Urate oxidase (UOx) surrounded by synthetic macromolecules, such as polyethyleneimine (PEI), poly(allylamine hydrochloride) (PAH), and poly(sodium 4-styrenesulfonate) (PSS) is a convenient model of redox-active biomacromolecules in a crowded environment and could display high enzymatic activity towards uric acid, an important marker of COVID-19 patients. In this work, the carbon fiber electrode was modified with Prussian blue (PB) redox mediator, UOx layer, and a layer-by-layer assembled polyelectrolyte film, which forms a complex coacervate consisting of a weakly charged polyelectrolyte (PEI or PAH) and a highly charged one (PSS). The film deposition process was controlled by cyclic voltammetry and scanning electron microscopy coupled with energy-dispersive X-ray analysis (at the stage of PB deposition) and through quartz crystal microbalance technique (at latter stages) revealed uniform distribution of the polyelectrolyte layers. Variation of the polyelectrolyte film composition derived the following statements. (1) There is a linear correlation between electrochemical signal and concentration of uric acid in the range of 10−4–10−6 M. (2) An increase in the number of polyelectrolyte layers provides more reproducible values for uric acid concentration in real urine samples of SARS-CoV-2 patients measured by electrochemical enzyme assay, which are comparable to those of spectrophotometric assay. (3) The PAH/UOx/PSS/(PAH/PSS)2-coated carbon fiber electrode displays the highest sensitivity towards uric acid. (4) There is a high enzyme activity of UOx immobilized into the hydrogel nanolayer (values of the Michaelis–Menten constant are up to 2 μM) and, consequently, high affinity to uric acid.

To improve the effectiveness of the viral infection diagnostic, we offer a new approach of immunochemical biosensors to determine single viral particles by specific antibodies. The antibodies are immobilized on the electrodes in a three-dimensional polymer matrix with several layers of polyelectrolytes on the screen-printed carbon electrode. Non-covalent immobilization of antibodies in successive layers of positively charged polyethyleneimine (PEI) and negatively charged polystyrene sulfonate (PSS) achieves the effect of macromolecular crowding. Such an immobilization approach promotes the preservation of the optimal conformation and antibody active center mobility for interaction with large virion particles. We established an electrochemical biosensor for tickborne encephalitis virus (TBEV) detection to demonstrate the method's applicability. Under the optimized architecture of the 3D-matrix, including a combination of two layers of a positively charged PEI with antibodies and the last layer of a negatively charged PSS, the assay is characterized by an extremely low limit of detection (LOD). This LOD could be as few as five viral particles in a sample volume of 5 μl, which is two orders of magnitude lower than conventional ELISA with the same reagents. The advantage of the biosensor is also a wide linear range of detection from 10 3 to 10 9 viral particles/ml. The proposed principle for determining virion particles is well suited to novel express diagnostics and Point-of-Care viral infections detection.