Search

An ultrasensitive electrochemical biosensor is described for the determination of microRNAs. It is based on the use of DNA-templated copper nanoparticles (Cu NPs) as signalling probe. MicroRNA-222 was selected as the model analyte. The probe was obtained from two different oligonucleotides (containing complementary bases) via hybridization chain reaction to form long DNA concatemers as template. The Cu NPs were formed by reaction of ascorbate with copper sulfate. The biosensor was fabricated as follows: (a) Capture probe (cDNA) with a thiolated group was immobilized on reduced graphene oxide modified with gold nanoparticles (rGO/Au NPs), (b) materials was placed on a glassy carbon electrode (GCE); (c) the modified electrode (cDNA/rGO/Au NPs/GCE) was sequentially hybridized with microRNA-222 and signal probe; this results in the formation of a sandwich structure of cDNA-microRNA-signal probe on surface of the modified electrode. Differential pulse voltammetry was employed to record the electrochemical response of biosensor in pH 6.0 solution. As a result, a sensitive oxidation current with a peak potential at 0.10 V (vs. SCE) was obtained corresponding to Cu NPs. The experimental conditions were optimized. Under optimal conditions, the biosensor exhibited wide linear response range (0.5 fM to 70 nM) and low limit of detection (0.03 fM; at S/N = 3). The assay possesses high selectivity and can discriminate analyte microRNA from single-base mismatched microRNA. Graphical abstractA sensitive electrochemical biosensor is described for the determination of microRNA-222 by using a dsDNA-templated Cu NPs as signalling probe. (A) represents the preparation of signal probe, and (B) represents the fabrication of electrochemical microRNA sensor.

A DNAzyme-embedded hyperbranched DNA dendrimer is used as a colorimetric signal amplifier in an ultrasensitive detection scheme for nucleic acids. The hyperbranched DNA dendrimers were constructed by single-step autonomous self-assembly of three structure-free DNA monomers. A cascade of self-assembly reactions between the first and second strands leads to the formation of linear DNA concatemers containing overhang flank fragments. The third strand, which bears a peroxidase-mimicking DNAzyme domain, serves as a bridge to trigger self-assembly between the first and second strands across the side chain direction. This results in a chain branching growth of the DNAzyme-embedded DNA dendrimer. This signal amplifier was incorporated into the streptavidin-biotin detection system which comprises an adaptor oligonucleotide and a biotinylated capture probe. The resulting platform is capable of detecting a nucleic acid target with an LOD as low as 0.8 fM. Such sensitivity is comparable if not superior to most of the reported enzyme-free (and even enzyme-assisted) signal amplification strategies. The DNA dendrimer based method is expected to provide a universal platform for extraordinary signal enhancement in detecting other nucleic acid biomarkers by altering the respective sequences of adaptor and capture probe. Graphical abstract Schematic of an assembly of a DNAzyme-embedded hyperbranched DNA dendrimer which operates as a signal amplifier for nucleic acids detection. The nanostructure is constructed by autonomous self-assembly of three DNA monomers. Colored letters represent each domain, and complementary domains are marked by asterisk. Domain d represents the DNAzyme sequence.

An enzyme-free signal amplification strategy for colorimetric detection of RNA molecules was developed. The detecting process was started by the hybridization of the target RNA via two helper oligonucleotides to bi-functionalized (initiator and linker oligonucleotides-modified) gold nanoparticles. Afterwards, in presence of two auxiliary oligonucleotides, the nanoparticle-confined initiators triggered the formation of DNA concatemers containing hemin-binding aptamers through a modified hybridization chain reaction [HCR] strategy. In addition to the mediatory role, the helper oligonucleotides, with a toehold-mediated strand displacement [TMSD] ability, helped unwind the secondary structures of the RNA molecule and provided a binding site for a biotinylated capture probe. Upon binding, the complex was harvested on the streptavidin-coated microwell, and subsequently the formation of HRP-mimicking DNAzymes was stimulated by adding hemin molecules. The assay was successfully employed for detecting target nucleic acids, which bears secondary structures, in isothermal conditions (room temperature) without heat denaturation. The assay detected DNA molecule with LOD value of 1 pM with the ability to differentiate a spurious target containing a single-nucleotide substitution. On real RNA samples, the assay truly discriminated Escherichia coli O157:H7's 16s rRNA from closely related bacteria with a detection limit of ≥ 5 × 10(5)CFU. Compared to enzyme-based signal amplifications, the assay can detect the target RNA with a sensitivity similar to the one already found for RT-PCR, NASBA, and RT-LAMP assays. Overall, the analytical platform eliminates the need for enzymatic reactions, heat denaturation, and complicated instruments during the detection process., (Copyright © 2016 Elsevier B.V. All rights reserved.)