Au-X (X=Pt/Ru)-decorated magnetic nanocubes as bifunctional nanozyme labels in colorimetric, magnetically-enhanced, one-step sandwich CRP immunoassay

Scientific interest in the investigation and application of multifunctional nanomaterials in medical diagnostics has been increasing. The employment of magnetocatalytic immunoconjugates as both analyte-capturing agents and enzyme-like catalytic labels may enable rapid preconcentration and determination of relevant antigens. In this work, we synthesized and comprehensively characterized two types of noble metal-decorated magnetic nanocubes (MDMCs) which were subsequently applied in the one-step, sandwich nanozyme-linked immunosorbent assay (NLISA). Magnetic cores allow for rapid separation from complex samples of biological origin. The catalytically active shell composed of Au-decorated Pt or Ru can effectively mimic the activity of horseradish peroxididase, retaining at the same time the ability to form stable bioconstructs through self-assembly of thiolated ligands. As a result, hybrid multifunctional nanoparticles were synthesized and used to detect C-reactive protein (CRP) in serum samples. We have also paid considerable attention to the mechanistic studies of the formation of sandwich immunocomplexes with nanoparticle labels by means of immunoenzymatic methods and surface plasmon resonance. Analytical parameters of the Pt-MDMCs-labeled NLISA (detection limit LOD = 0.336 ng mL^-1, recovery = 98.0%, linear response window covering two logarithmic units) turned out to be superior to the classical, one-step ELISA based on a horseradish peroxidase. In addition, our method offers further possibility of sensitivity adjustment by changing the parameters of magnetic preconcentration, together with good long-term stability of MDMCs conjugates and their good resistance to common interferences. We believe that the proposed simple synthetic protocol will guide a new approach to applying metal-decorated magnetic nanozymes as versatile and multifunctional labels for application in subsequent pre-analytical analyte concentration and immunoassays towards clinical applications.