Upgrading the Toolbox: Two-Photon Absorption Induced Cleavage of Coumarin Dimers for Light-Based 4D Printing.

The use of light for shaping and changing matter is of high relevance in polymer and material science. Herein, a photopolymer method is presented, which comprises the combination of 3D photo-printing at 405 nm light and subsequent modification under two-photon absorption (TPA) conditions at 532 nm light, adding the fourth dimension. The TPA-triggered cycloreversion reaction of an intramolecular coumarin dimer (ICD) structure occurs within the absorbing material. The 3D-printable matrix does not show any degradation under the TPA conditions. With the presented photochemical tool of TPA processes inside absorbing 3D photo-printable matrices, new possibilities for post-printing modification, e. g. for smart materials, are added.

Oxidative formation of bis-N-methylquinolinone from anti-head-to-head N-methylquinolinone cyclodimer.

The light-driven formation and cleavage of cyclobutane structural motifs resulting from [2 + 2]-pericyclic reactions, as found in thymine and coumarin-type systems, is an important and intensively studied photochemical reaction. Various applications are reported utilizing these systems, among others, in cross-linked polymers, light-triggered drug release, or other technical applications. Herein coumarin is most frequently used as the photoactive group. Quite often, a poor quantum yield for dimerization and cyclobutane-cleavage and a lack of reversibility are described. In this work, we present the identification of a heterogeneous pathway of dimer cleavage found in a rarely studied coumarin analog molecule, the N-methyl-quinolinone (NMQ). The monomer was irradiated in a tube flow-reactor and the reaction process was monitored using online HPLC measurements. We found the formation of a pseudo-equilibrium between monomeric and dimeric NMQ and a continuous rise of a side product via oxidative dimer splitting and proton elimination which was identified as 3,3'-bis-NMQ. Oxidative conversion by singlet oxygen was identified to be the cause of this non-conventional cyclobutane cleavage. The addition of antioxidants suppressing singlet oxygen enables achieving a 100% photochemical conversion from NMQ to the anti-head-to-head-NMQ-dimer. Using dissolved oxygen upon light activation to singlet oxygen limits the reversibility of the photochemical [2 + 2]-cycloaddition and cycloreversion of NMQ and most likely comparable systems. Based on these findings, the development of highly efficient cycloaddition-cycloreversion systems should be enabled.

Cycloreversion performance of coumarin and hetero-coumarin dimers under aerobic conditions: unexpected behavior triggered by UV-A light.

Photochemical [2+2]-cycloadditions of coumarin-like monomers are the textbook paradigms of photo-formation and photo-cleavage reactions. The electronic conjugation length of monomers and dimers is quite different which results in almost fully separated UV/Vis absorption bands in the UV-A and UV-C. This feature enables the selective light-controlled conversion between monomeric and dimeric forms by the choice of the appropriate wavelengths. Several applications are based on this kind of reversible photo linker without absorption in the visible range. But which is the best molecule from the coumarin family for such an application? Within this study, we compared the photochemical cleavage behavior of twelve coumarin-type cyclobutane dimers. In particular, the influence of isomer structure and substitution pattern was studied. Two dimers with an unexpected high quantum yield for cyclobutane cleavage were identified. This behavior is explained through the differing ring strain of the cyclobutane moiety. Electron donating substitutions of the framework, e.g. with a methoxy function (+M-effect), leads to a decreased oxidation potential, making the dimers sensitive towards oxidative dimer splitting. This result disqualifies coumarins, e.g. attached to a polymer backbone via an ether bond, often in the 7-position, because of their instabilities and side reactions in an aerobic environment. The methylated dimers (+I-effect) show excellent stability towards this undesired side reaction as well as a high cleavage efficiency upon irradiation with 265 nm. All twelve investigated dimers are ranked for their quantum efficiency and rate constant for cleavage at 265 nm, as well as their oxygen tolerance. As the most promising derivative within our scope for applications the methylated coumarin dimer was identified.

The role of phosphopeptides in the mineralisation of silica.

We investigated the silicification activity of hyperphosphorylated peptides in combination with long-chain polyamines (LCPA). The bioinspired in vitro silicification experiments with peptides containing different amounts of phosphorylated serines showed structure-activity dependence by altering the amount and morphology of the silica precipitate. Our study provides an explanation for the considerable metabolic role of diatoms in the synthesis of hyperphosphorylated poly-cationic peptides such as natSil-1A1. The efficient late-stage phosphorylation of peptides yielded a synthetic heptaphosphopeptide whose silicification properties resemble those of natSil-1A1. As opposed to this, unphosphorylated poly-cationic peptides or LCPA require concentrations above 1 mM for silicification. Hyperphosphorylated peptides showed a linear dependence between the amount of dissolved peptides and the amount of precipitated silica in the concentration range below 1 mM. Under mildly acidic conditions and short precipitation times, the concentration of the added LCPA determined the size of the silica spheres.

Applications of magnetic materials separation in biological nanomedicine.

As a result of their advantages for superparamagnetic properties, good biocompatibility, and high binding capacity, functionalized magnetic materials became widely popular over the past couple of decades, being applied on large scale in various processes of sample preparation for biomedicine. In this work, we perform an in-depth review on the current progress in the field of magnetic bead separation, discussing in detail the physical basis of this process, various synthesis methods and surface modification strategies. We place special focus of attention as well on the latest applications of magnetic polymer microspheres in cell separation, protein purification, immobilized enzyme, nucleic acid separation, and extraction of bioactive compounds with low molecular weight. Existing problems are highlighted and possible trends of magnetic separation techniques for biomedicine in the future are proposed.

Mechanically metastable structures generated by single pulse laser-induced periodic surface structures (LIPSS) in the photoresist SU8.

Laser-induced periodic surface structures (LIPSS) with a periodicity of 351 nm are generated in the negative photoresist SU8 by single nanosecond laser pulse impact. Friction scans indicate the periodic pattern to comprise alternating regions of crosslinked and non-crosslinked SU8. Intriguingly, even minor mechanical stimuli in the order of nanonewtons cause the unfolding or rather the deletion of the characteristic periodic pattern similarly to the release of a pre-loaded spring. This feature combined with high resilience to heat and photon irradiation makes SU8-LIPSS attractive for applications such as mechanical stress monitors, self-destructing memory and passive micro actuators.

Substituting Coumarins for Quinolinones: Altering the Cycloreversion Potential Energy Landscape.

The light-activated cleavage of cyclobutane-based systems via [2 + 2] cycloreversions, such as thymine and coumarin dimers, is an important but still poorly understood ultrafast photochemical reaction. Systems displaying reversible cycloreversion have found various uses in cross-linked polymers, enhancing gas adsorption affinities in inorganics, and light-activated medical therapies. We report the identification of a heterogeneous mode of cycloreversion for a rarely examined coumarin analogue system. Quinolinone monomers and dimers were probed using ultraviolet pumped, transient absorption spectroscopy and demonstrated radically different photophysical properties than coumarins. Monomers displayed enhanced intersystem crossing at almost 1:1 versus the combined nonradiative and radiative singlet decay, while the dimers underwent cycloreversion to a one excited-one ground state monomer photoproduct pair. The change in both systems was directly linked to the lactame group in the quinolinone motif. This discovery highlights the dramatic effects that small chemical changes can have on photoreaction pathways and opens up a new means to produce and develop more efficient cycloaddition-cycloreversion systems.

Photocleavage of coumarin dimers studied by femtosecond UV transient absorption spectroscopy.

Coumarins are a class of UV absorbing compounds which exhibit fast, photoinduced cyclobutane ring formation and cleavage reactions. The photophysics behind such processes hold significant relevance for biomedical and photoresponsive materials research. In order to further understand the underlying dynamics of the cleavage reaction, and develop strategies for increasing the reaction efficiency, UV transient absorption spectroscopy was applied to three unsubstituted, isomeric coumarin dimers: anti-head-to-head (anti-hh), syn-head-to-head (syn-hh) and syn-head-to-tail (syn-ht). The experiments performed under 280 nm excitation and broadband (300-620 nm) probing revealed that the cleavage reaction of coumarin dimers occurs through non-radiative, short-lived (<200 fs) singlet states. From the data, two branched kinetic models were developed to describe the monomer formation and dimer relaxation dynamics, identify possible intermediate states, and determine the quantum yields of the dimer cleavage. The anti-hh dimer shows the highest cleavage efficiency with a value of about 20%. The differences in the cleavage efficiency for the three isomers are interpreted in terms of differing steric hindrances of the benzene groups attached to the cyclobutane ring and charge delocalisation of the intermediate state.

Real-time, label-free monitoring of cell viability based on cell adhesion measurements with an atomic force microscope.

BACKGROUND: The adhesion of cells to an oscillating cantilever sensitively influences the oscillation amplitude at a given frequency. Even early stages of cytotoxicity cause a change in the viscosity of the cell membrane and morphology, both affecting their adhesion to the cantilever. We present a generally applicable method for real-time, label free monitoring and fast-screening technique to assess early stages of cytotoxicity recorded in terms of loss of cell adhesion. RESULTS: We present data taken from gold nanoparticles of different sizes and surface coatings as well as some reference substances like ethanol, cadmium chloride, and staurosporine. Measurements were recorded with two different cell lines, HeLa and MCF7 cells. The results obtained from gold nanoparticles confirm earlier findings and attest the easiness and effectiveness of the method. CONCLUSIONS: The reported method allows to easily adapt virtually every AFM to screen and assess toxicity of compounds in terms of cell adhesion with little modifications as long as a flow cell is available. The sensitivity of the method is good enough indicating that even single cell analysis seems possible.

Basic Physicochemical Properties of Polyethylene Glycol Coated Gold Nanoparticles that Determine Their Interaction with Cells.

A homologous nanoparticle library was synthesized in which gold nanoparticles were coated with polyethylene glycol, whereby the diameter of the gold cores, as well as the thickness of the shell of polyethylene glycol, was varied. Basic physicochemical parameters of this two-dimensional nanoparticle library, such as size, ζ-potential, hydrophilicity, elasticity, and catalytic activity ,were determined. Cell uptake of selected nanoparticles with equal size yet varying thickness of the polymer shell and their effect on basic structural and functional cell parameters was determined. Data indicates that thinner, more hydrophilic coatings, combined with the partial functionalization with quaternary ammonium cations, result in a more efficient uptake, which relates to significant effects on structural and functional cell parameters.

Directed assembly of gold nanowires on silicon via reorganization and simultaneous fusion of randomly distributed gold nanoparticles.

Laser-induced reorganization and simultaneous fusion of nanoparticles is introduced as a versatile concept for pattern formation on surfaces. The process takes advantage of a phenomenon called laser-induced periodic surface structures (LIPSS) which originates from periodically alternating photonic fringe patterns in the near-field of solids. Associated photonic fringe patterns are shown to reorganize randomly distributed gold nanoparticles on a silicon wafer into periodic gold nanostructures. Concomitant melting due to optical heating facilitates the formation of continuous structures such as periodic gold nanowire arrays. Generated patterns can be converted into secondary structures using directed assembly or self-organization. This includes for example the rotation of gold nanowire arrays by arbitrary angles or their fragmentation into arrays of aligned gold nanoparticles.

Laser-Induced Au Catalyst Generation for Tailored ZnO Nanostructure Growth.

ZnO nanostructures, semiconductors with attractive optical properties, are typically grown by thermal chemical vapor deposition for optimal growth control. Their growth is well investigated, but commonly results in the entire substrate being covered with identical ZnO nanostructures. At best a limited, binary growth control is achieved with masks or lithographic processes. We demonstrate nanosecond laser-induced Au catalyst generation on Si(100) wafers, resulting in controlled ZnO nanostructure growth. Scanning electron and atomic force microscopy measurements reveal the laser pulse's influence on the substrate's and catalyst's properties, e.g., nanoparticle size and distribution. The laser-induced formation of a thin SiO2-layer on the catalysts plays a key role in the subsequent ZnO growth mechanism. By tuning the irradiation parameters, the width, density, and morphology of ZnO nanostructures, i.e., nanorods, nanowires, and nanobelts, were controlled. Our method allows for maskless ZnO nanostructure designs locally controlled on Si-wafers.

The development of rare-earth combined Fe-based magnetic nanocomposites for use in biological theranostics.

Iron (Fe)-based nanoparticles (NPs) represented by Fe3O4 exhibit attractive properties, such as high saturation magnetization, low magneto-crystalline anisotropy, and good biocompatibility, and are useful as magnetic resonance imaging (MRI) contrast agents. However, the existence of artifacts makes the single magnetic resonance imaging mode lack accuracy in tumor diagnosis. To overcome this limitation, a strategy where rare-earth elements are combined with Fe-based NPs is applied. Rare earth is the general name of Sc, Y, and elements with unique 4f electronic configurations. Some rare-earth elements like Gd and Lu exhibit magnetic properties due to unpaired electrons, while some, like Er and Ho, fluoresce under excitation ascribed to the electron transition at intermediate energy levels. In this manuscript, attention is focused on multimodal nanomaterials composed of rare-earth elements and Fe-based NPs. We provide an overview of the synthetic routes and current biomedical application of the nanocomposites that show potential for precise diagnosis and efficient treatment of cancers.

Laser-Driven One- and Two-Dimensional Subwavelength Periodic Patterning of Thin Films Made of a Metal-Organic MoS2 Precursor.

Laser-based surface processing is an established way for the maskless generation of surface structures and functionalities on a large variety of materials. Laser-driven periodic surface texturing and structuring of thin films is reported for metallic-, semiconductive-, and polymeric films. Here, we introduce subwavelength surface patterning of metal-organic thin films of [Mo2S4(S2CNnBu2)2], a MoS2 precursor. Accurate control of one- and two-dimensional (1D and 2D) periodic patterns is achieved on silicon wafers with a pulsed 532 nm ns laser. With suitable combinations of laser polarization, laser pulse energy, the thickness of the SiO2 passivation layer, and the MoS2 precursor's thin film thickness, high-quality 1D and 2D self-organized periodic structures are obtained in virtually unlimited areas. The material redistribution related to the pattern formation is thermally driven at low laser energies. Increasing pulse energies beyond a threshold level, in our experiments a factor of 2, fully converts the precursor to MoS2.

Ground- and excited-state vibrational coherence dynamics in Bacteriorhodopsin probed with degenerate four-wave-mixing experiments.

Vibrational coherence dynamics in the all-trans retinal chromophore in Bacteriorhodopsin (BR) are investigated by means of temporally and spectrally resolved degenerate four-wave-mixing experiments. The coherence dynamics depend on the excitation wavelength when BR samples are excited at different wavelengths in a spectral range between 520 nm to above 620 nm. The trends in the dynamics observed by tuning of the excitation wavelength allow an assignment of the wave packet dynamics to ground- and excited-state potential energy surfaces. Specifically, the intensity of so-called "out-of-plane" modes of polyene-chain substituents increases for excitation wavelengths near 500 nm. It is shown that this is consistent with the assignment of out-of-plane modes to excited-state coherence dynamics. Moreover, intense low-frequency coherence dynamics around 200 cm(-1) are observed for signal detection in two different spectral regions of excited-state absorption. These modulations are assigned to excited-state dynamics due to the observed dependence on the excitation wavelength. In addition, we show that generally high-frequency modes (>1010 cm(-1)) originate from wave packet motion in the electronic ground state of all-trans retinal.

Photochemistry of coumarin-functionalized SiO2 nanoparticles.

We describe the synthesis and photochemistry of coumarin-functionalized silica nanoparticles, which were prepared utilizing 7-[3-(triethoxysilyl)propanyloxy]coumarin (TPC) to attach coumarin as a photoactive group to the silica nanoparticle surface. The nanoparticle size and morphology were investigated by scanning electron microscopy, atomic force microscopy, and dynamic light scattering. The diameter of the spherical nanoparticles was determined by all three methods to be about 40 nm. The surface functionalization was characterized in the bulk by ζ-potential measurements and on the single-nanoparticle level by electrostatic force microscopy, where the difference in surface potential between TPC-modified and unmodified silica nanoparticles is measured. The degree of surface functionalization was determined by thermogravimetric analysis (TGA), and a theoretical limit of about 23,000 coumarin entities per nanoparticle was calculated. The photochemistry, and its reversibility, of the nanoparticle-attached coumarin entities was found to be quite different from the coumarin photochemistry in solution or on flat surfaces. Photodimerization with light of 355 nm and photocleavage with light of 254, 266, and 280 nm were analyzed by absorption and fluorescence spectroscopy. Following several cycles of photodimerization and photocleavage showed that the absorption change at 320 nm decreases from cycle to cycle. The coumarin layer on the nanoparticles was proven to be unchanged by TGA. The apparent loss of absorption change is due to the formation of interlinked nanoparticles during the dimerization-cleavage cycles. Because the coumarin groups on the inside of the obtained nanoparticle clusters are inaccessible to light, the amount of "uncleavable" dicoumarins increases, thus lowering the obtainable absorption change from cycle to cycle.

Fusion of purple membranes triggered by immobilization on carbon nanomembranes.

A freestanding ultrathin hybrid membrane was synthesized comprising two functional layers, that is, first, a carbon nanomembrane (CNM) produced by electron irradiation-induced cross-linking of a self-assembled monolayer (SAM) of 4'-nitro-1,1'-biphenyl-4-thiol (NBPT) and second, purple membrane (PM) containing genetically modified bacteriorhodopsin (BR) carrying a C-terminal His-tag. The NBPT-CNM was further modified to carry nitrilotriacetic acid (NTA) terminal groups for the interaction with the His-tagged PMs forming a quasi-monolayer of His-tagged PM on top of the CNM-NTA. The formation of the Ni-NTA/His-tag complex leads to the unidirectional orientation of PM on the CNM substrate. Electrophoretic sedimentation was employed to optimize the surface coverage and to close gaps between the PM patches. This procedure for the immobilization of oriented dense PM facilitates the spontaneous fusion of individual PM patches, forming larger membrane areas. This is, to our knowledge, the very first procedure described to induce the oriented fusion of PM on a solid support. The resulting hybrid membrane has a potential application as a light-driven two-dimensional proton-pumping membrane, for instance, for light-driven seawater desalination as envisioned soon after the discovery of PM.

An intelligent tumor microenvironment responsive nanotheranostic agent for T1/T2 dual-modal magnetic resonance imaging-guided and self-augmented photothermal therapy.

Photothermal therapy (PTT), as a promising antineoplastic therapeutic strategy, has been harnessed to restrain tumor growth through near-infrared (NIR) irradiation mediated thermal ablation. Nevertheless, its biological applications are hampered by thermal diffusion and up-regulated heat shock proteins (HSPs). Herein, a versatile nanotheranostic agent is developed via integrating Zn0.2Fe2.8O4 nanoparticles (NPs), polydopamine (PDA), and MnO2 NPs for T1/T2 dual-modal magnetic resonance (MR) imaging-guided and self-augmented PTT. The as-designed Zn0.2Fe2.8O4@PDA@MnO2 NPs adequately serve as a PTT agent to realize effective photothermal conversion and obtain local hyperthermia. Additionally, the Zn0.2Fe2.8O4@PDA@MnO2 NPs can significantly consume overexpressed glutathione (GSH) and generate Mn2+ in the tumor microenvironment (TME), thus destroying redox homeostasis and catalytically generating hydroxyl radicals (˙OH) for HSP suppression and PTT enhancement. Meanwhile, Mn2+ and Zn0.2Fe2.8O4 NPs significantly strengthen T1- and T2-weighted MR contrast for tumor imaging and PTT guidance. Hence, this study offers proof of concept for self-augmented PTT and T1/T2 dual-modal MR imaging for tumor elimination.

Synthesis and photochemistry of coumarin-based self-assembled monolayers on silicon oxide surfaces.

In this study, we report on a system consisting of self-assembled monolayers (SAMs) formed by 7-(11-trichlorosilylundecyloxy)coumarin on mica and on quartz glass. For the first time, in the absence of an inert atmosphere or a stabilizing matrix, we demonstrate by means of absorption and fluorescence spectroscopy that the photochemical cycloaddition of coumarin head groups is completely reversible in SAMs. Photodimerization and photocleavage were monitored for five cycles of alternating irradiation with light of wavelengths 355 and 254 nm, respectively. SAM formation was analyzed using atomic force microscopy and contact angle measurements. The quantum yield of the single photon absorption induced photocleavage of coumarin dimers in a SAM was determined to be Phi = 0.29. Asymmetrical photocleavage after lactone ring-opening of the coumarin dimer SAM causes a change in contact angle from about 70 degrees to about 55 degrees. This may be observed, for example, as a significant change in surface wettability.

Ultrathin conductive carbon nanomembranes as support films for structural analysis of biological specimens.

Ultrathin carbon nanomembranes (CNM) have been tested as supports for both cryogenic high-resolution transmission electron microscopy (cryo-EM) as well as atomic force microscopy (AFM) of biological specimens. Purple membrane (PM) from Halobacterium salinarum, a 2-D crystalline monolayer of bacteriorhodopsin (BR) and lipids, was used for this study. Due to their low thickness of just 1.6 nm CNM add virtually no phase contrast to the transmission pattern. This is an important advantage over commonly used amorphous carbon support films which become instable below a thickness of approximately 20 nm. Moreover, the electrical conductivity of CNM can be tuned leading to conductive carbon nanomembranes (cCNM). cCNM support films were analyzed for the first time and were found to ideally meet all requirements of cryo-EM of insulating biological samples. A projection map of PM on cCNM at 4 A resolution has been calculated which proves that the structural integrity of biological samples is preserved up to the high-resolution range. CNM have also proven to be suitable supports for AFM analysis of biological samples. PM on CNM was imaged at molecular resolution and single molecule force spectra were recorded which show no differences compared to force spectra of PM obtained with other substrates. This is the first demonstration of a support film material which meets the requirements of both, cryo-EM and AFM, thus enabling comparative structural studies of biomolecular samples with unchanged sample-substrate interactions. Beyond high-resolution cryo-EM of biological samples, cCNM are attractive new substrates for other biophysical techniques which require conductive supports, i.e. scanning tunneling microscopy (STM) and electrostatic force microscopy (EFM).