Functionalized ultra-fine bimetallic PtRu alloy nanoparticle with high peroxidase-mimicking activity for rapid and sensitive colorimetric quantification of C-reactive protein

The in situ synthesis is reported of citric acid-functionalized ultra-fine bimetallic PtRu alloy nanoparticles (CA@PtRu ANPs) through a simple one-pot wet chemical method. The cost-efficient CA@PtRu ANPs with an average diameter of 3.2 nm revealed to have enhanced surface area, peroxidase-like activity, high stability, and adequate availability of functional groups to bind biomolecules. Along with nanoparticle surface area, the surface charge has also significantly affected the peroxidase-like activity and the colloidal suspension stability. As an excellent immobilization matrix and peroxidase mimic, the CA@PtRu ANPs were utilized to develop non-enzymatic colorimetric immunoassay for rapid, selective, and sensitive quantification of C-reactive protein (CRP) biomarkers. In this immunoassay, CA@PtRu ANPs serve as enzyme mimic that significantly amplifies the color signals, and amine-functionalized silica-coated magnetic microbeads (APTES/SiO₂@Fe₃O₄) act as CRP-recognizing capture probes. The absorbance curves of colorimetric immunoassay were measured in wavelengths between 550 and 750 nm, and the maximum absorbance at 652 nm was used to establish a linear relationship between absorbance and CRP concentrations. The developed colorimetric immunoassay showed rapid and sensitive quantification of CRP levels from 0.01 to 180 μg mL⁻¹ with a LOD of 0.01 μg mL⁻¹. Moreover, the mean recovery of CRP from spiked human serum samples lies between 97 and 109% (n = 3), which indicates that the proposed nanozyme-linked immunoassay has the potential to be used in rapid point-of-care applications. Graphical abstract: