Nanofluidic sensing platform for PNK assay using nonlinear hybridization chain reaction and its application in DNA...

In this study, a polyethyleneimine (PEI)/Zr⁴⁺-functionalized nanofluidic sensing platform based on nonlinear hybridization chain reaction (NHCR) was developed for PNK activity assay. With the existence of PNK, the hairpin H_PNK was cleaved by λ exonuclease, liberating the initiator T-DNA. Then T-DNA triggered the nonlinear HCR in solution and the reaction products were absorbed onto the nanopore, which changed the surface charge of nanofluidic device and could be detected by current-voltage characteristic curves. Compared to traditional linear HCR, the nonlinear HCR exhibits a higher sensitivity and order of growth kinetics, making it a powerful signal amplifier in bioanalysis. Due to the powerful amplification efficiency of nonlinear HCR, high sensitivity of the nanopore and specific recognition site of PNK/λ-Exo, an ultrasensitive and selective PNK sensing approach had been developed and applied to precisely quantitate the PNK activity with a LOD of 0.0001 U/mL. Moreover, utilizing this nanofluidic system as a foundation, we constructed a logic circuit that utilized PNK, adenosine diphosphate (ADP), and (NH₄)₂SO₄ as input elements. ADP and (NH₄)₂SO₄ had a crucial function in facilitating the PNK to regulate the DNA logic gate. By modifying the target and inhibitors, the nanofluidic device could detect a variety of stimuli and execute more advanced logical operations.