Duplex-specific nuclease-based electrochemical biosensor for the detection of microRNAs by conversion of homogeneous assay into surface-tethered electrochemical analysis

Lin Liu; Dehua Deng; Daohong Wu; Weilin Hou; Lu Wang; Ning Li; Zhifang Sun

doi:10.1016/j.aca.2021.338199

Duplex-specific nuclease-based electrochemical biosensor for the detection of microRNAs by conversion of homogeneous assay into surface-tethered electrochemical analysis

Anal Chim Acta. 2021 Mar 8:1149:338199. doi: 10.1016/j.aca.2021.338199. Epub 2021 Jan 7.

Authors

Lin Liu¹, Dehua Deng², Daohong Wu³, Weilin Hou², Lu Wang², Ning Li², Zhifang Sun⁴

Affiliations

¹ Henan Province of Key Laboratory of New Optoelectronic Functional Materials, Anyang Normal University, Anyang, Henan, 455000, PR China. Electronic address: liulin@aynu.edu.cn.
² Henan Province of Key Laboratory of New Optoelectronic Functional Materials, Anyang Normal University, Anyang, Henan, 455000, PR China.
³ College of Chemistry and Chemical Engineering, Central South University, Changsha, Hunan, 410083, PR China.
⁴ Henan Province of Key Laboratory of New Optoelectronic Functional Materials, Anyang Normal University, Anyang, Henan, 455000, PR China; College of Chemistry and Chemical Engineering, Central South University, Changsha, Hunan, 410083, PR China. Electronic address: allensune@gmail.com.

PMID: 33551055
DOI: 10.1016/j.aca.2021.338199

Abstract

We proposed a simple and sensitive strategy for the detection of microRNAs (miRNAs) by converting homogeneous assay into surface-tethered electrochemical analysis. Specifically, the biotinylated detection probes (biotin-DNA-biotin) can trigger the in-situ assembly of tetrameric streptavidin (SA) proteins on an electrode surface via the SA-biotin interactions. The (SA-biotin-DNA-biotin)_n assemblies electrically insulated the electrode interface, thereby blocking the electron transfer of [Fe(CN)₆]^3-/4-. When the probe was hybridized with the target miRNA, it would be cleaved into small fragments (denoted as biotin-DNA) by duplex-specific nuclease (DSN). The released target miRNA can enter into the next hybridization-enzymolysis cycle, thus leading to the generation of considerable amounts of biotin-DNA fragments. The released biotin-DNA competed with the detection probe to bind SA, thus limiting the in-situ formation of (SA-biotin-DNA-biotin)_n assemblies. The surface-tethered electrochemical analysis by the dual signal amplification of DSN and (SA-biotin-DNA-biotin)_n assemblies has been used for the determination of miRNAs in cell lysate with a satisfactory result. The method showed a detection limit down to 10 aM. The "one-step" immobilization-free strategy can be used to design novel biosensors for the detection of other biomarkers.

Keywords: Duplex-specific nuclease; Electrochemical biosensors; Streptavidin; Surface-tethered analysis; microRNA.

MeSH terms

Biosensing Techniques*
Electrochemical Techniques
Endonucleases
Limit of Detection
MicroRNAs*
Nucleic Acid Hybridization

Substances

MicroRNAs
Endonucleases