Immunochemical biosensor for single virus particle detection based on molecular crowding polyelectrolyte system

Abstract

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.

Ultrasensitive Detection of PSA Using Antibodies in Crowding Polyelectrolyte Multilayers on a Silicon Nanowire Field-Effect Transistor

“…The concentrations of the reagents were used in accordance with the method developed earlier for the formation of a polyelectrolyte matrix of PEI and PSS on the surface of screenprinted electrodes [30]. A total of 5 µL of PEI solution (1 mg/mL in water) was spotted onto the silicon surface, dried, and rinsed three times with water to remove excess PEI.…”

“…Due to the small volume of the hydrogel nanolayer as well as the "tightness" effect, the interaction between molecules in such an environment occurs more quickly than in the volume of the solution. In the process of developing an electrochemical biosensor for the detection of the encephalitis virus, the benefits of immobilizing specific antibodies into the matrix of successive layers of positively charged polyethylenimine (PEI) and negatively charged polystyrene sulfonate (PSS) were recently demonstrated [30].…”

Uricase Crowding via Polyelectrolyte Layers Coacervation for Carbon Fiber-Based Electrochemical Detection of Uric Acid

“…We have previously suggested an approach for electrochemical immunosensors with a quasi-mobile polyelectrolyte–protein hydrogel interface [ 22 ], including nanofilms [ 23 ]. Hydrogel interfaces based on neutral polyelectrolytes provide reproducible results in complex systems and simple variation of hydrogel composition, which enables facile data collection for machine learning to decrease the limit of detection [ 22 , 24 ].…”

“…It proves polyelectrolyte effectiveness for the sensitivity increase of the immunosensors compared to the antibody-only modified electrode. Hydrophilic properties of polyelectrolytes in a crowded medium suppressed nonspecific protein adsorption [ 23 ]. We have shown that LbL assembly effectively controls the adsorption of antigens and antibodies conjugated with horseradish peroxidase on the electrode surface.…”