Ultrasensitive Ratiometric Fluorescence Bioassay for Accurate Detection of Covid-19-Specific Nucleocapsid Protein in Clinical Serum Samples Using Modified Cleavable Mesoporous SiO₂ Satellite-Enriched Carbon Dots

Due to the presence of various autofluorescent compounds in biological samples like serum and the photobleaching of organic fluorophores, fluorescence sensing has limited practical applicability. This study describes the development of an improved ratiometric fluorescence assay to determine the nucleocapsid protein (N protein), one of the most conserved biomarkers of Covid-19 in spiked and serum samples using highly stable buffer-based near IR-dual emission carbon dots (CDs) encapsulated into the cavities of cleavable silica nanocapsule (SNCs) nanocomposite. The cavities of cleavable silica nanocapsules (SNCs) and the formed core–shell CDs@ SNCs were used as a superior reservoir of fluorescent markers produced by cohydrolyzing tetraethyl orthosilicate and diiminosilane linker, which held hundreds of CDs in silica shell frameworks. The SiO₂ nanocomposite was modified with an N protein antibody that specifically paired to the receptor binding region of the Cov-19 spike protein subunit. CDs were taken out of SNCs by NaBH₄ reduction, and the released CDs exhibited dual emission at 475 and 675 nm when excited at 400 nm. Ratiometric detection is completed over a binding-induced, concentration-dependent immuno-affinity of the N protein that drives the fluorescence quenching phenomenon between the CDs as fluorophore and the AuNPs as quencher. As the N protein concentration increased, the intensity of the red emission (675 nm) dropped, whereas the intensity of the green emission (475 nm) already remained constant, which is due to sandwich immunoassays of CDs around AuNPs. Using the exceptional fluorescent characteristics of CDs and the high selectivity of nanocomposite functionalized with N-protein antibody, the developed assay efficiently eliminates the autofluorescence background interference of serum samples. The fluorescence ratio (I₄₇₅/I₆₇₅) provides a limit of detection of 2 pg mL^–1 over a linear range of 0.01 to 5 ng mL^–1 and exhibits an amplified sensitivity of 54 times compared to conventional immunoassay using CDs as fluorescent labels. With one-step signal amplification and requiring small sample quantities (only 20 μL), this sensing platform can be effectively used for the accurate detection of N protein, and no cross-reactivity is detected in the presence of different interfering agents.