Nanomechanical Measurements Research Articles

Utilizing collagen-coated hydrogels with defined stiffness as calibration standards for AFM experiments on soft biological materials: the case of lung cells and tissue

Abstract Atomic Force Microscopy (AFM) is crucial in mechanobiology for high-resolution imaging and nanomechanical measurements of biological samples, providing insights into their mechanical properties. However, AFM faces challenges such as tip damage and cantilever selection errors, impacting measurement accuracy. This study proposes a methodology using collagen-coated hydrogels with predefined stiffness for calibrating AFM measurements on soft biological materials. By facilitating appropriate cantilever selection, assessing systematic errors, and evaluating tip damage, this approach ensures reliable Young’s modulus measurements. The proof of concept with human lung cells and tissue specimens demonstrates improved accuracy and reliability of AFM-based nanomechanical characterizations, essential for understanding cellular mechanics and disease progression.

Development of Halloysite Nanohybrids-Based Films: Enhancing Mechanical and Hydrophilic Properties for Wound Healing.

Most of the therapeutic systems developed for managing chronic skin wounds lack adequate mechanical and hydration properties, primarily because they rely on a single component. This study addresses this issue by combining organic and inorganic materials to obtain hybrid films with enhanced mechanical behavior, adhesion, and fluid absorption properties. To that aim, chitosan/hydrolyzed collagen blends were mixed with halloysite/antimicrobial nanohybrids at 10% and 20% (w/w) using glycerin or glycerin/polyethylene glycol-1500 as plasticizers. The films were characterized through the use of Fourier-transform infrared (FTIR) spectroscopy, thermogravimetric analysis (TGA), and electron microscopy. The mechanical properties were evaluated macroscopically using tensile tests, and at a nanoscale through atomic force microscopy (AFM) and nanoindentation. Thermodynamic studies were conducted to assess their hydrophilic or hydrophobic character. Additionally, in vitro cytocompatibility tests were performed on human keratinocytes. Results from FTIR, TGA, AFM and electron microscopy confirmed the hybrid nature of the films. Both tensile tests and nanomechanical measurements postulated that the nanohybrids improved the films' toughness and adhesion and optimized the nanoindentation properties. All nanohybrid-loaded films were hydrophilic and non-cytotoxic, showcasing their potential for skin wound applications given their enhanced performance at the macro- and nanoscale.

Automated High-Throughput Atomic Force Microscopy Single-Cell Nanomechanical Assay Enabled by Deep Learning-Based Optical Image Recognition.

Mechanical forces are essential for life activities, and the mechanical phenotypes of single cells are increasingly gaining attention. Atomic force microscopy (AFM) has been a standard method for single-cell nanomechanical assays, but its efficiency is limited due to its reliance on manual operation. Here, we present a study of deep learning image recognition-assisted AFM that enables automated high-throughput single-cell nanomechanical measurements. On the basis of the label-free identification of the cell structures and the AFM probe in optical bright-field images as well as the consequent automated movement of the sample stage and AFM probe, the AFM probe tip could be accurately and sequentially moved onto the specific parts of individual living cells to perform a single-cell indentation assay or single-cell force spectroscopy in a time-efficient manner. The study illustrates a promising method based on deep learning for achieving operator-independent high-throughput AFM single-cell nanomechanics, which will benefit the application of AFM in mechanobiology.

Nanoscopic gel particle for intra-articular injection formulation

Hyaluronic acid (HA) based nanogels showed effective intracellular delivery efficacy for anti-cancer and anti-inflammatory drugs, characterized by their ability targeting relevant cell receptors. In the present study, we demonstrate the ability of hyaluronic acid-polyethyleneimine (HA-PEI) nanogels as a promising dual-functional interfacial active for intra-articular injection to intervene arthritis. Nanomechanical measurements on both model substrates and human cartilage samples confirm that the HA-PEI nanogels can significantly improve interfacial lubrication, in comparison to HA molecules, or silica-based nanoparticles. We show that the Coefficient of Friction significantly decreases with a decreasing nanogel size. The exceptional lubricating performance, coupled with the proven drug delivery capability, evidences the great potential of nanoscopic hydrogels for early-stage arthritis treatment. The flexibility in choosing the chemical nature, molecular architecture, and structural characteristics of nanogels makes it possible to modulate both drug delivery kinetics and interfacial lubrication, thus representing an innovative approach to treat degenerative joint diseases.

A nanonewton force sensor using a U-shape tapered microfiber interferometer.

Nanomechanical measurements, especially the detection of weak contact forces, play a vital role in many fields, such as material science, micromanipulation, and mechanobiology. However, it remains a challenging task to realize the measurement of ultraweak force levels as low as nanonewtons with a simple sensing configuration. In this work, an ultrasensitive all-fiber nanonewton force sensor structure based on a single-mode-tapered U-shape multimode-single-mode fiber probe is proposed and experimentally demonstrated with a limit of detection of ~5.4 nanonewtons. The use of the sensor is demonstrated by force measurement on a human hair sample to determine the spring constant of the hair. The results agree well with measurements using an atomic force microscope for the spring constant of the hair. Compared with other force sensors based on optical fiber in the literature, the proposed all-fiber force sensor provides a substantial advancement in the minimum detectable force possible, with the advantages of a simple configuration, ease of fabrication, and low cost.

Chemical bonds and hair behaviour-A review.

When undertaking any review of the structure of the hair and its mechanical properties it becomes apparent that the overall behaviour of keratin fibres is commonly attributed to the presence of hydrogen, disulfide and ionic bonds. The action of physico-chemical agents used during various cosmetic treatments is viewed as the result of an interaction with these bonds. Thus, the breaking of bonds by chemical agents, or via mechanical or thermal stresses, affects the relative balance of disulfide and hydrogen bonds and the contribution of hydrophobic interactions, which are all important factors that may alter hair behaviour. Generally, these chemical bonds are considered as responding homogeneously to the environmental and cosmetic factors. This unitary image is challenged, however, by evaluating the results of chemical, nanomechanical, tensile and thermal measurements, which suggest that disulfide bonds may be grouped into several types, according to their location within the fibre and the way they respond to various agents. A compensatory effect of newly formed hydrogen bonds for broken disulfide bonds may also be seen, and additionally involves different types of hydrogen bonds. As a result, the picture of chemical bonding in hair appears to be far from a homogeneous one. In addition, it is apparent that further investigation is required for clarifying the action of ionic bonds and hydrophobic interactions within the hair fibre. The present review aims, thus, at offering a deeper background for understanding how the hair behaves under various conditions.

Abstract CT273: Unique multicenter trial design to investigate ARTIDIS nanomechanical generated measurements for early breast lesions (ANGEL)

Abstract Background: The conventional diagnosis of breast cancer followed by molecular subtyping for therapy selection is a lengthy process and associated with patient and clinician anxiety. Efficient, bedside approaches are desirable to drive fast diagnosis and early planning of clinical management. This clinical trial (NCT06085833) tackles the novel concept of automated and reliable tissue diagnostics (ARTIDIS) based on the measurement of nanomechanical signature of fresh clinical biopsies from patients with breast lesions. This study is based on our previous single center clinical study (Basel NANO) where tissue nanomechanical signature can: 1) reliably distinguish benign and malignant breast lesions, 2) predict the presence of metastatic disease based on imminent aggressiveness rating and 3) predict the response to neoadjuvant chemotherapy from baseline biopsies. The current study aims to prospectively validate the efficacy of ARTIDIS system as an aid to diagnosis, assessment of breast cancer prognosis and prediction of therapy response, particularly in the context of neoadjuvant therapy (Natira). Methods: A cohort of 2,705 patients with breast lesions, who are clinically indicated for core needle biopsy (CNB) are being enrolled, from diverse ethnic and socioeconomic backgrounds. Single fresh CNB will be transferred into tissue preservation buffer and measured by ARTIDIS device within 1-3 hours to collect thousands of nanomechanical measurements. Undisrupted CNB will be fixed and returned to standard diagnostic workflow, remaining unaltered for downstream assessments. Nanomechanical measurement data are prospectively analyzed in a blind manner before the collection of histopathology and follow up clinical data. The primary endpoint of the study is to validate ARTIDIS potential in diagnosis of breast lesions (benign vs malignant), secondary endpoints address whether nanomechanical signature can differentiate subtypes of breast cancer, associate with clinical outcome and guide treatment decisions in NAT setting. Follow up data will be collected regularly up to 10 years after patient enrollment. Results: The multicenter ANGEL study is a prospective validation of the NANO (University Hospital Basel, Switzerland) - first ever performed clinical study which investigated the diagnostic, prognostic, and predictive value of tissue nanomechanical signature in patients with breast cancer. The ANGEL study takes advantage of ARTIDIS non-disruptive technology and is fully integrated into routine clinical workflow. The study started enrollment in 11/02/2023, and is expected to complete by 2025. The seamless integration of ARTIDIS within the standard clinical settings, its automatization, bedside implementation, and the lack of additional burden to patients enables effective reach of a diverse population through local hospitals and communities. Conclusion: This study presents the first of its kind as a direct prospective, multicenter- based translation of the tissue nanomechanical signature into clinical settings. This unique trial design allows the prospective development of ARTIDIS as an aid to diagnosis, prognosis and therapy optimization in patients with breast cancer. The innovative trial design of this study may also further the development of strategies to increase reach to underrepresented communities. Citation Format: Karla A. Sepulveda, Ashley Roark, Sagar Dhamne, Chandandeep Nagi, Ivan Marin, Iqbal Tabish, Sara Nizzero, Mariam Gachechiladze, Tobias Appenzeller, Philipp Oertle, Marko Loparic, Susan G. Hilsenbeck, Marija Plodinec, Alastair Thompson. Unique multicenter trial design to investigate ARTIDIS nanomechanical generated measurements for early breast lesions (ANGEL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT273.

Nanomechanical and nano-FTIR analysis of polyester coil coatings before and after artificial weathering experiments

Local heterogeneities can have significant effects on the performance of anti-corrosion coatings. Even small features can act as initiation points for damage and result in corrosion of the substrate material. Analysis methods with high spatial resolution and the ability to collect information relevant to crosslinking and degradation behavior of these coatings are therefore highly relevant. In this work, we demonstrate the utility of nanomechanical AFM measurements and nano-FTIR in investigating the nanoscale mechanical and chemical properties of two polyester coil coating clearcoats before and after weathering. On the nanoscale, weathering led to a stiffer and less deformable coating with less variation in the nanomechanical properties. Chemical degradation was quantified using changes in band ratios in the IR-spectra. Macro and nano-scale measurements showed similar trends with the latter measurements showing larger heterogeneity. Our results demonstrate the usefulness of the described analysis techniques and will pave the way for future studies of local properties in other coating systems and formulations.

Nanomechanical mass measurements through feature-based time series clustering

Recent years have seen explosive growth in miniaturized sensors that can continuously monitor a wide variety of processes, with applications in healthcare, manufacturing, and environmental sensing. The time series generated by these sensors often involves abrupt jumps in the detected signal. One such application uses nanoelectromechanical systems (NEMS) for mass spectrometry, where analyte adsorption produces a quick but finite-time jump in the resonance frequencies of the sensor eigenmodes. This finite-time response can lead to ambiguity in the detection of adsorption events, particularly in high event-rate mass adsorption. Here, we develop a computational algorithm that robustly eliminates this often-encountered ambiguity. A moving-window statistical test together with a feature-based clustering algorithm is proposed to automate the identification of single-event jumps. We validate the method using numerical simulations and demonstrate its application in practice using time-series data that are experimentally generated by molecules adsorbing onto NEMS sensors at a high event rate. This computational algorithm enables new applications, including high-throughput, single-molecule proteomics.

Characterization of nanoscale morphology and mechanical properties of conjugated polymer thin films dynamically exposed to a secondary solvent

Solution processing techniques are often used to enhance intra and interchain order in semiconducting conjugated polymer thin films used in the active layer of organic optoelectronic devices. While there has been extensive research into the impact of solution processing on the local structural and electronic properties of these films, the nanomechanical properties of the films are less well understood and are challenging to characterize. We investigate the nanomechanical properties of conjugated polymer thin films arising from solution processing techniques. Our study relies on dynamic secondary solvent dripping to induce subtle changes to the film morphology. We examine thin films of P3HT, PCDTBT, PTB7, and PBDB-T-SF conjugated polymers, commonly used in organic photovoltaics, with the bimodal amplitude modulation-frequency modulation (AM-FM) imaging mode of the atomic force microscopy (AFM) viscoelastic method to probe their local physical and mechanical properties. We find that Young's Modulus data measured by AM-FM AFM can detect additional changes in film properties not discernible by other commonly used bulk thin-film characterization techniques. For PBDB-T-SF, we detect an increase in molecular order that is not noticable by UV–visible absorption spectroscopy, X-ray diffraction or nanoindentation. PCDTBT, the most amorphous of the polymers studied, shows no changes in absorption or X-ray diffraction data, yet clear changes in Young's Modulus were detected by AFM. On the other hand, increases in P3HT order that occurred due to dynamic secondary solvent dripping are detected in both the bulk and AFM measurements. Our study demonstrates the applicability of nanomechanical measurements for characterizing local structural variations in conjugated polymer films. This work is relevant to ongoing efforts to control and understand the complex structure-property-processing relationships of conjugated polymer thin films.

Real-Time Monitoring of Cell Adhesion onto a Soft Substrate by a Graphene Impedance Biosensor.

Soft substrates are interesting for many applications, ranging from mimicking the cellular microenvironment to implants. Conductive electrodes on such substrates allow the realization of flexible, elastic, and transparent sensors. Single-layer graphene as a candidate for such electrodes brings the advantage that the active area of the sensor is transparent and conformal to the underlying substrate. Here, we overcome several challenges facing the routine realization of graphene cell sensors on a canonical soft substrate, namely, poly(dimethylsiloxane) (PDMS). We have systematically studied the effect of surface energy before, during, and after the transfer of graphene. Thus, we have identified a suitable support polymer, optimal substrate (pre)treatment, and an appropriate solvent for the removal of the support. Using this procedure, we can reproducibly obtain stable and intact graphene sensors on a millimeter scale on PDMS, which can withstand continuous measurements in cell culture media for several days. From local nanomechanical measurements, we infer that the softness of the substrate is slightly affected after the graphene transfer. However, we can modulate the stiffness using PDMS with differing compositions. Finally, we show that graphene sensors on PDMS can be successfully used as soft electrodes for real-time monitoring of the cell adhesion kinetics. The routine availability of single-layer graphene electrodes on a soft substrate with tunable stiffness will open a new avenue for studies, where the PDMS-liquid interface is made conducting with minimal alteration of the intrinsic material properties such as softness, flexibility, elasticity, and transparency.

Integrative microscopy to explore physical and nanomechanical properties of eggshells of diapausing embryos in Rotifera: a proof-of-concept study

ABSTRACTDiapausing embryos of invertebrates represent investments in future populations. Thus, they must be capable of withstanding a variety of environmental assaults. Consequently, their eggshells should be adapted to resist injuries from predators, sediments, or excessive shrinkage if desiccated. To date, there have been no direct nanomechanical measurements of the eggshells of most invertebrates that produce diapausing eggs. Here, we used three approaches to understand how the eggshells of two rotifers, a freshwater species (Brachionus calyciflorus) and a brackish-water species (B. plicatilis), tolerate harsh conditions: (1) atomic force microscopy to measure elasticity and hardness; (2) transmission electron microscopy to study ultrastructure; (3) scanning electron microscopy to examine surface features. We compared these values to measurements of brine shrimp (Artemia salina) cysts and mosquito (Aedes aegypti) overwintering eggs. Our results revealed that rotifer eggshells are structurally similar and have comparable nanomechanical values. While rotifer eggshells had lower Young’s moduli (ca 13–16 MPa) and hardness values (1.84–1.85 × 10−2 GPa) than eggshells of Artemia and Aedes, eggshells of all species were relatively elastic and not particularly resistant to deformation. Pliancy of shells that form egg banks (ie Artemia, Brachionus) may be an adaptation to resist cracking under the physical forces associated with burial in sediments. Although there are no obvious relationships among eggshell thickness, ultrastructure, ornamentation, or nanomechanical values in rotifer eggshells, we hypothesise that eggshell composition may play an important role in determining elasticity and hardness. Future studies should consider an integrative approach to understand the importance of eggshell structure, chemistry, and mechanics in protecting diapausing embryos.

Impact of the sizing reactivity of glass fibers on composites hydrothermal aging

Epoxy composites were prepared with reactive and non-reactive sizings deposited on glass fibers. The resulting interphase properties have been studied during hydrothermal aging. The purpose here is to evaluate at nano and macroscale the dependence of aging, interfacial strength, and mechanical properties of model composites on the glass sizing reactivity. SEM/AFM, DMA and 3 points bending experiments have been done before and after water immersion at 70 °C for several weeks. Water diffusion kinetics have been found to be related only to the presence of sized glass fibers but not to the sizing reactivity. The SEM observations demonstrated a cohesive fracture at matrix/fiber in the case of composite elaborated with reactive sizing and an adhesive one in the case of non-reactive sizing, and this remains true with aging. Depending on the sizing nature and on the aging time, the evolution of interphase zones around fibers has been identified by a specific α-relaxation (Tan δ) and confirmed by AFM nanomechanical measurements. The results showed that the interphase thickness was almost the same at initial state (around 200 nm) for both composites, then it progresses with aging time to reach 400 nm and 500 nm (at 42 days) for reactive and non-reactive systems respectively. It suggests that water molecules diffusion is related to the hydrophilic character of the sizing and to the degree of cross-linking/or plasticization of the interphase during hydrothermal aging.

In Situ Probing of the Intrinsic Adhesion Strength of Single Anaerobic Microbial Cells.

Probing the single-cell mechanobiology in situ is imperative for microbial processes in the medical, industrial, and agricultural realms, but it remains a challenge. Herein, we present a single-cell force microscopy method that can be used to measure microbial adhesion strength under anaerobic conditions in situ. This method integrates atomic force microscopy with an anaerobic liquid cell and inverted fluorescence microscopy. We obtained the nanomechanical measurements of the single anaerobic bacterium Ethanoligenens harbinense YUAN-3 and the methanogenic archaeon Methanosarcina acetivorans C2A and their nanoscale adhesion forces in the presence of sulfoxaflor, a successor of neonicotinoid pesticides. This study presents a new tool for in situ single-cell force measurements of various anoxic and anaerobic species and provides new perspectives for evaluating the potential environmental risk of neonicotinoid applications in ecosystems.

Investigation of Soft Matter Nanomechanics by Atomic Force Microscopy and Optical Tweezers: A Comprehensive Review

Soft matter exhibits a multitude of intrinsic physico-chemical attributes. Their mechanical properties are crucial characteristics to define their performance. In this context, the rigidity of these systems under exerted load forces is covered by the field of biomechanics. Moreover, cellular transduction processes which are involved in health and disease conditions are significantly affected by exogenous biomechanical actions. In this framework, atomic force microscopy (AFM) and optical tweezers (OT) can play an important role to determine the biomechanical parameters of the investigated systems at the single-molecule level. This review aims to fully comprehend the interplay between mechanical forces and soft matter systems. In particular, we outline the capabilities of AFM and OT compared to other classical bulk techniques to determine nanomechanical parameters such as Young's modulus. We also provide some recent examples of nanomechanical measurements performed using AFM and OT in hydrogels, biopolymers and cellular systems, among others. We expect the present manuscript will aid potential readers and stakeholders to fully understand the potential applications of AFM and OT to soft matter systems.

In-Situ EC-AFM Study of Electrochemical P-Doping of Polymeric Nickel(II) Complexes with Schiff base Ligands

Conductive electrochemically active metallopolymers are outstanding materials for energy storage and conversion, electrocatalysis, electroanalysis, and other applications. The hybrid inorganic–organic nature of these materials ensures their rich chemistry and offers wide opportunities for fine-tuning their functional properties. The electrochemical modulation of the nanomechanical properties of metallopolymers is rarely investigated, and the correlations between the structure, stiffness, and capacitive properties of these materials have not yet been reported. We use electrochemical atomic force microscopy (EC-AFM) to perform in-situ quantitative nanomechanical measurements of two Schiff base metallopolymers, poly[NiSalphen] and its derivative that contains two methoxy substituents in the bridging phenylene diimine unit poly[NiSalphen(CH3O)2], during their polarization in the electrolyte solution to the undoped and fully doped states. We also get insight into the electrochemical p-doping of these polymers using electrochemical quartz crystal microgravimetry (EQCM) coupled with cyclic voltammetry (CV). Combined findings for the structurally similar polymers with different interchain interactions led us to propose a correlation between Young’s modulus of the material, its maximum doping level, and ion and solvent fluxes in the polymer films upon electrochemical oxidation.

Micropipette-assisted atomic force microscopy for single-cell 3D manipulations and nanomechanical measurements.

Mechanical cues play a crucial role in regulating physiological and pathological processes, and atomic force microscopy (AFM) has become an important and standard tool for measuring the mechanical properties of single cells. In particular, providing a capability to manipulate cells in a three-dimensional (3D) space benefits enhancing the applications of AFM measurements in cell biology. Here, we present the complementary integration of AFM and micropipette micromanipulation, which allows precise 3D manipulations and nanomechanical measurements of single living cells. A micropipette micromanipulation system under the guidance of optical microscopy was established to isolate single living cells, and polydimethylsiloxane (PDMS) micropillar substrates were used to physically immobilize the isolated living cells for downstream AFM detection. The viscoelastic properties (Young's modulus, relaxation time, viscosity) of cells were quantitatively measured by AFM-based indentation assay. The effectiveness of micropipette-assisted AFM in single-cell analysis was confirmed on both living animal suspended cells and living animal adherent cells, showing dramatic changes in cell mechanics in different states and revealing the dynamics of single cells grown on micropillar arrays. The study demonstrates the great potential of a micropipette to aid AFM in single-cell manipulations for better accessing the mechanical cues involved in cellular processes, which will allow additional studies of single-cell mechanics and will benefit the field of mechanobiology.

Quantifying the local mechanical properties of twisted double bilayer graphene.

Nanomechanical measurements of minimally twisted van der Waals materials remained elusive despite their fundamental importance for device realisation. Here, we use Ultrasonic Force Microscopy (UFM) to locally quantify the variation of out-of-plane Young's modulus in minimally twisted double bilayer graphene (TDBG). We reveal a softening of the Young's modulus by 7% and 17% along single and double domain walls, respectively. Our experimental results are confirmed by force-field relaxation models. This study highlights the strong tunability of nanomechanical properties in engineered twisted materials, and paves the way for future applications of designer 2D nanomechanical systems.

Exceptionally strong boron nitride nanotube aluminum composite interfaces

We report the direct strength property measurements along boron nitride nanotube (BNNT) aluminum (Al) composite interface using in situ scanning electron microscopy single-nanotube pullout techniques. The nanomechanical measurements reveal that the BNNT-Al interface possesses an average interfacial shear strength of ∼46 MPa and a maximum shear load of ∼340 nN, and is over 60% stronger than the comparable carbon nanotube (CNT) -Al interface. This strong interface enables significant loading of the nanotube during pull-out from the metal matrix with a generated maximum tensile stress close to its intrinsic strength limit. Density functional theory (DFT) calculations reveal stronger interfacial physio- and chemisorption interactions on an oxidized Al interface with hexagonal boron nitride (hBN) as compared to graphene, which are in contrast to comparable binding properties of hBN and graphene with pure Al. The exceptional Al-BNNT strength properties can thus be attributed to a partially oxidized metal-nanotube binding interface, which has important implications for optimizing the local interfacial load transfer and bulk properties of BNNT-metal nanocomposites.

Frequency-dependent nanomechanical profiling for medical diagnosis.

Atomic force microscopy (AFM), developed in the early 1980s, has become a powerful characterization tool in micro- and nanoscale science. In the early 1990s, its relevance within biology and medicine research became evident, although its incorporation into healthcare applications remains relatively limited. Here, we briefly explore the reasons for this low level of technological adoption. We also propose a path forward for the incorporation of frequency-dependent nanomechanical measurements into integrated healthcare strategies that link routine AFM measurements with computer analysis, real-time communication with healthcare providers, and medical databases. This approach would be appropriate for diseases such as cancer, lupus, arteriosclerosis and arthritis, among others, which bring about significant mechanical changes in the affected tissues.