Measuring the Radius of Gyration and Intrinsic Flexibility of Viral Proteins in Buffer Solution Using Small-Angle X-ray Scattering

Measuring structural features of proteins dispersed in buffer solution, in contrast to crystal form, is indispensable in understanding morphological characteristics of the biomolecule in a native environment. We report on the structure and apparent viscosity of unfolded α and β variants of SARS-CoV-2 spike proteins dispersed in buffer solutions. The radius of gyration of the β variant is found to be larger than that of the α variant, while the ab initio computation of one of the possible particle-like bodies is consistent with the small-angle X-ray scattering (SAXS) profiles resembling a conformation similar to the three-dimensional structure of the folded state of the corresponding α and β spike variant. However, a smaller radius of gyration with respect to the predicted folded state of 2.4 and 2.7 is observed for both α and β variants, respectively. Our work complements the structural characterization of spike proteins using cryo-electron microscopy techniques. The measurement/analysis discussed here might be useful for quick and cost-effective evaluation of several protein structures, let alone mutated viral proteins, which is useful for drug discovery/development applications

Detecting Escherichia coli Biofilm Development Stages on Gold and Titanium by Quartz Crystal Microbalance

Bacterial biofilms are responsible for persistent infections and biofouling, raising serious concerns in both medical and industrial processes. These motivations underpin the need to develop methodologies to study the complex biological structures of biofilms and prevent their formation on medical implants, tools, and industrial apparatuses. Here, we report the detailed comparison of Escherichia coli biofilm development stages (adhesion, maturation, and dispersion) on gold and titanium surfaces by monitoring the changes in both frequency and dissipation of a quartz crystal microbalance (QCM) device, a cheap and reliable microgravimetric sensor which allows the real-time and label-free characterization of various stages of biofilm development. Although gold is the most common electrode material used for QCM sensors, the titanium electrode is also readily available for QCM sensors; thus, QCM sensors with different metal electrodes serve as a simple platform to probe how pathogens interact with different metal substrates. The QCM outcomes are further confirmed by atomic force microscopy and crystal violet staining, thus validating the effectiveness of this surface sensitive sensor for microbial biofilm research. Moreover, because QCM technology can easily modify the substrate types and coatings, QCM sensors also provide well-controlled experimental conditions to study antimicrobial surface treatments and eradication procedures, even on mature biofilms

Sensing Dynamically Evolved Short‐Range Nanomechanical Forces in Fast‐Mutating Single Viral Spike Proteins

Understanding changes in the mechanical features of a single protein from a mutated virus while establishing its relation to the point mutations is critical in developing new inhibitory routes to tackle the uncontrollable spread of the virus. Addressing this, herein, the chemomechanical features of a single spike protein are quantified from alpha, beta, and gamma variants of SARS-CoV-2. Integrated amplitude-modulation atomic force microscopy is used with dynamic force–distance curve (FDC) spectroscopy, in combination with theoretical models, to quantify Young's modulus, stiffness, adhesion forces, van der Waals forces, and the dissipative energy of single spike proteins. These obtained nanomechanical properties can be correlated with mutations in the individual proteins. Therefore, this work opens new possibilities to understand how the mechanical properties of a single spike protein relate to the viral functions. Additionally, single-protein nanomechanical experiments enable a variety of applications that, collectively, may build up a new portfolio of understanding protein biochemistry during the evolution of viruses

PSO-based tuning of MURAME parameters for creditworthiness evaluation of Italian SMEs

In this work we use a MultiCriteria Decision Analysis (MCDA) model to evalu- ate the creditworthiness of a sample of Italian Small and Medium-sized Enterprises (SMEs), on the basis of their balance sheet data provided by the AIDA database. Our methodology is able to consider simultaneously different factors affecting the firms’ solvency level, and can produce results in terms of scoring, classification into homogeneous rating classes and migration probabilities. In this contribution we compare the results obtained considering two scenarios. On one hand, we experience an exogenous specification of the parameters that describe the preference structure implicit in the used MCDA model. On the other hand, we consider the results obtained using a preference disaggregation method to endogenously determine some of the model parameters. Because of the complexity of the obtained math- ematical programming problem, we use an heuristic methodology, namely Particle Swarm Optimization (PSO), which provides a reasonable compromise between the quality of the solution and the computational burden

Detection of parathion and patulin by quartz-crystal microbalance functionalized by the photonics immobilization technique

Oriented antibodies are tethered on the gold surface of a quartz crystal microbalance through the photonics immobilization technique so that limit of detection as low as 50 nM and 140 nM are achieved for parathion and patulin, respectively. To make these small analytes detectable by the microbalance, they have been weighed down through a “sandwich protocol” with a second antibody. The specificity against the parathion has been tested by checking the immunosensor response to a mixture of compounds similar to parathion, whereas the specificity against the patulin has been tested with a real sample from apple puree. In both cases, the results are more than satisfactory suggesting interesting outlook for the proposed device

Rheology of the Electric Double Layer in Electrolyte Solutions

Electric double layers (EDLs) are ionic structures formed on charged surfaces and play an important role in various biological and industrial processes. An extensive study in the past decade has revealed the structure of the EDL in concentrated electrolyte solutions of both ordinary salts and ionic liquids. However, how the EDL structure affects their material properties remains a challenging topic due to technical difficulties of these measurements at nanoscale. In this work, we report the first detailed characterization of the viscoelasticity of the EDL formed over a wide range of ion concentrations, including concentrated electrolyte solutions. Specifically, we investigate the complex shear modulus of the EDL by measuring the resonant frequency and the energy dissipation of a quartz crystal microbalance (QCM), a surface-sensitive device, immersed in aqueous solutions containing three types of solutes: an ionic liquid, 1-butyl-3-methylimidazolium chloride (BmimCl); an ordinary salt, sodium chloride (NaCl); and a nonelectrolyte, ethylene glycol (EG). For the two electrolyte solutions, we observe a monotonic decrease in the resonant frequency and a monotonic increase in the energy dissipation with increasing ion concentrations due to the presence of the EDL. The complex shear modulus of the EDL is estimated through a wave propagation model in which the density and shear modulus of the EDL decay exponentially toward those of the bulk solution. Our results show that both the storage and the loss modulus of the EDL increase rapidly with increasing ion concentrations in the low ion concentration regime (<1 M) but reach saturation values with similar magnitude at a sufficiently high ion concentration. The shear viscosity of the EDL near the charged QCM surface is approximately 50 times for NaCl solutions and 500 times for BmimCl solutions of the bulk solution value at the saturation concentration. We also demonstrate that QCM can be utilized for analyzing the rheological properties of the EDL, thus providing a complementary, low-cost, and portable alternative to conventional laboratory instruments such as the surface force apparatus. Our results elucidate new perspectives on the viscoelastic properties of the EDL and can potentially guide device optimization for applications such as biosensing and fast charging of batteries

Nanoplasmonics for Real-Time and Label-Free Monitoring of Microbial Biofilm Formation

Microbial biofilms possess intrinsic resistance against conventional antibiotics and cleaning procedures; thus, a better understanding of their complex biological structures is crucial in both medical and industrial applications. Existing laboratory methodologies have focused on macroscopic and mostly indirect characterization of mechanical and microbiological properties of biofilms adhered on a given substrate. However, the kinetics underlying the biofilm formation is not well understood, while such information is critical to understanding how drugs and chemicals influence the biofilm formation. Herein, we report the use of localized surface plasmon resonance (LSPR) for real-time, label-free monitoring of E. coli biofilm assembly on a nanoplasmonic substrate consisting of gold mushroom-like structures. Our LSPR sensor is able to capture the signatures of biofilm formation in real-time by measuring the wavelength shift in the LSPR resonance peak with high temporal resolution. We employ this sensor feature to elucidate how biofilm formation is affected by different drugs, including conventional antibiotics (kanamycin and ampicillin) as well as rifapentine, a molecule preventing cell adhesion yet barely affecting bacterial viability and vitality. Due to its flexibility and simplicity, our LSPR based platform can be used on a wide variety of clinically relevant bacteria, thus representing a valuable tool in biofilm characterization and drug screening

High sensitive sensing by effective immobilization of UV photo-activated antibodies

Nowadays there is a strong interest in precise and reliable measurement tools suitable for quantifying physical, chemical, and biological properties. Biosensors face this problem by exploiting the intrinsic specificity provided by biological sensitive molecules to reveal the presence of the compound of interest. In particular, proteins like antibodies have a dominant role in biosensor development since they are selected by host immune system to efficiently bind foreign species including bacteria, viruses and toxins. Biomolecules involved in biosensing are usually characterized by a recognition site responsible for the selective detection of the analyte. This portion of the macromolecule has to be accessible when the sensitive element is coupled with the inorganic transducer, thus making surface functionalization a crucial phase of biosensor development. This issue strongly motivates the research of new immobilization and functionalization techniques allowing the control on both amount and orientation of the biomolecules thus resulting in better sensitivity and lower limit of detection. Conventional functionalization strategies are based on covalent and non-covalent interactions between the biological element and the surface of the transducer. Even if covalent approaches provide an effective immobilization of the biomolecules, these methods are laborious and time-consuming since several chemical treatments and purification steps are needed. In addition, the high toxicity of some chemicals and the complexity of the procedure require trained operators. On the other side, non-covalent immobilization is much easier to realize since it involves the spontaneous adsorption of the biomolecules onto the substrate. It is worth mentioning that in this case uncontrolled adsorption usually results in irregular layers and compromised recognition of the analyte due to steric hindrance of the binding sites. In addition, weak connections like van der Waals and hydrogen bonding interactions sometimes do not provide a stable immobilization onto the sensor surface. To face this issue, at the Physics Department of University of Naples "Federico II", an all optical technique (PIT, Photonic Immobilization Technique) based on the interaction of ultrashort UV pulses with antibodies has been proposed as a simple and rapid approach capable to effectively functionalize the sensitive surface of a quartz crystal microbalance. In this thesis, PIT has been used to realize immunosensors for the detection of a group of analytes of practical interest. This functionalization technology provides an effective immobilization of antibodies onto the gold sensor surface upon activation of the protein sample through the selective photoreduction of the disulphide bridge in the triad cysteine-cysteine/tryptophan, a typical structural feature of the immunoglobulins. The absorption of ultrashort UV laser pulses required for this activation process does not affect the recognition properties of the antibodies. On the other side, the free thiol groups so produced interact with gold surface thus leading to the effective exposure of the sensitive portions of the protein, the so-called antigen binding sites, thus greatly improving the detection efficiency. The effects of this unconventional functionalization approach on immunoglobulins have been investigated by means of optical techniques, atomic force microscopy and the so-called Ellman's assay, a chemical method used to quantify the thiol groups in a protein sample. PIT based immunosensors have proven to be effective in the detection of small toxic molecules like parathion (pesticide) and patulin (micotoxin). The issue of revealing these light molecules using a microgravimetric transducer like a quartz crystal microbalance have been overcome by ballasting the analytes using two labelling procedures involving either bovine serum albumin or an antibody in a sandwich-type configuration. PIT has been used also to realize an immunosensor for the detection of gliadin, the principal responsible for the coeliac disease. In all these cases, both sensitivity and limit of detection (usually in nanomolar concentration range) result to be in line with the limits set by current regulations and comparable or even better than other techniques used to quantify these harmful molecules. These promising results make PIT a valuable functionalization method for technologies involving gold surfaces for sensing and detection purposes

Detection and Characterization of Bacterial Biofilms and Biofilm-Based Sensors

Microbial biofilms have caused serious concerns in healthcare, medical, and food industries because of their intrinsic resistance against conventional antibiotics and cleaning procedures and their capability to firmly adhere on surfaces for persistent contamination. These global issues strongly motivate researchers to develop novel methodologies to investigate the kinetics underlying biofilm formation, to understand the response of the biofilm with different chemical and physical treatments, and to identify biofilm-specific drugs with high-throughput screenings. Meanwhile microbial biofilms can also be utilized positively as sensing elements in cell-based sensors due to their strong adhesion on surfaces. In this perspective, we provide an overview on the connections between sensing and microbial biofilms, focusing on tools used to investigate biofilm properties, kinetics, and their response to chemicals or physical agents, and biofilm-based sensors, a type of biosensor using the bacterial biofilm as a biorecognition element to capture the presence of the target of interest by measuring the metabolic activity of the immobilized microbial cells. Finally we discuss possible new research directions for the development of robust and rapid biofilm related sensors with high temporal and spatial resolutions, pertinent to a wide range of applications