Target-InducedEnzymatic Cascade Reaction Method:Integrating Strand Displacement Amplification and CRISPR/Cas12a forHomogeneous Assays of Biotin

The important biological role of biotin emphasizes the need for a sensitive method to detect it in foodstuffs. This article introduces a homogeneous and sensitive biotin analysis method that leverages a target-induced enzymatic cascade reaction, incorporating strand-displacement amplification (SDA) and the CRISPR/Cas12a system. Without target biotin, streptavidin (SA) specifically binds to the biotinylated probe DNA, hindering Klenow polymerase from extending the primer single-stranded DNA (ssDNA) due to the steric hindrance created by the SA-biotin complex, resulting in low fluorescence. Conversely, competition between the target biotin and the biotin label for binding to SA reduces the amount of SA captured on the primer ssDNA. The SDA process, which involves Klenow polymerase and the Nb.BbvCI enzyme, proceeds smoothly, thereby activating the CRISPR/Cas12a system and producing an intense fluorescence signal. Utilizing this principle, precise and sensitive biotin detection in food matrices was achieved, with a limit of detection of 0.01 nM.