Quantitative Nanomechanical Atomic Force Microscopy Research Articles

Nanomechanical mapping of rejuvenated asphalt binders

To recycle reclaimed asphalt pavement (RAP) derived from porous asphalt (PA) surfacing into a new PA mix, a fundamental understanding of RAP binder properties is needed. In this paper, properties of two PA RAP binders, with and without rejuvenation, were evaluated using quantitative nanomechanical atomic force microscopy (QNM AFM) and performance grading (PG) testing. A model that describes the effect of a bio-rejuvenator on RAP microstructure was developed. A strong correlation was found between the properties measured at nanoscale and macroscale, indicating that the macro properties of RAP binders are driven by the level of polar associations and intermolecular interactions in the bitumen microstructure. This indicates that the microstructures observed in AFM are representative of the bulk properties. Addition of bio-rejuvenator in RAP resulted in the dispersion of polar associations. Finally, it was found that one percent of the rejuvenator can reduce PG critical temperatures by approximately 2 degrees.

Nanomechanical mapping reveals localized stiffening of the basilar membrane after cochlear implantation

Cochlear implantation leads to many structural changes within the cochlea which can impair residual hearing. In patients with preserved low-frequency hearing, a delayed hearing loss can occur weeks-to-years post-implantation. We explore whether stiffening of the basilar membrane (BM) may be a contributory factor in an animal model. Our objective is to map changes in morphology and Young’s modulus of basal and apical areas of the BM after cochlear implantation, using quantitative nanomechanical atomic force microscopy (QNM-AFM) after cochlear implant surgery. Cochlear implantation was undertaken in the guinea pig, and the BM was harvested at four time-points: 1 day, 14 days, 28 days and 84 days post-implantation for QNM-AFM analysis. Auditory brainstem response thresholds were determined prior to implantation and termination. BM tissue showed altered morphology and a progressive increase in Young’s modulus, mainly in the apex, over time after implantation. BM tissue from the cochlear base demonstrated areas of extreme stiffness which are likely due to micro-calcification on the BM. In conclusion, stiffening of the BM after cochlear implantation occurs over time, even at sites far apical to a cochlear implant.

Membrane-Disrupting Nanofibrous Peptide Hydrogels.

Self-assembled peptide nanofibers can form biomimetic hydrogels at physiological pH and ionic strength through noncovalent and reversible interactions. Inspired by natural antimicrobial peptides, we designed a class of cationic amphiphilic self-assembled peptides (CASPs) that self-assemble into thixotropic nanofibrous hydrogels. These constructs employ amphiphilicity and high terminal charge density to disrupt bacterial membranes. Here, we focus on three aspects of the self-assembly of these hydrogels: (a) the material properties of the individual self-assembled nanofibers, (b) emergence of bulk-scale elasticity in the nanofibrous hydrogel, and (c) trade-off between the desirable material properties and antimicrobial efficacy. The design of the supramolecular nanofibers allows for higher-order noncovalent ionic cross-linking of the nanofibers into a viscoelastic network. We determine the stiffness of the self-assembled nanofibers via the peak force quantitative nanomechanical atomic force microscopy and the bulk-scale rheometry. The storage moduli depend on peptide concentration, ionic strength, and concentration of multivalent ionic cross-linker. CASP nanofibers are demonstrated to be effective against Pseudomonas aeruginosa colonies. We use nanomechanical analysis and microsecond-time scale coarse-grained simulation to elucidate the interaction between the peptides and bacterial membranes. We demonstrate that the membranes stiffen, contract, and buckle after binding to peptide nanofibers, allowing disruption of osmotic equilibrium between the intracellular and extracellular matrix. This is further associated with dramatic changes in cell morphology. Our studies suggest that self-assembled peptide nanofibrils can potentially acts as membrane-disrupting antimicrobial agents, which can be formulated as injectable hydrogels with tunable material properties.

Spatiotemporal PFQNM visualization of the effect of suicide dendriplexes on dividing HeLa cells

Suicide gene delivery is significant in cancer therapy but has not been fully investigated on a cellular scale. Here, Peak Force Quantitative Nanomechanical atomic force microscopy (PFQNM-AFM) was applied to visualize the effect of herpes simplex virus thymidine kinase dendriplexes (G4AcFaHSTK) on the morphological and nanomechanical properties of individual live and dividing HeLa cells. Cells were then exposed to G4AcFaHSTK, followed by ganciclovir, and directly imaged by real-time PFQNM-AFM. Cell membrane liquefaction, cytoplasmic shrinkage, and cytoskeleton structure loss were observed during cell division. The average Young's modulus of the nuclear region increased with time as the cell continued from metaphase (6.29 kPa) to telophase (13.6 kPa) and then decreased (2.25 kPa) upon apoptosis. In contrast, cells exposed to either ganciclovir or G4AcFaHSTK alone have no changes. Thus, understanding the real-time effects of suicide dendriplexes on the cytoskeletal and nanomechanical behaviors of cancer cells may provide new methods for cancer treatment.

Atomic Force Microscopy of Triple Negative Breast Cancer Cells: A Predictive Value of Mechanical Phenotype

We applied Quantitative Nanomechanical Atomic Force Microscopy (QNM-AFM) to test a response of physical phenotype of triple negative breast cancer cells to treatment with proteasome inhibitors. Genetic screens identified TNBCs as addicted to the activity of the ubiquitin-proteasome pathway, the major intracellular venue of regulated protein degradation, served by the essential protease, the proteasome. We investigated two types of proteasome inhibitors: the drug bortezomib (Velcade®) targeting active sites of the enzyme and a novel allosteric inhibitor B1 interfering with stability of the 26S proteasome. Mechanical phenotype constitutes a sensitive indicator of physiological status of cells. The most explored mechanical parameters are cell elasticity (“softness”) and surface adhesiveness (“stickiness”). It has been established that cancerous transformation makes cells softer and less sticky. These differences are attributed to remodeling of the cytoskeleton and altered expression of membrane proteins. Therefore, the physical phenotype should have a predictive value as the early indicator of the cells’ response to a drug treatment and disease stage. Indeed, it has been shown that exposure of cancer cells to anticancer drugs at least partially reverses their mechanical phenotype resembling healthy cells. Here we found that subjecting breast cancer cells to low doses of bortezomib increased their stiffness and adhesiveness about two times. Moreover, similar treatment with low doses of B1 induced almost a threefold increase of stiffness and a twofold increase in cell adhesion. Surprisingly, much higher doses of the drugs were required to inflict detectable changes in cells viability or morphology. The results point at the extraordinary sensitivity of the mechanical phenotype to detect cells’ response to anticancer drugs. We are exploring the potential predictive value of AFM-based cell surface studies in a search for effective drugs or drug combinations.

Amyloid-like fibrils formed from intrinsically disordered caseins: physicochemical and nanomechanical properties.

Amyloid-like fibrils are studied because of their significance in understanding pathogenesis and creating functional materials. Amyloid-like fibrils have been studied by heating globular proteins at acidic conditions. In the present study, intrinsically disordered α-, β-, and κ-caseins were studied to form amyloid-like fibrils at pH 2.0 and 90 °C. No fibrils were observed for α-caseins, and acid hydrolysis was found to be the rate-limiting step of fibrillation of β- and κ-caseins. An increase of β-sheet structure was observed after fibrillation. Nanomechanic analysis of long amyloid-like fibrils using peak-force quantitative nanomechanical atomic force microscopy showed the lowest and highest Young's modulus for β-casein (2.35 ± 0.29 GPa) and κ-casein (4.14 ± 0.66 GPa), respectively. The dispersion with β-casein fibrils had a viscosity more than 10 and 5 times higher than those of κ-casein and β-lactoglobulin, respectively, at 0.1 s(-1) at comparable concentrations. The current findings may assist not only the understanding of amyloid fibril formation but also the development of novel functional materials from disordered proteins.

Study Sub-Membrane Structure and Corresponding Functions of Conductive Bacteria Cable by SPMs

As a kind of surface characterization tool, traditional scanning probe microscopy (SPM), especially atomic force microscopy (AFM), is hard to explore the structures underneath sample surface and correlate them with the corresponding functions. With the development of advanced AFMs, this obstacle has been overcome gradually, for example, with the application of quantitative dynamic AFM mapping, the hollow helical amyloid self-assembly fibrils have been identified;[1] and the protein structure flexibility on inner/outer sides of membrane is also possible to determine[2]. However, distinguishing the sub-surface features and corresponding function of macro-size biological samples, such as bacteria and cells, is still challenging to SPM. In this presentation, we combined quantitative dynamic AFM, AFM based manipulation, electrostatics force microscopy and scanning ion conductance microscopy to illustrate the inside membrane feature of recently identified conductive bacteria cable[3]. Basing on the SPM results, we proposed the model to explain the reason of the bacterial function to transport electrons over centimeter distance along cable direction. On one hand, this work will help biologists to understand the bacterial cable and promote the future application in nano-conductive cable field; on the other hand, it will inspire the further applications of SPM to beyond surface limitation on macro-size bio-systems.1. Zhang, S., et al., Coexistence of ribbon and helical fibrils originating from hIAPP20-29 revealed by quantitative nanomechanical atomic force microscopy. PNAS, 2013.2. Dong, M.D., S. Husale, and O. Sahin, Determination of protein structural flexibility by microsecond force spectroscopy. Nat. Nano., 2009. 4(8): p. 514-517.3. Pfeffer, C., et al., Filamentous bacteria transport electrons over centimetre distances. Nature, 2012. 491(7432).

Coexistence of ribbon and helical fibrils originating from hIAPP 20–29 revealed by quantitative nanomechanical atomic force microscopy

Uncontrolled misfolding of proteins leading to the formation of amyloid deposits is associated with more than 40 types of diseases, such as neurodegenerative diseases and type-2 diabetes. These irreversible amyloid fibrils typically assemble in distinct stages. Transitions among the various intermediate stages are the subject of many studies but are not yet fully elucidated. Here, we combine high-resolution atomic force microscopy and quantitative nanomechanical mapping to determine the self-assembled structures of the decapeptide hIAPP(20-29), which is considered to be the fibrillating core fragment of the human islet amyloid polypeptide (hIAPP) involved in type-2 diabetes. We successfully follow the evolution of hIAPP(20-29) nanostructures over time, calculate the average thickening speed of small ribbon-like structures, and provide evidence of the coexistence of ribbon and helical fibrils, highlighting a key step within the self-assembly model. In addition, the mutations of individual side chains of wide-type hIAPP(20-29) shift this balance and destabilize the helical fibrils sufficiently relative to the twisted ribbons to lead to their complete elimination. We combine atomic force microscopy structures, mechanical properties, and solid-state NMR structural information to build a molecular model containing β sheets in cross-β motifs as the basis of self-assembled amyloids.

A Novel Inspection for Deformation Phenomenon of Reduced-graphene Oxide via Quantitative Nano-mechanical Atomic Force Microscopy

The deformation and stacking of graphene significantly affect the overall electronic and mechanical properties. The graphene sheets are easily stacked each other due to van der Waals attraction. The folded characteristic may prevent graphene stacking and increase the d-spacing between graphenes. It can easily form dense graphene structures with high surface area and be applied on electrode materials of battery, nano-composites and so on. The traditional topographic mapping of atomic force microscope (AFM) is hard to measure the characters of surface deformation and distinguish the folding and stacking. In this study, we apply a novel quantitative nano-mechanical AFM to analyze the reduced-graphene oxide (r-GO). The results indicate that the novel AFM system could recognize the difference between folding and stacking of r-GO effectively. Nevertheless, the images including topography, deformation, adhesion, and elastic modulus show the different phenomena between measuring region, which appears that status on measuring region are folding and stacking, respectively.

Single-step direct measurement of amyloid fibrils stiffness by peak force quantitative nanomechanical atomic force microscopy

We present an original application of a new atomic force microscopy mode called peak force tapping for the investigation of the mechanical properties of β-lactoglobulin amyloid fibrils. The values of Young’s modulus obtained by this technique are in perfect agreement with the indirect evaluation of fibrils stiffness obtained by combining polymer physics and topological statistical analysis on fibrils’ structural conformations. This technique shows great promise in the estimation of the elastic properties of nanostructured objects relevant in biology, soft matter, and nanotechnology.