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

Abstract

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.

Dual functional Phi29 DNA polymerase-triggered exponential rolling circle amplification for sequence-specific detection of target DNA embedded in long-stranded genomic DNA

“…In the past few decades, significant and substantial progress has been made in this field 1–5 . In order to realize ultrasensitive detection of target DNA, many promising methods have been designed on the basis of various DNA amplification techniques, including polymerase chain reaction (PCR) 6, 7 , rolling circle amplification (RCA) 8–10 , exonuclease III-aided target recycling 11, 12 , strand-displacement signal amplification 13–16 , hybridization chain reaction 17, 18 and nicking endonuclease signal amplification 19, 20 . Of all these DNA amplification techniques, PCR is the most well-known and frequently used.…”

“…As an important isothermal DNA amplification technique, RCA has attracted more and more attention in biosensing applications and various RCA-based DNA detection strategies have been reported 19, 22–32 . However, most of these works are restricted to the detection of artificial synthetic short-stranded DNA oligonucleotides 19, 27–32 .…”

“…However, most of these works are restricted to the detection of artificial synthetic short-stranded DNA oligonucleotides 19, 27–32 . These short-stranded target DNA might be used as indicators for the genomic DNA of interest (e.g.…”

“…RCA-based DNA sensors mainly work in three ways: (1) Target DNA is used to trigger the formation of the circular RCA template, and an additional RCA primer is needed 19, 28 . (2) Target DNA is used as the RCA primer, and preformed circular RCA template is needed 29, 30 .…”

Ultrasensitive Electrochemiluminescence Biosensor for mRNA Based on Polymerase Assisted Signal Amplification

“…Basically, it was essential to initiate exponential circular hybridization involving probe nucleic acid and target, leading to the release of target and more than once utilization of target strand. For example, exonuclease or endonuclease has been applied to achieve ultrasensitive detection by nicking at specific recognition sites, which limits its versatile application. Rolling circle amplification needs complex process to accomplish the reproduction of both nicked and unicked nucleic acid strands.…”