Concatemeric dsDNA-Templated Copper Nanoparticles Strategy with Improved Sensitivity and Stability Based on Rolling Circle Replication and Its Application in MicroRNA Detection

Abstract

DNA-templated copper nanoparticles (CuNPs) have emerged as promising fluorescent probes for biochemical assays, but the reported monomeric CuNPs remain problematic because of weak fluorescence and poor stability. To solve this problem, a novel concatemeric dsDNA-templated CuNPs (dsDNA-CuNPs) strategy was proposed by introducing the rolling circle replication (RCR) technique into CuNPs synthesis. In this strategy, a short oligonucleotide primer could trigger RCR and be further converted to a long concatemeric dsDNA scaffold through hybridization. After the addition of copper ions and ascorbate, concatemeric dsDNA-CuNPs could effectively form and emit intense fluorescence in the range of 500-650 nm under a 340 nm excitation. In comparison with monomeric dsDNA-CuNPs, the sensitivity of concatemeric dsDNA-CuNPs was greatly improved with ~10,000 folds amplification. And their fluorescence signal was detected to reserve ~60% at 2.5 h after formation, revealing ~2 times enhanced stability. On the basis of these advantages, microRNA let-7d was selected as the model target to testify this strategy as a versatile assay platform. By directly using let-7d as the primer in RCR, the simple, low-cost, and selective microRNA detection was successfully achieved with a good linearity between 10 and 400 pM and a detection limit of 10 pM. The concatemeric dsDNA-CuNPs strategy might be widely adapted to various analytes that can directly or indirectly induce RCR.

Photoinduced Electron Transfer-Based Fluorescence Quenching Combined with Rolling Circle Amplification for Sensitive Detection of MicroRNA

“…The detection limit was calculated to be 6 aM by evaluating the average response of the blank plus 3 times the standard deviation, which was much lower that of the some recently reported RCA-based fluorescent methods for miRNA quantification. [18,20,23] It is still a great challenge to distinguish miRNA family members with sequence homology. To evaluate the specificity of the strategy for mmu-miR-433-5p, we check the response of the strategy to other three miRNAs (oar-miR-433-5p, mmu-miR-433-3p and ssc-miR-4334-3p) in the same family of mmu-miR-433-5p.…”

An electrochemical aptasensor for thrombin detection based on the recycling of exonuclease III and double-stranded DNA-templated copper nanoparticles assisted signal amplification

“…Since it was reported by Mokhir et al in 2010, dsDNA-templated CuNPs have attracted great attention in fluorescence analysis of biomolecules for its facile synthesis and excellent optical properties [26][27][28][29]. In this paper, we have employed electrochemical technique as a substitution of fluorescent technique to characterize the formation of dsDNA-templated CuNPs for the development of an improved new aptasensor.…”

Hairpin-shaped DNA Templated Copper Na nopar tides for Fluorescence Detection of Adenosine Triphosphate Based on Ligation-mediated Exonuclease Cleavage

“…21,22 Furthermore, several reports have combined dsDNA-CuNPs with DNA amplification techniques to improve sensitivity. 23,24 Therefore, it would be necessary and of great importance to expand the biochemical applications of DNA-templated CuNPs.…”