The use of quantum dot nanoparticles (QDs) in bio-applications has gained quite some interest and requires a deep understanding of their interactions with model cell membranes. This involves assessing the extent of nanoparticle disruption of the membrane and how it depends on both nanoparticle and membrane physicochemical properties. Surface charge plays an important role in nanoparticle adsorption, which is primarily driven by electrostatic interactions; yet, once adsorbed, most reported works overlook the subsequent spatial nanoparticle insertion and location within the membrane. There is therefore a need for studies to assess the mutual role of membrane and nanoparticle charge into membrane structure and stability at the nanoscale, with a view to better design and control the functionality of these nanomaterials. In this work, we have resolved the extent of the interactions between hydrophilic, negatively charged CdTe QDs and positively charged lipid bilayers. A multiscale combination of surface-sensitive techniques enabled probing how surface charge mediates QD adsorption and membrane reorganization. Increasing membrane surface charge results into a larger adsorption of oppositely charged QDs, concomitantly inducing structural changes. Hydration of the membrane hydrophobic parts by QDs goes deeper into the inner leaflet with increasing membrane charge, resulting in supported lipid bilayers with decreased nanomechanical stability.

Over the past decades, advances in lipid nanotechnology have shown that self-assembled lipid structures providing ease of preparation, chemical stability, and biocompatibility represent a landmark on the development of multidisciplinary technologies. Lipid nanotubes (LNTs) are a unique class of lipid self-assembled structures, bearing unique properties such as high-aspect ratio, tunable diameter size, and precise molecular recognition. They can be obtained either by the action of external factors to already formed vesicles or spontaneously, the latter depending strongly on subtle molecular features. Here, we report on the spontaneous formation of supported lipid nanotubes of a particular type of glycolipid, ohmline, whose hydrophobic core displays remarkable asymmetry. The combination of bulk and surface-sensitive techniques indicates that below its main transition, ohmline displays an interdigitated gel phase, likely driven by the unique asymmetry in its hydrophobic core. Enhanced order packing by interdigitation favors the formation of ohmline nanotubes in agreement with chiral-based models of nanotube formation. The findings presented in this work call for additional studies to link lipid molecular structure-assembly relationships, whose understanding is relevant for the controlled design of lipid nanotubes networks in particular and controlled design of soft-matter nanomaterials in general.

The human body contains a few trillions of cells of different types and functions, which are crucial for living. The study of cell membranes is important for understanding biolog-ical events and developing new medicines. As cell membranes are very complex, research-ers often create and study simple models, referred to as biomimetic membranes, in order to understand their behavior and properties. In this paper, we briefly present quartz crys-tal microbalance with dissipation monitoring (QCMD) as a surface-sensitive technique to study biomimetic membranes. By measuring changes in the oscillation frequency and dis-sipation energy of quartz crystal sensors, QCMD can monitor in real time molecular events occurring at the quartz sensor-sample interface, such as formation of supported layers, changes in thickness and structural properties, as well as biomolecular interactions. We present a concise description of the basic QCMD principles, followed by a few examples on the adsorption of model membranes, namely, solid-supported lipid bilayers and vesi-cles, as well as on studying lipid phase transitions. Keywords: supported lipid bilayers, supported lipid vesicles, phase transitions, quartz crystal microbalance with dissipation monitoring (QCMD)

Solid supported lipid bilayers (SLBs) are excellent platforms for studying the biophysical properties of cell membranes, as well as versatile biomimetic films for biotechnology applications. Among the existing approaches used to form SLBs, vesicle fusion and rupture onto solid supports is the most commonly employed one owing to its straightforward procedure. SLBs are typically formed on atomically flat and very hydrophilic surfaces, overlooking the influence of roughness and topography on membrane formation and organization. As a matter of fact, lipid bilayers in vivo are corrugated at the nanoscale level, as a result of interactions with proteins, fibrils, and other components within the intracellular and extracellular environment. Fundamental studies of the effect of surface roughness on SLBs are scarce and restricted to few contributions, where nanoroughness has shown to affect lipid mobility by a 5-fold decrease and inhibit domain growth in phase-separated membranes. In this work, the impact of nanoroughness on the formation and stability of SLBs onto SiO2 surfaces with different degrees of vertical and lateral surface roughness is studied. Combining quartz crystal microbalance with dissipation monitoring (QCM-D) and atomic force microscopy with force spectroscopy (AFM-FS), it is shown that nanoroughness affects the formation of SLBs by increasing the activation energy of vesicle fusion, rupture and spreading, and weakens the stability and lateral organization of the formed SLBs.

Supported lipid bilayers (SLBs) are low-complexity biomimetic membranes, serving as popular experimental platforms to study membrane organization and lipid transfer, membrane uptake of nanoparticles and biomolecules, and many other processes. Quartz crystal microbalance with dissipation monitoring has been utilized to probe the influence of several parameters on the quality of SLBs formed on Au- and SiO2-coated sensors. The influence of the aqueous medium (i.e., buffer type) and the adsorption temperature, above and below the lipid melting point, is neatly explored for SLBs of 1,2-dimyristoyl-sn-glycero-3-phosphocholine and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine formed by a solvent exchange. Below the lipid melting temperature, quality variations are observed upon the formation on Au and SiO2 surfaces, with the SLBs being more homogeneous for the latter. We further investigate how the buffer affects the detection of lipid melting in SLBs, a transition that necessitates high-sensitivity and time-consuming surface-sensitive techniques to be detected.

The kinetics of spontaneous monomer transfer between vesicles consisting of zwitterionic phospholipids is dictated by the difference in desorption rate of lipid monomers from their donor vesicles and the concentration imbalance in the dispersion. In a system with two lipid species with the same headgroup, transfer is asymmetric, and takes place from the population of donor vesicles consisting of shorter chain lipids to acceptor ones of longer chain. Transfer typically proceeds until equilibrium is reached, resulting in populations of vesicles consisting of a binary mixture of both lipid species, whose concentration depends on the number of lipids in the precursor donor and acceptor vesicles before transfer.Upon the introduction of a second lipid species in the donor vesicle population, the desorption rate of monomers should change with time, since the composition of donor vesicles changes when monomers of a given lipid type desorb. To tackle this problem, we added a cationic lipid, 1,2-dimyristoyl-3-trimethylammonium-propane (DMTAP), into donor zwitterionic lipid vesicles and assessed how the concentration of DMTAP affects the lipid transfer process. Lipid transfer is the result of the interplay between the initial concentration of DMTAP in the donor vesicles (and related probability of desorption at short transfer times) and their concomitant time-dependent concentration (and thus desorption rate) change due to the depletion of monomer species as the transfer process proceeds.

The influence of peptides over the thermodynamic and mechanic stability of lipid membranes is studied. To this end, diphenylalanine and 1,2-dimyristoyl-sn-glycero-3-phosphocholine are selected as model systems for peptide and lipid bilayers, respectively. Vesicles at different peptide-lipid ratios were prepared and characterized using different experimental methodologies. Densitometry and differential scanning calorimetry were used to determine the influence of the peptide over the melting transition properties (temperature, volume and enthalpy) for bulk multilamellar vesicles, whereas viscoelastic and nanomechanical properties of supported vesicle layers and supported lipid bilayers were studied using quartz crystal microbalance with dissipation monitoring and atomic force microscopy, respectively. The influence of the sample preparation methodology -solvents for obtaining the lipid films and hydration media- is also characterized. The analysis of the experimental data reveals that diphenylalanine affects the lipid membrane, decreasing lipid molecular cooperativity in a concentration-dependent manner and inducing an overall weakening of membrane organization.

Solid-supported lipid bilayers (SLBs) provide biomimetic platforms for multi disciplinary research encompassing material science, biology, sensing and medical applications. Apart from well-established methods to produce SLBs, such as the vesicle fusion and Langmuir-Blodgett deposition, a new approach based on solvent exchange has been recently developed and shown to be very versatile. In this chapter, we describe in detail the solvent-exchange method and refer to a number of factors, such as the solvent type, the substrate nature and the lipid concentration and phase, which influence the so-produced SLBs. Furthermore, we briefly discuss the recently reported extension of the method towards solid-supported polymer and hybrid membranes.

Hybrid lipid bilayers are a particular case of supported lipid bilayers with the two monolayer leaflets composed by different types of molecules. These nanostructures can be produced in a well-controlled array fashion and are suitable for the study of biomembrane-related phenomena via electrochemical or plasmonic sensing. Understanding how the underlying solid surface affects the supported membrane formation and organization is necessary for the potential use of these hybrid platforms in applications for which surfaces are not flat and topographically complex. Here we assess the role of lipid phase, substrate surface energy and topography on the formation and stability of hybrid supported membranes from vesicle precursors using complementary surface-sensitive techniques, namely quartz crystal microbalance with dissipation and atomic force microscopy. The stability of hybrid bilayers against thermal and osmotic changes is evaluated and compared to standard supported lipid bilayers formed onto hydrophilic SiO2. Force spectroscopy measurements reveal an overall weaker lateral organization of hybrid membranes as a result of the underlying self-assembled monolayer being not optimally organized. Hybrid bilayers display a decoupled behavior between the two leaflets when vertically compressed at constant speed. On microcontact printed Au surfaces, hybrid bilayers were formed over printed patches, while surprisingly, supported lipid bilayers were observed on non-patterned Au regions suggesting a non-trivial self-assembled monolayer reorganization when in aqueous environment.

Solid‐supported lipid bilayers (SLBs) are excellent platforms for studying the biophysical properties of cell membranes. Among the existing approaches used to form SLBs, vesicle fusion and rupture onto solid supports are most commonly employed owing to their straightforward procedure. The current understanding of the mechanisms behind this approach has greatly benefited from the use of surface‐sensitive techniques, especially quartz crystal microbalance with dissipation monitoring (QCM‐D) in combination with other analytical techniques, such as atomic force microscopy (AFM) or localized surface plasmon resonance (LSPR). Herein, an overview of the pathways of vesicle adsorption and rupture under various experimental conditions is provided. Examples including recent findings of how the variation of the properties of lipid vesicles (size, charge), aqueous buffer (pH, ionic strength, osmotic pressure), and solid support (surface energy) affect the pathway mechanism of adsorption and rupture are provided. Recent reports on poorly understood properties such as surface roughness and topography are provided, together with the need for further studies relevant to biomimetic and sensing purposes.

The electrochemical nucleation and growth (EN&G) on active surface sites has been a concept of fundamental and technological interest for several decades. Here, we have studied the EN&G of Cu on glassy carbon with a new perspective using the Scanning Electrochemical Cell Microscopy (SECCM), in combination with scanning electron microscopy, atomic force microscopy, and X‐ray photo-electron spectroscopy. Unlike the conventional macroscopic approach, we leveraged the spatial resolution of the SECCM to probe individual sites on the same surface, independently from each other, revealing regions with different energy barriers for nucleation and a distribution of activities for EN&G at the microscopic scale. This site-dependent activity can be modified with common surface pretreatments (i.e., polishing and preanodization). We addressed the electrochemical diversity through multiple descriptors and used them to conduct statistical analysis, supported by surface characterization techniques, bringing forward information that is simply unavailable with the conventional macroscopic approach. This work serves as a departure point to conceive new analysis strategies and address the real nature of active sites for nucleation.

Rapid antigen tests are currently used for population screening of COVID-19. However, they lack sensitivity and utilize antibodies as receptors, which can only function in narrow temperature and pH ranges. Consequently, molecularly imprinted polymer nanoparticles (nanoMIPs) are synthetized with a fast (2 h) and scalable process using merely a tiny SARS-CoV-2 fragment (∼10 amino acids). The nanoMIPs rival the affinity of SARS-CoV-2 antibodies under standard testing conditions and surpass them at elevated temperatures or in acidic media. Therefore, nanoMIP sensors possess clear advantages over antibody-based assays as they can function in various challenging media. A thermal assay is developed with nanoMIPs electrografted onto screen-printed electrodes to accurately quantify SARS-CoV-2 antigens. Heat transfer-based measurements demonstrate superior detection limits compared to commercial rapid antigen tests and most antigen tests from the literature for both the alpha (∼9.9 fg mL-1) and delta (∼6.1 fg mL-1) variants of the spike protein. A prototype assay is developed, which can rapidly (∼15 min) validate clinical patient samples with excellent sensitivity and specificity. The straightforward epitope imprinting method and high robustness of nanoMIPs produce a SARS-CoV-2 sensor with significant commercial potential for population screening, in addition to the possibility of measurements in diagnostically challenging environments.

Photosensitizers that gather high photo-oxidizing power and strong visible-light absorption are of great interest in the development of new photo-chemotherapeutics. Indeed, such compounds constitute attractive candidates for the design of type I photosensitizers that are not dependent on the presence of oxygen. In this paper, we report on the synthesis and studies of new ruthenium(II) complexes that display strong visible-light absorption and can oxidize guanine residues under visible-light irradiation, as evidenced by nanosecond transient absorption spectroscopy. The reported compounds also tightly bind to G-quadruplex DNA structures from the human telomeric sequence (TTAGGG repeat). The kinetic and thermodynamic parameters of the interaction of these Ru(II) complexes with G-quadruplex and duplex DNA were studied thanks to luminescence titrations and bio-layer interferometry measurements, which revealed higher affinities towards the non-canonical G-quadruplex architecture. Docking experiments and non-covalent ionic analysis allowed us to gain information on the mode and the strength of the interaction of the compounds towards G-quadruplex and duplex DNA. The different studies emphasize the substantial influence of the position and the number of non-chelating nitrogen atoms on the interaction with both types of DNA secondary structures.

Lipid transfer between vesicle systems by monomer diffusion is typically investigated using deterministic kinetic models and fluorescent label-based experiments that yield averaged dynamic information of changes in vesicle properties like size and concentration. Obtaining quantitative information about the distribution of sizes and concentrations at each time remains not straightforward and, in many cases, inaccessible. In this work we have resolved inter-vesicle lipid transfer for asymmetric systems via monomer diffusion under non-equilibrium conditions. Our approach consists of departing from a well-defined non equilibrium state, where zwitterionic vesicles differing in hydrophobic chain length are incubated, and experimentally monitoring the temporal evolution of size and concentration. For this purpose, we have combined complementary techniques, namely dynamic light scattering and quartz crystal microbalance with dissipation monitoring. The size and concentration behavior agree well with the predictions of a kinetic deterministic model and motivate the study of monomer transfer in the framework of stochastic simulations. Furthermore, we have introduced the use of equilibrium Monte Carlo simulations to unravel the quantitative distribution of vesicles with a given concentration present at different times during the transfer process. Results show the temporal evolution of the system from a non-equilibrium state at short incubation times consisting of many coexisting lipid concentrations towards a steady state of a single concentration at long incubation times. The methodology used acts as a reference point to more complex systems, i.e., lipid monomer transfer between confined lipid systems and in the presence of external stimuli.(c) 2022 Elsevier B.V. All rights reserved.

The interest in nano-sized lipid vesicles in nano-biotechnology relies on their use as mimics for endosomes, exosomes, and nanocarriers for drug delivery. The interactions between nanoscale size lipid vesicles and cell membranes involve spontaneous interbilayer lipid transfer by several mechanisms, such as monomer transfer or hemifusion. Experimental approaches toward monitoring lipid transfer between nanoscale-sized vesicles typically consist of transfer assays by fluorescence microscopy requiring the use of labels or calorimetric measurements, which in turn require a large amount of sample. Here, the capability of a label-free surface-sensitive method, quartz crystal microbalance with dissipation monitoring (QCM-D), was used to monitor lipid transfer kinetics at minimal concentrations and to elucidate how lipid physicochemical properties influence the nature of the transfer mechanism and dictate its dynamics. By studying time-dependent phase transitions obtained from nanoviscosity measurements, the transfer rates (unidirectional or bidirectional) between two vesicle populations consisting of lipids with the same head group and differing alkyl chain length can be estimated. Lipid transfer is asymmetric and unidirectional from shorter-chain lipid donor vesicles to longer-chain lipid acceptor vesicles. The transfer is dramatically reduced when the vesicle populations are incubated at temperatures below the melting of one of the vesicle populations.

Quantum dots (QDs) are semiconductor nanoparticles with unique optical and electronic properties, whose interest as potential nano-theranostic platforms for imaging and sensing is increasing. The design and use of QDs requires the understanding of cell-nanoparticle interactions at a microscopic and nanoscale level. Model systems such as supported lipid bilayers (SLBs) are useful, less complex platforms mimicking physico-chemical properties of cell membranes. In this work, we investigated the effect of topographical homogeneity of SLBs bearing different surface charge in the adsorption of hydrophilic QDs. Using quartz-crystal microbalance, a label-free surface sensitive technique, we show significant differences in the interactions of QDs onto homogeneous and inhomogeneous SLBs formed following different strategies. Within short time scales, QDs adsorb onto topographically homogeneous, defect-free SLBs is driven by electrostatic interactions, leading to no layer disruption. After prolonged QD exposure, the nanomechanical stability of the SLB decreases suggesting nanoparticle insertion. In the case of inhomogeneous, defect containing layers, QDs target preferentially membrane defects, driven by a subtle interplay of electrostatic and entropic effects, inducing local vesicle rupture and QD insertion at membrane edges.

Antibodies not only play a major role in clinical diagnostics and biopharmaceutical analysis but also are a class of drugs that are regularly used to treat numerous diseases. The identification of antibody-epitope binding sites is then of great interest to many emerging medical and bioanalytical applications, particularly to design monoclonal antibodies (mAb) mimics taking advantage of amino acid residues involved in the binding. Among relevant antibodies, the monoclonal antibody rituximab has received significant attention as it is exploited to treat several cancers including non-Hodgkin's lymphoma and chronic lymphocytic leukemia, as well as some autoimmune disorders such as rheumatoid arthritis. The binding of rituximab to the targeted cells occurs via the recognition of the CD20 epitope. A crystallographic study has shown that the binding area, named paratope, is located at the surface of rituximab. Combining the SPOT method and the complementary surface plasmon resonance technique allowed us to detect an extended recognition domain buried in the pocket of the rituximab Fab formed by four β-sheets. More generally, the present study offers a comprehensive approach to identify antibody-epitope binding sites.

Lipid phase behavior in model membranes has been the subject of intensive research aiming to unravel fundamental mechanisms behind membrane function. Among the experimental techniques used for characterizing lipid phase transitions at different scale levels, quartz crystal microbalance with dissipation monitoring (QCM-D) has emerged as a useful approach for detecting phase transitions in solid-supported lipid membranes at the nano-mesoscale. In this chapter, we provide examples of how phase transitions in supported lipid membranes, detected by QCM-D, can be used as descriptors for monitoring specific lipid-membrane related mechanisms such as vesicle adsorption, changes in lipid organization upon addition of inclusions of small molecules and lipid transfer kinetics by monomer diffusion. Combined with other complementary techniques at a multiscale, QCM-D stands as a promising approach to be employed in monitoring the phase behavior of supported lipid membranes in particular and biomolecules in general.