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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.

Electron paramagnetic resonance (EPR) spectroscopy is a widely used tool to detect and characterize paramagnetic species, such as some metal ions and organic radicals, in various science fields such as physics, chemistry, material science and medicine. However, conventional EPR systems only allow studying macroscopic samples due to the weak coupling between the spins and the microwave photons. The detection and identification of certain proteins and molecules in samples of biological and environmental interest is then limited, due to the low spin concentration. New alternatives are being investigated for pushing the detection limit to its minimum. Among them, using superconducting circuits as quantum sensors opens a new path for these developments. In fact, lumped element resonators (LERs), which consist of a series inductance-capacitance circuit, can act as very high efficient microwave cavities. By a correct circuit design, the microwave photon within the cavity can be localized within a microscopic volume pushing the limits of sensitivity of the EPR spectroscopy by several orders of magnitude. Moreover, frequency multiplexing and tuneability of LERs allow characterizing spin transitions in the low magnetic field regime and the simultaneous characterization of different samples in a single chip. As a proof of concept, we have developed an on-chip EPR device based on Nb superconducting LERs to sense the iron- and oxygen-binding protein metmyoglobin (Fe(III)-porphyrin) (from equine heart). We have checked the spectrometer sensitivity by depositing different amounts of these proteins on different LERs, going from microdroplets to a single molecular layer, deposited by means of dip pen nanolithography based on AFM. Low temperature spectroscopy measurements show that we can get weak coupling for all myoglobin protein samples. This sensitivity opens the path to the detection and characterization of micro-sized samples and even individual proteins. Further optimization o

Biomolecular interactions underpin most processes inside the cell. Hence, a precise and quantitative understanding of molecular association and dissociation events is crucial, not only from a fundamental perspective, but also for the rational design of biomolecular platforms for state-of-the-art biomedical and industrial applications. In this context, atomic force microscopy (AFM) appears as an invaluable experimental technique, allowing the measurement of the mechanical strength of biomolecular complexes to provide a quantitative characterization of their interaction properties from a single molecule perspective. In the present review, the most recent methodological advances in this field are presented with special focus on bioconjugation, immobilization and AFM tip functionalization, dynamic force spectroscopy measurements, molecular recognition imaging and theoretical modeling. We expect this work to significantly aid in grasping the principles of AFM-based force spectroscopy (AFM-FS) technique and provide the necessary tools to acquaint the type of data that can be achieved from this type of experiments. Furthermore, a critical assessment is done with other nanotechnology techniques to better visualize the future prospects of AFM-FS.

Innovative materials are needed to produce scaffolds for various tissue engineering and regenerative medicine (TERM) applications, including tissue models. Materials derived from natural sources that offer low production costs, easy availability, and high bioactivity are highly preferred. Chicken egg white (EW) is an overlooked protein-based material. Whilst its combination with the biopolymer gelatin has been investigated in the food technology industry, mixed hydrocolloids of EW and gelatin have not been reported in TERM. This paper investigates these hydrocolloids as a suitable platform for hydrogel-based tissue engineering, including 2D coating films, miniaturized 3D hydrogels in microfluidic devices, and 3D hydrogel scaffolds. Rheological assessment of the hydrocolloid solutions suggested that temperature and EW concentration can be used to fine-tune the viscosity of the ensuing gels. Fabricated thin 2D hydrocolloid films presented globular nano-topography and in vitro cell work showed that the mixed hydrocolloids had increased cell growth compared with EW films. Results showed that hydrocolloids of EW and gelatin can be used for creating a 3D hydrogel environment for cell studies inside microfluidic devices. Finally, 3D hydrogel scaffolds were fabricated by sequential temperature-dependent gelation followed by chemical cross-linking of the polymeric network of the hydrogel for added mechanical strength and stability. These 3D hydrogel scaffolds displayed pores, lamellae, globular nano-topography, tunable mechanical properties, high affinity for water, and cell proliferation and penetration properties. In conclusion, the large range of properties and characteristics of these materials provide a strong potential for a large variety of TERM applications, including cancer models, organoid growth, compatibility with bioprinting, or implantable devices.

The Apoptosis-Inducing Factor (AIF) is a moonlighting flavoenzyme involved in the assembly of mitochondrial respiratory complexes in healthy cells, but also able to trigger DNA cleavage and parthanatos. Upon apoptotic-stimuli, AIF redistributes from the mitochondria to the nucleus, where upon association with other proteins such as endonuclease CypA and histone H2AX, it is proposed to organize a DNA–degradosome complex. In this work, we provide evidence for the molecular assembly of this complex as well as for the cooperative effects among its protein components to degrade genomic DNA into large fragments. We have also uncovered that AIF has nuclease activity that is stimulated in the presence of either Mg2+ or Ca2+. Such activity allows AIF by itself and in cooperation with CypA to efficiently degrade genomic DNA. Finally, we have identified TopIB and DEK motifs in AIF as responsible for its nuclease activity. These new findings point, for the first time, to AIF as a nuclease able to digest nuclear dsDNA in dying cells, improving our understanding of its role in promoting apoptosis and opening paths for the development of new therapeutic strategies.

Magnetism plays a pivotal role in many biological systems. However, the intensity of the magnetic forces exerted between magnetic bodies is usually low, which demands the development of ultra-sensitivity tools for proper sensing. In this framework, magnetic force microscopy (MFM) offers excellent lateral resolution and the possibility of conducting single-molecule studies like other single-probe microscopy (SPM) techniques. This comprehensive review attempts to describe the paramount importance of magnetic forces for biological applications by highlighting MFM's main advantages but also intrinsic limitations. While the working principles are described in depth, the article also focuses on novel micro- and nanofabrication procedures for MFM tips, which enhance the magnetic response signal of tested biomaterials compared to commercial nanoprobes. This work also depicts some relevant examples where MFM can quantitatively assess the magnetic performance of nanomaterials involved in biological systems, including magnetotactic bacteria, cryptochrome flavoproteins, and magnetic nanoparticles that can interact with animal tissues. Additionally, the most promising perspectives in this field are highlighted to make the reader aware of upcoming challenges when aiming toward quantum technologies.

Microorganisms interplay with their neighbors according their intrinsic cellular adhesion and membrane rigidity. The protein receptors located at the cellular membrane mediate specific recognition processes with biomolecules and others cells. This topic has generated great interest based on the huge variability displayed at different environmental conditions. Aquatic ecosystems present notably versatility of microorganisms and dissolved molecules that mutually influence in their viability, proliferation and biological performance. Biodiversity preservation of the current water reservoirs strongly depends on the gathering of these properties and their accurate interpretation. Bulk measurements from meso-scale techniques provide average information of aquatic microorganisms hidden relevant details. For this reason, it is opportune to devote high-throughput single molecule tools as atomic force microscopy (AFM) to determine the physico-chemical properties exhibited by individual aquatic microorganisms and thus, establish a full comprehension of all parameters that influence the state of aquatic ecosystems. The present work aims to highlight the potential applications and future perspectives of AFM for the study of all living entities involved in aquatic ecosystems to better understand their inherent characteristics at the single molecule level.

Soft matter encompasses multitude of systems like biomolecules, living cells, polymers, composites or blends. The increasing interest to better understand their physico-chemical properties has significantly favored the development of new techniques with unprecedented resolution. In this framework, atomic force microscopy (AFM) can act as one main actor to address multitude of intrinsic sample characteristics at the nanoscale level. AFM presents many advantages in comparison to other bulk techniques as the assessment of individual entities discharging thus, ensemble averaging phenomena. Moreover, AFM enables the visualization of singular events that eventually can provide response of some open questions that still remain unclear. The present manuscript aims to make the reader aware of the potential applications in the employment of this tool by providing recent examples of scientific studies where AFM has been employed with success. Several operational modes like AFM imaging, AFM based force spectroscopy (AFM-FS), nanoindentation, AFM-nanoscale infrared spectroscopy (AFM-nanoIR) or magnetic force microscopy (MFM) will be fully explained to detail the type of information that AFM is capable to gather. Finally, future prospects will be delivered to discern the following steps to be conducted in this field.

Intermittent jumping force is an operational atomic-force microscopy mode that produces simultaneous topography and tip-sample maximum-adhesion images based on force spectroscopy. In this work, the operation conditions have been implemented scanning in a repulsive regime and applying very low forces, thus avoiding unspecific tip-sample forces. Remarkably, adhesion images give only specific rupture events, becoming qualitative and quantitative molecular recognition maps obtained at reasonably fast rates, which is a great advantage compared to the force–volume modes. This procedure has been used to go further in discriminating between two similar protein molecules, avidin and streptavidin, in hybrid samples. The adhesion maps generated scanning with biotinylated probes showed features identified as avidin molecules, in the range of 40–80 pN; meanwhile, streptavidin molecules rendered 120–170 pN at the selected working conditions. The gathered results evidence that repulsive jumping force mode applying very small forces allows the identification of biomolecules through the specific rupture forces of the complexes and could serve to identify receptors on membranes or samples or be applied to design ultrasensitive detection technologies.

Plastidic ferredoxin-NADP+ reductase (FNR) transfers two electrons from two ferredoxin or flavodoxin molecules to NADP+, generating NADPH. The forces holding the Anabaena FNR:NADP+ complex were analyzed by dynamic force spectroscopy, using WT FNR and three C-terminal Y303 variants, Y303S, Y303F, and Y303W. FNR was covalently immobilized on mica and NADP+ attached to AFM tips. Force–distance curves were collected for different loading rates and specific unbinding forces were analyzed under the Bell–Evans model to obtain the mechanostability parameters associated with the dissociation processes. The WT FNR:NADP+ complex presented a higher mechanical stability than that reported for the complexes with protein partners, corroborating the stronger affinity of FNR for NADP+. The Y303 mutation induced changes in the FNR:NADP+ interaction mechanical stability. NADP+ dissociated from WT and Y303W in a single event related to the release of the adenine moiety of the coenzyme. However, two events described the Y303S:NADP+ dissociation that was also a more durable complex due to the strong binding of the nicotinamide moiety of NADP+ to the catalytic site. Finally, Y303F shows intermediate behavior. Therefore, Y303, reported as crucial for achieving catalytically competent active site geometry, also regulates the concerted dissociation of the bipartite nucleotide moieties of the coenzyme.

Flavin and redox-active disulfide domains of ferredoxin-dependent flavin thioredoxin reductase (FFTR) homodimers should pivot between flavin-oxidizing (FO) and flavin-reducing (FR) conformations during catalysis, but only FR conformations have been detected by X-ray diffraction and scattering techniques. Atomic force microscopy (AFM) is a single-molecule technique that allows the observation of individual biomolecules with sub-nm resolution in near-native conditions in real-time, providing sampling of molecular properties distributions and identification of existing subpopulations. Here, we show that AFM is suitable to evaluate FR and FO conformations. In agreement with imaging under oxidizing condition, only FR conformations are observed for Gloeobacter violaceus FFTR (GvFFTR) and isoform 2 of Clostridium acetobutylicum FFTR (CaFFTR2). Nonetheless, different relative dispositions of the redox-active disulfide and FAD-binding domains are detected for FR homodimers, indicating a dynamic disposition of disulfide domains regarding the central protein core in solution. This study also shows that AFM can detect morphological changes upon the interaction of FFTRs with their protein partners. In conclusion, this study paves way for using AFM to provide complementary insight into the FFTR catalytic cycle at pseudo-physiological conditions. However, future approaches for imaging of FO conformations will require technical developments with the capability of maintaining the FAD-reduced state within the protein during AFM scanning

Single-molecule force spectroscopy is a powerful technique based on the application of controlled forces to macromolecules. In order to relate the measured response of the molecule to its equilibrium and dynamic properties, a suitable physical picture of the involved process is necessary. In this work, we introduce a plausible model for mechanical unbinding of some molecular complexes, based on a novel free energy profile. We combine two standard theoretical frameworks for analyzing force spectroscopy experiments on two protein:protein complexes, obtaining key magnitudes of the underlying free energy profile, which are only understood within the mentioned model. Additionally, we carry out detailed stochastic dynamics simulations to prove the validity of the analysis protocol and the reliability of the free energy profile. Remarkably, we can compare directly the obtained unbinding free energies with the previously known bulk binding free energies, bridging the gap between bulk and single molecule techniques.