary ion mass spectrometry (ToF-SIMS) and XPS imaging.

Reversible Surface Engineering via Nitrone-Mediated Radical Coupling.

Efficient and simple polymer conjugation reactions are critical for introducing functionalities on surfaces. For polymer surface grafting, postpolymerization modifications are often required, which can impose a significant synthetic hurdle. Here, we report two strategies that allow for reversible surface engineering via nitrone-mediated radical coupling (NMRC). Macroradicals stemming from the activation of polymers generated by copper-mediated radical polymerization are grafted via radical trapping with a surface-immobilized nitrone or a solution-borne nitrone. Since the product of NMRC coupling features an alkoxyamine linker, the grafting reactions can be reversed or chain insertions can be performed via nitroxide-mediated polymerization (NMP). Poly( n-butyl acrylate) ( Mn = 1570 g·mol-1, D̵ = 1.12) with a bromine terminus was reversibly grafted to planar silicon substrates or silica nanoparticles as successfully evidenced via X-ray photoelectron spectroscopy (XPS), time-of-flight secondary ion mass spectrometry, and grazing angle attenuated total reflection Fourier-transform infrared spectroscopy (GAATR-FTIR). NMP chain insertions of styrene are evidenced via GAATR-FTIR. On silica nanoparticles, an NMRC grafting density of close to 0.21 chains per nm2 was determined by dynamic light scattering and thermogravimetric analysis. Concomitantly, a simple way to decorate particles with nitroxide radicals with precise control over the radical concentration is introduced. Silica microparticles and zinc oxide, barium titanate, and silicon nanoparticles were successfully functionalized.

Engineering Nitroxide Functional Surfaces Using Bioinspired Adhesion.

We pioneer a versatile surface modification strategy based on mussel-inspired oxidative catecholamine polymerization for the design of nitroxide-containing thin polymer films. A 3,4-dihydroxy-l-phenylalanine (l-DOPA) monomer equipped with a 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO)-derived oxidation-labile hydroxylamine functional group is employed as a universal coating agent to generate polymer scaffolds with persistent radical character. Various types of materials including silicon, titanium, ceramic alumina, and inert poly(tetrafluoroethylene) (PTFE) were successfully coated with poly(DOPA-TEMPO) thin films in a one-step dip-coating procedure under aerobic, slightly alkaline (pH 8.5) conditions. Steadily growing polymer films (∼1.1 nm h-1) were monitored by ellipsometry, and their thicknesses were critically compared with those obtained from atomic force microscopic cross-sectional profiles. The heterogeneous composition of surface-adherent nitroxide scaffolds examined by X-ray photoelectron spectroscopy was correlated to that examined by in-solution polymer analysis via high-resolution electrospray ionization mass spectrometry, revealing oligomeric structures with up to six repeating units, mainly composed of covalently linked dihydroxyindole along the polymer backbone. Critically, the reversible redox-active character of the nitroxide-containing polymer scaffolds was investigated by cyclic voltammetric measurements, revealing a convenient and facile access route to electrochemically active nitroxide polymer coatings with potential application in electronic devices such as organic radical batteries.

Polylutidines: Multifunctional Surfaces through Vapor-Based Polymerization of Substituted Pyridinophanes.

We report a new class of functionalized polylutidine polymers that are prepared by chemical vapor deposition polymerization of substituted [2](1,4)benzeno[2](2,5)pyridinophanes. To prepare sufficient amounts of monomer for CVD polymerization, a new synthesis route for ethynylpyridinophane has been developed in three steps with an overall yield of 59 %. Subsequent CVD polymerization yielded well-defined films of poly(2,5-lutidinylene-co-p-xylylene) and poly(4-ethynyl-2,5-lutidinylene-co-p-xylylene). All polymers were characterized by infrared reflection-absorption spectroscopy, ellipsometry, contact angle studies, and X-ray photoelectron spectroscopy. Moreover, ζ-potential measurements revealed that polylutidine films have higher isoelectric points than the corresponding poly-xylylene surfaces owing to the nitrogen atoms in the polymer backbone. The availability of reactive alkyne groups on the surface of poly(4-ethynyl-2,5-lutidinylene-co-p-xylylene) coatings was confirmed by spatially controlled surface modification by means of Huisgen 1,3-dipolar cycloaddition. Compared to the more hydrophobic poly-p-xylylyenes, the presence of the heteroatom in the polymer backbone of polylutidine polymers resulted in surfaces that supported an increased adhesion of primary human umbilical vein endothelial cells (HUVECs). Vapor-based polylutidine coatings are a new class of polymers that feature increased hydrophilicity and increased cell adhesion without limiting the flexibility in selecting appropriate functional side groups.

Bioinstructive Coatings for Hematopoietic Stem Cell Expansion Based on Chemical Vapor Deposition Copolymerization.

We report the chemical vapor deposition (CVD) of dual-functional polymer films for the specific and orthogonal immobilization of two biomolecules (notch ligand delta-like 1 (DLL1) and an RGD-peptide) that govern the fate of hematopoietic stem and progenitor cells. The composition of the CVD polymer and thus the biomolecule ratio can be tailored to investigate and optimize the influence of the relative surface concentrations of biomolecules on stem cell behavior. Prior to cell experiments, all surfaces were characterized by infrared reflection adsorption spectroscopy, time-of-flight secondary ion mass spectrometry, and X-ray photoelectron spectroscopy to confirm the presence of both biomolecules. In a proof-of-principle stem cell culture study, we show that all polymer surfaces are cytocompatible and that the proliferation of the hematopoietic stem and progenitor cells is predominantly influenced by the surface concentration of immobilized DLL1.

Organocatalyzed Photo-Atom Transfer Radical Polymerization of Methacrylic Acid in Continuous Flow and Surface Grafting.

The organocatalyzed photo-atom transfer radical polymerization (photoATRP) using 10-phenylphenothiazine as catalyst is studied toward its use in methacrylic acid (MAA) polymerization and surface grafting. The organocatalyzed photoATRP of methyl methacrylate (MMA) is first optimized for continuous flow synthesis in order to assess the livingness of the polymerization. MMA can be polymerized in batch and in flow; however, conversions are limited by the loss of bromine functionality and hence high conversions have to be traded in with increasing dispersities. Also, MAA is polymerized successfully in continuous flow with similar limitations. Flow conditions are transferred to surface grafting from silanized silicon wafers. The presence of ATRP initiators after silanization is confirmed by secondary ion mass spectrometry and X-ray photoelectron spectroscopy. Dense polymethacrylic acid brush films are successfully produced, which is not directly accessible via classical copper-mediated ATRP techniques.

Single-Molecule Encapsulation: A Straightforward Route to Highly Stable and Printable Enzymes.

A mild, fast, and sequence-independent method for controlled enzyme immobilization is presented. This novel approach involves the encapsulation of single-enzyme molecules and the covalent attachment of these nanobiocatalysts onto surfaces. Fast and mild immobilization conditions, combined with low nonspecific adsorption on hydrophobic substrates, enables well-defined surface patterns via microcontact printing. The biohybrid materials show enhanced activity in organic solvents.

Thermoresponsive Agarose Based Microparticles for Antibody Separation.

We report the development of thermoresponsive 4-mercaptoethylpyridine (MEP)-based chromatographic microsphere based resins for antibody separation that show switchable release abilities by adsorbing immunoglobulins at 40 °C and releasing the proteins at 5 °C. The thermoswitchable release properties were introduced to the porous resins by the grafting of linear poly(N-isopropylacrylamide) (PNIPAM) chains synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization, which were modified to possess MEP end functionalities. Adsorption of γ-globulins as a model antibody on the shortest PNIPAM-MEP (3 kDa) grafted microparticles display binding capacities of up to 20 g L(-1) at 40 °C and a significant decrease in binding capacity to less than 2.5 g L(-1) at 5 °C. By switching the temperature to 5 °C, the release of bound γ-globulins is shown to be as high as 90%. The effects of polymer chain length on the binding capacity are studied in detail and found to be critical as they influence the density of MEP functionalities on the particle surfaces.

A photolithographic approach to spatially resolved cross-linked nanolayers.

The preparation of cross-linked nanosheets with 1-2 nm thickness and predefined shape was achieved by lithographic immobilization of trimethacryloyl thioalkanoates onto the surface of Si wafers, which were functionalized with 2-(phenacylthio)acetamido groups via a photoinduced reaction. Subsequent cross-linking via free radical polymerization as well as a phototriggered Diels-Alder reaction under mild conditions on the surface led to the desired nanosheets. Electrospray ionization mass spectrometry (ESI-MS), X-ray photoelectron spectroscopy (XPS), time-of-flight secondary ion mass spectrometry (ToF-SIMS), as well as infrared reflection-absorption spectroscopy (IRRAS) confirmed the success of individual surface-modification and cross-linking reactions. The thickness and lateral size of the cross-linked structures were determined by atomic force microscopy (AFM) for samples prepared on Si wafers functionalized with a self-assembled monolayer of 1H,1H,2H,2H-perfluorodecyl groups bearing circular pores obtained via a polymer blend lithographic approach, which led to the cross-linking reactions occurring in circular nanoareas (diameter of 50-640 nm) yielding an average thickness of 1.2 nm (radical cross-linking), 1.8 nm (radical cross-linking in the presence of 2,2,2-trifluoroethyl methacrylate as a comonomer), and 1.1 nm (photochemical cross-linking) of the nanosheets.

Surface Grafting via Photo-Induced Copper-Mediated Radical Polymerization at Extremely Low Catalyst Concentrations.

Surface-initiated photo-induced copper-mediated radical polymerization is employed to graft a wide range of polyacrylate brushes from silicon substrates at extremely low catalyst concentrations. This is the first time that the controlled nature of the reported process is demonstrated via block copolymer formation and re-initiation experiments. In addition to unmatched copper catalyst concentrations in the range of few ppb, film thicknesses up to almost 1 µm are achieved within only 1 h.

Macromolecular surface design: photopatterning of functional stable nitrile oxides.

The efficient trapping of photogenerated thioaldehydes with functional shelf-stable nitrile oxides in a 1,3-dipolar cycloaddition is a novel and versatile photochemical strategy for polymer end-group functionalization and surface modification under mild and equimolar conditions. The modular ligation in solution was followed in detail by electrospray ionization mass spectrometry (ESI-MS). X-ray photoelectron spectroscopy (XPS) was employed to analyze the functionalized surfaces, whereas time-of-flight secondary-ion mass spectrometry (ToF-SIMS) confirmed the spatial control of the surface functionalization using a micropatterned shadow mask. Polymer brushes were grown from the surface in a spatially confined regime by surface-initiated atom transfer radical polymerization (SI-ATRP) as confirmed by TOF-SIMS, XPS as well as ellipsometry.

Temperature responsive cellulose-graft-copolymers via cellulose functionalization in an ionic liquid and RAFT polymerization.

Well-defined cellulose-graft-polyacrylamide copolymers were synthesized in a grafting-from approach by reversible addition-fragmentation chain transfer polymerization (RAFT). A chlorine moiety (degree of substitution DS(Cl) ≈ 1.0) was introduced into the cellulose using 1-butyl-3-methylimidazolium chloride (BMIMCl) as solvent before being substituted by a trithiocarbonate moiety resulting in cellulose macro-chain transfer agents (cellulose-CTA) with DS(RAFT) of 0.26 and 0.41. Poly(N,N-diethylacrylamide) (PDEAAm) and poly(N-isopropylacrylamide) (PNIPAM) were subsequently grafted from these cellulose-CTAs and the polymerization kinetics, the molecular weight characteristics and the product composition were studied by nuclear magnetic resonance spectroscopy, X-ray photoelectron spectroscopy, and size exclusion chromatography of the polyacrylamides after cleavage from the cellulose chains. The number-average molecular weights, Mn, of the cleaved polymers ranged from 1100 to 1600 g mol(-1) for PDEAAm (dispersity D = 1.4-1.8) and from 1200 to 2600 g mol (-1) for PNIPAM (D = 1.7-2.1). The LCST behavior of the cellulose-graft-copolymers was studied via the determination of cloud point temperatures, evidencing that the thermoresponsive properties of the hybrid materials could be finely tuned between 18 and 26 °C for PDEAAm and between 22 and 26 °C for PNIPAM side chains.

Ambient temperature ligation of diene functional polymer and peptide strands onto cellulose via photochemical and thermal protocols.

In the present contribution, two novel ambient temperature avenues are introduced to functionalize solid cellulose substrates in a modular fashion with synthetic polymer strands (poly(trifluoro ethyl methacrylate), PTFEMA, Mn = 4400 g mol(-1) , D = 1.18) and an Arg-Gly-Asp (RGD) containing peptide sequence. Both protocols rely on a hetero Diels-Alder reaction between an activated thiocarbonyl functionality and a diene species. In the first-thermally activated-protocol, the cellulose features surface-expressed thiocarbonylthio compounds, which readily react with diene terminal macromolecules at ambient temperature. In the second protocol, the reactive ene species are photochemically generated based on a phenacyl sulfide-decorated cellulose surface, which upon irradiation expresses highly reactive thioaldehyde species. The generated functional hybrid surfaces are characterized in-depth via ToF-SIMS and XPS analysis, revealing the successful covalent attachment of the grafted materials, including the spatially resolved patterning of both synthetic polymers and peptide strands using the photochemical protocol. The study thus provides a versatile platform technology for solid cellulose substrate modification via efficient thermal and photochemical ligation strategies.