Label-Free and Sensitive Electrochemical Biosensor for Amplification Detection of Target Nucleic Acids Based on Transduction Hairpins and Three-Leg DNAzyme Walkers

Abstract

Nucleic acids are regarded as reliable biomarkers for the early diagnosis of various diseases. By ingeniously combining a transduction hairpin (THP) with the toehold-mediated strand displacement reaction (TSDR) to form three-leg DNAzyme walkers, for the first time, we constructed a label-free and sensitive electrochemical sensing system for the amplification detection of target nucleic acids. With microRNA-155 (miR-155) as a model target, the feasibility of the biosensing strategy and the conformational states of DNA in the recognition process were studied in detail on the basis of electrochemical and dual polarization interferometry techniques. With the assistance of THP, miR-155 indirectly triggered the TSDR between three hairpins (H1, H2, and H3), then massive Mg2+-dependent three-leg DNAzyme walkers were formed in aqueous solutions. After the binding/cleaving/moving process of three-leg DNAzyme walkers on the electrode surface modified with substrate hairpins (SHPs), a number of single-stranded DNAs (ssDNAs) were generated. Hence, the interaction of methylene blue (MB) with the duplex section of SHPs was impeded, which brought about a decreased electrochemical signal. Benefiting from the cyclic amplification of the TSDR and the higher cleavage activity of three-leg DNAzyme walkers, the proposed sensing strategy showed remarkable improvement in sensitivity with a low detection limit of 0.27 fM for miR-155. Owing to the precise design of the THP, this method exhibited excellent specificity to distinguish miR-155 from the single-base and triplex-base mismatched sequences. This sensing strategy importing the flexible THP can be utilized to detect various nucleic acid biomarkers by only redesigning the THP without changing the main circuit or reporter constructs, showing the great versatility and potential for the early diagnostics and biological analysis.

Nucleic acid isothermal amplification-based soft nanoarchitectonics as an emerging electrochemical biosensing platform

“…38 To further achieve higher reaction efficiency, the captured hairpin DNA could also be modified on nanomaterials, such as gold nanomaterials (AuNPs or gold nanorods) and magnetic nanobeads to construct 3D DNA nanomachine-mediated CHA 39 or without using nanomaterials, a transduction hairpin (THP) DNA with stem-loop structure is used to form three-leg DNA nanomachine. 40 Benefitting from the improved reaction rate and the conversion efficiency, 3D DNA nanomachine-mediated CHA realized a detection limit of 0.27 fM miRNA-155 on gold electrode, 40 0.139 fM miRNA-21 on magnetic glassy carbon electrode (MGCE), 39 0.17 fM miRNA-21 in enzymatic biofuel cells (EBFCs) 41 and 0.32 aM miRNA-21 on magnetic AuNFs/ITO electrodes. Further combining loop-mediated isothermal amplification (LAMP) with CHA, which takes full advantage of the ultrasensitive feature of nucleic acid amplification, an extremely small amount of gene targets (down to 2 copies per μL) was analyzed (Fig.…”

Enzyme-Free Autocatalysis-Driven Feedback DNA Circuits for Amplified Aptasensing of Living Cells

“…Hence, signal amplification approaches are urgently needed to improve the sensitivity of ATP aptasensors and are expected to facilitate the real-time visualization of intracellular ATP. To date, various enzyme-free self-assembly DNA nanodevices have been designed for biomolecule analysis, including hybridization chain reaction (HCR), , catalytic hairpin assembly (CHA), − entropy-driven catalysis (EDC), , and other methods. , The efficient signal amplification occurs via the autonomous and successive DNA hybridization and strand-displacement processes for analyzing biomolecules of trace amount in living cells. Therefore, these nonenzymatic DNA amplifiers represent excellent candidates to visualize ATP molecules in living cells.…”

Photoactivated DNA Walker Based on DNA Nanoflares for Signal-Amplified MicroRNA Imaging in Single Living Cells

“…28−31 Enzymes such as EXO III or DNAzyme are generally used as an energy source that complements and enhances the strand displacement strategy to enhance the efficiency of DNA walker motion. 22,32,33 DNAzyme has sequence dependence, which sometimes limits its applications. EXO III, in contrast, is more versatile.…”

“…For signal amplification during detection, a DNA walker which could constantly move along a designed DNA track has also been used to achieve sensitive target nucleic acid detection. ,− Particularly, a DNA walker based on DNA nanoflares − is widely used for signal amplification imaging of target miRNA in living cells. − Enzymes such as EXO III or DNAzyme are generally used as an energy source that complements and enhances the strand displacement strategy to enhance the efficiency of DNA walker motion. ,, DNAzyme has sequence dependence, which sometimes limits its applications. EXO III, in contrast, is more versatile.…”