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. The detection limit of microRNA-155 assay was 0.4 pM. The coefficient of variation of the chemiluminescent signal, formed upon the heterogeneous determination of miRNA-155, was less than 12 % within the working range. The efficiency of CHAOR as an amplification method was similar to that of traditional CHA, as miRNA-155 assays based on CHAOR and CHA had similar analytical parameters. In addition, the proposed assay was highly specific. Contrary to traditional CHA, CHAOR, one of whose products is a single-stranded oligonucleotide, can be used in analytical methods based on cascade amplification.

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. In addition, the high specificity of the assay was demonstrated. The proposed assay allowed assessment of the content of microRNA-155 in human cancer lines such as HepG2, Caco2, MCF7, and HeLa. The quantitation of microRNA-155 was performed after purification of short RNAs (less than 200 nt) from cell lysates since a high matrix effect was observed without this pre-treatment. The results of the quantitative determination of the microRNA content in cells were normalized using nematode microRNA-39, the concentration of which was determined using a heterogeneous assay developed by us using a strategy identical to that of the microRNA-155 assay.

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. The mCHA reaction produced double-stranded (ds) DNA labeled at one end with fluorescein (Flu) for capture with anti-Flu antibodies immobilized on a solid carrier, on the other end with biotin for recognition by streptavidin-polyperoxidase conjugate. The conditions of immobilization of anti-Flu antibody on MBs (a diameter of 440 nm) performed using a carbodiimide method were optimized by varying the antibody concentration in the reaction solution. It was shown that the dependence of chemiluminescent signal as a function of the concentration of anti-FluAb-MBs conjugates had a bell-shaped character. The maximum chemiluminescence was produced at the concentration of the conjugates of 2 × 109 particles/mL, with a surface area of 65 mm2. The identical surface area was used upon the assay performance with polystyrene microplates. Comparison of MBs- and microplate-assays for miRNA-141 determination showed that the obtained calibration curves and their detection limit values were the same and did not depend on the used carrier. The results showed that the choice of a carrier for heterogeneous assays should be guided by the convenience of the assay performance, not its surface area.

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 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.

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. The one-pot format of the assay, where all steps of the DNA determination are performed in the same well without transfer of samples from one test tube to another, increased its precision. The proposed assay detected the target DNA in the fM range and distinguished the target DNA from related DNAs, demonstrating its high sensitivity and high selectivity. Moreover, the assay was applied successfully for the quantitative determination of the target in spiked samples of human plasma. A microplate format of the assay was convenient for the analysis of a large number of samples. This study provides a prospective tool for DNA detection. Graphical abstract.

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). The catalytic activity of the produced PMDNAzyme was measured towards luminol/H2O2. Under the optimized conditions the limit of detection and sensitivity of the one-step chemiluminescent assay of ExoIII were 7.3 nM and 1.7 × 108 M-1, respectively. The coefficient of variation (CV) was lower than 6%.

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. Under the favorable conditions the limit of detection and a linear range of the assay were 3 pM and 0.07-2.0 nM, respectively. The coefficient of variation (CV) for determination of HBV DNA concentrations within the working range was lower than 4%. The obtained results demonstrated that the developed assay had high sensitivity and precision.