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.

An ultrasensitive bunge bedstraw herb type DNA machine for absolute quantification of mRNA in single cell.

Messenger ribonucleic acids (mRNAs) comprise a class of small nucleic acids carrying genetic information, which exhibit very important role in medical research and diagnosis. If only the mean mRNA expression levels of the mRNA population are considered in medical research, important information linking mRNA expression and cellular function may be lost. Single-cell analysis provides valuable insights into studying its heterogeneity, signaling, and stochastic gene expression. In this study, a "bunge bedstraw herb"-type DNA machine based on DNAzyme catalyzing coupled clamping hybrid chain reaction (c-HCR) is presented. In the DNA machine, a bunge bedstraw herb-type DNA structure was first formed by hybridizing a core junction scaffold cruciform probe to a hairpin probe that can trigger the c-HCR via a target molecule in four directions. This approach can reduce the detection limit of mRNA to 5 × 10-15 M. Absolute quantification of survivin mRNA in individual cells was achieved using the DNA machine on a microfluidic chip electrophoresis platform. The reported method represents an unprecedented single-cell analysis platform for single-cell biology studies.

Comparison of chemiluminescent heterogeneous and homogeneous-heterogeneous assays coupled with isothermal circular strand-displacement polymerization reaction amplification for the quantification of miRNA-141.

Heterogeneous and homogeneous-heterogeneous assays for the quantitation of hsa-miR-141-3p (miRNA-141) were constructed. Both microplate assays were based on the use of the isothermal circular strand-displacement polymerization reaction (ICSDPR), which was carried out in heterogeneous and homogeneous media, respectively. In addition, a streptavidin-polyperoxidase conjugate and enhanced chemiluminescence were used to increase the assay's sensitivity. A comparison of the developed assays showed that the sensitivity of the heterogeneous assay was higher than that of the homogeneous-heterogeneous assay. The detection limit values of the heterogeneous and homogeneous-heterogeneous assays were 51 fM and 10 pM, respectively. The amplification index for the ICSDPR used in the heterogeneous assay of miRNA-141 was 100. Using miRNAs of the miRNA-200 family, the high specificity of the assay was demonstrated. MiRNA-141 in human cultured cells was determined by the heterogeneous ICSDPR-assisted assay with chemiluminescence detection. To assess the purification yield of miRNAs from cellular lysates, the heterogeneous assay of miRNA-39 developed on the same platform was used. The intracellular content of miRNA-141 in Caco-2, HepG2, MCF-7 and HeLa was shown to be 3400, 1400, 1300 and 470 copies per cell, respectively.

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.

An ultrasensitive multivariate signal amplification strategy based on microchip platform tailored for simultaneous quantification of multiple microRNAs in single cell.

MicroRNAs (miRNAs) play a very important regulatory role in life activities. Abnormal expression levels of miRNAs in cells are associated with various diseases, especially human cancer. Nevertheless, accurate detection of the copy numbers of various miRNA molecules in single cell is still a great challenge. In this study, an intracellular multivariate signal amplification strategy based on microchip platform was proposed, and an ultrasensitive single-cell analysis method was established for simultaneous quantification of absolute copy numbers of multiple miRNAs in a single cell. Using miRNA-21 and miRNA-141 as the analytical models of miRNAs, the detection limits of 1.0 and 2.0 fM were obtained. Based on the developed method, an analysis of 600 randomly acquired different types of cells was performed. The distribution of absolute copy numbers of miRNA-21 and miRNA-141 in six types of cells was obtained. It was found that the number of copies of miRNA-21 and miRNA-141 in different types of cancer cells showed different expression characteristics. The study results can help us more accurately understand cell-to-cell heterogeneity and the relationship between different miRNAs and different types of cancer at the single cell level.

Absolute Quantification of MicroRNAs in a Single Cell with Chemiluminescence Detection Based on Rolling Circle Amplification on a Microchip Platform.

The absolute quantification of miRNAs in a single cell allows to better understand the heterogeneity of cells and the relationship between miRNAs and diseases. However, seldom methods for miRNA quantification in a single cell have been reported because the miRNA content in a single cell is very low. Herein, an ultrasensitive chemiluminescence assay strategy based on rolling circle amplification (RCA) on a microchip platform was proposed for the absolute quantification of miRNAs in a single cell. In this strategy, a ring probe with specificity was designed and synthesized, which could perform RCA for target miRNAs to improve the sensitivity and satisfy the need of absolute quantification of miRNAs in a single cell. The 20 liver cancer cells (HepG2) and 20 normal liver cells (HL-7702) were analyzed using this method; it is found that the miRNA-21 contents varied among cells, and miRNA-21 was overexpressed in HepG2 cells. Compared with traditional methods, the proposed strategy has many advantages such as low cost, simple operation, short analysis time, good specificity, and lower probability of false positives. This method is expected to be one of the powerful tools for the absolute quantification of miRNAs in a single cell.

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.

Isothermal chemiluminescent assay based on circular stand-displacement polymerization reaction amplification for cel-miRNA-39-3p determination in cell extracts.

A sensitive and specific heterogeneous assay for quantitation of cel-miRNA-39-3p (miRNA-39) was constructed. To improve the assay sensitivity an amplification strategy based on the use of isothermal circular strand-displacement polymerization reaction (ICSDPR), polyperoxidase conjugated with streptavidin and enhanced chemiluminescence was used. The detection limit of the proposed assay was 4 × 10-13 M. The coefficient of variation (CV) for quantitation of miRNA-39 within the working range was below 8%. The study of cross-reactivity of different miRNAs including miRNA-39 demonstrated high specificity of the proposed assay. Comparison of the calibration curves of miRNA-39 dissolved in the buffer and the lysate of MCF-7 cells (prepared by lysis of the cells with phenol/guanidine thiocyanate mixture and purified using silica membrane spin column) has demonstrated a negligible matrix effect. The proposed assay makes it possible to estimate the yield of purification of miRNAs from cells, which is necessary for the quantitative calculation of the intracellular content of miRNAs measured with the isothermal assay coupled with ICSDPR.

Chemiluminescent microplate-based assay of DNA based on isothermal circular strand-displacement polymerization reaction (ICSDPR).

Sensitive microplate-based chemiluminescent assay coupled with isothermal circular strand-displacement polymerization reaction (ICSDPR) for DNA detection was developed. The assay sensitivity was improved using a triple amplification strategy based on employment of ICSDPR, streptavidin-polyperoxidase conjugate and an enhanced chemiluminescent reaction. To increase the accuracy all stages of the assay (one-pot format) were carried out in the same microplate well. The proposed assay detected the target DNA in fM-pM ranges and distinguish target DNA from related mismatched DNAs, demonstrating high sensitivity and high selectivity. This format is highly suitable for the development of sensitive and high-throughput kits of nucleic acids which can be easily automated.

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.

Suicide inactivation of covalent peroxidase-mimicking DNAzyme with hydrogen peroxide and its protection by a reductant substrate.

Recently a covalent peroxidase-mimicking DNAzyme (cPMDNAzyme) with the improved catalytic activity was prepared. Here we demonstrate that hydrogen peroxide, the oxidant substrate of cPMDNAzyme is an inactivating agent of this catalyst. Presence of the reductant substrate, 2,2'-azino-bis(3-ethylbenthothiazoline-6-sulfonic acid (ABTS) prevents the inactivation of cPMDNAzyme. The experimental conditions (pH-optimum, concentrations of ABTS and H2O2) for the determination of cPMDNAzyme activity were optimized that allows a construction of the colorimetric cPMDNAzyme-based biosensors and assays with improved sensitivity.

Fiber-optic immunosensor for detection of Crimean-Congo hemorrhagic fever IgG antibodies in patients.

Crimean-Congo hemorrhagic fever (CCHF) is a severe viral disease with high fatality rate. CCHF virus is endemic in parts of Africa, Asia, the Middle East, and southeastern Europe. Rapid diagnostics of CCHF is vital for appropriate clinical management and prevention of secondary spread from human-to-human. Currently, diagnostics relies on real-time RT-PCR and antibody or antigen detection using ELISA. These methods require trained personnel and expensive equipment and are not appropriate for point-of-care (POC) diagnostics. Furthermore, there are no POC assays available for CCHF. We developed a fiber-optic biosensor for the detection of CCHF IgG antibodies. In order to improve sensitivity, we optimized both the bioreceptor immobilization protocol and the chemiluminescence substrate formulation. The resulting protocol showed a 100-fold greater sensitivity for detection of CCHF antibodies. Finally, we evaluated the fiber-optic biosensor with two CCHF patient sera. We showed that the fiber-optic biosensor is 10-times more sensitive than colorimetric ELISA and is able to detect both patients with high and low levels of IgG antibodies. We believe that the fiber-optic biosensor is a suitable alternative to ELISA as it is much more sensitive and makes it possible to detect a small amount of antibodies at an early stage of infection and can be integrated as a point-of-care diagnostic system of CCHF.

A WS2 nanosheet based chemiluminescence resonance energy transfer platform for sensing biomolecules.

We developed a WS2 nanosheet based chemiluminescence resonance energy transfer (CRET) platform for sensing biomolecules. This platform exhibits high detection sensitivity and high specificity for target molecules.

An enhanced chemiluminescence resonance energy transfer system based on target recycling G-guadruplexes/hemin DNAzyme catalysis and its application in ultrasensitive detection of DNA.

An enhanced chemiluminescence resonance energy transfer (CRET) system based on target recycling G-guadruplexes/hemin DNAzyme catalysis was developed for ultrasensitive detection of DNA. CRET system consists of luminol as chemiluminescent donor, and fluorescein isothiocyanate (FITC) as acceptor. The sensitive detection was achieved by using the system consisted of G-riched DNA, blocker DNA, and the Nb.BbvCI biocatalyst. Upon addition of target DNA to the system, target DNA hybridizes with the quasi-circular DNA structure, and forms a DNA duplex. The formation of DNA duplex triggers selective enzymatic cleavage of quasi-circular DNA by Nb.BbvCI, resulting in the release of target DNA and two G-riched DNAzyme segments. Released target DNA then hybridizes with another quasi-circular DNA structure to initiate the cleavage of the quasi-circular DNA structure. Eventually, each target DNA can go through many cycles, resulting in the digestion of many quasi-circular DNA structures, generating many G-riched DNAzyme segments. G-riched DNAzyme segment products assemble with hemin to form stable hemin/G-quadruplexes that exhibit peroxidase-like activity which can catalyze the oxidation of luminol by H2O2 to produce CL signals. In the presence of FITC, CL of luminol can excite FITC molecules, and thus produced CRET between the luminol and FITC. This unique analysis strategy gives a detection limit down to 80 fM, which is at least four orders of magnitude lower than that of unamplified DNA detection methods.

High chemiluminescence activity of an Fe(III)-TAML activator in aqueous-organic media and its use in the determination of organic peroxides.

High activity of Fe(III)-TAML, peroxidase mimic, upon the catalytic oxidation of luminol in aqueous-organic media (ethanol, isopropanol and acetonitrile) was determined. Using Fe(III)-TAML the sensitive chemiluminescence assays for the determination of benzoyl peroxide and tert-butyl hydroperoxide in the presence of organic solvents were performed.

Amino acid sequence of anionic peroxidase from the windmill palm tree Trachycarpus fortunei.

Palm peroxidases are extremely stable and have uncommon substrate specificity. This study was designed to fill in the knowledge gap about the structures of a peroxidase from the windmill palm tree Trachycarpus fortunei. The complete amino acid sequence and partial glycosylation were determined by MALDI-top-down sequencing of native windmill palm tree peroxidase (WPTP), MALDI-TOF/TOF MS/MS of WPTP tryptic peptides, and cDNA sequencing. The propeptide of WPTP contained N- and C-terminal signal sequences which contained 21 and 17 amino acid residues, respectively. Mature WPTP was 306 amino acids in length, and its carbohydrate content ranged from 21% to 29%. Comparison to closely related royal palm tree peroxidase revealed structural features that may explain differences in their substrate specificity. The results can be used to guide engineering of WPTP and its novel applications.

Improved method for chemiluminescent determination of peroxidase-mimicking DNAzyme activity.

The optimization of experimental conditions for the chemiluminescent determination of peroxidase-mimicking DNAzyme (PMDNAzyme) formed at the interaction of hemin and its aptamer EAD2 was performed. The effect of concentrations of hydrogen peroxide and luminol, acidity of the substrate solution, and composition and concentration of the assay buffer was estimated. Under optimized conditions, a value of detection limit for the PMDNAzyme was 350 pM. A comparison of the conditions determined in this work with those reported previously showed that the optimization of the composition of the substrate solution improved the sensitivity of the chemiluminescent determination of the PMDNAzyme. The obtained results open up promising perspectives for using the proposed method to improve the sensitivity of PMDNAzyme-based assays.