The response of coral skeletal nano structure and hardness to ocean acidification conditions.

Ocean acidification typically reduces coral calcification rates and can fundamentally alter skeletal morphology. We use atomic force microscopy (AFM) and microindentation to determine how seawater pCO2 affects skeletal structure and Vickers hardness in a Porites lutea coral. At 400 µatm, the skeletal fasciculi are composed of tightly packed bundles of acicular crystals composed of quadrilateral nanograins, approximately 80-300 nm in dimensions. We interpret high adhesion at the nanograin edges as an organic coating. At 750 µatm the crystals are less regular in width and orientation and composed of either smaller/more rounded nanograins than observed at 400 µatm or of larger areas with little variation in adhesion. Coral aragonite may form via ion-by-ion attachment to the existing skeleton or via conversion of amorphous calcium carbonate precursors. Changes in nanoparticle morphology could reflect variations in the sizes of nanoparticles produced by each crystallization pathway or in the contributions of each pathway to biomineralization. We observe no significant variation in Vickers hardness between skeletons cultured at different seawater pCO2. Either the nanograin size does not affect skeletal hardness or the effect is offset by other changes in the skeleton, e.g. increases in skeletal organic material as reported in previous studies.

Combining SPR with atomic-force microscopy enables single-molecule insights into activation and suppression of the complement cascade.

Activation and suppression of the complement system compete on every serum-exposed surface, host or foreign. Potentially harmful outcomes of this competition depend on surface molecules through mechanisms that remain incompletely understood. Combining surface plasmon resonance (SPR) with atomic force microscopy (AFM), here we studied two complement system proteins at the single-molecule level: C3b, the proteolytically activated form of C3, and factor H (FH), the surface-sensing C3b-binding complement regulator. We used SPR to monitor complement initiation occurring through a positive-feedback loop wherein surface-deposited C3b participates in convertases that cleave C3, thereby depositing more C3b. Over multiple cycles of flowing factor B, factor D, and C3 over the SPR chip, we amplified C3b from ∼20 to ∼220 molecules·µm-2 AFM revealed C3b clusters of up to 20 molecules and solitary C3b molecules deposited up to 200 nm away from the clusters. A force of 0.17 ± 0.02 nanonewtons was needed to pull a single FH molecule, anchored to the AFM probe, from its complex with surface-attached C3b. The extent to which FH molecules stretched before detachment varied widely among complexes. Performing force-distance measurements with FH(D1119G), a variant lacking one of the C3b-binding sites and causing atypical hemolytic uremic syndrome, we found that it detached more uniformly and easily. In further SPR experiments, KD values between FH and C3b on a custom-made chip surface were 5-fold tighter than on commercial chips and similar to those on erythrocytes. These results suggest that the chemistry at the surface on which FH acts drives conformational adjustments that are functionally critical.

Direct Organocatalytic Enantioselective Functionalization of SiOx Surfaces.

Traditional methods to prepare chiral surfaces involve either the adsorption of a chiral molecule onto an achiral surface, or adsorption of a species that forms a chiral template creating lattices with long range order. To date only limited alternative strategies to prepare chiral surfaces have been studied. In this manuscript a "bottom-up" approach is developed that allows the preparation of chiral surfaces by direct enantioselective organocatalytic reactions on a functionalized silicon oxide supported self-assembled monolayer (SAM). The efficient catalytic generation of enantiomerically enriched organic surfaces is achieved using a commercially available homogeneous isothiourea catalyst that promotes an enantioselective Michael-lactonization process upon a silicon-oxide supported SAM functionalized with a reactive trifluoroenone group. Chiral atomic force microscopy (χ-AFM) is used to probe the enantiomeric enrichment of the organic films by measurement of the force distributions arising from interaction of d- or l-cysteine-modified AFM tips and the organic films.

Contact-free experimental determination of the static flexural spring constant of cantilever sensors using a microfluidic force tool.

Micro- and nanocantilevers are employed in atomic force microscopy (AFM) and in micro- and nanoelectromechanical systems (MEMS and NEMS) as sensing elements. They enable nanomechanical measurements, are essential for the characterization of nanomaterials, and form an integral part of many nanoscale devices. Despite the fact that numerous methods described in the literature can be applied to determine the static flexural spring constant of micro- and nanocantilever sensors, experimental techniques that do not require contact between the sensor and a surface at some point during the calibration process are still the exception rather than the rule. We describe a noncontact method using a microfluidic force tool that produces accurate forces and demonstrate that this, in combination with a thermal noise spectrum, can provide the static flexural spring constant for cantilever sensors of different geometric shapes over a wide range of spring constant values (≈0.8-160 N/m).

Isothiourea-Mediated Organocatalytic Michael Addition-Lactonization on a Surface: Modification of SAMs on Silicon Oxide Substrates.

Tailoring the functionality of self-assembled monolayers (SAMs) can be achieved either by depositing prefunctionalized molecules with the appropriate terminal groups or by chemical modification of an existing SAM in situ. The latter approach is particularly advantageous to allow for diversity of surface functionalization from a single SAM and if the incorporation of bulky groups is desired. In the present study an organocatalytic isothiourea-mediated Michael addition-lactonization process analogous to a previously reported study in solution is presented. An achiral isothiourea, 3,4-dihydro-2H-pyrimido[2,1-b]benzothiazole (DHPB), promotes the intermolecular Michael addition-lactonization of a trifluoromethylenone terminated SAM and a variety of arylacetic acids affording C(6)-trifluoromethyldihydropyranones tethered to the surface. X-ray photoelectron spectroscopy, atomic force microscopy, contact angle, and ellipsometry analysis were conducted to confirm the presence of the substituted dihydropyranone. A model study of this approach was also performed in solution to probe the reaction diastereoselectivity as it cannot be measured directly on the surface.

Calibration of the torsional and lateral spring constants of cantilever sensors.

A method suitable for the calibration of the spring constants of all torsional and lateral eigenmodes of micro- and nanocantilever sensors is described. Such sensors enable nanomechanical measurements and the characterization of nanomaterials, for example with atomic force microscopy. The method presented involves the interaction of a flow of fluid from a microchannel with the cantilever beam. Forces imparted by the flow cause the cantilever to bend and induce a measurable change of the torsional and lateral resonance frequencies. From the frequency shifts the cantilever spring constants can be determined. The method does not involve physical contact between the cantilever or its tip and a hard surface. As such it is non-invasive and does not risk damage to the cantilever. Experimental data is presented for two rectangular microcantilevers with fundamental flexural spring constants of 0.046 and 0.154 N m(-1). The experimentally determined torsional stiffness values are compared with those obtained by the Sader method. We demonstrate that the torsional spring constants can be readily calibrated using the method with an accuracy of around 15%.

Bis(trifluoromethyl)methylene addition to vinyl-terminated SAMs: a gas-phase C-C bond-forming reaction on a surface.

Vinyl-terminated self-assembled monolayers (SAMs) on silicon oxide substrates were chemically modified by the addition of a bis(trifluoromethyl)methylene group in a rare gas-phase C-C bond-forming reaction to directly generate films carrying terminal CF3 groups. The vinyl-terminated films were treated with hexafluoroacetone azine (HFAA) for modification. The films were characterized with ellipsometry, contact angle measurements, atomic force microscopy (AFM), and X-ray photoelectron spectroscopy (XPS). In this study, we find that for optimized conditions clean reactions occur on a surface between SAMs with terminal olefins and HFAA, and the product is consistent with bis(trifluoromethyl)cyclopropanation formation after nitrogen extrusion.

Organic chemistry on surfaces: Direct cyclopropanation by dihalocarbene addition to vinyl terminated self-assembled monolayers (SAMs).

C11-Vinyl-terminated self-assembled monolayers (SAMs) on silica surfaces are successfully modified in C-C bond forming reactions with dihalocarbenes to generate SAMs, terminated with dihalo- (fluoro, chloro, bromo) cyclopropane motifs with about 30% surface coverage.

Determination of the spring constants of the higher flexural modes of microcantilever sensors.

A method for the simultaneous calibration of the spring constants of all flexural modes of microcantilevers is presented. It is based on a flow of gas from a microchannel that interacts with the microcantilever. The gas flow causes a measurable shift in the resonance frequencies of thermal noise spectra of the flexural modes. From the magnitude of the frequency shifts of the individual modes the spring constants can be determined with high accuracy and precision. The method is non-invasive and does not risk damage to the cantilever. Experimental data are presented for several V-shaped and rectangular cantilevers with nominal fundamental spring constants in the range of 0.03-1.75 N m(-1). The spring constants of the fundamental modes compare favorably to those obtained using the Sader method. The higher modes of oscillation are readily calibrated with experimental uncertainties of 5-10%.

Electron-beam patterned self-assembled monolayers as templates for Cu electrodeposition and lift-off.

Self-assembled monolayers (SAMs) of 4'-methylbiphenyl-4-thiol (MBP0) adsorbed on polycrystalline gold substrates served as templates to control electrochemical deposition of Cu structures from acidic solution, and enabled the subsequent lift-off of the metal structures by attachment to epoxy glue. By exploiting the negative-resist behaviour of MBP0, the SAM was patterned by means of electron-beam lithography. For high deposition contrast a two-step procedure was employed involving a nucleation phase around -0.7 V versus Cu(2+)/Cu and a growth phase at around -0.35 V versus Cu(2+)/Cu. Structures with features down to 100 nm were deposited and transferred with high fidelity. By using substrates with different surface morphologies, AFM measurements revealed that the roughness of the substrate is a crucial factor but not the only one determining the roughness of the copper surface that is exposed after lift-off.

Mass determination and sensitivity based on resonance frequency changes of the higher flexural modes of cantilever sensors.

Micro- and nanocantilevers are increasingly employed as mass sensors. Most studies consider the first flexural mode and adsorbed masses that are either discretely attached or homogeneously distributed along the entire length of the cantilever. We derive general expressions that allow for the determination of the total attached mass with any mass distribution along the cantilever length and all flexural modes. The expressions are valid for all cantilevers whose flexural deflection can be described by a one-dimensional function. This approach includes the most common types of microcantilevers, namely, rectangular, picket, and V-shaped. The theoretical results are compared with experimental data up to the fourth flexural mode obtained from thermal noise spectra of rectangular and V-shaped cantilevers.

Protein adsorption onto CF(3)-terminated oligo(ethylene glycol) containing self-assembled monolayers (SAMs): the influence of ionic strength and electrostatic forces.

Oligo(ethylene glycol) (OEG) containing self-assembled monolayers (SAMs) on gold are known for their protein resistant properties. The underlying molecular mechanisms and the contributions of the interactions involved, however, are still not completely understood. It is known that electrostatic, van der Waals, hydrophobic, and hydration forces all play a role in the interaction between proteins and surfaces, but it is difficult to study their influence separately and to quantify their contributions. In the present study we investigate five different OEG containing SAMs and the influence of the ionic strength and the electrostatic component on the amount of a negatively charged protein (fibrinogen) that adsorbs onto them. Atomic force microscopy (AFM) was employed to record force-distance curves with hydrophobic probes depending on the ion concentration, and the amount of the protein that adsorbs relative to a hydrophobic surface was quantified using ellipsometry. The findings suggest that electrostatic forces can create a very low energy barrier thus only slightly decreasing the number of negatively charged proteins in solution with sufficient energy to approach the surface closely, and have a rather small influence on the amount that adsorbs. The films we investigated were not protein resistant. This supports other studies, reporting that a strong short-range repulsion as for example caused by hydration forces is required to make these films resistant to the non-specific adsorption of proteins.

Dynamic spring constants for higher flexural modes of cantilever plates with applications to atomic force microscopy.

In atomic force microscopy (AFM) a sharp tip fixed close to the free end of a cantilever beam interacts with a surface. The interaction can be described by a point-mass model of an equivalent oscillator with a single spring located at the position of the tip. However, other spring constants have to be used to describe the oscillation behavior correctly if forces are acting on the cantilever over an extended lateral range. A point-mass model is then no longer valid. In the present study we derive expressions for the spring constants of cantilevers that can interact with any part of their plan view area along the beam and for all flexural modes. The equations describe the oscillation behavior in the corresponding mass model and are based on the eigenfrequencies and modal shapes of the free cantilever. The results are of high practical relevance, for example if an AFM is operated in a higher flexural mode, if the tip is not located at the free end of the cantilever beam, or if the external conservative forces affecting cantilever movement are not restricted to a single point. The limitations of the approach are discussed.

Hydrodynamic methods for calibrating the normal spring constant of microcantilevers.

Knowledge of the spring constants of microcantilevers is vital in atomic force microscopy and for cantilever-based devices that are, for example, employed as probes in biomedical applications. We compare two recently developed hydrodynamic methods for the determination of the normal spring constant of microcantilevers. Both approaches are non-invasive when determining the spring constant and require only knowledge of the thermal noise response of the cantilever in a fluid and its plan view dimensions. The methods do not bear the risk of damaging the cantilever and are therefore attractive for example in mass sensing applications in cases where the cantilever has been modified, e.g. with a coating. The specific strengths of the methods are discussed and the results for a variety of cantilevers are presented and compared.

Ionic strength mediated hydrophobic force switching of CF3-terminated ethylene glycol self-assembled monolayers (SAMs) on gold.

We have synthesised novel oligo(ethylene glycol), CF3-terminated switching self-assembled monolayers, which allow the force experienced by a hydrophobic object to be controlled via the ionic strength of the environment.

Calibration of the normal spring constant of microcantilevers in a parallel fluid flow.

We demonstrate a novel approach to determine the normal spring constant of microcantilevers. The cantilevers are placed parallel to a fluid flow thus establishing one of the walls of the flow channel. Resonance frequencies are recorded depending on the velocity of the fluid. The pressure gradient resulting from the flow causes the resonance frequency to change. This change can be exploited to deduce the cantilever spring constant with high precision. The method we present can be performed in situ and does not involve any contact of the cantilever with a surface thus having great potential for the calibration of modified probes and for being incorporated in microfluidic systems. In case the spring constant is known, the setup can also be employed to determine the velocity of fluid flows and the flow rate with high precision and up to high speeds.

Temperature dependence of viscosity and density of viscous liquids determined from thermal noise spectra of uncalibrated atomic force microscope cantilevers.

We demonstrate that the thermal response of uncalibrated atomic force microscope cantilevers can be used to extract the density and the viscosity of viscous liquids with good accuracy. Temperature dependent thermal noise spectra were measured in water/poly(ethylene glycol) mixtures. Empirical parameters characteristic of the resonance behavior of the system were extracted from data recorded for one of the solutions at room temperature. These parameters were then employed to determine both viscosity and density values of the solutions simultaneously at different temperatures. In addition, activation energies for viscous flow were determined from the viscosity values obtained. The method presented is both fast and reliable and has the potential to be applied in connection with microfluidic systems, making macroscopic amounts of liquid and separate measurements with a viscometer and a densimeter redundant.

Near edge X-ray absorption fine structure spectroscopy as a tool to probe electronic and structural properties of thin organic films and liquids.

Synchrotron-based spectroscopic techniques have contributed significantly to a better understanding of the properties of materials on the macroscopic and microscopic scale over the last decades. They can be applied to samples from a diversity of fields, including Biology, Life Sciences, Chemistry and Materials. One of these techniques is Near Edge X-Ray Absorption Fine Structure (NEXAFS) spectroscopy, revealing electronic structure and information on the orientation of adsorbed molecules. The present article describes the basics of the technique and the progress it has made over the last three decades, and summarizes some of its more recent developments and applications. This tutorial review article should be accessible for novices to the field from Physics, Chemistry, Biology, Materials, and the Life Sciences, interested in thin organic films and liquid systems.

Solvent dependence of the molecular order in ion-exchanged self-assembled dialkylammonium monolayers on mica studied with soft X-ray absorption.

Dialkyldimethylammonium films on mica prepared via ion exchange from solution have been reported to be of high quality in terms of their density and molecular orientation. Different preparation procedures are described in the literature. The molecular order and the inclination of the alkyl chains, however, are often deduced from indirect experimental evidence such as the wettability and the film thickness. In the present study we employed near edge X-ray absorption fine structure spectroscopy (NEXAFS) to determine directly the order of the molecules adsorbed from different solvents (water, methanol, water/methanol 1:1, cyclohexanol, and chloroform). It was found that films prepared from different solvents are displaying large differences in the established surface coverage and orientation. In particular, NEXAFS disclosed that the orientation of the alkyl chains can differ significantly even when similar water contact angle values are observed.

Chain-length dependence of the conformational order in self-assembled dialkylammonium monolayers on mica studied with soft X-ray absorption.

Self-assembled alkyl chain based monolayers on mica are important for industrial and technological processes since they can be employed for an organic modification of the inorganic substrate. The conformational structure and orientational order of the films determine the interaction of the modified substrate with the environment and the chemical character and stability of its surface. We have studied the conformational order in ion exchanged dialkylammonium monolayers adsorbed on mica depending on the length of the alkyl chains systematically with near-edge X-ray absorption fine structure spectroscopy (NEXAFS). In addition, films were characterized by water contact angle measurements. The experimentally determined average tilt angles of the chains are discussed in terms of the degree of order. It was found that the absolute number of gauche defects in the films increases with decreasing chain length.