Framework nucleic acid-wrapped protein-inorganic hybrid nanoflowers with three-stage amplified fluorescence polarization for terminal deoxynucleotidyl transferase activity biosensing

Abstract

Herein, we proposed a terminal deoxynucleotidyl transferase (TdT), a potential biomarker of lymphoid tumors, responsive fluorescence polarization (FP)- sensing protocol based on framework nucleic acid (FNA)-wrapped protein-inorganic hybrid nanoflowers. To achieve this goal, a pair of poly-A-composed extension primers (EPa and EPb) was designed, and protein-inorganic hybrid nanoflowers were synthesized by a biomineralization reaction. EPa was labeled with carboxyfluorescein (FAM) fluorophore to create the preliminary FP signal. EPb was labeled with biotin to conjugate with hybrid nanoflowers. Upon introduction of TdT into the dTTP pool, both EPa and EPb can be catalyzed by TdT to incorporate numerous T bases, thereby facilitating intermolecular hybridization between ‘A’ and ‘T’ bases. The final assembled FNA-wrapped hybrid nanoflowers with greatly enhanced molecular volume and weight restrict the free rotation of attached FAMs, causing a great FP enhancement from a designated three-stage FP amplification. Under optimized conditions, the TdT can be detected with a detection limit of 0.023 U/mL and a linear detection from 0.1 U/mL to 100 U/mL within 20 min. As a proof-of-concept study, the first exploitation of FNA and protein-inorganic nanoflowers to improve the FP signal and the merit of FP without sample separation and washing opens a new avenue for biochemical study and disease diagnosis.