Triggered Polycatenated DNA Scaffolds for DNA Sensors and Aptasensors by a Combination of Rolling Circle Amplification and DNAzyme Amplification

Abstract

The concept of triggered polycatenated DNA scaffolds has been elegantly introduced into ultrasensitive biosensing applications by a combination of rolling circle amplification (RCA) and DNAzyme amplification. As compared to traditional methods in which one target could only initiate the formation of one circular template for RCA reaction, in the present study two species of linear single-stranded DNA (ssDNA) monomers are self-assembled into mechanically interlocked polycatenated nanostructures on capture probe-tagged magnetic nanoparticles (MNPs) only upon the introduction of one base mutant DNA sequence as initiator for single-nucleotide polymorphisms (SNPs) analysis. The resultant topologically polycatenated DNA ladder is further available for RCA process by using the serially ligated circular DNA as template for the synthesis of hemin/G-quadruplex HRP-mimicking DNAzyme chains, which act as biocatalytic labels for the luminol-H(2)O(2) chemiluminescence (CL) system. Notably, the problem of high background induced by excess hemin itself is circumvented by immobilizing the biotinylated RCA products on streptavidin-modified MNPs via biotin-streptavidin interaction. Similarly, a universal strategy is contrived by substitutedly employing aptamer as initiator for the construction of polycatenated DNA scaffolds to accomplish ultrasensitive detection of proteins based on structure-switching of aptamer upon target binding, which is demonstrated by using thrombin as a model analyte in this study. Overall, with two successive amplification steps and one magnetic separation procedure, this flexible biosensing system exhibits not only high sensitivity and specificity with the detection limits of SNPs and thrombin as low as 71 aM and 6.6 pM, respectively, but also excellent performance in real human serum assay with no PCR preamplification for SNPs assay. Given the unique and attractive characteristics, this study illustrates the potential of DNA nanotechnology in bioanalytical applications for both fundamental and practical research.

Ultrasensitive and Signal‐on Electrochemiluminescence Aptasensor Using the Multi‐tris(bipyridine)ruthenium(II)‐β‐cyclodextrin Complexes

“…As expected, the addition of thrombin [61] or lysozyme [62] did not affect the samples. Thus, the serum sample was first diluted with binding buffer and spiked with protein (thrombin or lysozyme) at different concentrations.…”

In vitroselection of DNA-cleaving deoxyribozyme with site-specific thymidine excision activity

“…Many of these so-called single-nucleotide polymorphisms (SNPs) have been linked to human diseases, including phenylketonuria, hemophilia and certain cancers (6). Significant advances have been made in the past several years to develop accurate, rapid and cost-effective technologies for SNP detection, such as denaturing gradient gel electrophoresis (7), microfluidic devices (8), technologies based upon chip (9), allele-specific polymerase chain reaction (PCR) (10), strand displacement amplification (11), rolling circle amplification (12) and ligase chain reaction (13). …”

Novel One-Tube-One-Step Real-Time Methodology for Rapid Transcriptomic Biomarker Detection: Signal Amplification by Ternary Initiation Complexes

“…[42][43][44][45] However, these methods typically included three main steps with different conditions; e.g., RCA in 50 mM tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) buffer (pH 7.5) containing 20 mM MgCl2 and 1 mM dithiothreitol at 37°C for 2-3 h, formation of hemin/G4 complexes in 10 mM 4-(2- 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (pH 7.4) containing 200 mM NaCl at room temperature for 1-4 h, and subsequent colorization in the presence of 2,2'-azinobis(3-ethylbenzthiazoline)-6-sulfonic acid (ABTS) and hydrogen peroxide (H2O2) at room temperature for several minutes or more.…”