Silica-coated magnetic particles for efficient RNA extraction for SARS-CoV-2 detection.

Molecular diagnostic methods to detect and quantify viral RNA in clinical samples rely on the purification of the genetic material prior to reverse transcription polymerase chain reaction (qRT-PCR). Due to the large number of samples processed in clinical laboratories, automation has become a necessity in order to increase method processivity and maximize throughput per unit of time. An attractive option for isolating viral RNA is based on the magnetic solid phase separation procedure (MSPS) using magnetic microparticles. This method offers the advantage over other alternative methods of making it possible to automate the process. In this study, we report the results of the MSPS method based on magnetic microparticles obtained by a simple synthesis process, to purify RNA from oro- and nasopharyngeal swab samples of patients suspected of COVID-19 provided by three diagnostic laboratories located in the Buenos Aires Province, Argentina. Magnetite nanoparticles of Fe3O4 (MNPs) were synthesized by the coprecipitation method and then coated with silica (SiO2) produced by hydrolysis of tetraethyl orthosilicate (TEOS). After preliminary tests on samples from the A549 human lung cell line and swabs, an extraction protocol was developed. The quantity and purity of the RNA obtained were determined by gel electrophoresis, spectrophotometry, and qRT-PCR. Tests on samples from naso- and oropharyngeal swabs were performed in order to validate the method for RNA purification in high-throughput SARS-CoV-2 diagnosis by qRT-PCR. The method was compared to the spin columns method and the automated method using commercial magnetic particles. The results show that the method developed is efficient for RNA extraction from nasal and oropharyngeal swab samples, and also comparable to other extraction methods in terms of sensitivity for SARS-CoV-2 detection. Of note, this procedure and reagents developed locally were intended to overcome the shortage of imported diagnostic supplies as the sudden spread of COVID-19 required unexpected quantities of nucleic acid isolation and diagnostic kits worldwide.

Redox phase transformations in magnetite nanoparticles: impact on their composition, structure and biomedical applications.

Magnetite nanoparticles (NPs) are one of the most investigated nanomaterials so far and modern synthesis methods currently provide an exceptional control of their size, shape, crystallinity and surface functionalization. These advances have enabled their use in different fields ranging from environmental applications to biomedicine. However, several studies have shown that the precise composition and crystal structure of magnetite NPs depend on their redox phase transformations, which have a profound impact on their physicochemical properties and, ultimately, on their technological applications. Although the physical mechanisms behind such chemical transformations in bulk materials have been known for a long time, experiments on NPs with large surface-to-volume ratios have revealed intriguing results. This article is focused on reviewing the current status of the field. Following an introduction on the fundamental properties of magnetite and other related iron oxides (including maghemite and wüstite), some basic concepts on the chemical routes to prepare iron oxide nanomaterials are presented. The key experimental techniques available to study phase transformations in iron oxides, their advantages and drawbacks to the study of nanomaterials are then discussed. The major section of this work is devoted to the topotactic oxidation of magnetite NPs and, in this regard, the cation diffusion model that accounts for the experimental results on the kinetics of the process is critically examined. Since many synthesis routes rely on the formation of monodisperse magnetite NPs via oxidation of wüstite counterparts, the modulation of their physical properties by crystal defects arising from the oxidation process is also described. Finally, the importance of a precise control of the composition and structure of magnetite-based NPs is discussed and its role in their biomedical applications is highlighted.

Shell-mediated control of surface chemistry of highly stoichiometric magnetite nanoparticles.

Magnetite (Fe3O4) nanoparticles are one of the most studied nanomaterials for different nanotechnological and biomedical applications. However, Fe3O4 nanomaterials gradually oxidize to maghemite (γ-Fe2O3) under conventional environmental conditions leading to changes in their functional properties that determine their performance in many applications. Here we propose a novel strategy to control the surface chemistry of monodisperse 12 nm magnetite nanoparticles by means of a 3 nm-thick Zn-ferrite epitaxial coating in core/shell nanostructures. We have carried out a combined Mössbauer spectroscopy, dc magnetometry, X-ray photoelectron spectroscopy and spatially resolved electron energy loss spectroscopy study on iron oxide and Fe3O4/Zn0.6Fe2.4O4 core/shell nanoparticles aged under ambient conditions for 6 months. Our results reveal that while the aged iron oxide nanoparticles consist of a mixture of γ-Fe2O3 and Fe3O4, the Zn-ferrite-coating preserves a highly stoichiometric Fe3O4 core. Therefore, the aged core/shell nanoparticles present a sharp Verwey transition, an increased saturation magnetization and the possibility of tuning the effective anisotropy through exchange-coupling at the core/shell interface. The inhibition of the oxidation of the Fe3O4 cores can be accounted for in terms of the chemical nature of the shell layer and an epitaxial crystal symmetry matching between the core and the shell.

The Role of a Double Molecular Anchor on the Mobility and Self-Assembly of Thiols on Au(111): The Case of Mercaptobenzoic Acid.

The dynamics of the self-assembly process of thiol molecules on Au(111) is affected by the interplay between molecule-substrate and molecule-molecule interactions. Therefore, it is interesting to explore the effect of a second anchor to the gold surface, in addition to the S atom, on both the order and the feasibility of phase transitions in self-assembled monolayers. To assess the role of an additional O anchor, we have compared the adsorption of two mercaptobenzoic acid isomers, 2-mercaptobenzoic acid (2-MBA) and 4-mercaptobenzoic acid (4-MBA), on Au(111). Results from scanning tunneling microscopy, X-ray photoelectron spectroscopy, electrochemical techniques, and density functional theory calculations show that the additional O anchor in 2-MBA hinders surface mobility, reducing domain size and impeding the molecular reorganization involved in phase transition to denser phases on the Au(111) substrates. This knowledge can help to predict the range order and molecular density of the thiol SAM depending on the chemical structure of the adsorbate.

The role of the crystalline face in the ordering of 6-mercaptopurine self-assembled monolayers on gold.

Well-ordered molecular films play an important role in nanotechnology, from device fabrication to surface patterning. Self-assembled monolayers (SAMs) of 6-mercaptopurine (6MP) on the Au(100)-(1 × 1) and Au(111)-(1 × 1) have been used to understand the interplay of molecule-substrate interactions for heterocyclic thiols capable of binding to the surface by two anchors, which spontaneously form a highly disordered film on Au(111). Our results reveal that for the same surface coverage the simple change of the substrate from Au(111)-(1 × 1) to Au(100)-(1 × 1) eliminates molecular disorder and yields well-ordered SAMs. We discuss these findings in terms of differences in the surface mobility of 6MP species on these surfaces, the energetics of the adsorption sites, and the number of degrees of freedom of these substrates for a molecule with reduced surface mobility resulting from its two surface anchors. These results reveal the presence of subtle molecule-substrate interactions involving the heteroatom that drastically alter SAM properties and therefore strongly impact on our ability to control physical properties and to build devices at the nanoscale.

Optical Nanoparticle Sorting Elucidates Synthesis of Plasmonic Nanotriangles.

We investigate the optical and morphological properties of gold nanoparticles grown by reducing a gold salt with Na2S. Lasers are tuned to the observed plasmon resonances, and the optical forces exerted on the nanoparticles are used to selectively print individual nanoparticles onto a substrate. This enables us to combine dark-field spectroscopy and scanning electron microscopy to compare the optical properties of single nanoparticles with their morphology. By arresting the synthesis at different times, we are able to investigate which type of nanoparticle is responsible for the respective resonances. We find that thin Au nanotriangles are the source of the observed near infrared (NIR) resonance. The initial lateral growth of these triangles causes the plasmon resonance to redshift into the NIR, whereas a subsequent thickening of the triangles and a concomitant truncation lead to a blueshift of the resonance. Furthermore, we find that the nanotriangles produced have extremely narrow line widths (187 ± 23 meV), show nearly isotropic scattering, and are stable for long periods of time. This shows their vast potential for applications such as in vivo imaging and bio(chemical) sensing. The method used here is generally applicable to other syntheses, and shows how complex nanostructures can be built up on substrates by selectively printing NPs of varying plasmonic resonances.

Citrate-capped silver nanoparticles showing good bactericidal effect against both planktonic and sessile bacteria and a low cytotoxicity to osteoblastic cells.

A common problem with implants is that bacteria can form biofilms on their surfaces, which can lead to infection and, eventually, to implant rejection. An interesting strategy to inhibit bacterial colonization is the immobilization of silver (Ag) species on the surface of the devices. The aim of this paper is to investigate the action of citrate-capped silver nanoparticles (AgNPs) on clinically relevant Gram-positive (Staphylococcus aureus) and Gram-negative (Pseudomonas aeruginosa) bacteria in two different situations: (i) dispersed AgNPs (to assess the effect of AgNPs against planktonic bacteria) and (ii) adsorbed AgNPs on titanium (Ti) substrates, a material widely used for implants (to test their effect against sessile bacteria). In both cases, the number of surviving cells was quantified. The small amount of Ag on the surface of Ti has an antimicrobial effect similar to that of pure Ag surfaces. We have also investigated the capability of AgNPs to kill planktonic bacteria and their cytotoxic effect on UMR-106 osteoblastic cells. The minimum bactericidal concentration found for both strains is much lower than the AgNP concentration that leads to cytotoxicity to osteoblasts. Planktonic P. aeruginosa show a higher susceptibility to Ag than S. aureus, which can be caused by the different wall structures, while for sessile bacteria, similar results are obtained for both strains. This can be explained by the presence of extracellular polymeric substances in the early stages of P. aeruginosa biofilm formation. Our findings can be important to improving the performance of Ti-based implants because a good bactericidal action is obtained with very small quantities of Ag, which are not detrimental to the cells involved in the osseointegration process.

New insight into the electrochemical desorption of alkanethiol SAMs on gold.

A combination of Polarization Modulation Infrared Reflection Absorption Spectroscopy (PMIRRAS) under electrochemical control, Electrochemical Scanning Tunneling Microscopy (ECSTM) and Molecular Dynamics (MD) simulations has been used to shed light on the reductive desorption process of dodecanethiol (C12) and octadecanethiol (C18) SAMs on gold in aqueous electrolytes. Experimental PMIRRAS, ECSTM and MD simulations data for C12 desorption are consistent with formation of randomly distributed micellar aggregates stabilized by Na(+) ions, coexisting with a lying-down phase of molecules. The analysis of pit and Au island coverage before and after desorption is consistent with the thiolate-Au adatoms models. On the other hand, PMIRRAS and MD data for C18 indicate that the desorbed alkanethiolates adopt a Na(+) ion-stabilized bilayer of interdigitated alkanethiolates, with no evidence of lying down molecules. MD simulations also show that both the degree of order and tilt angle of the desorbed alkanethiolates change with the surface charge on the metal, going from bilayers to micelles. These results demonstrate the complexity of the alkanethiol desorption in the presence of water and the fact that chain length and counterions play a key role in a complex structure.

The chemistry of the sulfur-gold interface: in search of a unified model.

Over the last three decades, self-assembled molecular films on solid surfaces have attracted widespread interest as an intellectual and technological challenge to chemists, physicists, materials scientists, and biologists. A variety of technological applications of nanotechnology rely on the possibility of controlling topological, chemical, and functional features at the molecular level. Self-assembled monolayers (SAMs) composed of chemisorbed species represent fundamental building blocks for creating complex structures by a bottom-up approach. These materials take advantage of the flexibility of organic and supramolecular chemistry to generate synthetic surfaces with well-defined chemical and physical properties. These films already serve as structural or functional parts of sensors, biosensors, drug-delivery systems, molecular electronic devices, protecting capping for nanostructures, and coatings for corrosion protection and tribological applications. Thiol SAMs on gold are the most popular molecular films because the resulting oxide-free, clean, flat surfaces can be easily modified both in the gas phase and in liquid media under ambient conditions. In particular, researchers have extensively studied SAMs on Au(111) because they serve as model systems to understand the basic aspects of the self-assembly of organic molecules on well-defined metal surfaces. Also, great interest has arisen in the surface structure of thiol-capped gold nanoparticles (AuNPs) because of simple synthesis methods that produce highly monodisperse particles with controllable size and a high surface/volume ratio. These features make AuNPs very attractive for technological applications in fields ranging from medicine to heterogeneous catalysis. In many applications, the structure and chemistry of the sulfur-gold interface become crucial since they control the system properties. Therefore, many researchers have focused on understanding of the nature of this interface on both planar and nanoparticle thiol-covered surfaces. However, despite the considerable theoretical and experimental efforts made using various sophisticated techniques, the structure and chemical composition of the sulfur-gold interface at the atomic level remains elusive. In particular, the search for a unified model of the chemistry of the S-Au interface illustrates the difficulty of determining the surface chemistry at the nanoscale. This Account provides a state-of-the-art analysis of this problem and raises some questions that deserve further investigation.

Electrochemical preparation and delivery of melanin-iron covered gold nanoparticles.

Attractive combination: Biopolymer-modified nanoparticles which combine magnetic properties with biocompatibility are prepared and delivered following a three-step strategy (see figure): i) Adsorption of thiol-capped metal nanoparticles on graphite, ii) electrochemical modification, iii) potential-induced delivery of the modified nanoparticles to the electrolyte. Thiol-capped gold nanoparticles modified with iron-melanin are attractive because they combine magnetic properties and biocompatibility. The biopolymer modified nanoparticles are prepared and delivered following a three step strategy: i) adsorption of thiol-capped metal nanoparticles on graphite, ii) electrochemical deposition of melanin-iron, iii) potential-induced delivery of the modified nanoparticles to the electrolyte.

Mechanical properties of alkanethiol monolayers studied by force spectroscopy.

The mechanical properties of alkanethiol monolayers on Au(111) in KOH solution have been studied by force spectroscopy. The analysis of the vertical force versus penetration curves showed that monolayer penetration is a stepped process that combines elastic regions with sudden penetration events. The structural meaning of these events can be explained both by the creation of gauche defects on the hydrocarbon chains and by a cooperative molecular tilting model proposed by Barrena et al. [J. Chem. Phys. 113, 2413 (2000)]. The validity of these models for alkanethiol monolayers of different compactness and chain length has been discussed. The Young's modulus (E) of the monolayers has been calculated by using a recently developed model which considers the thickness of the monolayer as a parameter, thus allowing a decoupling of the mechanical properties of the thiol layer from those of the Au(111) substrate. As a result, the calculated E values are in the range of 50-150 Pa, which are remarkably lower than those previously reported in the literature.

Two-site adsorption model for the (square root 3 x square root 3)-R30 degrees dodecanethiolate lattice on Au(111) surfaces.

The surface structure of dodecanethiolate self-assembled monolayers (SAMs) on Au(111) surfaces, formed from the liquid phase, have been studied by grazing incidence X-ray diffraction (GIXRD), scanning tunneling microscopy (STM), and electrochemical techniques. STM images show that the surface structure consists of (square root 3 x square root 3)-R30 degrees domains with only a few domains of the c(4 x 2) lattice. The best fitting of GIXRD data for the (square root 3 x square root 3)-R30 degrees lattice is obtained with alkanethiolate adsorption at the top sites, although good fittings are also obtained for the fcc and hcp hollow sites. On the basis of this observation, STM data, electrochemical measurements, and previously reported data, we propose a two-site model that implies the formation of incoherent domains of alkanethiolate molecules at top and fcc hollow sites. This model largely improves the fitting of the GIXRD data with respect to those observed for single adsorption sites and, also, for the other possible two-site combinations. The presence of alkanethiolate molecules adsorbed at the less favorable top sites could result from the adsorption pathway that involves an initial physisorption step which, for steric reasons, takes place at on top sites. Once the molecules are chemisorbed, the presence of energy barriers for alkanethiolate surface diffusion, arising mostly from chain-chain interactions, "freezes" some of them at the on top sites, hindering their movement toward fcc hollow sites. By considering the length of the hydrocarbon chain and the adsorption time, the two-site model could be a tool to explain most of the controversial results on this matter reported in the literature.

Exploring three-dimensional nanosystems with Raman spectroscopy: methylene blue adsorbed on thiol and sulfur monolayers on gold.

Resonant Raman and surface-enhanced Raman scattering (SERS) spectroscopies, complemented with scanning tunnel microscopy and electrochemical techniques, have been used to obtain information about the amount and spatial distribution of methylene blue (MB) molecules immobilized on sulfur and four ultrathin molecular alkanethiolate films self-assembled on Au(111) and rough Au electrodes. The intensity of the Raman signals allow one to estimate the amount of immobilized MB at different organic films, whereas the decrease in the SERS intensity as a function of distance for the rough Au electrodes is used to locate the average position of the MB species with respect to the Au substrate. We found that significant amounts of cationic MB species are able to diffuse into methyl-terminated thiols, but they are stopped at the outer plane of the self-assembled monolayer (SAM) by negatively charged carboxylate groups. The relative shift of C-N stretching Raman modes indicates that the binding of MB to S is different from that found for MB on thiols. Most of the molecules immobilized on methyl- and carboxylate-terminated thiols are electrochemically inactive, suggesting that strong coupling between the Au electrode and the MB molecules is needed for charge transfer. Our results are consistent with a small population of electrochemically active MB species very close to the Au surface that reach this position driven by their lipophilic (hydrophobic) character through defects at SAMs.