SMARTEN - Mechanical Properties of Soft Materials at the NanoScale
programme INTER-EXCELLENCE, sub-programme INTER-COST
The aim of the project "Mechanical properties of soft materials at the nanoscale" is to contribute to the standardization of the measurement of mechanical properties of soft samples at the nanoscale, an area that has a huge potential for advances in the so-called bio-disciplines, with potential overlap to medicine, but also to other technologies. The project is proposed under the COST MecaNano program (European Network for the Mechanics of Matter at the Nano-Scale,CA21121). Soft materials, i.e., materials exhibiting Young's modulus of elasticity typically below 1 MPa, such as biopolymers, gels, and biological tissues, have unique mechanical properties that differ significantly from their hard material counterparts. Understanding these properties can open up new possibilities in a variety of fields, including biomedical engineering, materials science, and nanotechnology.
The main focus of the project is to standardize measurement procedures using an atomic force microscope (AFM) and a nanoindenter designed for sensitive indentation of soft samples. The reference methods will be optical spectroscopy, turbidimetry, Raman and fluorescence microscopy. Reference samples based on defined phospholipid bilayers, biomolecules, hydrogels, and tissue cultures will be prepared to standardize the measurements. The performance of the standardized methods and reference methods will be verified in intra- and inter-laboratory comparison tests to determine validation parameters
In accordance with the COSTMecaNano Terms of Reference, we will be actively involved in collaboration, education, dissemination of openly available data and methodologies (Open/Fair Data), discussion of priorities and appropriate procedures for biomechanical studies. From a gender perspective, we consider it important to increase the proportion of female researchers in nanomechanical science through career development. From a technological point of view, the project should contribute to the improvement and standardisation of nanomechanical testing methods through interaction with members of the COSTMecaNano consortium, as well as other experts and or instrument manufacturers. The result should then be more efficient methods for characterising the mechanical properties of complex materials and understanding their properties at the nanoscale, but also their relation to the molecular nature of certain processes, with overlap to biological and biochemical sciences, where such characterisation can lead to understanding the links between physiological or disease states and changes in mechanical properties at the molecular, cellular or tissue level.
Stage 1 - Standard method for characterization of mechanical properties of materials
1.9.2024 - 31.8.2025
Use of nanoindentation devices (nanoindentor, AFM) and correlative microscopy techniques (combined with light and Raman microscopy) in the research, optimisation and standardisation of measurement procedures for nanomechanical studies. In the field of mechanical analysis of soft and composite materials, there is essentially no standardised measurement procedure covering materials with stiffness below 1 MPa or their combination with stiffer materials. Therefore, we consider it essential to better characterize and standardize these methods if we want to promote further research and dissemination of these methods. The choice of measurement samples (e.g. spherical indenters) as well as all measurement parameters need to be standardised. It is also important to find a consensus on the processing of the measurement data, i.e. the so-called force-distance (FDC) curves. There are a large number of mathematical models that try to cover measurements on different kinds of materials. The close collaboration within the COST MecaNano project is an ideal opportunity to discuss, find and then independently test, compare and disseminate such solutions to colleagues and other users.
- Standardization of the AFM measurement procedure - full indentation approaches, hybrid modes (off-resonance tapping): influence of measurement parameters, geometry of the indenter (tip, ball), influence of the environment.
- Standardization of the measurement procedure on nanoindenter: influence of measurement parameters, indenter geometry (tip, bead), influence of environment.
- Standardization of complementary methods for characterization of standard samples: absorbance, turbidimetry, confocal microscopy, Raman microscopy.
- Standardization of data processing: selection of suitable software for processing of measured FD curves (instrument manufacturer's software, Gwyddion, AtomicJ, etc.), selection of mathematical models suitable for robust and repeatable processing of experimental data (Hertz, Sneddon, Maugis, etc.).
Stage 2 - Preparation and characterization of standard samples for nanomechanical studies
1.9.2024 - 31.8.2025
The motivation in this step is to prepare well-characterised samples suitable for calibration of nanoindentation measurements and also suitable for analysis by reference methods (spectroscopy, turbidimetry, fluorescence, Raman microscopy). Such samples should also be prepared in other laboratories (collaborative and comparative studies), and reference methods should help to monitor their temporal stability and reproducibility of preparation. In the field of mechanical analysis of soft and hydrated materials, there is essentially no well-defined calibration material that covers the Young's modulus range below 1 MPa. Therefore, we consider it essential, if we want to promote further research and dissemination of these methods, to prepare such a material and well validate its properties and stability.The prepared standardisation samples will be further tested in the framework of the COST MecaNano consortium, where the project partners participate in inter-laboratory comparison tests. The following types of calibration samples will be prepared to standardise the measurements at different levels of sample sizes and stiffnesses:
- Phospholipid bilayers (PLB): for the study of biological membranes in-vitro, it is good to have a standardized bilayer model based on a stable structure. The chemical composition of the PCPG(phosphocoline:phosphoglycerol) membrane will be used, providing a stable and homogeneous structure. Stability can also be studied by a reference method - confocal fluorescence microscopy - using specific staining with PlasMem BrightGreen. This will result in a standardized preparation of a bilayer with defined thickness and Young's modulus values.-
- Biomolecules: the advantage of AFM is the ability to observe dynamic processes at the level of biomolecules, including changes in their mechanical properties. For calibration at this level, collagen samples will be used, which will be polymerised in-situ in an acidic environment from precursors. D-band relief will be used here for size calibration, while mathematical analysis of measured curves with a well-defined indenter will be used primarily for stiffness calibration. Similarly, a plasmid DNA sample will be used which can be well isolated and is clearly defined by its composition. The known width of the DNA strand and the geometry of the small and large groove in its structure can be used to calibrate the size of the molecules. Liquid chromatography-agel electrophoresis can be used as reference methods to verify the composition of the sample used and its quality.
- Hydrogels: important for growing cells, 3D printing cells, targeted drug delivery and enhancing biocompatibility of implants. Characterization of their mechanical properties is essential for their perception by the cells they interact with. This section will use chemically cross-linked hyaluronan, polyacrylamide-based hydrogels (PAGE), but also inorganic hydrogels based on a single component, such as nanostructured sheets of aluminum hydroxide, which are highly homogeneous and eliminate the problem of cross-linker-induced heterogeneity. The reference methods here are nanoindentation and rheology, which characterize the material in larger units.
- Live cells, cell clusters and tissue sections: the standardisation of measurements on sections and cell clusters can be approached in principle as heterogeneous hydrogels. Therefore, for this sub-region, standardisation by hydrogels will also be applied, see previous section. However, single and isolated cells are inherently more complex systems to standardize. For one, it is a relatively heterogeneous system by nature - soft heterogeneous single cells growing on a rigid substrate. Add to this the fact that they grow on a rigid substrate, typically plastic or glass. Their influence is felt at the periphery. To do this, the distinctness of cells growing in different generations, called passages, must be eliminated. All this will be subject to standardisation work. As a reference method, we will use indentor characterization and monitoring of cellular compartment composition by confocal staining of the cytoskeleton, nucleus, and extracellular matrix.
Stage 3 - Carrying out comparative tests
1.3.2025 - 1.3.2026
Within this stage, comparative tests are to be carried out, both intra- and inter-laboratory. Thus, in the third stage, we will test the reproducibility and robustness of the combination of the two components of nanomechanical studies, i.e. the methodological approach and the preparation of standardised and calibration samples. This testing will first be done intra-laboratory, i.e. testing robustness and stability over time, user influence, etc. This will be followed by testing in the form of round-robin inter-laboratory tests between the participants of the COST MecaNano consortium. A good comparative approach may be simulations of the measurements in question, for which we have arranged with Dr. Mathesan's group within the consortium. The comparative tests will be divided into the following main phases:
- In-laboratory benchmarking - i.e. validation of methods and preparation of standard samples (robustness, precision, accuracy, repeatability, reproducibility) within a single laboratory.
- Inter-laboratory comparative tests including ring tests, analogous to the description in the first point.
- Comparative studies of experimental measurements vs. simulated data, within the COST MecaNano consortium.
Stage 4 - Application of methods within the COST consortium (MecaNano)
1.8.2025 - 31.8.2026
Nanoindentation and reference methods, including standardised samples, will be prepared, optimised and validated in intra- and inter-laboratory activities, including data simulation. In the preparatory phase of this project, two groups of sites have been formed - one of which intends to focus more on standardisation work as described in the Stage 3 plan. The other group of scientists is more interested in the application of these methods to the characterisation of samples that these groups are preparing or applying in their research. These are mostly liposome complexes, nanoparticles, hydrogels and cells in tissue culture. Of course, here too we need to remember to share ideas and education, see the description of the next stage.
Stage 5 - Educational and networking events, open data publication
1.1.2025 - 31.8.2026
An important part of the COST MecaNano projects should be events where further work on the project will be consulted, problems encountered will be solved, but also education of participants and dissemination of knowledge and know-how gained in the field of nanomechanical studies. The organisation of these events will be guided by the needs of the collaborating sites and by the progress of the project - once certain stages have been completed, an educational campaign will be launched. The COST MecaNano project's Terms of Reference place a strong emphasis on events of this type at various times (symposia, workshops, participation in conferences, networking events), and we concur. This is an essential condition for the successful development and, most importantly, standardization and dissemination of nanomechanical testing methods. The COST MecaNano consortium bodies designated for this purpose, i.e. SFC and TSOC, will be consulted on the progress of this phase.
Outcomes: workshops, symposia, networking events
An important part of the COST MecaNano project is the "barrier-free" publication of data and measurement procedures for nanomechanical studies (Open/Fair Data). Closely following on from Stages 1-3, we will not only publish data and procedures in an impact journal, but also actively participate in the creation of the COST MecaNano project website, which will be built for this purpose. In order to effectively disseminate the findings from Stages 1-3 (i.e. standardisation of methods, sample preparation and also validation methods) not only to the consortium members but also to the wider scientific community, they need to be made freely available and also popularised in some way. This will be done preferably by presenting them at smaller, highly specialised workshops and symposia that will be organised by the project, but also at large conferences where we will try to convince scientists outside the field of nanomechanics of the usefulness and suitability of nanomechanical mapping. For researchers in the biological and biochemical fields, examples of the use of this technology may be a good argument to incorporate these methods into the portfolio of methods they will use as standard. The free sharing of data and workflows, including procedures for their evaluation and wider implementation in ongoing projects, will certainly contribute to this.
Outcomes: publication of data and procedures under the OPEN/FAIR data policy on the project website and on our department's website.
