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

Bodulev, Oleg L.; Burkin, Konstantin M.; Ефремов, Е. Е.; Sakharov, I Iu

doi:10.1007/s00216-020-02438-6

Cited by 16 publications

(9 citation statements)

References 26 publications

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“…The calibration curve obtained under the optimized conditions obeyed a double rectangular hyperbola equation with the formula ( R 2 0.9979), where a , b , c , and d were 2.03 × 10 6 , 0.12, 5.86 × 10 7 , and 365, respectively, and may be used to calculate the miRNA-141 concentration in the samples of interest. The curves with similar behaviors were previously reported for assays coupled with CHA/mCHA. ,, The analysis of the calibration curve showed that when the target concentration changed from 1 to 100 pM, the chemiluminescence intensity increased by 8 times, while in many of the miRNA assays reported previously, the signal value changed only by 3–4 times with changing the analyte concentration by 6–7 orders of magnitude. − …”

Section: Results and Discussionsupporting

confidence: 80%

“…In further work, the annealing of HP1 and HP2 was carried out using 30 nM and 100 nM solutions in 10 mM Tris–HCl, pH 7.2, containing 20 mM and 10 mM MgCl 2 , respectively. In the case of HP1, lower concentrations were not used because it was previously shown that a suitable concentration of HP1 for its immobilization on anti-FITC antibodies adsorbed in the wells of a microplate is 30 nM . The annealing of the biotin-HP2 probe with concentrations below 100 nM did not show any additional effect on the background value.…”

Section: Results and Discussionmentioning

confidence: 99%

“…In the case of HP1, lower concentrations were not used because it was previously shown that a suitable concentration of HP1 for its immobilization on anti-FITC antibodies adsorbed in the wells of a microplate is 30 nM. 28 The annealing of the biotin-HP2 probe with concentrations below 100 nM did not show any additional effect on the background value. Optimization of MgCl 2 Concentration in the Assay Reaction Solution.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

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

2021

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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.

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Section: Results and Discussionsupporting

confidence: 80%

Section: Results and Discussionmentioning

confidence: 99%

Section: ■ Results and Discussionmentioning

confidence: 99%

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

2021

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“…Single-stranded target DNA serves as a trigger and a signal amplifier to generate a large number of self-assembled DNA products from catalytic hairpin DNAs. These amplified DNA products can then be incorporated into a variety of detection techniques including colorimetry [ 7 – 9 ], fluorescence [ 10 – 12 ], chemiluminescence [ 13 , 14 ], and electrochemistry [ 15 ], providing for convenient and sensitive detection of very low abundance target DNA. But despite such advantages, specific and expensive instruments are often still required, and therefore, the development of enzyme- and instrument-free sensing systems is in demand for cost-effective POC diagnostics.…”

Section: Introductionmentioning

confidence: 99%

Target DNA- and pH-responsive DNA hydrogel–based capillary assay for the optical detection of short SARS-CoV-2 cDNA

2021

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Graphical abstract DNA is recognized as a powerful biomarker for clinical diagnostics because its specific sequences are closely related to the cause and development of diseases. However, achieving rapid, low-cost, and sensitive detection of short-length target DNA still remains a considerable challenge. Herein, we successfully combine the catalytic hairpin assembly (CHA) technique with capillary action to develop a new and cost-effective method, a target DNA- and pH-responsive DNA hydrogel–based capillary assay, for the naked eye detection of 24 nt short single-stranded target DNA. Upon contact of target DNA, three individual hairpin DNAs hybridize with each other to sufficiently amplify Y-shaped DNA nanostructures (Y-DNA) until they are completely consumed via CHA cycling reactions. Each arm of the resultant Y-DNA contains sticky ends with i-motif DNA structure-forming sequences that can be self-assembled in an acidic environment (pH 5.0) to form target DNA- and pH-responsive DNA hydrogels by means of i-motif DNA-driven crosslinking. When inserting a capillary tube in the resultant solution, the liquid level inside clearly reduces due to the decrease in capillary force induced by the gels. In this way, the developed assay demonstrates sensitive and quantitative detection, with a detection limit of approximately 10 pM of 24 nt short complementary DNA (cDNA) targeting SARS-CoV-2 RNA genes at room temperature within 1 h. The assay is further shown to successfully detect target cDNA in serum, and it is also applied to detect several types of target sequences. Requiring no analytic equipment, precise temperature control, or enzymatic reactions, the developed DNA hydrogel–based capillary assay has potential as a promising naked eye detection platform for target DNA in resource-limited clinical settings. Supplementary Information The online version contains supplementary material available at 10.1007/s00604-021-05138-9.

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“…With the addition of a molecular beacon (MB), CHA can generate the fluorescent signal to achieve signal detection, thus realizing sensitive analysis of target. 39,40 In this work, a fluorescent biosensor template containing functional AuNP-based 3D DNA walker and CHA was proposed for pathogen detection. As a track of the 3D DNA walker, AuNPs can provide an appropriate anchoring area for the DNA ligand; the distance and conformation between the adjacent PolyA-DNA probes have been systematically studied by changing the length of PolyA with real-time fluorescence intensity monitoring.…”

Section: Introductionmentioning

confidence: 99%

An enzyme-mediated universal fluorescent biosensor template for pathogen detection based on a three-dimensional DNA walker and catalyzed hairpin assembly

Yang

Luo

et al. 2021

Nanoscale

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

Cited by 16 publications

References 26 publications

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

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

Target DNA- and pH-responsive DNA hydrogel–based capillary assay for the optical detection of short SARS-CoV-2 cDNA

An enzyme-mediated universal fluorescent biosensor template for pathogen detection based on a three-dimensional DNA walker and catalyzed hairpin assembly

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