UV-Laser Interference Lithography for Local Functionalization of Plasmonic Nanostructures with Responsive Hydrogel.

A novel approach to local functionalization of plasmonic hotspots at gold nanoparticles with biofunctional moieties is reported. It relies on photocrosslinking and attachment of a responsive hydrogel binding matrix by the use of a UV interference field. A thermoresponsive poly(N-isopropylacrylamide)-based (pNIPAAm) hydrogel with photocrosslinkable benzophenone groups and carboxylic groups for its postmodification was employed. UV-laser interference lithography with a phase mask configuration allowed for the generation of a high-contrast interference field that was used for the recording of periodic arrays of pNIPAAm-based hydrogel features with the size as small as 170 nm. These hydrogel arrays were overlaid and attached on the top of periodic arrays of gold nanoparticles, exhibiting a diameter of 130 nm and employed as a three-dimensional binding matrix in a plasmonic biosensor. Such a hybrid material was postmodified with ligand biomolecules and utilized for plasmon-enhanced fluorescence readout of an immunoassay. Additional enhancement of the fluorescence sensor signal by the collapse of the responsive hydrogel binding matrix that compacts the target analyte at the plasmonic hotspot is demonstrated.

Resveratrol-Induced Temporal Variation in the Mechanical Properties of MCF-7 Breast Cancer Cells Investigated by Atomic Force Microscopy.

Atomic force microscopy (AFM) combined with fluorescence microscopy has been used to quantify cytomechanical modifications induced by resveratrol (at a fixed concentration of 50 µM) in a breast cancer cell line (MCF-7) upon temporal variation. Cell indentation methodology has been utilized to determine simultaneous variations of Young's modulus, the maximum adhesion force, and tether formation, thereby determining cell motility and adhesiveness. Effects of treatment were measured at several time-points (0-6 h, 24 h, and 48 h); longer exposures resulted in cell death. Our results demonstrated that AFM can be efficiently used as a diagnostic tool to monitor irreversible morpho/nano-mechanical changes in cancer cells during the early steps of drug treatment.

In-situ 2D bacterial crystal growth as a function of protein concentration: An atomic force microscopy study.

The interplay between protein concentration and (observation) time has been investigated for the adsorption and crystal growth of the bacterial SbpA proteins on hydrophobic fluoride-functionalized SiO2 surfaces. For this purpose, atomic force microscopy (AFM) has been performed in real-time for monitoring protein crystal growth at different protein concentrations. Results reveal that (1) crystal formation occurs at concentrations above 0.08 µM and (2) the compliance of the formed crystal decreases by increasing protein concentration. All the crystal domains observed presented similar lattice parameters (being the mean value for the unit cell: a = 14.8 ± 0.5 nm, b = 14.7 ± 0.5 nm, γ = 90 ° ± 2). Protein film formation is shown to take place from initial nucleation points which originate a gradual and fast extension of the crystalline domains. The Avrami equation describes well the experimental results. Overall, the results suggest that protein-substrate interactions prevail over protein-protein interactions. RESEARCH HIGHLIGHTS: AFM enables to monitor protein crystallization in real-time. AFM high-resolution determines lattice parameters and viscoelastic properties. S-layer crystal growth rate increases with protein concentration. Avrami equation models protein crystal growth.

Bacillus thuringiensis Cyt2Aa2 binding on lipid/cholesterol bilayer depends on protein concentration and time.

Bacillus thuringiensis produces cytolytic proteins (Cyt) that show toxicity against dipteran insect larvae acting directly on the cell membrane. Up to now, two different models have been proposed to explain the interaction mechanism of the cytolytic protein Cyt2Aa2 on lipid membranes: pore-forming and detergent-like action. Here we report on the interaction of Cyt2Aa2 with lipid/cholesterol bilayers at early stage (far from equilibrium) as a function of protein concentration. Quartz crystal microbalance with dissipation (QCM-D) measurements showed that the rate of protein adsorption increased with concentration, although the mass of the final protein-lipid was similar after two hours. In addition, the dissipation (compliance of the hybrid lipid/protein layer) increased with decreasing protein concentration. Furthermore, atomic force microscopy (AFM) revealed that the structure of the protein-lipid layer was concentration and time dependent. A rigid hybrid homogeneous layer was observed at protein concentrations of 50 µg/ml and 100 µg/ml after 30 min. At lower concentrations, 10 µg/ml and 17.5 µg/ml, protein adsorption on the lipid layer led to the formation of small aggregates. Interestingly, at 25 µg/ml a transition of a hole-like structure into a homogeneous layer was observed. This suggests that 25 µg/ml is a threshold concentration for the binding mechanism of Cyt2Aa2 on to lipid membranes.

Cation-chelation and pH induced controlled switching of the non-fouling properties of bacterial crystalline films.

We report the controlled loss of the anti-fouling activity of the S-layer protein SbpA from Lysinibacillus sphaericus (CCM2177). This protein forms crystal-like films with square lattice (p4) via self-assembly on almost any type of surfaces. Such engineered bioinspired nanometric membranes are known by their excellent preventive performance under biological conditions. However, their exposure to certain treatments can lead to gradual degradation of the S-protein layer. In this work, two distinctive approaches are studied for understanding either specific or non-specific degradation of the film, by treatment with a chelating agent (EDTA), which interacts with inner Ca2+ ions, or Citrate buffer (with pH

Impact of surface wettability on S-layer recrystallization: a real-time characterization by QCM-D.

Quartz crystal microbalance with dissipation monitoring (QCM-D) has been employed to study the assembly and recrystallization kinetics of isolated SbpA bacterial surface proteins onto silicon dioxide substrates of different surface wettability. Surface modification by UV/ozone oxidation or by vapor deposition of 1H,1H,2H,2H-perfluorododecyltrichlorosilane yielded hydrophilic or hydrophobic samples, respectively. Time evolution of frequency and dissipation factors, either individually or combined as the so-called Df plots, showed a much faster formation of crystalline coatings for hydrophobic samples, characterized by a phase-transition peak at around the 70% of the total mass adsorbed. This behavior has been proven to mimic, both in terms of kinetics and film assembly steps, the recrystallization taking place on an underlying secondary cell-wall polymer (SCWP) as found in bacteria. Complementary atomic force microscopy (AFM) experiments corroborate these findings and reveal the impact on the final structure achieved.

Investigating cell-substrate and cell-cell interactions by means of single-cell-probe force spectroscopy.

Cell adhesion forces are typically a mixture of specific and nonspecific cell-substrate and cell-cell interactions. In order to resolve these phenomena, Atomic Force Microscopy appears as a powerful device which can measure cell parameters by means of manipulation of single cells. This method, commonly known as cell-probe force spectroscopy, allows us to control the force applied, the area of interest, the approach/retracting speed, the force rate, and the time of interaction. Here, we developed a novel approach for in situ cantilever cell capturing and measurement of specific cell interactions. In particular, we present a new setup consisting of two different half-surfaces coated either with recrystallized SbpA bacterial cell surface layer proteins (S-layers) or integrin binding Fibronectin, on which MCF-7 breast cancer cells are incubated. The presence of a clear physical boundary between both surfaces benefits for a quick detection of the region under analysis. Thus, quantitative results about SbpA-cell and Fibronectin-cell adhesion forces as a function of the contact time are described. Additionally, the importance of the cell spreading in cell-cell interactions has been studied for surfaces coated with two different Fibronectin concentrations: 20 µg/mL (FN20) and 100 µg/mL (FN100), which impact the number of substrate receptors. Microsc. Res. Tech. 80:124-130, 2017. © 2016 Wiley Periodicals, Inc.

Time influence on the interaction between Cyt2Aa2 and lipid/cholesterol bilayers.

Protein-membrane interactions are still an important topic of investigation. One of the suitable experimental techniques used by the scientific community to address such question is atomic force microscopy. In a previous work, we have reported that the binding mechanism between the cytolytic and antimicrobial protein (Cyt2Aa2) and lipid/cholesterol bilayers was concentration-dependent, leading to either the formation of holes in the bilayer or aggregates. Here we study such binding mechanism as a function of time at low protein concentrations (10 µg/mL). We demonstrate that although holes are formed during the first stages of the protein-lipid interaction, a reparation process due to molecular mobility in the bilayer leads to a homogenous and isotropic protein-lipid/cholesterol layer within 3 hr. The combination of imaging, force spectroscopy, and phase contrast delivered information about topography dynamics (molecular mobility), layer thickness, and mechanical properties of the protein-lipid/cholesterol system. These results highlight the importance of the observation time in (such type of) protein-lipid interactions (at low protein concentrations).

Effect of the Concentration of Cytolytic Protein Cyt2Aa2 on the Binding Mechanism on Lipid Bilayers Studied by QCM-D and AFM.

Bacillus thuringiensis is known by its insecticidal property. The insecticidal proteins are produced at different growth stages, including the cytolytic protein (Cyt2Aa2), which is a bioinsecticide and an antimicrobial protein. However, the binding mechanism (and the interaction) of Cyt2Aa2 on lipid bilayers is still unclear. In this work, we have used quartz crystal microbalance with dissipation (QCM-D) and atomic force microscopy (AFM) to investigate the interaction between Cyt2Aa2 protein and (cholesterol-)lipid bilayers. We have found that the binding mechanism is concentration dependent. While at 10 µg/mL, Cyt2Aa2 binds slowly on the lipid bilayer forming a compliance protein/lipid layer with aggregates, at higher protein concentrations (100 µg/mL), the binding is fast, and the protein/lipid layer is more rigid including holes (of about a lipid bilayer thickness) in its structure. Our study suggests that the protein/lipid bilayer binding mechanism seems to be carpet-like at low protein concentrations and pore forming-like at high protein concentrations.