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Highly Sensitive Fluorescence Assay for miRNA Detection: Investigation of the DNA Spacer Effect on the DSN Enzyme Activity toward Magnetic-Bead-Tethered Probes.

ABSTRACT: Researchers have recently designed various biosensors combining magnetic beads (MBs) and duplex-specific nuclease (DSN) enzyme to detect miRNAs. Yet, the interfacial mechanisms for surface-based hybridization and DSN-assisted target recycling are relatively not well understood. Thus, herein, we developed a highly sensitive and selective fluorescent biosensor to study the phenomenon that occurs on the local microenvironment surrounding the MB-tethered DNA probe via detecting microRNA-21 as a model. Using the above strategy, we investigated the influence of different DNA spacers, base-pair orientations, and surface densities on DSN-assisted target recycling. As a result, we were able to detect as low as 170 aM of miR-21 under the optimized conditions. Moreover, this approach exhibits a high selectivity in a fully matched target compared to a single-base mismatch, allowing the detection of miRNAs in serum with improved recovery. These results are attributed to the synergetic effect between the DSN enzyme activity and the neutral DNA spacer (triethylene glycol: TEG) to improve the miRNA detection's sensitivity. Finally, our strategy could create new paths for detecting microRNAs since it obliterates the enzyme-mediated cascade reaction used in previous studies, which is more expensive, more time-consuming, less sensitive, and requires double catalytic reactions.

SUBMITTER: Djebbi K

PROVIDER: S-EPMC8771974 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Accurate detection of SARS-CoV-2 neutralizing antibody (nAb) is critical for assessing the immunity levels after virus infection or vaccination. As fast, cost-effective alternatives to viral infection-based assays, competitive binding (CB) assays were developed to quantitate nAb by monitoring the ability of sera to inhibit the binding of viral spike (S) protein to the angiotensin converting enzyme 2 (ACE2) receptor. Herein, we established a bead-based flow cytometric CB assay and tested the detection performance of six combination models, i.e. immobilized ACE2 and soluble Fc-tagged S1 subunit of S protein (iACE2/S1-Fc), immobilized ACE2 and soluble Fc-tagged receptor binding domain (RBD) of S protein (iACE2/RBD-Fc), immobilized S1 and soluble Fc-tagged ACE2 (iS1/ACE2-Fc), immobilized S1 and soluble His-tagged ACE2 (iS1/ACE2-His), immobilized RBD and soluble Fc-tagged ACE2 (iRBD/ACE2-Fc), and immobilized RBD and soluble His-tagged ACE2 (iRBD/ACE2-His). Using SARS-CoV-2 monoclonal antibodies and sera of convalescent COVID-19 patients and vaccinated subjects, the combination models iACE2/RBD-Fc, iACE2/S1-Fc and iS1/ACE2-His were identified to be able to specifically detect SARS-CoV-2 nAb, among which iACE2/RBD-Fc model showed the highest sensitivity, superior to a commercial SARS-CoV-2 surrogate virus neutralization test (sVNT) ELISA kit. Further studies demonstrated that the sensitivity and specificity of CB assays were affected by the tag of ACE2, type of spike and method of measuring binding rate between ACE2 and spike. Moreover, the iACE2/RBD-Fc model showed good performance in detecting kinetic development of nAb against both the prototype SARS-CoV-2 strain and an omicron variant of SARS-CoV-2 in people immunized by an inactivated SARS-CoV-2 vaccine, and the results of iACE2/RBD-Fc model are correlated well with those of live virus-based and pseudovirus-based neutralization tests, demonstrating the potential to be developed into a highly sensitive, specific, versatile and high-throughput method for detecting SARS-CoV-2 nAb in clinical practice.

Dataset Information

Highly Sensitive Fluorescence Assay for miRNA Detection: Investigation of the DNA Spacer Effect on the DSN Enzyme Activity toward Magnetic-Bead-Tethered Probes.

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