Chromium-Doped Zinc Gallate Nanoparticles for Enhanced Enzyme-Linked Immunosorbent Assay Sensitivity: Optimization of Synthesis and Functionalization Strategies for Ultra-Low IgG Detection

The use of zinc gallate nanoparticles (ZnGa₂O₄:Cr³⁺) (ZGO-NPs) presents significant potential for improving the sensitivity in enzyme-linked immunosorbent assays (ELISA). The persistent luminescence signal increase of these nanoparticles in the presence of hydrogen peroxide (H₂O₂) offers advantages for the sensitive detection of biomolecules. Herein, different conditions of ZGO synthesis have been investigated by varying the hydrothermal reaction duration (6, 12, and 24 h) and examining its impact in the presence of H₂O₂. These nanoparticles have been integrated into ELISA assays, using as target antigen IgG. The lowest limit of detection (LOD) of 0.2 pg mL⁻¹ is observed for ZGO-NPs prepared during 12 h (ZGO2), and with a detection range from 1 to 1000 pg mL⁻¹. The impact of covalently functionalizing these nanoparticles has then been assessed. First using glucose oxidase (GOx) and the detection antibody (Ab_D) linked to PEGylated ZGO-NPs, named ZGO-GOx-Ab_D. Alternatively, only the detection antibody is linked to the PEG ZGO-NPs, named ZGO-Ab_D. The results show a significant lowering of the LOD when using the functionalized ZGO2 NPs and also highlight the impact of the signal amplification by H₂O₂. Specifically, when using ZGO2-GOx-Ab_D incubated with glucose to produce H₂O₂, or with ZGO2-Ab_D to which H₂O₂ was added, the LODs are ≈98 and 56 fg mL⁻¹ respectively, with detection ranges from 0.01 to 100 pg mL⁻¹.