Chemiluminescent plate assay of microRNA-155 coupled with catalytic hairpin assembly with oligonucleotide release (CHAOR).

A heterogeneous sensitive microRNA-155 assay based on a new isothermal amplification method, called catalytic hairpin assembly with oligonucleotide release (CHAOR), was developed. The principle of CHAOR was studied by non-denaturing electrophoresis. To detect the amplification product, a polyperoxidase-streptavidin conjugate (molar ratio 1:80) and an enhanced chemiluminescence reaction were used, which made it possible to increase assay sensitivity.

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

In the present work, we describe the development of a chemiluminescent enzyme-linked oligonucleotide assay coupled with mismatched catalytic hairpin assembly (mCHA) amplification for the quantitative determination of microRNA-155. To improve its sensitivity, a polymerase-free mCHA reaction was applied as an isothermal amplification method. The detection limit of the proposed assay was 400 fM.

Modern Methods for Assessment of microRNAs.

The review discusses modern methods for the quantitative and semi-quantitative analysis of miRNAs, which are small non-coding RNAs affecting numerous biological processes such as development, differentiation, metabolism, and immune response. miRNAs are considered as promising biomarkers in the diagnosis of various diseases.

Comparative study of magnetic beads and microplates as supports in heterogeneous amplified assay of miRNA-141 by using mismatched catalytic hairpin assembly reaction.

Magnetic beads (MBs) are often considered as an effective carrier in heterogeneous assays due to the simplicity of separation and washing, and the ability to increase and control the surface area. However, the effect of the MBs surface on the analytical parameters is poorly characterized and is often postulated from intuitive considerations. Herein, experimental evaluation through the comparison of MBs and microwell plate was carried out using the miRNA-141 (biomarker for cancer) as a target, the detection of which was performed by chemiluminescent assay with a homogeneous mismatched catalytic hairpin assembly (mCHA) reaction.

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

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 MgCl to HP1/HP2 annealing solutions sharply reduces the background in mCHA and simultaneously increases the signal-to-background (S/B) ratio.

One-pot microplate-based chemiluminescent assay coupled with catalytic hairpin assembly amplification for DNA detection.

Nowadays, considerable efforts are focused on advancing DNA detection methods, which are extremely important in clinical diagnostics, pathogen determination, gene therapy, and forensic analysis. A one-pot sensitive microplate-based chemiluminescent assay coupled with catalytic hairpin assembly (CHA) amplification for detection of a 35-mer DNA oligonucleotide was developed. To improve the assay sensitivity, a triple amplification strategy based on the application of CHA (1), streptavidin-polyperoxidase conjugate (Stp-polyHRP) (2), and an enhanced chemiluminescent reaction (3) was used.

One-step label-free chemiluminescent assay for determination of exonuclease III activity towards hairpin oligonucleotides.

Fast label-free chemiluminescent assay for determination of exonuclease III (ExoIII) activity measured towards hairpin oligonucleotide substrates was developed. The designed substrates consisted of EAD2 aptamer to hemin which was associated with DNA sequence complementary to 5'-terminus fragment of EAD2. In the presence of ExoIII the associated sequence of the hairpin stem was digested, producing EAD2 aptamer which reacted with hemin with the formation of peroxidase-mimicking DNAzyme (PMDNAzyme).

Microplate chemiluminescent assay for HBV DNA detection using 3-(10'-phenothiazinyl)propionic acid/N-morpholinopyridine pair as enhancer of HRP-catalyzed chemiluminescence.

A sensitive sandwich assay for hepatitis B virus (HBV) DNA detection based on use of commercial CL-ELISA microplates was developed. To reveal the target the covalent conjugate of reporter oligonucleotide and horseradish peroxidase (HRP) was synthesized. An employment of enhanced chemiluminescence reaction, where 3-(10'-phenothiazinyl)propionic acid/N-morpholinopyridine pair was used as enhancer of HRP-catalyzed chemiluminescence, permitted to measure the enzyme activity of the conjugate with high sensitivity.