ABSTRACT
The advantages of the complex of recombinant human lactoferrin (rhLF) with europium ions have been used to establish quantitative parameters of specific interaction of rhLF with immobilized heparin-protein conjugate as a model of cell-surface heparan sulfate proteoglycans. Heparin coupled through terminal formyl by reductive amination to an inert protein was adsorbed through the protein part in the wells of a polystyrene microplate. The rhLF-Eu3+ complex obtained from native rhLF contains 0.8 mol of lanthanide ion per mol of protein (40 % saturation level). Equilibrium in the heterophase binding system is established within 1 min at room temperature, and the calculated association constant of the rhLF-heparin complex is 2.1. 107 M-1. The reversible and saturable character of binding rhLF labeled by Eu3+ at the active site to heparin was confirmed by the transition of rhLF-Eu3+ into the liquid phase when a 1000-fold molar excess of unlabeled rhLF was added to the system. Based on the affinity of rhLF for glycosaminoglycan, a blocking effect of this protein on the binding of the SARS-CoV-2 virus to the immobilized heparin-protein conjugate that imitates proteoglycan on the host cell surface was revealed. Pretreatment of the adsorbed conjugate with a solution of rhLF (10 mu g per well) reduces the specific binding of 100 ng of viral particles added to the well by approximately 80 %. The presented results allow one, in particular, to evaluate the integrity of the structure and activity of rhLF as a possible substance in food supplements and pharmaceuticals and may be useful in developing combined drugs for corona virus infection.
ABSTRACT
We report two novel genosensors for the quantification of SARS-CoV-2 nucleic acid using glassy carbon electrodes modified with a biocapture nanoplatform made of multi-walled carbon nanotubes (MWCNTs) non-covalently functionalized with avidin (Av) as a support of the biotinylated-DNA probes. One of the genosensors was based on impedimetric transduction offering a non-labelled and non-amplified detection of SARS-CoV-2 nucleic acid through the increment of [Fe(CN)6]3-/4- charge transfer resistance. This biosensor presented an excellent analytical performance, with a linear range of 1.0 × 10-18 M - 1.0 × 10-11 M, a sensitivity of (5.8 ± 0.6) x 102 Ω M-1 (r2 = 0.994), detection and quantification limits of 0.33 aM and 1.0 aM, respectively; and reproducibilities of 5.4% for 1.0 × 10-15 M target using the same MWCNTs-Av-bDNAp nanoplatform, and 6.9% for 1.0 × 10-15 M target using 3 different nanoplatforms. The other genosensor was based on a sandwich hybridization scheme and amperometric transduction using the streptavidin(Strep)-biotinylated horseradish peroxidase (bHRP)/hydrogen peroxide/hydroquinone (HQ) system. This genosensor allowed an extremely sensitive quantification of the SARS-CoV-2 nucleic acid, with a linear range of 1.0 × 10-20 M - 1.0 × 10-17 M, detection limit at zM level, and a reproducibility of 11% for genosensors prepared with the same MWCNTs-Av-bDNAp1 nanoplatform. As a proof-of-concept, and considering the extremely high sensitivity, the genosensor was challenged with highly diluted samples obtained from SARS-CoV-2 RNA PCR amplification.
ABSTRACT
Respiratory illness caused by influenza virus is a serious public health problem worldwide. As the symptoms of influenza virus infection are similar to those of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, it is essential to distinguish these two viruses. Therefore, to properly respond to a pathogen, a detection method that is capable of rapid and accurate diagnosis in a hospital or at home is required. To satisfy this need, we applied loop-mediated isothermal amplification (LAMP) and an isothermal nucleic acid amplification technique, along with a system to analyze the results without specialized equipment, a lateral flow assay (LFA). Using the platform developed in this study, all processes, from sample preparation to detection, can be performed without special equipment. Unlike existing PCR methods, the nucleic acid amplification can be performed in the field because hot packs do not require electricity. Thus, the designed platform can provide rapid results without the need to transport the samples to a laboratory or hospital. These advantages are not limited to operations in developing countries with poor access to medical systems. In conclusion, the developed technology is a promising tool for infectious disease management that allows for rapid identification of infectious diseases and appropriate treatment of patients.