Amplification-Free Quantification of Donkey DNA for Authenticating Colla Corii Asini with a Time-Resolved Fluorescent...

Colla Corii Asini (CCA), a traditional food-medicine product, is vulnerable to food safety and market integrity due to frequent adulteration. This study developed a lateral flow assay based on polyethylenimine-functionalized europium metal-organic frameworks (PEI-Eu-MOFs-LFA) for quantitatively detecting donkey DNA to assess the authenticity of CCA, without the need for knowing the types of adulterants. The protonated amino groups on the PEI‑Eu-MOFs surface formed stable complexes with negatively charged single‑stranded DNA (ssDNA) probes. Electrostatic attraction and hydrogen bonding facilitated conjugate formation, leading to fluorescence quenching of the PEI-Eu-MOFs via the inner-filter effect (IFE). This method enables direct analysis after DNA extraction, eliminating the need for additional nucleic acid amplification steps, and completes the entire process in just 60 min. Furthermore, a calibration curve was established between the T/C ratio and the logarithmic value of donkey hide content (2% to 100%). The PEI-Eu-MOFs-LFA exhibited excellent sensitivity and accuracy, with a detection limit of 1.86%, recoveries ranging from 85.70% to 114.63%, and relative standard deviations from 2.48% to 12.59%. Consequently, PEI-Eu-MOFs-LFA demonstrates high sensitivity and accuracy, significantly improving detection efficiency and providing an innovative approach for quality control and market regulation of CCA.