Microgels display specific features attracting a high research interest in several applications such as bioactives, drug delivery or protein purification. Whey protein microgels (WPM) are obtained by promoting whey protein intramolecular cross-linking through heat treatment. Temperature-induced protein denaturation and aggregation involves several kinds of interactions such as electrostatic repulsions and disulfide bonds. In the present work, the WPM particles topography and nanomechanical properties were investigated at native pH (6.5) and acid pH (5.5 and 3.0) by atomic force microscopy (AFM) in contact mode in a liquid environment. Prior to AFM analysis, WPM particles were captured on a gold substrate via low energy interactions by means of specific monoclonal antibodies. The 2D AFM images clearly showed a swelling of WPM particles induced by pH decrease. At native pH, they displayed an average width and height of 192 ± 27 nm and 44 ± 9 nm, respectively. A decrease in pH to 5.5 led to a significant (p<0.05) increase in WPM particles width (282 ± 9 nm) and height (101 ± 1 nm). At pH 3.0, a further increase in WPM size (a width of 420 ± 25 nm and a height of 78 ± 1 nm). The AFM elasticity (E) data showed a significant (p<0.05) increase in stiffness at pH 5.5 (E: 199 ± 9 kPa) and pH 3.0 (E: 187 ± 12 kPa) compared to native pH (E: 12 ± 1 kPa). These findings indicate that the mechanical profiles of WPM network directly varied with pH decrease. The WPM topographic and nanomechanical changes induced by acidification were most likely due to substantial changes in the shape and the structure of WPM particles. These strengthened internally crosslinked structures, modified by acidification, could display interesting encapsulation properties, providing an additional proof for their use as nanovectors monitoring bioactives release to the desired target after ingestion.

Atomic force microscopy-based nanomechanical methods were directly applied in order to elucidate important questions related to brain metastasis formation and amyotrophic lateral sclerosis (ALS). Force mapping with single cell force spectroscopy was combined in order to gain direct insight into the surface “screening” process of tumor cells during their extravasation into the brain parenchyma. Furthermore, comparative SCFS measurements were performed between three melanoma cell types (WM35, A2058 and A375), showing altered invasive characteristics, and blood vessel lining endothelials in order to examine how the metastatic potential relates to tumor cell’s autonomous and inter-cellular nanomechanical properties. In case of ALS by comparing the adhesion of CD8+ cytotoxic T cells isolated from wild-type as well as SOD1G93A mutant mice against wild-type motor neurons we found that cytotoxic T cells are active contributors of neurodegeneration in the development of ALS. In addition, nanomechanical mapping of wild-type and SOD1G93A mutant mice derived single elongated myoblasts and multinuclear myotubes revealed differences in elasticity during early differentiation stage into myotubes. These findings provide valuable knowledge to the understanding of brain metastasis formation and ALS pathogenesis.

Cost-effective health care technologies for detection of disease biomarkers at ultralow concentrations can significantly improve the quality of life in resource-constrained countries. However, current techniques require expensive instruments, complex conjugation protocols and tedious laboratory procedures that may not be affordable for the major part of the world population. Here, we propose to sidestep the problem by establishing a simple, relatively inexpensive sensing method employing a photonic substrate, a material affinity peptide, and a smart phone CCD detector to achieve detection of clinically relevant proteins in serum at concentrations much lower than standard enzyme-linked immunosorbent assay (ELISA). Easy to process porous silicon (PSi) micro-cavities were employed as substrates that provide a three-dimensionality, large surface area, and convenient light enhancement properties for molecular detection. Anti-histone H2B antibodies and biomarkers of severe illnesses are detected in whole serum at concentrations as low as 10 fg mL−1 by using the proposed PSi-ELISA protocol. Due to its easy use, cost effectiveness and high sensitivity, the proposed method has potential large- scale applications in ultrasensitive sensing of different other clinically relevant molecules for early stage diagnostics.

Casein micelle (CM), porous colloidal phosphoprotein-mineral complex, naturally present in milk to deliver minerals, also has several features, which could ensure its use as nanocarrier for bioactives. CM structure being not steady according to the physico-chemical conditions, its stability can be improved by intra-micellar cross-linking using transglutaminase (TGase) inducing a strengthened structure called casein nanogel. The aim of this research was to investigate the morphology and nanomechanics of casein nanogel particles cross-linked by TGase (TG-CM) using atomic force microscopy (AFM) in native-like liquid environment (lactose-free simulated milk ultrafiltrate, SMUF). Prior to AFM, TG-CM were captured by anti-phospho-Ser/Thr/Tyr monoclonal antibodies covalently bound to a gold-coated slide via carbodiimide chemistry. Surface topography and size properties evaluation revealed an increase in size of TG-CM compared to native CM, TG-CM being characterized by a mean width of 264 ± 7 nm and a mean height of 111 ± 5 nm. TG-CM displayed a relatively high contact angle (62°) indicating a limited flattening of these particles after adsorption on the substrate. The TG-CM elasticity was then evaluated applying low indentation forces on single TG-CM. The TGase treatment led to a significant modification of CM nanomechanics attributed to intramolecular rearrangements within the micellar structure. The elasticity distribution of TG-CM revealed three elasticity peaks centered at 219±14kPa, 536±14kPa and 711 ± 11 kPa. The lower elasticity peak is related to the native CM nanomechanical signature and the two stiffer peaks were attributed to the substantial changes in the TG-CM structure.

Dentin carious lesion is a dynamic process that involves de- mineralization and collagen denaturation. Collagen type I is the major protein in dentin and it has been investigated based on its optical properties. Multiphoton microscopy (MPM) is a nonlinear imaging technique that reveals the car- ies process using the collagen two-photon excitation fluo- rescence (2PEF) and its second-harmonic generation (SHG). Combining the histological and the International Caries De- tection and Assessment System (ICDAS) classifications with nonlinear optical spectroscopy (NLOS), 2PEF and SHG inten- sities of enamel and dentin were highly altered during the caries process. It has been proven that the ratio SHG/2PEF is a relevant indicator of the organic matrix denaturation [Ter- rer et al.: J Dent Res 2016;96:574–579]. In the present study, a series of measurable signals is made to detect early stages of carious lesion according to the ICDAS classification and to explore the relationship between these measures and theICDAS scale. Comparison of the efficiency of nonlinear opti- cal signals for caries detection with the ICDAS classification is essential to evaluate their potential for clinical applica- tion. In our study, the use of the NLOS measured by MPM allowed us to monitor a quantitative parameter (SHG/2PEF ratio) according to the dentin carious lesion state (ICDAS and histological examination). Three coherent new groups were defined (ICDAS 0/1; ICDAS 2/3; ICDAS 4/5/6), where the carious process can be clearly described with a statistically significant decrease of the SHG/2PEF ratio.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that a ects the motorsystem leading to generalized paralysis and death of patients. The understanding of early pathogenic mechanisms will help to de ne early diagnostics criteria that will eventually provide basis for e cient therapeutics. Early symptoms of ALS usually include muscle weakness or sti ness. Therefore, mechanical response of di erentiated myotubes from primary cultures of mice, expressing the ALS- causing SOD1G93A mutation, was examined by atomic force microscopy. Simultaneous acquisition of topography and cell elasticity of ALS myotubes was performed by force mapping method, compared with healthy myotubes and supplemented with immuno uorescence and qRT-PCR studies. Wildtype myotubes reveal a signi cant di erence in elasticity between a narrow and a wide population, consistent with maturation occurring with higher actin expression relative to myosin together with larger myotube width. However, this is not true for SOD1G93A expressing myotubes, where a signi cant shift of thin population towards higher elastic modulus values was observed. We provide evidence that SOD1 mutant induces structural changes that occurs very early in muscle development and well before symptomatic stage of the disease. These ndings could signi cantly contribute to the understanding of the role of skeletal muscle in ALS pathogenesis.

Background: Understanding stem cell behavior as a delivery tool in cancer therapy is essential for evaluating their future clinical potential. Previous in-vivo studies proved the use of mesenchymal stem cells (MSCs) for local delivery of the commonest anticancer drug, paclitaxel (PTX). Dental pulp is a relatively abundant noninvasive source of MSCs. We assess dental pulp stem cells (DPSCs), for the first time, as anticancer drug carriers. Confocal Raman microscopy is a unique tool to trace drug and cell viability without labeling.Methods: Drug uptake and cell apoptosis are identified through confocal Raman microscope. We traced translocation of cytochrome c enzyme from the mitochondria, as a biomarker for apoptosis, after testing both cancer and stem cells. The viability of stem cells was checked by means of confocal Raman microscope and by cytotoxicity assays.Results: In this study, we prove that DPSCs can be loaded in vitro with the anticancerous drug without affecting their viability, which is later released in the culture medium of breast cancer cells (MCF-7 cells) in a time-dependent fashion. The induced cytotoxic damage in MCF-7 cells was observed consequently after PTX release by DPSCs. Additionally, quantitative Raman images of intracellular drug uptake in DPSCs and MCF-7 cells were obtained. Cytotoxic assays prove the DPSCs to be more resistant to PTX as compared to bone marrow-derived MSCs, provided similar conditions.Conclusions: Applications of dental stem cells for targeted treatment of cancer could be a revolution to reduce morbidity due to chemotherapy, and to increase the efficacy of systemic cancer treatment.