Purpose: To develop a general method for NP fabrication from various proteins with maintenance of biological activity. Methods: A novel general approach for producing protein nanoparticles (NP) by nanoprecipitation of the protein solutions in 1,1,1,3,3,3-hexafluoroisopropanol is described. Protein NP sizes and shapes were analyzed by dynamic light scattering, scanning electron and atomic force microscopy (SEM and AFM). Chemical composition of the NP was confirmed using ultraviolet (UV) spectroscopy, energy-dispersive X-ray spectroscopy (EDX) and circular dichroism (CD). Biological properties of the NP were analyzed in ELISA, immunofluorescent analysis and lysozyme activity assay. Results: Water-insoluble NP were constructed from globular (bovine serum albumin (BSA), lysozyme, immunoglobulins), fibrillar (fibrinogen) proteins and linear polylysines by means of nanoprecipitation of protein solutions in fluoroalcohols. AFM and SEM revealed NP sizes of 20–250 nm. The NP chemical structure was confirmed by UV spectroscopy, protease digestion and EDX spectroscopy. CD spectra revealed a stable secondary structure of proteins in NP. The UV spectra, microscopy and SDS-PAA gel electrophoresis (PAGE) proved the NP stability at +4°C for 7 months. Co-precipitation of proteins with fluorophores or nanoprecipitation of pre-labeled BSA resulted in fluorescent NP that retained antigenic structures as shown by their binding with specific antibodies. Moreover, NP from monoclonal antibodies could bind with the hepatitis B virus antigen S. Besides that, lysozyme NP could digest bacterial cellular walls. Conclusion: Thus, the water-insoluble, stable protein NP were produced by nanoprecipitation without cross-linking and retained ligand-binding and enzymatic activities.

Here, we propose and study several types of quartz surface coatings designed for the high-performance sorption of biomolecules and their subsequent detection by a photonic crystal surface mode (PC SM) biosensor. The deposition and sorption of biomolecules are revealed by analyzing changes in the propagation parameters of optical modes on the surface of a photonic crystal (PC). The method makes it possible to measure molecular and cellular affinity interactions in real time by independently recording the values of the angle of total internal reflection and the angle of excitation of the surface wave on the surface of the PC. A series of dextrans with various anchor groups (aldehyde, carboxy, epoxy) suitable for binding with bioligands have been studied. We have carried out comparative experiments with dextrans with other molecular weights. The results confirmed that dextran with a Mw of 500 kDa and anchor epoxy groups have a promising potential as a matrix for the detection of proteins in optical biosensors. The proposed approach would make it possible to enhance the sensitivity of the PC SM biosensor and also permit studying the binding process of low molecular weight molecules in real time.

The process of stone formation in the human body remains incompletely understood, which requires clinical and laboratory studies and the formulation of a new endogenous, nanotechnological concept of the mechanism of origin and formation of crystallization centers. Previously, the mechanism of sialolithiasis was considered a congenital disease associated with the pathology of the ducts in the structure of the glands themselves. To date, such morphological changes of congenital nature can be considered from the position of the intrauterine formation of endogenous bacterial infections complicated by the migration of antigenic structures initiating the formation of crystallization centers. The present work is devoted to the study of the morphology and composition of stones obtained as a result of surgical interventions for sialolithiasis. Presumably, nanoparticles of metals and other chemical compounds can be structural components of crystallization centers or incorporated into the conditions of chronic endogenous inflammation and the composition of antigenic structures, in complexes with protein and bacterial components. X-ray microtomography, X-ray fluorescence analysis, scanning transmission electron microscopy and microanalysis, mass spectrometry, and Raman spectroscopy were used to study the pathogenesis of stone formation. Immunoglobulins (Igs) of classes A and G, as well as nanoparticles of metals Pb, Fe, Cr, and Mo, were found in the internal structure of the stones. The complex of antigenic structures was an ovoid calcified layered matrix of polyvid microbial biofilms, with the inclusion of metal nanoparticles and chemical elements, as well as immunoglobulins. The obtained results of clinical and laboratory studies allow us to broaden the view on the pathogenesis of stone formation and suggest that the occurrence of the calcification of antigenic structures may be associated with the formation of IgG4-associated disease.

Background: The pandemic of the new coronavirus infection, COVID-19, is currently ongoing in the world. Over the years, the pathogen, SARS-CoV-2, has undergone a series of mutational genome changes, which has led to the spread of various genetic variants of the virus. Meanwhile, the methods used to diagnose SARS-CoV-2, to establish the disease stage and to assess the immunity, are nonspecific to SARS-CoV-2 variants and time-consumable. Thus, the development of new methods for diagnosing COVID-19, as well as their implementation in practice, is currently an important direction. In particular, application of systems based on chemically modified fluorescent microspheres (with a multiplex assay for target protein molecules) opens great opportunities. Aim: development of a microfluidic diagnostic test system based on fluorescent microspheres for the specific detection of immunoglobulins G (IgG) to SARS-CoV-2. Methods: A collection of human serum samples was characterized using enzyme-linked immunosorbent assay (ELISA) and commercially available reagent kits. IgG to SARS-CoV-2 in the human serum were detected by the developed immunofluorescent method using microspheres containing the chemically immobilized RBD fragment of the SARS-CoV-2 (Kappa variant) viral S-protein. Results: The level of IgG in the blood serum of recovered volunteers was 9-300 times higher than that in apparently healthy volunteers, according to ELISA (p0.001). Conjugates of fluorescent microspheres with the RBD-fragment of the S-protein, capable of specifically binding IgG from the blood serum, have been obtained. The immune complexes formation was confirmed by the fluorescence microscopy data; the fluorescence intensity of secondary antibodies in the immune complexes formed on the surface of microspheres was proportional to the content of IgG (r 0.963). The test system had a good predictive value (AUC 70.3%). Conclusion: A test system has been developed, based on fluorescent microspheres containing the immobilized RBD fragment of the SARS-CoV-2 S-protein, for the immunofluorescent detection of IgG in the human blood serum. When testing the system on samples with different levels of IgG to SARS-CoV-2, its prognostic value was shown. The obtained results allow us to present the test system as a method to assess the level of immunoglobulins to SARS-CoV-2 in the human blood serum for the implementation in clinical practice. The test system can also be integrated into various microfluidic systems to create chips and devices for the point-of-care diagnostics.

Nowadays, biosensor platforms based on various organic electrolytic transistors are in great demand due to their ability to specifically determine a wide range of biological analytes with extreme sensitivity. The main drawback of such platforms is their disposability at relatively high costs, preventing widespread application. In this work, we elaborate and successfully demonstrate a proof of concept for a universal approach for the fabrication of a reusable EGOFET-based aptasensor using polymer track-etched membranes as cheap disposable elements for biorecognition layers. We defined the most suitable pore size of track-etched membranes, which was enough for the penetration of viral particle and their capture near the current-carrying layer of EGOFET. The sensitivity of the fabricated EGOFET-based aptasensor to influenza A virus was comparable with disposal EGOFET-based biosensors having a biorecognition layer placed directly on the semiconducting layer. The limit of detection of the fabricated device was 8 × 10⁴ VP mL⁻¹, which was superior to those of antibody-based rapid analysis test systems (1 × 10⁶–4 × 10⁸ VP mL⁻¹) but inferior to those of the PCR method (3 × 10²–1.2 × 10³ VP mL⁻¹). The elaborated approach paves the way for the simple development of universal point-of-care tools consisting of rapid, non-invasive miniaturized sensors for the detection of a wide range of analytes, which are recognizable by aptamers.

We propose and demonstrate dendrimer-based coatings for a sensitive biochip surface that enhance the high-performance sorption of small molecules (i.e., biomolecules with low molecular weights) and the sensitivity of a label-free, real-time photonic crystal surface mode (PC SM) biosensor. Biomolecule sorption is detected by measuring changes in the parameters of optical modes on the surface of a photonic crystal (PC). We describe the step-by-step biochip fabrication process. Using oligonucleotides as small molecules and PC SM visualization in a microfluidic mode, we show that the PAMAM (poly-amidoamine)-modified chip’s sorption efficiency is almost 14 times higher than that of the planar aminosilane layer and 5 times higher than the 3D epoxy-dextran matrix. The results obtained demonstrate a promising direction for further development of the dendrimer-based PC SM sensor method as an advanced label-free microfluidic tool for detecting biomolecule interactions. Current label-free methods for small biomolecule detection, such as surface plasmon resonance (SPR), have a detection limit down to pM. In this work, we achieved for a PC SM biosensor a Limit of Quantitation of up to 70 fM, which is comparable with the best label-using methods without their inherent disadvantages, such as changes in molecular activity caused by labeling.