3'-Terminal Repair-Powered Dendritic Nanoassembly of Polyadenine Molecular Beacons for One-Step Quantification of Alkaline Phosphatase in Human Serum.

Abstract

Alkaline phosphatase (ALP) is an important hydrolase with crucial roles in biological processes, and the dysregulation of ALP may cause various human diseases. The conventional ALP assays usually involve cumbersome procedures with poor sensitivity. Herein, taking advantage of intrinsic superiorities of molecular beacons (MBs) and unique features of terminal deoxynucleotidyl transferase (TdT), we demonstrate for the first time the 3'-terminal repair-powered dendritic nanoassembly of polyadenine (A) MBs for one-step quantification of ALP in human serum. When ALP is present, it catalyzes 3'-terminal dephosphorylation of poly-A MBs to induce TdT-mediated template-free polymerization, generating long chains of polythymidine (T) sequences. The long poly-T chains can function as the anchoring templates to hybridize with many poly-A MBs, leading to the unfolding of loop structures and the dissociation of FAM/BHQ1 pairs (the 1st amplification stage). Subsequently, all 3'-hydroxylated poly-A MBs can be extended with the assistance of TdT to generate the branched long poly-T chains, leading to the hybridization of more poly-A MBs and the dissociation of more FAM/BHQ1 pairs (the 2nd amplification stage). Through multiple rounds of extension, assembly, and activation of poly-A MBs, dendritic DNA nanostructures are automatically formed, resulting in the dissociation of abundant fluorophores from the FAM/BHQ1 pairs to generate an exponentially amplified fluorescence signal (the nth amplification stage). This strategy possesses high sensitivity and excellent specificity, and the detection limit can reach 1 cell. Moreover, it can evaluate kinetic parameters, screen inhibitors, estimate cellular inhibition effects, and measure ALP in human serums.