The mechanical properties of biological nanofibers such as collagen fibrils are important in many applications, ranging from tissue-engineering to cancer treatment. However, mechanical testing is not straightforward at the nanometer scale. Here, we use the theory of column-buckling to determine the bending properties of individual collagen fibrils. To achieve this, fibrils were deposited on a manually pre-stretched foil, which was then released with the fibrils attached. Atomic Force Microscopy (AFM) imaging was used to determine the tensile modulus by measuring the buckling-wavelength and the radius for each fibril. Comparison with data obtained by AFM nanoindentation and other, more sophisticated methods, shows that our results are in very good agreement. The great advantage of this simple approach is that it can be used to quickly determine mechanical properties without force or stress-strain measurements, which are challenging to obtain accurately and at high throughput at the nanoscale. The method could be applied to any nanofibers, not just collagen fibrils. Statement of significance Collagen fibrils are the main constituent of the extracellular matrix, and alterations of their mechanical properties can have significant effects on cell adhesion and motility. This has, ultimately, implications in age-related diseases and cancer. Furthermore, tuning the mechanical properties of collagen fibrils could be an important tool in the design of artificial cell scaffolds in tissue-engineering. For these reasons, it is important to have methods that can be used to determine the mechanical properties of fibrils at the single-fibril level and, therefore, at the nanometer scale. The method presented here has the advantage of being easy to use and avoids some of the fundamental issues of more established methods. (C) 2022 The Author(s). Published by Elsevier Ltd on behalf of Acta Materialia Inc.

Mapping charged chemical groups at the solidliquid interface is important in many areas, ranging from colloidal systems to biomolecular interactions. However, classical methods to measure surface charges either lack spatial resolution or-like Kelvin-probe force microscopy (KPFM)-cannot be applied in aqueous solutions because a DC bias voltage is used. Here, we show that using AC Kelvin probe force microscopy (AC-KPFM), in which the DC bias is replaced with an AC voltage of sufficiently high frequency, the surface potential of spatially fixated, charged surface groups can be mapped in aqueous solution. We demonstrate this with micropatterned, functionalized alkanethiol layers which expose ionized amino- and carboxy-groups. These groups are representative of the charged groups of most biomolecules such as proteins. By adjusting the pH of the solution, the charge of the groups was reversibly altered, demonstrating the electrostatic nature of the measured signal. The influence of the electric double layer (EDL) on the measurement is discussed, and we, furthermore, show how charged, micropatterned layers can be used to spatially direct the deposition of nanoparticles of opposite charge.

Collagen fibrils act like nanoscale cables in the extracellular matrix of vertebrate tissues and provide a scaffold for cells to attach to. However, beyond this mechanical function, the surface charge of collagen fibrils is also likely to play an important role. Here, we show that native, type I collagen fibrils from a mammal tendon exhibit a particular dependence of surface charge on longitudinal strain. Fibrils first become more positive with strain of up to 10% and then become more negative again with strain between 10 and 17%. The effect correlates with the stiffness of fibrils and can be explained by structural rearrangements, which expose hidden, ionizable residues. Fibrils treated with glutaraldehyde did not show any change in surface charge when strained. The electrical surface potential, which is directly related to the number ratio of exposed amine and carboxy groups on the surface, was determined by Kelvin-probe force microscopy of fibrils attached on an extensible, thin polymer film. By stretching the film, a large number of individual fibrils could be strained simultaneously without resorting to sophisticated nanomechanical devices. It is conceivable that cells react to such changes of the fibril charge and that this effect is an additional contributor, besides mechanics, to a number of physiological processes. It may also need to be considered in the design of tissue-engineering scaffolds.

Muscovite mica is a widely accepted substrate for scanning probe microscopy (SPM) investigations. However, mica has intrinsic properties that alter samples and obstruct their analysis due to free charges build-up, ionic exchange and water adsorption taking place at the surface. In addition to interfacial phenomena, there is a growing interest in electrostatic charges on insulators as they are crucial in diverse applications. Despite the high demand for studies of this nature, experimental set-ups capable of resolving charge build-up at the micro-scale are still scarce and technically limited. Here, we report the imaging of surface charge dissipation on freshly cleaved mica by Kelvin-probe Force Microscopy (KPFM). A local electrostatic charge micro-domain was generated by friction between an atomic force microscope (AFM) tip and mica, and its decay was tracked by two-dimensional mapping using KPFM. We found time-dependent charge dissipation, which is attributed to the adsorption of water molecules on mica surface.

The mechanical properties of collagen fibrils play an important role in cell-matrix interactions and are a manifestation of their molecular structure. Using a, to our knowledge, novel combination of uniaxial, longitudinal straining and radial nanoindentation, we found that type I collagen fibrils show a pronounced nonlinear behavior in the form of strain stiffening at strains from 0 to 15%, followed by strain softening at strains from 15 to 25%. At the molecular scale, this surprising phenomenon can be explained by the combination of unfolding of disordered domains and breaking of native cross-links at different stages of strain. Fibrils cross-linked artificially by glutaraldehyde do not show such a behavior, and nanoindentation allowed us to measure the mechanics of the overlap and gap regions in the D-banding individually. The results could have consequences for our understanding of matrix mechanics and the influence of excessive glycation, which has been linked with age-related diseases such as diabetes. Furthermore, the simplicity of the straining method could be attractive in other areas of biophysics at the nanometer scale because it does not require any bespoke instrumentation and is easy to use.

Collagen fibrils are central to the molecular organization of the extracellular matrix (ECM) and to defining the cellular microenvironment. Glycation of collagen fibrils is known to impact on cell adhesion and migration in the context of cancer and in model studies, glycation of collagen molecules has been shown to affect the binding of other ECM components to collagen. Here we use TEM to show that ribose-5-phosphate (R5P) glycation of collagen fibrils – potentially important in the microenvironment of actively dividing cells, such as cancer cells – disrupts the longitudinal ordering of the molecules in collagen fibrils and, using KFM and FLiM, that R5P-glycated collagen fibrils have a more negative surface charge than unglycated fibrils. Altered molecular arrangement can be expected to impact on the accessibility of cell adhesion sites and altered fibril surface charge on the integrity of the extracellular matrix structure surrounding glycated collagen fibrils. Both effects are highly relevant for cell adhesion and migration within the tumour microenvironment.

Detection methods and analytical devices have drawn increasing attention in recent years due to their direct impact on early detection, monitoring and diagnosis of disease in medical research. In this work, we describe a simple but so far unrecognized label-free method for surface-bound analyte detection, which could be applicable to a wide range of substances. In this respect, the feasibility and practical aspects of a micrometer-scale, poly-l-lysine-based, solid-phase assay for label-free analyte detection with electrostatic read-out are investigated. Micropatterned poly-l-lysine layers were produced using soft-lithography on mica and their electrostatic surface potential was determined using Kelvin-probe Force Microscopy. Ribose, a natural sugar, was used as analyte. Upon exposure to ribose, the surface potential changed from positive to negative in a reversible manner. We report for the first time the use of an electrostatic principle for assay read-out. This purely physical effect could be used to develop label- and marker-free assays for sugars, various other substances or, possibly, biosensors.

Kelvin-probe force microscopy is a measurement mode of atomic force microscopy, which is used to quantitatively map the electrical surface potential of a sample. Inadequate hardware and electronic design can lead to signal cross talk and, in consequence, false results. Here, we show that certain cross talk artifacts not only do manifest themselves in additional noise, reduced resolution, or an offset of the measured surface potential but can also lead to an inverted signal scale and, crucially, cannot be diagnosed with a known reference signal. We show experimental data on an electrically homogeneous sample, describe a method to detect the artifact, and propose simple remedies, which should be well within the reach of most research and industrial laboratories.

This paper investigates the benefit of active mechanical Q-control for electrical surface potential measurements in atomic force microscopy. The effective quality-factor of the electrically stimulated cantilever oscillation is additionally increased mechanically at the same frequency with an active analog Q-controller, while in lift-mode. It is shown experimentally that the cantilever oscillation amplitude and therefore the measurement sensitivity is improved by a factor of 15. By spectral noise analysis around the resonance frequency of the cantilever, an increase of the signal-to-noise ratio from 2.5dB to 23dB is achieved.

Nanoparticles or similar, nanoscale objects such as proteins or biological fibrils usually have to be deposited from aqueous suspension onto a solid support surface for further characterization by atomic force microscopy (AFM) and related methods such as Kelvin-probe force microscopy (KFM). Here we show, on the examples of functionalized nanoparticles and collagen fibrils, that water desorption after sample preparation affects their electrostatic potential determined by KFM in a predictable manner. We explain this effect with a simple, analytical model based on the capacitance of the partially dielectric-filled tip-sample system. We also propose practical measures to avoid false interpretation of electrical AFM-based experiments. As the phenomenon is very generic it may have significant implications in the application of AFM to nanoparticles and other nanostructures including biological ones.

Collagen fibrils are a major component of the extracellular matrix. They form nanometer-scale "cables" acting as a scaffold for cells in animal tissues and are widely used in tissue-engineering. Besides controlling their structure and mechanical properties, it is crucial to have information of their surface charge, as this affects how cells attach to the scaffold. Here, we employed Kelvin-probe Force Microscopy to determine the electrostatic surface potential at the single-fibril level and investigated how glutaraldehyde, a well-established protein cross-linking agent, shifts the surface charge to more negative values without disrupting the fibrils themselves. This shift can be interpreted as the result of the reaction between the carbonyl groups of glutaraldehyde and the amine groups of collagen. It reduces the overall density of positively charged amine groups on the collagen fibril surface and, ultimately, results in the observed negative shift of the surface potential measured. Reactions between carbonyl-containing compounds and proteins are considered the first step in glycation, the nonenzymatic reaction between sugars and proteins. It is conceivable that similar charge shifts happen in vivo caused by sugars, which could have serious implications on age-related diseases such as diabetes and which has been hypothesised for many years.

This paper presents a novel feedback based Scanning Probe Microscopy method which enables quantitative surface potential measurements without the need of the DC bias of Kelvin Probe Force Microscopy. In addition to the sinusoidal excitation signal at frequency ω, a sinusoidal signal with the frequency 2ω is applied to the conductive cantilever. By modulating the amplitude of the signal at 2ω, the resulting electric force component at the frequency ω can be nullified by a feedback controller. When the force and, hence, the cantilever oscillation is zero, the required amplitude represents the quantitative surface potential. Recording this amplitude while scanning over the sample allows to acquire a two dimensional map of the surface potential. The AC-KPFM method, shown analytically and with experimental results, keeps the compensation based principle of classical KPFM, resulting in quantitative measurements but without the need of a DC bias.

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Kelvin-probe Force Microscopy (KFM) is an established method to map surface potentials or surface charges at high, spatial resolution. However, KFM does not work in water, which restricts its applicability considerably, especially when considering common, functional chemical groups in biophysics such as amine or carboxy groups, whose charge depends on pH. Here, we demonstrate that the KFM signal of such groups taken in air after exposure to water correlates qualitatively with their expected charge in water for a wide range of pH values. The correlation was tested with microcontact-printed thiols exposing amine and carboxy groups. Furthermore, it was shown that collagen fibrils, as an example of a biological material, exhibit a particular, pH-sensitive surface charge pattern, which could be caused by the particular arrangement of ionizable residues on the collagen fibril surface.

There is continued debate as to how nanomaterials enhance the passive diffusion of drugs through the skin. This study examined if drug-nanoparticle surface interactions, which occurred during topical application, had the capability to enhance percutaneous penetration. Atomic force microscopy force adhesion measurements were used to demonstrate that a model drug, tetracaine, strongly adsorbed to the surface of a negatively charged carboxyl-modified polystyrene nanoparticle (NanoPSCOOH) through both its methyl and amine functionalities (up to a 6- and 16-fold greater adhesion force respectively compared with the CH3-CH3 control). These drug-particle adhesion forces were significantly reduced (p < 0.05) to values that were lower than the CH3-CH3 control measurements when tetracaine interacted with a silica nanoparticle (NanoSiO2). This reduction in adhesion was attributed to the lower surface charge of the NanoSiO2 (ca. -23 mV) compared to the NanoPSCOOH (ca. -40 mV), which diminished the electrostatic interactions between positive amine of tetracaine and the negative particle. Mixing NanoPSCOOH with tetracaine on the skin retarded percutaneous drug penetration compared to the control (tetracaine saturated solution without nanoparticles), but the NanoSiO2, which still adsorbed the tetracaine, produced a 3.6-fold enhancement in percutaneous penetration compared to the same control. These data demonstrated the capability of moderate nanoparticle surface interactions that occurred within the application vehicle to promote drug percutaneous penetration.

Understanding the molecular aggregation of therapeutic agents is particularly important when applying low doses of a drug to the surface of the skin. The aim of this study was to understand how the concentration of a drug influenced its molecular aggregation and its subsequent percutaneous penetration after topical application. A model drug tetracaine was shown to form a series of different aggregates across the μM (fluorescence spectroscopy) to mM (light scattering analysis) concentration range. The aggregate formation process was sensitive to the pH of the vehicle in which the drug was dissolved (pH 4, critical aggregation concentration (CAC) - 11 μM; pH 8, CAC - 7 μM) and it appeared to have an impact upon the drug's percutaneous penetration. At pH 4, increasing the concentration of the drug in the donor solution decreased the skin permeability coefficient (Kp) of tetracaine (13.7 ± 4.3 × 10(-3)cm/h to 0.06 ± 0.02 × 10(-3)cm/h), whilst at pH 8, it increased the Kp (29.9 ± 9.9 × 10(-3)cm/h to 75.1 ± 41.7 × 10(-3)cm/h). These data trends were reproduced in a silicone membrane and this supported the notion that the more polar aggregates formed at pH 4 acted to decrease the proportion of species available to pass through the skin, whilst the more hydrophobic aggregates formed in pH 8 increased the membrane diffusing species.

Nanoparticle-loaded topical formulations can disrupt drug aggregation through controlled drug-nanoparticle interactions to enhance topical drug delivery. However, the complex relationship between the drug, nanoparticle and formulation vehicle requires further understanding. The aim of this study was to use nanoparticle-loaded hydroxypropyl methylcellulose (HPMC) and xanthan gum gels to probe how the drug, nanoparticle and formulation vehicle interactions influenced the delivery of an aggregated drug into the skin. Tetracaine was chosen as a model drug. It was loaded into HPMC and xanthan gum gels, and it was presented to porcine skin using infinite and finite dosing protocols. Gel infinite doses showed no important differences in tetracaine skin permeation rate, but HPMC gel finite doses delivered the drug more efficiently (46.99±7.96μg/cm2/h) compared to the xanthan gum (1.16±0.14μg/cm2/h). Finite doses of the nanoparticle-loaded HPMC gel generated a 10-fold increase in drug flux (109.95±28.63μg/cm2/h) compared to the equivalent xanthan gum system (14.19±2.27μg/cm2/h). Rheology measurements suggested that the differences in the gels ability to administer the drug into the skin were not a consequence of gel-nanoparticle interactions rather, they were a consequence of the dehydration mediated diffusional restriction imparted on the drug by xanthan gum compared to the viscosity independent interactions of HPMC with the drug.

Drug self-association can hinder percutaneous penetration. The aim of this study was to investigate if disrupting drug-drug interactions using charged nanoparticle surfaces could promote delivery of a pharmaceutically active agent into the skin. Tetracaine was chosen as a model drug. It was presented to the skin as a hydroxyl methylcellulose (HPMC) gel and Ametop gel™ with and without the addition of –ve charged silica nanoparticles ( Nano SiO 2 ). When the nanoparticles were added to the topical formulations it resulted in the fastest drug permeation (109.95 ± 28.63 µg/cm 2 /h), highest accumulative mass at 45 min (76.83 ± 18.92 µg) and shortest lag time (2.02 ± 0.79 min) in porcine skin. Nano SiO 2 disrupted the tetracaine aggregates in the HPMC gel whilst not interacting with the polymer to increase the formulation macroviscosity. When Nano SiO 2 were added to the commercial Ametop gel they also enhanced penetration through the skin. Despite the very different composition of the Ametop system compared to the HPMC the nanoparticles appeared to function in a very similar manner, i.e., they broke up the tetracaine aggregates without interacting the formulation. The similar results for both the gel systems suggested that, provided the drug-nanoparticle interactions were optimised to facilitate drug aggregate break up, the addition of solid nanomaterials to semi-solid topical preparations could be a novel penetration enhancement method in a number of different types of skin product. The transport studies were carried out using upright individually calibrated Franz diffusion cells with an average of 2.1 ± 0.1 cm 2 surface areas and 9.2 ± 0.5 mL receptor compartment volume. The porcine skin was cut, mounted and sealed with parafilm between two chambers of the glass diffusion cell with a 13 mm magnetic flea in the receiver chamber. The cell was inverted and filled with previously filtered and sonicated receiver fluid. Phosphate buffered saline (pH 7.4) was employed as a receiver fluid for the porcine epidermis transport studies to mimic the skin environment. The transport studies were performed on a submergible magnetic stirrer plate in a pre-heated water bath set at 37°C to provide a membrane surface temperature of 32°C. After cell equilibration for 1 h, the cells were checked for leaks by inversion and visual inspection for back diffusion. The tetracaine test systems were prepared and adjusted to 8.0 using hydrochloric acid and equilibrated at 32°C unless stated otherwise. Solutions were stirred for at least 24 h and the pH rechecked prior to analysis to ensure they were at equilibrium. The vehicle containing the nanoparticles was corrected to the necessary pH using hydrochloric acid prior to addition to the tetracaine solutions. The infinite dosing studies used 1 mL of tetracaine formulations, which were applied uniformly to the surface of the test membrane and the donor compartment was covered with a parafilm to minimise donor phase evaporation. In the infinite studies, a saturated tetracaine solution of pH 8 was compared to a HPMC formulation (developed in the study) and the commercial preparation Ametop. None of the systems contained nanoparticles as the objective was to understand the drug-vehicle interactions. The finite dosing tested the addition of nanoparticles on the formulations ability to deliver tetracaine in to the skin. The nanoparticles were a 50 mg/mL