Introduction
Nanoconjugates are widely used as recognizing agents in different immunoassays, targeted therapies and biochemical methods of analysis. The performance of such conjugates strongly depends on their kinetic properties in interaction with the analyte. Various techniques such as surface plasmon resonance (SPR), bio-layer interferometry (BLI) and spectral correlation interferometry (SCI) allow for the kinetic characterization of protein–protein interactions, but there is still a lack of methods for screening nanoconjugate kinetics. Here, we show a novel approach for the characterization of magnetic nanoconjugates binding kinetics, which is based on the magnetometric detection of conjugates in the analytical zone of lateral flow assay (LFA) test strips. The developed method was used to screen different magnetic nanoconjugates against biotin, which was further used for its sensitive detection.
Methods
Magnetic nanocojugates were synthesized by adding 200 nm magnetic particles to a carbodiimide solution. After incubation, the particles were washed three times with water, and the solution of anti-biotin antibodies was added to the coupling mixture. After incubation, BSA solution was also added to block the excess of activated carboxyl groups. Then, the suspension of magnetic conjugates was washed with water again, and then used for further analysis.
Synthesized nanoconjugates were tested on LFA test strips with BSA-biotin conjugates immobilized in the analytical zone. The interaction between the target and labels was registered with the magnetic particle quantification (MPQ) technique during the test.
Results
Different amounts of nanoconjugates were taken every test. The experimental relation between the nanoconjugate concentration and saturation MPQ signal was fitted with a Langmuir adsorption model to determine the KD constant. After that, real-time interaction data were used to measure the kon and koff rate constants.
Based on determined kinetic properties, the most efficient conjugate was chosen in the magnetic LFA test system for biotin detection.
Conclusions
The developed approach was used to provide the kinetics screening of magnetic nanoconjugates. This method could be particularly advantageous in point-of-care diagnostics and the development of rapid screening assays for biomedical applications.
