Highly Selective and Sensitive Electrochemiluminescence Biosensor for p53 DNA Sequence Based on Nicking Endonuclease Assisted Target Recycling and Hyperbranched Rolling Circle Amplification.

An ultrasensitive and specific electrochemiluminescence (ECL) biosensor has been designed for the p53 DNA sequence, which is based on cascade signal amplification of nicking endonuclease assisted target recycling and hyperbranched rolling circle amplification (HRCA). First of all, biotin modified hairpin capture DNA (HP) probe was immobilized on the surface of streptavidin magnespheres paramagnetic particles (PMPs). Target DNA hybridized with the loop portion of the HP probe, therefore unfolding HP to form a double-stranded DNA (dsDNA) containing the specific nicking site of the nicking endonuclease. Then, the nicking endonuclease recognized the specific nicking site and cleaved the HP into two pieces, liberating target DNA and the complementary sequence piece for the padlock probe. The intact target DNA would initiate the next cycle of hybridization and cleavage, thereby releasing multiple complementary sequences for the padlock probes. The liberated complementary sequences hybridized with the padlock probes, subsequently inducing the HRCA reaction and generating numerous dsDNA segments. Herein, Ru(phen)3(2+) was embedded into dsDNA and worked as ECL signal reporter. The reaction products were eventually pretreated by dialysis tube with the cutoff membrane to remove the residual Ru(phen)3(2+) in the solution for the following ECL measurements. Using this cascade amplification strategy, an ultrasensitive p53 DNA sequence detection method was developed with a wide linear range from 0.05 to 100 fM and a low detection limit of 0.02 fM. Moreover, this cascade amplified ECL biosensor had specific recognition capacity for noncomplementary and single- and double-base mismatched DNA. The proposed ECL biosensor might have a great potential in biomedical research and clinic analysis.