Simple, Amplified, and Multiplexed Detection of MicroRNAs Using Time-Gated FRET and Hybridization Chain Reaction

Abstract

The hybridization chain reaction (HCR) is a simple and sensitive method for quantifying nucleic acids. Current approaches cannot combine a washing-free sensing format with multiplexed target quantification at low concentrations, which would be highly desirable for detection both in solution and in situ. Here, we demonstrate the implementation of time-gated Forster resonance energy transfer (TG-FRET) between terbium donors and dye acceptors into HCR for multiplexed quantification of microRNAs (miR-20a and miR-21) and their DNA analogues. HCR-TG-FRET provided washing-free nucleic acid quantification with very low limits of detection down to 240 amol (1.7 pM) of microRNA and 123 amol (0.88 pM) of DNA. Efficient distinction from very homologous microRNAs demonstrated high target specificity. Multiplexing with a single measurement, a single excitation wavelength, and a single FRET pair allowed for a simultaneous quantification of miR-20a and miR-21 at concentrations between 30 and 300 pM from the same sample. HCR-TG-FRET showed similar performance for serum-free and serum-containing samples without the use of RNase inhibitors. Our results present a significant improvement in current HCR approaches regarding simplicity, sensitivity, and multiplexing. The versatile diagnostic performance of HCR-TG-FRET even in challenging biological environments presents an important advantage for advanced nucleic acid biosensing.

A Janus 3D DNA nanomachine for simultaneous and sensitive fluorescence detection and imaging of dual microRNAs in cancer cells

“…[10][11][12][13][14][15][16][17] Although the current method for simultaneous uorescence detection and imaging of dual miRNAs in cells could efficiently eliminate "false positive" results obtained in single type miRNA detection, [18][19][20][21][22][23] it is difficult to achieve sensitive detection and accurate co-location imaging of miRNAs in living cells due to the use of two different separate molecular beacons (MBs). [24][25][26][27][28][29] Recently, some 3D DNA nanostructures have been employed in simultaneous detection and imaging of dual miRNA targets via stochastically immobilizing two different signal probes on the surface of the homogeneous nanoparticles to realize relatively precise co-location in the same position of a cell. [30][31][32][33][34] However, the uorescence signal interference could be still caused by the proximity of the two different signal probes immobilized on the homogeneous nanoparticle-based 3D DNA nanostructure, resulting in low sensitivity for the simultaneous detection and uorescence imaging of miRNAs in cells.…”

Mechanically Triggered Hybridization Chain Reaction

“…Note that mechano-HCR reflects the traction forces present at earlier time points because of the kinetics of HCR. Assays such as the "exchange-mechano-HCR assay", where the hairpin monomers are barcoded with different dyes and washed into the sample periodically,may offer the ability to time-stamp the mechano-HCR signals.F urthermore, advances in HCR, such as controlled HCR, [22] branched HCR, [37] and FRET HCR, [38] suggest future directions to improve mechano-HCR S/N,c onvenience by reducing wash steps,a nd other advantages in simplifying quantification to address the challenge.F inally,m echano-HCR offers important potential in the adoption for applications such as medium-throughput drug and cell screening in both biological research and clinics.…”

A Novel Colorimetric Nano Aptasensor for Ultrasensitive Detection of Aflatoxin B1 Based on the Exonuclease III-Assisted Signal Amplification Approach

“…The AFB1-induced displacement of the cDNA from the aptamer is the key not only for the recognition unit of the target but also for the starting unit of the amplification reaction. We tested the feasibility of this process for AFB1 detection employing a FRET analysis [28,29]. We labeled the anti-AFB1 aptamer and its complementary DNA with a fluorescence probe (FAM) and quencher probe (BHQ1), respectively.…”