SPR and Double Resonance LPG Biosensors for Helicobacter pylori BabA Antigen Detection.

Given the medical and social significance of Helicobacter pylori infection, timely and reliable diagnosis of the disease is required. The traditional invasive and non-invasive conventional diagnostic techniques have several limitations. Recently, opportunities for new diagnostic methods have appeared based on the recent advance in the study of H. pylori outer membrane proteins and their identified receptors. In the present study we assess the way in which outer membrane protein-cell receptor reactions are applicable in establishing a reliable diagnosis. Herein, as well as in other previous studies of ours, we explore the reliability of the binding reaction between the best characterized H. pylori adhesin BabA and its receptor, the blood antigen Leb. For the purpose we developed surface plasmon resonance (SPR) and double resonance long period grating (DR LPG) biosensors based on the BabA-Leb binding reaction for diagnosing H. pylori infection. In SPR detection, the sensitivity was estimated at 3000 CFU/mL-a much higher sensitivity than that of the RUT test. The DR LPG biosensor proved to be superior in terms of accuracy and sensitivity-concentrations as low as 102 CFU/mL were detected.

Binding of SARS-CoV-2 Structural Proteins to Hemoglobin and Myoglobin Studied by SPR and DR LPG.

One of the first clinical observations related to COVID-19 identified hematological dysfunctions. These were explained by theoretical modeling, which predicted that motifs from SARS-CoV-2 structural proteins could bind to porphyrin. At present, there is very little experimental data that could provide reliable information about possible interactions. The surface plasmon resonance (SPR) method and double resonance long period grating (DR LPG) were used to identify the binding of S/N protein and the receptor bind domain (RBD) to hemoglobin (Hb) and myoglobin (Mb). SPR transducers were functionalized with Hb and Mb, while LPG transducers, were only with Hb. Ligands were deposited by the matrix-assisted laser evaporation (MAPLE) method, which guarantees maximum interaction specificity. The experiments carried out showed S/N protein binding to Hb and Mb and RBD binding to Hb. Apart from that, they demonstrated that chemically-inactivated virus-like particles (VLPs) interact with Hb. The binding activity of S/N- and RBD proteins was assessed. It was found that protein binding fully inhibited heme functionality. The registered N protein binding to Hb/Mb is the first experimental fact that supports theoretical predictions. This fact suggests another function of this protein, not only binding RNA. The lower RBD binding activity reveals that other functional groups of S protein participate in the interaction. The high-affinity binding of these proteins to Hb provides an excellent opportunity for assessing the effectiveness of inhibitors targeting S/N proteins.

A Surface Plasmon Resonance Biosensor Based on Directly Immobilized Hemoglobin and Myoglobin.

Immobilization of proteins on a surface plasmon resonance (SPR) transducer is a delicate procedure since loss of protein bioactivity can occur upon contact with the untreated metal surface. Solution to the problem is the use of an immobilization matrix having a complex structure. However, this is at the expense of biosensor selectivity and sensitivity. It has been shown that the matrix-assisted pulsed laser evaporation (MAPLE) method has been successfully applied for direct immobilization (without a built-in matrix) of proteins, preserving their bioactivity. So far, MAPLE deposition has not been performed on a gold surface as required for SPR biosensors. In this paper we study the impact of direct immobilization of heme proteins (hemoglobin (Hb) and myoglobin (Mb)) on their bioactivity. For the purpose, Hb and Mb were directly immobilized by MAPLE technique on a SPR transducer. The bioactivity of the ligands immobilized in the above-mentioned way was assessed by SPR registration of the molecular reactions of various Hb/Mb functional groups. By SPR we studied the reaction between the beta chain of the Hb molecule and glucose, which shows the structural integrity of the immobilized Hb. A supplementary study of films deposited by FTIR and AFM was provided. The experimental facts showed that direct immobilization of an intact molecule was achieved.

Dataset of MAPLE Parameters for Hemoglobin Deposition upon long period gratings.

Matrix-assisted pulsed laser evaporation (MAPLE) is an alternative and complimentary method to pulsed laser deposition. MAPLE has been demonstrated to be a less harmful approach for transporting and depositing delicate, highly sensitive molecules. Metalloproteins are considered sensitive molecules since their bioactivity is determined not only by their chemical structure but also by conformational changes that can be altered by deposition methods. Here we report a dataset of MAPLE deposition parameters of haemoglobin (Hb) that ensures the retention of its bioactivity. Methods for parameters optimization are also described. The data and analysis should be valuable for researchers interested in application of MAPLE techniques for metalloprotein immobilization since it provides a unique opportunity for direct immobilization. The data presents the results of previously conducted experiments on the basis of which is based the research article entitled "A Highly Efficient Biosensor based on MAPLE Deposited Hemoglobin on LPGs Around Phase Matching Turning Point" [1].

Crystalline inverted membranes grown on surfaces by electrospray ion beam deposition in vacuum.

Crystalline inverted membranes of the nonvolatile surfactant sodium dodecylsulfate are found on solid surfaces after electrospray ion beam deposition (ES-IBD) of large SDS clusters in vacuum. This demonstrates the equivalence of ES-IBD to conventional molecular beam epitaxy.

A close look at proteins: submolecular resolution of two- and three-dimensionally folded cytochrome c at surfaces.

Imaging of individual protein molecules at the single amino acid level has so far not been possible due to the incompatibility of proteins with the vacuum environment necessary for high-resolution scanning probe microscopy. Here we demonstrate electrospray ion beam deposition of selectively folded and unfolded cytochrome c protein ions on atomically defined solid surfaces in ultrahigh vacuum (10(-10) mbar) and achieve unprecedented resolution with scanning tunneling microscopy. On the surface folded proteins are found to retain their three-dimensional structure. Unfolded proteins are observed as extended polymer strands displaying submolecular features with resolution at the amino acid level. On weakly interacting surfaces, unfolded proteins refold into flat, irregular patches composed of individual molecules. This suggests the possibility of two-dimensionally confined folding of peptides of an appropriate sequence into regular two-dimensional structures as a new approach toward functional molecular surface coatings.

The quantum magnetism of individual manganese-12-acetate molecular magnets anchored at surfaces.

The high intrinsic spin and long spin relaxation time of manganese-12-acetate (Mn(12)) makes it an archetypical single molecular magnet. While these characteristics have been measured on bulk samples, questions remain whether the magnetic properties replicate themselves in surface supported isolated molecules, a prerequisite for any application. Here we demonstrate that electrospray ion beam deposition facilitates grafting of intact Mn(12) molecules on metal as well as ultrathin insulating surfaces enabling submolecular resolution imaging by scanning tunneling microscopy. Using scanning tunneling spectroscopy we detect spin excitations from the magnetic ground state of the molecule at an ultrathin boron nitride decoupling layer. Our results are supported by density functional theory based calculations and establish that individual Mn(12) molecules retain their intrinsic spin on a well chosen solid support.

Experimental and theoretical investigations of the thermodynamic stability of Ba-c(60) and K-C(60) compound clusters.

A novel experimental set-up was used to study superstable (magic) Ba-C(60) and K-C(60) compound clusters. The most stable systems observed cannot be rationalized by simple electronic or by geometrical shell filling arguments. Annealing the clusters past the temperature necessary for the fragmentation of the initial metastable clusters formed at the source reveals information about their thermodynamic stability. Higher temperatures yield larger species, suggesting that similar experiments may be used to rationally produce nanoscale clusters with highly desirable properties. Density functional calculations reveal ionic (K, Ba) and covalent (Ba) bonding between C(60) and the metal atoms. The entropic contribution to the Gibbs free energy is shown to be essential in determining absolute and relative cluster stabilities. In particular, we demonstrate that at higher temperatures the entropy favors the formation of larger clusters. A simple criterion which may be used to determine the absolute and relative stabilities of general multicomponent clusters is proposed.