The atomic force microscope (AFM) has become a powerful tool for the visualization, probing and manipulation of RNA at the single molecule level. AFM measurements can be carried out in buffer solution in a physiological medium, which is crucial to study the structure and function of biomolecules, also allowing studying them at work. Imaging the specimen in its native state is a great advantage compared to other high resolution methods such as electron microscopy and X-ray diffraction. There is no need to stain, freeze or crystallize biological samples. Moreover, compared to NMR spectroscopy for instance, for AFM studies the size of the biomolecules is not limiting. Consequently the AFM allows one also to investigate larger RNA molecules. In particular, structural studies of nucleic acids and assemblies thereof, have been carried out by AFM routinely including ssRNA, dsRNA and nucleoprotein complexes thereof, as well as RNA aggregates and 2D RNA assemblies. These are becoming increasingly important as novel unique building blocks in the emerging field of RNA nanotechnology. In particular by AFM unique information can be obtained on these RNA based assemblies. Moreover, the AFM is of fundamental relevance to study biological relevant RNA interactions and dynamics. In this short review a brief overview will be given on structural studies that have been done related to AFM topographic imaging of RNA, RNA assemblies and aggregates. Finally, an overview on AFM beyond imaging will be provided. This includes force spectroscopy of RNA under physiological conditions in aqueous buffer to probe RNA interaction with proteins and ligands as well as other AFM tip based RNA probing. Important applications include the detection and quantification of RNA in biological samples. A selection of recent highlights and breakthroughs will be provided related to structural and functional studies by AFM. The main intention of this short review to provide the reader with a flavor of what AFM is able to contribute to RNA research and engineering.

A segmented polyurethane elastomer (PU) was synthesized in poly(lactic acid) (PLA) melt by reactive processing. The isocyanate component was anticipated to react with the end-groups of PLA resulting in the formation of block-copolymers. The stoichiometry of the functional groups was optimized in the preliminary experiments. Two different processing methods were compared in the further experiments: conventional mixing of PU with PLA (PLA/PU), and reactive blending (PLA-b-PU). The comparison of the structure and properties of compatibilized reactive blends and conventional physical blends clearly shows the benefits of reactive processing. Coupling resulted in a finer dispersion of the particles in the matrix leading to better mechanical properties in the reactive blend. The successful synthesis of PEA-b-PU block copolymers was confirmed by NMR spectroscopy. The isocyanate component was found to react only with the hydroxyl end-groups of PLA, while the formation of amide and acylurea groups was not detected on the carboxyl end.

Ligand–receptor interactions are fundamental in life sciences and include hormone–receptor, protein–protein, pathogen–host, and cell–cell interactions, among others. Atomic force microscopy (AFM) proved to be invaluable for scrutinizing ligand–receptor interactions at the single molecular level. Basically, a ligand is attached to the AFM tip while its cognate receptor is immobilized on a surface or vice versa, and interactions are studied following triggered ligand–receptor binding. However, with rising biological complexity it becomes increasingly challenging to attach a single intact biomolecule to the tip and ensure interaction‐specific orientation. This study presents a novel strategy of inducible in situ tip functionalization with complex multiprotein nanoparticles exemplified by viral capsids, termed photopicking. It ensures a firm attachment of single 125 nm large capsids to the tip. Specific orientation is attained by weak immunosorption of capsids to the substrate and strong photoinducible covalent cross‐linking to the tip. Validation of the tip functionalization success is immediate in situ. The versatility of the strategy is further demonstrated on 20–60 nm large amino‐modified nanoparticles. In conclusion, considering the size range of the tested biomolecules, the presented strategy is applicable to viruses, viral particles, cellular organelles, multiprotein ligands/receptors, and therapeutic nanoparticles, among others. It therefore opens up exciting new avenues in broad biomedical research fields.

The redox active metalloprotein Cu-azurin was directly chemisorbed on bare gold electrodes through disulfide forming groups (Cys3Cys26). Topological and electrochemical properties of the immobilized molecules were investigated by electrochemical atomic force microscopy (EC-AFM) in Peak Force Tapping mode and cyclic voltammetry (CV). Cu-azurin layers showed surface confined reversible electrochemical behavior. In-situ EC-AFM studies revealed a height difference of 0.32 nm between the oxidized (+0.45 V) and the reduced state (-0.10 V) for Cu-azurin by imaging single molecules independent of the ionic strength of the buffer solution. In contrast, no height change was detected for Zn-azurin which is non-redox active and served as control. The observed height changes of the Cu-azurin upon electrochemical redox switching are thought to originate from conformational changes of the protein and the variation in the orientation of immobilized proteins between the oxidized and reduced states. In this manner, the height of the Cu-azurin could be modulated reversibly by the applied potential. (C) 2015 Elsevier Ltd. All rights reserved.

In the past 30 years, the atomic force microscope (AFM) has become a true enabling platform in the life sciences opening entire novel avenues for structural and dynamic studies of biological systems. It enables visualization, probing and manipulation across the length scales, from single molecules to living cells in buffer solution under physiological conditions without the need for labeling or staining of the specimen. In particular, for structural studies of nucleic acids and assemblies thereof, the AFM has matured into a routinely used tool providing nanometer spatial resolution. This includes ssRNA, dsRNA and nucleoprotein complexes thereof, as well as RNA aggregates and 2D RNA assemblies. By AFM unique information can be obtained on RNA based assemblies which are becoming increasingly important as novel unique building blocks in the emerging field of RNA nanotechnology. In addition, the AFM is of fundamental relevance to study biological relevant RNA interactions and dynamics. In this short review first the basic functioning principles of commonly used AFM modes including AFM based force spectroscopy will be briefly described. Next a brief overview will be given on structural studies that have been done related to AFM topographic imaging of RNA, RNA assemblies and aggregates. Finally, an overview on AFM beyond imaging will be provided. This includes force spectroscopy of RNA under physiological conditions in aqueous buffer to probe RNA interaction with proteins and ligands as well as other AFM tip based RNA probing. The main intention of this short review to give the reader a flavor of what AFM contributes to RNA research and engineering.

Poly(ferrocenylsilane) (PFS) is a redox-active polymer that can be utilized in a variety of applications, such as sensing, nanoparticle foundries or dual-responsive hydrogels, due to the fact that it can be partially or completely oxidized, reversibly, by (electro)chemical means. In this research, PFS is tethered to a cysteamine-modified gold-coated microchannel electrode via amine alkylation, following a recent published procedure for conventional macro-sized electrodes. The PFS-coated microfluidic device is successfully employed as an electrochemical sensor for sodium 1-ascorbate, or vitamin C, with a detection limit of 0.5 mM in aqueous solution. The fabrication of the device, that comprises gold-coated electrodes of micrometers in width, is discussed, as well as the static and in-line detection of sodium 1-ascorbate. The analyte concentration is sensed through an electrocatalytic process in both static and in-line cases, by cyclic voltammetry and amperometry, respectively. The findings presented in this work open novel opportunities for local surface modification of chip-integrated gold electrodes by redox-responsive polymers used, e.g., as electrochemical sensors. (C) 2016 Elsevier Ltd. All rights reserved.

Tip-enhanced nanoscale optical imaging techniques such as apertureless scanning near-field optical microscopy (a-SNOM) and scanning near-field ellipsometric microscopy (SNEM) applications can suffer from a steady degradation in performance due to adhesion of atmospheric contaminants to the metal coated tip. Here, we demonstrate that a self-assembled monolayer (SAM) of ethanethiol (EtSH) is an effective means of protecting gold-coated atomic force microscopy (AFM) probe tips from accumulation of surface contaminants during prolonged exposure to ambient air. The period over which they yield consistent and reproducible results for scanning near-field ellipsometric microscopy (SNEM) imaging is thus extended. SNEM optical images of a microphase separated polystyrene-block-poly (methylmethacrylate) (PS-b-PMMA) diblock copolymer film, which were captured with bare and SAM-protected gold-coated AFM probes, both immediately after coating and following five days of storage in ambient air, were compared. During this period the intensity of the optical signals from the untreated gold tip fell by 66%, while those from the SAM protected tip fell by 14%. Additionally, gold coated AFM probe tips were modified with various lengths of alkanethiols to measure the change in intensity variation in the optical images with SAM layer thickness. The experimental results were compared to point dipole model calculations. While a SAM of 1-dodecanethiol (DoSH) was found to strongly suppress field enhancement we find that it can be locally removed from the tip apex by deforming the molecules under load, restoring SNEM image contrast.

Striped-phase of oligo(phenylene ethynylene) molecules on Au(111).

We describe a novel approach to quantitatively visualize sub nm height changes occurring in thin films of redox active polymers upon reversible electrochemical oxidation/reduction in situ and in real-time with electrochemical imaging ellipsometry (EC-IE). Our approach is based on the utilization of a micro-patterned substrate containing circular patterns of passive (non-redox active) 11-mercapto-1-undecanol (MCU) within a redox-responsive oligoethylene sulfide end-functionalized poly(ferrocenyldimethylsilane) (ES-PFS) film on a gold substrate. The non-redox responsive MCU layer was used as a molecular reference layer for the direct visualization of the minute thickness variations of the ES-PFS film. The ellipsometric microscopy images were recorded in aqueous electrolyte solutions at potentials of -0.1 V and 0.6 V vs. Ag/AgCl corresponding to the reduced and oxidized redox states of ES-PFS, respectively. The ellipsometric contrast images showed a 37 (±2)% intensity increase in the ES-PFS layer upon oxidation. The thickness of the ES-PFS layer reversibly changed between 4.0 (±0.1) nm and 3.4 (±0.1) nm upon oxidation and reduction, respectively, as determined by IE. Additionally, electrochemical atomic force microscopy (EC-AFM) was used to verify the redox controlled thickness variations. The proposed method opens novel avenues to optically visualize minute and rapid height changes occurring e.g. in redox active (and other stimulus responsive) polymer films in a fast and non-invasive manner.

Polymer brushes lead to small friction and wear and thus hold great potential for industrial applications. However, interdigitation of opposing brushes makes them prone to damage. Here we report molecular dynamics simulations revealing that immiscible brush systems can form slick interfaces, in which interdigitation is eliminated and dissipation strongly reduced. We test our findings with friction force microscopy experiments on hydrophilic and hydrophobic brush systems in both symmetric and asymmetric setups. In the symmetric setup both brushes are chemically alike, while the asymmetric system consists of two different brushes that each prefer their own solvent. The trends observed in the experimentally measured force traces and the friction reduction are similar to the simulations and extend to fully immersed contacts. These results reveal that two immiscible brush systems in mechanical contact slide at a fluid-fluid interface while having load-bearing ability. This makes them ideal candidates for tribological applications.

The integration of smart stimulus-responsive polymers as functional elements within microfluidic devices has greatly improved the performance capabilities of controlled fluid delivery. For their use as actuators in microfluidic systems, reversible expansion and shrinking are unique mechanisms which can be utilized as both passive and active fluid control elements to establish gate and valve functions (passive) and pumping elements (active). Various constituents in microfluidic glass channels based on stimulus-responsive elements have been reported based on pH-responsive, thermoresponsive and photoresponsive coatings. Fluid control and robust performance have been demonstrated in microfluidic devices in a number of studies. Here we give a brief overview of selected examples from the literature reporting on the use of stimulus response polymers as active or passive elements for fluid control in microfluidic devices, with specific emphasis on glass-based devices. The remaining challenges include improving switching times and achieving local addressability of the responsive constituent. We envisage tackling these challenges by utilizing redox-responsive polymers which offer fast and reversible switching and local addressability in combination with nanofabricated electrodes.

Macromolecular networks consisting of homogeneously distributed covalently bonded inorganic and organic precursors are anticipated to show remarkable characteristics, distinct from those of the individual constituents. A novel hyper-cross-linked ultrathin membrane is presented, consisting of a giant molecular network of alternating polyhedral oligomeric silsesquioxanes and aromatic imide bridges. The hybrid characteristics of the membrane are manifested in excellent gas separation performance at elevated temperatures, providing a new and key enabling technology for many important industrial scale applications.

Using a combination of ellipsometry and friction force microscopy, we study the reversible swelling, collapse and variation in friction properties of covalently bound poly(N-isopropylacrylamide) (PNIPAM) layers on silicon with different grafting densities in response to exposure to good solvents and co-nonsolvent mixtures. Changes in the thickness and segment density distribution of grafted films are investigated by in situ ellipsometry. Based on quantitative modelling of the ellipsometry spectra, we postulate a structural model, which assumes that collapse takes place in the contacting layer between the brush and the co-nonsolvent and the top-collapsed brushes remain hydrated in the film interior. Using the structural model derived from ellipsometry spectra, we analyse the AFM based friction force microscopy data, which were obtained by silica colloidal probes. Results show a large increase of the friction coefficient of PNIPAM grafts when the grafts swollen by water are brought in contact with co-nonsolvents. For instance, the value of the friction coefficient for a medium density brush in water is four times lower than the value observed in a water-methanol (50% v/v) mixture. This increase of friction is accompanied by an increase in adherence between the PNIPAM chains and the silica colloidal probes, and is a result of chain collapse in the graft when contacted by a co-nonsolvent mixture in agreement with the model postulated on the basis of ellipsometric characterisation. The kinetic behaviour of the collapse is assessed by measuring the temporal variation of friction in situ as a function of elapsed time following contact with the co-nonsolvent as a function of graft density. In conclusion, the effect of co-nonsolvency influenced both the thickness of the PNIPAM brushes and the tribological behavior of the brush surfaces.

Our research is focused on the engineering of novel, highly sensitive and miniaturized hybrid materials from carbon nanotubes (CNTs) and DNA molecules for applications in biosensors and medical devices. These hybrid sensors allow for a high degree of miniaturization, a key factor in the design of lightweight components while maintaining the advantages of in-situ and real-time analysis capabilities. In the first phase of the sensor design process, we investigated the structural and electrical properties of the supramolecular complexes made of amide-functionalized CNTs and double-stranded DNA. The solubilization properties of the hybrid nanotubes in aqueous solutions with different concentrations of DNA were studied, and an optimal ratio of nanotubes and biomolecules to achieve a good level of dispersion was found. Complexes formed in aqueous solution from CNTs and DNA are highly stable and maintain their properties up to one month from preparation. The morphology of the CNT-DNA composites was investigated at the nanoscale level using atomic force microscopy (AFM) and electron microscopy (SEM). Results from these experiments show the strong affinity between the surface of the amide-functionalized CNTs and the DNA strands. Further, the CNT-DNA films were investigated by atomic force microscopy in the PeakForce TUNA mode to assess the suitability of this technique in determining the local conductive properties of the hybrid films.

Oligo(phenylene-ethynylene), self-assembled monolayers on Au(111) have been studied with scanning tunneling microscopy. The oligo(phenylene-ethynylene) molecules are adsorbed in a flat-lying orientation. Time-resolved scanning tunneling microscopy measurements reveal that the molecules continuously switch back and forth between two nearly degenerate configurations. The energy difference between the two configurations is 22±5meV and the activation barrier for the transition from the low-energy configuration to the high-energy configuration is 0.65±0.03eV. A statistical analysis of the residence times revealed that the switching process is stochastic. We propose that the two level switching is due to a torsional mode of the central phenyl ring.