Sensitive and Facile Detection of Vitamin D Based on Fluorescent Labeled Aptamer Probe and Exonuclease I-Assisted Signal Amplification.

This study presents a rapid, simple aptamer-based fluorescence sensor using fullerenes as a quencher for detecting vitamin D3. Fullerenes, with its π-electron cloud, acts as a powerful electron acceptor, facilitating Förster resonance energy transfer (FRET) from carboxyfluorescein (FAM). The aptamer, labeled with 5'6-FAM, adsorbs onto the fullerenes surface through hydrogen bonding and π-π stacking interactions, leading to fluorescence quenching. Upon vitamin D3 binding, the aptamer forms a hairpin structure that prevents adsorption onto the fullerenes surface, restoring fluorescence. The fullerenes were characterized using UV-Vis, FT-IR, Raman spectroscopy, SEM, TEM, and zeta potential measurements. The sensor's response to varying vitamin D3 concentrations was analyzed with a fluorescence spectrometer, revealing a linear detection range of 0-600 nM and a detection limit of 200 nM, which improved to 50 nM with exonuclease I. The sensor demonstrated a recovery rate of 88.4%-96.3% for vitamin D3 in water samples, confirming its feasibility for practical applications. In addition, our proposed sensor exhibited good repeatability (3.27%) and stability (90%). The innovative use of fullerenes as a fluorescence quencher, along with the aptamer's specific binding to vitamin D3, provides a novel and highly sensitive avenue for vitamin D3 detection.