Improving the Sensitivity of the miRNA Assay Coupled with the Mismatched Catalytic Hairpin Assembly Reaction by Optimization of Hairpin Annealing Conditions

Abstract

The mismatched catalytic hairpin assembly (mCHA), a programmable oligonucleotide circuit, is one of the promising isothermal amplification methods used in nucleic acid detection. Its limitations are related to a high background noise observed due to the target-independent hybridization of the reacting hairpins (HPs). In this work, it was shown that the introduction of salts such as NaCl and MgCl2 to HP1/HP2 annealing solutions sharply reduces the background in mCHA and simultaneously increases the signal-to-background (S/B) ratio. A comparison of the salts demonstrated the higher activity of MgCl2 as compared to NaCl. A similar effect of reducing the background was observed with a decrease in the concentration of H1/H2 probes in annealing solutions. Using the favorable annealing conditions allowed the development of an ultrasensitive chemiluminescence assay coupled with mCHA for miRNA quantitation. Except mCHA, the use of a streptavidin–polyHRP conjugate and an enhanced chemiluminescence reaction additionally increased the assay sensitivity. Notably, the optimization of the HP annealing diminished the detection limit of the assay by 2 orders of magnitude and increased the sensitivity and precision of miRNA-141 determination. The discovered fact of reducing the background by the variation of HP annealing conditions may be valuable not only for the mCHA performance but also likely for other HP-based biochemical methods.

Quantitation of MicroRNA-155 in Human Cells by Heterogeneous Enzyme-Linked Oligonucleotide Assay Coupled with Mismatched Catalytic Hairpin Assembly Reaction

“…Before being used in the assay, Flu-HP1 and B-HP2 were annealed at 88 °C for 15 min and cooled to room temperature for 1 h. The Flu-HP1 probe was annealed at a concentration of 30 nM in 10 mM Tris-HCl, pH 7.2 (TB) supplemented with 20 mM MgCl 2 . The B-HP2 probe was annealed at a concentration of 100 nM in TB supplemented with 10 mM MgCl 2 [ 21 ]. The determination of the miRNA concentration in the samples was carried out as follows: In total, 50 μL of Flu-HP1 (30 nM in TB supplemented with 20 mM MgCl 2 ) was added to the plate wells to bind with the adsorbed anti-FITC antibodies.…”

Dual Signal Amplification by Urease Catalysis and Silver Nanoparticles for Ultrasensitive Colorimetric Detection of Nucleic Acids

“…Solid biosensing interfaces, such as magnetic nanoparticles, well plates, , glass slides, and papers, established superiority in selectivity and low background signals due to their ease of separation from disrupters . Among them, magnetic nanoparticles are widely used to provide effective sensing interfaces and achieve ultrasensitive biosensing .…”

DNA Logic Nanodevices for the Sequential Imaging of Cancer Markers through Localized Catalytic Hairpin Assembly Reaction

“…Cancer biomarkers, such as nucleic acids, proteins, and glycans, play a significant role in early diagnosis, cancer progression monitoring, and treatment. , Up to now, these discovered biomarkers have shown different spatial distribution in tumor cells, for instance, some proteins located on the cell membrane and nucleic acids mostly distributed in the cytoplasm. − One interesting finding is that lots of cancer biomarkers are shared in different types of cells, for example, microRNA-21 (miR-21) present in breast and liver cancer cells, , that is, simultaneous monitoring of multiple and multilayer cancer biomarkers in living cells is crucial to acquire precise and multidimensional information in cell types and progression of cancers, which is a big challenge for conventional methods that usually focus on single biomarker detection or monitoring of several related targets with the same spatial locations.…”