3D Nanoprinting of All-Metal Nanoprobes for Electric AFM Modes.

3D nanoprinting via focused electron beam induced deposition (FEBID) is applied for fabrication of all-metal nanoprobes for atomic force microscopy (AFM)-based electrical operation modes. The 3D tip concept is based on a hollow-cone (HC) design, with all-metal material properties and apex radii in the sub-10 nm regime to allow for high-resolution imaging during morphological imaging, conductive AFM (CAFM) and electrostatic force microscopy (EFM). The study starts with design aspects to motivate the proposed HC architecture, followed by detailed fabrication characterization to identify and optimize FEBID process parameters. To arrive at desired material properties, e-beam assisted purification in low-pressure water atmospheres was applied at room temperature, which enabled the removal of carbon impurities from as-deposited structures. The microstructure of final HCs was analyzed via scanning transmission electron microscopy-high-angle annular dark field (STEM-HAADF), whereas electrical and mechanical properties were investigated in situ using micromanipulators. Finally, AFM/EFM/CAFM measurements were performed in comparison to non-functional, high-resolution tips and commercially available electric probes. In essence, we demonstrate that the proposed all-metal HCs provide the resolution capabilities of the former, with the electric conductivity of the latter onboard, combining both assets in one design.

Humidity Response of Cellulose Thin Films.

Cellulose-water interactions are crucial to understand biological processes as well as to develop tailor made cellulose-based products. However, the main challenge to study these interactions is the diversity of natural cellulose fibers and alterations in their supramolecular structure. Here, we study the humidity response of different, well-defined, ultrathin cellulose films as a function of industrially relevant treatments using different techniques. As treatments, drying at elevated temperature, swelling, and swelling followed by drying at elevated temperatures were chosen. The cellulose films were prepared by spin coating a soluble cellulose derivative, trimethylsilyl cellulose, onto solid substrates followed by conversion to cellulose by HCl vapor. For the highest investigated humidity levels (97%), the layer thickness increased by ca. 40% corresponding to the incorporation of 3.6 molecules of water per anhydroglucose unit (AGU), independent of the cellulose source used. The aforementioned treatments affected this ratio significantly with drying being the most notable procedure (2.0 and 2.6 molecules per AGU). The alterations were investigated in real time with X-ray reflectivity and quartz crystal microbalance with dissipation, equipped with a humidity module to obtain information about changes in the thickness, roughness, and electron density of the films and qualitatively confirmed using grazing incidence small angle X-ray scattering measurements using synchrotron irradiation.

High-Fidelity 3D Nanoprinting of Plasmonic Gold Nanoantennas.

The direct-write fabrication of freestanding nanoantennas for plasmonic applications is a challenging task, as demands for overall morphologies, nanoscale features, and material qualities are very high. Within the small pool of capable technologies, three-dimensional (3D) nanoprinting via focused electron beam-induced deposition (FEBID) is a promising candidate due to its design flexibility. As FEBID materials notoriously suffer from high carbon contents, the chemical postgrowth transfer into pure metals is indispensably needed, which can severely harm or even destroy FEBID-based 3D nanoarchitectures. Following this challenge, we first dissect FEBID growth characteristics and then combine individual advantages by an advanced patterning approach. This allows the direct-write fabrication of high-fidelity shapes with nanoscale features in the sub-10 nm range, which allow a shape-stable chemical transfer into plasmonically active Au nanoantennas. The here-introduced strategy is a generic approach toward more complex 3D architectures for future applications in the field of 3D plasmonics.

Three-Dimensional Nanothermistors for Thermal Probing.

Accessing the thermal properties of materials or even full devices is a highly relevant topic in research and development. Along with the ongoing trend toward smaller feature sizes, the demands on appropriate instrumentation to access surface temperatures with high thermal and lateral resolution also increase. Scanning thermal microscopy is one of the most powerful technologies to fulfill this task down to the sub-100 nm regime, which, however, strongly depends on the nanoprobe design. In this study, we introduce a three-dimensional (3D) nanoprobe concept, which acts as a nanothermistor to access surface temperatures. Fabrication of nanobridges is done via 3D nanoprinting using focused electron beams, which allows direct-write fabrication on prestructured, self-sensing cantilever. As individual branch dimensions are in the sub-100 nm regime, mechanical stability is first studied by a combined approach of finite-element simulation and scanning electron microscopy-assisted in situ atomic force microscopy (AFM) measurements. After deriving the design rules for mechanically stable 3D nanobridges with vertical stiffness up to 50 N m-1, a material tuning approach is introduced to increase mechanical wear resistance at the tip apex for high-quality AFM imaging at high scan speeds. Finally, we demonstrate the electrical response in dependence of temperature and find a negative temperature coefficient of -(0.75 ± 0.2) 10-3 K-1 and sensing rates of 30 ± 1 ms K-1 at noise levels of ±0.5 K, which underlines the potential of our concept.

Impact of Electron-Beam Heating during 3D Nanoprinting.

An artifact limiting the reproduction of three-dimensional (3D) designs using nanoprinting has been quantified. Beam-induced heating was determined through complementary experiments, models, and simulations to affect the deposition rate during the 3D nanoprinting of mesh objects using focused electron beam induced deposition (FEBID). The mesh objects are constructed using interconnected nanowires. During nanowire growth, the beam interaction driving deposition also causes local heating. The temperature at the beam impact region progressively rises as thermal resistance increases with nanowire growth. Heat dissipation resembles the classical mode of heat transfer from extended surfaces; heat must flow through the mesh object to reach the substrate sink. Simulations reveal that beam heating causes an increase in the rate of precursor desorption at the BIR, causing a concomitant decrease in the deposition rate, overwhelming an increase in the deposition rate driven by thermally enhanced precursor surface diffusion. Temperature changes as small as 10 K produce noticeable changes in deposit geometry; nanowires appear to deflect and curve toward the substrate because the vertical growth rate decreases. The 3D FEBID naturally ensues from the substrate surface upward, inducing a vertical temperature gradient along the deposit. Simulations, experiments, temperature-controlled studies, and process current monitoring all confirm the cause of nanowire distortion as beam-induced heating while also revealing the rate-determining physics governing the final deposit shape.

Multi-layered nanoscale cellulose/CuInS2 sandwich type thin films.

A generic procedure for the manufacturing of cellulose-metal sulfide multilayered sandwich type thin films is demonstrated at the example of copper indium sulfide. These multilayers were created by alternate spin coating steps of precursors, followed by their conversion using either acidic vapors, or heat treatment. As precursors, cellulose xanthate, a widely available cellulose derivative employed in viscose fiber manufacturing and commercial copper and indium xanthates were used. After conversion of the single layers into cellulose and copper indium sulfide, the film properties (structure, thickness, photoelectric activity) of the single and multilayer systems consisting of alternate layers of cellulose and copper indium sulfide were studied. For the proof of concept, up to five layers were built up, showing a clear separation of the cellulose and the metal sulfide layers as demonstrated using cross sectional analysis using ion slope beam cutting and SEM imaging. Finally, the conversion of xanthates was performed using UV light and a mask, allowing for the creation of 2D patterns.

Single-molecule study of oxidative enzymatic deconstruction of cellulose.

LPMO (lytic polysaccharide monooxygenase) represents a unique paradigm of cellulosic biomass degradation by an oxidative mechanism. Understanding the role of LPMO in deconstructing crystalline cellulose is fundamental to the enzyme's biological function and will help to specify the use of LPMO in biorefinery applications. Here we show with real-time atomic force microscopy that C1 and C4 oxidizing types of LPMO from Neurospora crassa (NcLPMO9F, NcLPMO9C) bind to nanocrystalline cellulose with high preference for the very same substrate surfaces that are also used by a processive cellulase (Trichoderma reesei CBH I) to move along during hydrolytic cellulose degradation. The bound LPMOs, however, are immobile during their adsorbed residence time ( ~ 1.0 min for NcLPMO9F) on cellulose. Treatment with LPMO resulted in fibrillation of crystalline cellulose and strongly ( ≥ 2-fold) enhanced the cellulase adsorption. It also increased enzyme turnover on the cellulose surface, thus boosting the hydrolytic conversion.Understanding the role of enzymes in biomass depolymerization is essential for the development of more efficient biorefineries. Here, the authors show by atomic force microscopy the real-time mechanism of cellulose deconstruction by lytic polysaccharide monooxygenases.

How Bound and Free Fatty Acids in Cellulose Films Impact Nonspecific Protein Adsorption.

The effect of fatty acids and fatty acid esters to impair nonspecific protein adsorption on cellulose thin films is investigated. Thin films are prepared by blending trimethylsilyl cellulose solutions with either cellulose stearoyl ester or stearic acid at various ratios. After film formation by spin coating, the trimethylsilyl cellulose fraction of the films is converted to cellulose by exposure to HCl vapors. The morphologies and surface roughness of the blends were examined by atomic force microscopy revealing different feature shapes and sizes depending on the blend ratios. Nonspecific protein adsorption at the example of bovine serum albumin toward the blend thin films was tested by means of surface plasmon resonance spectroscopy in real-time. Incorporation of stearic acid into the cellulose leads to highly protein repellent surfaces regardless of the amount added. The stearic acid acts as a sacrificial compound that builds a complex with bovine serum albumin thereby inhibiting protein adsorption. For the blends where stearoyl ester is added to the cellulose films, the cellulose:cellulose stearoyl ester ratios of 3:1 and 1:1 lead to much lower nonspecific protein adsorption compared to pure cellulose, whereas for the other ratios, adsorption increases. Supplementary results were obtained from atomic force microscopy experiments performed in liquid during exposure to protein solution and surface free energy determinations.

Star-shaped Polymers through Simple Wavelength-Selective Free-Radical Photopolymerization.

Star-shaped polymers represent highly desired materials in nanotechnology and life sciences, including biomedical applications (e.g., diagnostic imaging, tissue engineering, and targeted drug delivery). Herein, we report a straightforward synthesis of wavelength-selective multifunctional photoinitiators (PIs) that contain a bisacylphosphane oxide (BAPO) group and an α-hydroxy ketone moiety within one molecule. By using three different wavelengths, these photoactive groups can be selectively addressed and activated, thereby allowing the synthesis of ABC-type miktoarm star polymers through a simple, highly selective, and robust free-radical polymerization method. The photochemistry of these new initiators and the feasibility of this concept were investigated in unprecedented detail by using various spectroscopic techniques.

Direct-Write Fabrication of Cellulose Nano-Structures via Focused Electron Beam Induced Nanosynthesis.

In many areas of science and technology, patterned films and surfaces play a key role in engineering and development of advanced materials. Here, we introduce a new generic technique for the fabrication of polysaccharide nano-structures via focused electron beam induced conversion (FEBIC). For the proof of principle, organosoluble trimethylsilyl-cellulose (TMSC) thin films have been deposited by spin coating on SiO2 / Si and exposed to a nano-sized electron beam. It turns out that in the exposed areas an electron induced desilylation reaction takes place converting soluble TMSC to rather insoluble cellulose. After removal of the unexposed TMSC areas, structured cellulose patterns remain on the surface with FWHM line widths down to 70 nm. Systematic FEBIC parameter sweeps reveal a generally electron dose dependent behavior with three working regimes: incomplete conversion, ideal doses and over exposure. Direct (FT-IR) and indirect chemical analyses (enzymatic degradation) confirmed the cellulosic character of ideally converted areas. These investigations are complemented by a theoretical model which suggests a two-step reaction process by means of TMSC → cellulose and cellulose → non-cellulose material conversion in excellent agreement with experimental data. The extracted, individual reaction rates allowed the derivation of design rules for FEBIC parameters towards highest conversion efficiencies and highest lateral resolution.

Functional characterization of the native swollenin from Trichoderma reesei: study of its possible role as C1 factor of enzymatic lignocellulose conversion.

BACKGROUND: Through binding to cellulose, expansin-like proteins are thought to loosen the structural order of crystalline surface material, thus making it more accessible for degradation by hydrolytic enzymes. Swollenin SWO1 is the major expansin-like protein from the fungus Trichoderma reesei. Here, we have performed a detailed characterization of a recombinant native form of SWO1 with respect to its possible auxiliary role in the enzymatic saccharification of lignocellulosic substrates. RESULTS: The swo1 gene was overexpressed in T. reesei QM9414 Δxyr1 mutant, featuring downregulated cellulase production, and the protein was purified from culture supernatant. SWO1 was N-glycosylated and its circular dichroism spectrum suggested a folded protein. Adsorption isotherms (25 °C, pH 5.0, 1.0 mg substrate/mL) revealed SWO1 to be 120- and 20-fold more specific for binding to birchwood xylan and kraft lignin, respectively, than for binding to Avicel PH-101. The SWO1 binding capacity on lignin (25 µmol/g) exceeded 12-fold that on Avicel PH-101 (2.1 µmol/g). On xylan, not only the binding capacity (22 µmol/g) but also the affinity of SWO1 (K d = 0.08 µM) was enhanced compared to Avicel PH-101 (K d = 0.89 µM). SWO1 caused rapid release of a tiny amount of reducing sugars (<1 % of total) from different substrates (Avicel PH-101, nanocrystalline cellulose, steam-pretreated wheat straw, barley ß-glucan, cellotetraose) but did not promote continued saccharification. Atomic force microscopy revealed that amorphous cellulose films were not affected by SWO1. Also with AFM, binding of SWO1 to cellulose nanocrystallites was demonstrated at the single-molecule level, but adsorption did not affect this cellulose. SWO1 exhibited no synergy with T. reesei cellulases in the hydrolysis of the different celluloses. However, SWO1 boosted slightly (1.5-fold) the reducing sugar release from a native grass substrate. CONCLUSIONS: SWO1 is a strongly glycosylated protein, which has implications for producing it in heterologous hosts. Although SWO1 binds to crystalline cellulose, its adsorption to xylan is much stronger. SWO1 is not an auxiliary factor of the enzymatic degradation of a variety of cellulosic substrates. Effect of SWO1 on sugar release from intact plant cell walls might be exploitable with certain (e.g., mildly pretreated) lignocellulosic feedstocks.

Tunable Semicrystalline Thin Film Cellulose Substrate for High-Resolution, In-Situ AFM Characterization of Enzymatic Cellulose Degradation.

In the field of enzymatic cellulose degradation, fundamental interactions between different enzymes and polymorphic cellulose materials are of essential importance but still not understood in full detail. One technology with the potential of direct visualization of such bioprocesses is atomic force microscopy (AFM) due to its capability of real-time in situ investigations with spatial resolutions down to the molecular scale. To exploit the full capabilities of this technology and unravel fundamental enzyme-cellulose bioprocesses, appropriate cellulose substrates are decisive. In this study, we introduce a semicrystalline-thin-film-cellulose (SCFTC) substrate which fulfills the strong demands on such ideal cellulose substrates by means of (1) tunable polymorphism via variable contents of homogeneously sized cellulose nanocrystals embedded in an amorphous cellulose matrix; (2) nanoflat surface topology for high-resolution and high-speed AFM; and (3) fast, simple, and reproducible fabrication. The study starts with a detailed description of SCTFC preparation protocols including an in-depth material characterization. In the second part, we demonstrate the suitability of SCTFC substrates for enzymatic degradation studies by combined, individual, and sequential exposure to TrCel6A/TrCel7A cellulases (Trichoderma reesei) to visualize synergistic effects down to the nanoscale.