Highly Sensitive Detection of miR-21 through Target-Activated Catalytic Hairpin Assembly of X-Shaped DNA Nanostructures

Abstract

MicroRNAs (miRNAs) are found in extremely low concentrations in cells, so highly sensitive quantitation is a great challenge. Herein, a simple dual-amplification strategy involving target-activated catalytic hairpin assembly (CHA) coupled with multiple fluorophores concentrated on one X-shaped DNA is reported. In this strategy, four hairpin probes (H1, H2, H3, and H4) are modified with FAM and BHQ1 at both sticky ends, while a circulating hairpin probe (H0) is used to activate CHA circuits once it binds to complementary sequences in the target miR-21 (T). The powerful dual-amplification cascades in Förster resonance energy transfer (FRET)-based nonenzymatic nucleic acid circuits are triggered by T–H0-activated formation of the X-shaped DNA nanostructure, freeing T–H0 for the next CHA reaction cycle. CHA circuits increase the fluorescence due to the wide distance between FAM and BHQ1 in the formed X-shaped DNA nanostructure, resulting in signal amplification and highly sensitive detection of miR-21, with a limit of detection (LOD, 3σ) of 0.025 nM, which is 25.6 or 57.6 times lower than that obtained through a single-amplification strategy without multiple fluorophores on one X-shaped DNA or CHA circuit. Furthermore, this cascade reaction was completed in 45 min, effectively avoiding target degradation. This new enzyme-free signal amplification strategy holds promising potential for sensitively detecting different DNA or RNA sequences by simply adapting the fragment of the H0 sequence complementary to the target.

Sensitive, Highly Stable, and Anti-Fouling Electrode with Hexanethiol and Poly-A Modification for Exosomal microRNA Detection

“…The concentration of miR-21 showed a good linear relationship with the current signal and the limit of detection (LOD) of the proposed biosensor was calculated as 1.46 aM based on the 3σ/slope rule (Figure 5B). 29 Compared to other recently reported miRNA sensors, the proposed sensor had a lower LOD (Table S3). 2,30−38 The dual signal amplification steps ensured the sensitivity of this sensor, showing a great potential for tumor marker-based diagnosis.…”

“…The dynamic range of detection was from 100.00 aM to 1.00 nM. The concentration of miR-21 showed a good linear relationship with the current signal and the limit of detection (LOD) of the proposed biosensor was calculated as 1.46 aM based on the 3σ/slope rule (Figure B) . Compared to other recently reported miRNA sensors, the proposed sensor had a lower LOD (Table S3).…”

Entropy-Driven Three-Dimensional DNA Nanofireworks for Simultaneous Real-Time Imaging of Telomerase and MicroRNA in Living Cells

“…As illustrated by Figure a,b, a dynamic correlation between the Cy3 fluorescent signal and the concentration of miRNA-21 was observed in the range of 0–2000 pM. Calculated based on the 3σ method (Group I in Figure a,b), the limit of detection (LOD) of this method is 34 pM. , Compared to the reported strategies with fluorescence analysis based on nucleic acid nanomaterials (displayed in Table S2), our proposed DNFs showed a higher sensitivity for this target detection. Similarly, the Cy5 fluorescence intensity proportionally increased with varying concentrations of HeLa cell extracts (containing the corresponding level of telomerase) ranging from 0 to 1.6 × 10 4 cells/μL (Group II in Figure a,b), and LOD is calculated as 100 cells/μL according to the average signal of three times of standard deviation of the control, , showing a comparative sensitivity with the previous methods (Table S3).…”

Localized DNA tetrahedrons assisted catalytic hairpin assembly for the rapid and sensitive profiling of small extracellular vesicle-associated microRNAs

“…Despite their decent performances, these strategies are not rapid and simple enough for sEV-miRNAs measurement because of the tedious operation and harsh reaction conditions. Accordingly, metastable hairpindriven amplification, including catalytic hairpin assembly (CHA) [21][22][23], hybridization chain reaction (HCR) [24,25], have been proposed due to its high specificity, lowcost, and easy to use. Compared with the assembled concatemer products of HCR, the dangled ssDNA products of CHA are easier to capture and generate an enhanced electrochemical signal [18,26,27].…”