Peroxidase-mimicking nanozyme with surface-dispersed Pt atoms for the colorimetric lateral flow immunoassay of C-reactive protein

Abstract

Platinum-containing nanozymes with peroxidase-mimicking activity (PMA) have found a broad application in bioanalytical methods and are potentially able to compete with enzymes as the labels. However, traditionally used methods for the synthesis of nanozymes result in only a small fraction of surface-exposed Pt atoms, which participate in catalysis. To overcome this limitation, we propose a new approach for the synthesis of nanozymes with the efficient dispersion of Pt atoms on particles' surfaces. The synthesis of nanozymes includes three steps: the synthesis of gold nanoparticles (Au NPs), the overgrowth of a silver layer over Au NPs (Au@Ag NPs, 6 types of NPs with different thicknesses of Ag shell), and the galvanic replacement of silver with PtCl₆^2- leading to the formation of trimetallic Au@Ag-Pt NPs with uniformly deposited catalytic sites and high Pt-utilization efficiency. Au@Ag-Pt NPs (23 types of NPs with different concentrations of Pt) with various sizes, morphology, optical properties, and PMA were synthesized and comparatively tested. Using energy-dispersive spectroscopy mapping, we confirm the formation of core@shell Au@Ag NPs and dispersion of surface-exposed Pt. The selected Au@Ag-Pt NPs were conjugated with monoclonal antibodies and used as the colorimetric and catalytic labels in lateral flow immunoassay of the inflammation biomarker: C-reactive protein (CRP). The colorimetric signal enhancement was achieved by the oxidation of 3,3'-diaminobenzidine by H₂O₂ catalyzed by Au@Ag-Pt NPs directly on the test strip. The use of Au@Ag-Pt NPs as the catalytic label produces a 65-fold lower limit of CRP detection in serum (15 pg mL^-1) compared with Au NPs and ensures the lowest limit of detection for equipment-free lateral flow immunoassays. The assay shows a high correlation with data of enzyme-linked immunosorbent assay (R² = 0.986) and high recovery (83.7-116.2%) in serum and plasma. The assay retains all the benefits of lateral flow immunoassay as a point-of-care method.

Keywords: Cardiac markers; Core@shell nanoparticles; Galvanic replacement; Immunochromatography; Inflammatory markers; Multimetallic nanoparticles; Nanocatalyst; Point-of-care testing.