Fluorometric determination of ssDNA based on functionalized magnetic microparticles and DNA supersandwich self-assemblie
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ORIGINAL PAPER
Fluorometric determination of ssDNA based on functionalized magnetic microparticles and DNA supersandwich self-assemblies Yufei Liu 1 & Jingfang Shangguan 1 & Ningning He 1 & Haixia Lv 1 & Qianqian Wang 1 & Suping Bai 1 Received: 11 April 2019 / Accepted: 20 September 2019 # Springer-Verlag GmbH Austria, part of Springer Nature 2019
Abstract A method is described for the determination of DNA via nucleic acid amplification by using nucleic acid concatemers that result from DNA supersandwich self-assemblies (SSAs). The method employs two auxiliary probes to form self-assembled biotin SSAs. These exhibit strong fluorescence if labeled with intercalator SYBR Green I. In the presence of the target (as exemplified for a 30-mer), streptavidin is released from the surface of the functionalized magnetic microparticles (FMMPs) by competitive hybridization on the surface. However, the SSA products do not conjugate to the FMMPs. This leads to a large amount of SYBR Green I intercalated into the concatemers and eventually results in amplified fluorescence in the supernate. The SSA products can be prepared beforehand, and amplification therefore can be completed within 50 min. The method is more efficient than any other conventional amplification. The detection limit for the 30-mer is 26.4 fM which is better by a factor of 10 compared to other amplification methods. Conceivably, the method can be further extended to the determination of a wide variety of targets simply by replacing the sequences of the probes. Finally, this rapid and highly sensitive method was employed for detection of Ebola virus gene (≈30-mer) and ATP in spiked serum with satisfactory results. Keywords Ebola virus . ATP . Pre-prepared nucleic acid concatemers . SSA . HCR . Magnetic separation . Aptamer . Signal amplification . Detection time . Fluorescence
Introduction One of the most important issues in bioassay research is to increase the sensitivity with signal amplification technology such as ligase chain reaction (LCR) [2], rolling-circle amplification (RCA) [3], exonuclease III-aided target recycling (EATR) [4, 5], strand-displacement amplification (SDA) [6, 7], helicase-dependent amplification (HDA) [8, 9], catalytic hairpin assembly (CHA) [10, 11], and hybridization chain reaction (HCR) [12, 13].
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00604-019-3865-z) contains supplementary material, which is available to authorized users. * Yufei Liu [email protected] 1
School of Pharmacy, Xinxiang Medical University, Xinxiang, Henan 453003, People’s Republic of China
DNA supersandwich self-assembly(SSA) is a type of onedimensional DNA nanostructure signal amplification method, which contains two straight chain DNA nucleic acids [14–16]. These two hybridized nucleic acids were used as modified signal probes, which contains a Bsticky end^ to selfassemble into supersandwich structure and serves as carriers of signal amplification. However, since the supersandwich structure can be spont
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