Chemiluminescent Nanogels as Intensive and Stable Signal Probes for Fast Immunoassay of SARS-CoV-2 Nucleocapsid Protein

It is highly desired to exploit good nanomaterials as nanocarriers for immobilizing chemiluminescence (CL) reagents, catalysts and antibodies to develop signal probes with intensive and stable CL properties for immunoassays. In this work, N-(4-aminobutyl)-N-ethylisoluminol (ABEI) and Co²⁺ bifunctionalized polymethylacrylic acid nanogels (PMAANGs-ABEI/Co²⁺) were synthesized via a facile strategy by utilizing carboxyl group-rich PMAANGs as nanocarriers to immobilize ABEI and Co²⁺. The obtained PMAANGs-ABEI/Co²⁺ showed extraordinary CL performance. The CL intensity is 2 orders of magnitude higher than that of previously reported ABEI and Cu²⁺-cysteine complex bifunctionalized gold nanoparticles with high CL efficiency. This was attributed to the excellent catalytic ability of Co²⁺ and polymethylacrylic acid nanogels, as well as the improved CL catalytic efficiency from a decreased spatial distance between ABEI and the catalyst. The as-prepared nanogels also possess abundant surface reaction sites and good CL stability. On this basis, a sandwich immunoassay for the nucleocapsid protein of SARS-CoV-2 (N protein) was developed by using magnetic bead connected primary antibody as a capture probe and PMAANGs-ABEI/Co²⁺ connected secondary antibody as a signal probe. The linear range of the proposed method for N protein detection was 3.16-316 ng/mL, and its detection limit was 2.19 ng/mL (S/N = 3). Moreover, the developed immunoassay was performed with a short incubation time of 5 min, which greatly reduced the detection time for N protein. By using corresponding antibodies, the developed strategy might be applied to detect other biomarkers.