Ratiometric Fluorescence Detection of Colorectal Cancer-Associated Exosomal miR-92a-3p with DSN-Assisted Signal Amplification by a MWCNTs@Au NCs Nanoplatform

Abstract

The detection of miRNA shows great promise in disease diagnosis. In this work, a ratiometric fluorescent biosensor based on multi-walled carbon nanotubes@gold nanoclusters (MWCNTs@Au NCs) and duplex-specific nuclease (DSN)-assisted signal amplification was fabricated for miRNA detection. Colorectal cancer (CRC)-associated miR-92a-3p extracted from exosomes was selected as the target. MWCNTs@Au NCs performs the dual functions of fluorescence quencher and internal fluorescence reference. In the absence of miR-92a-3p, an Atto-425-modified single-stranded DNA probe is adsorbed on MWCNTs@Au NCs, resulting in the quenching of Atto-425. In the presence of miR-92a-3p, the duplex is formed by hybridization of the probe and miR-92a-3p and leaves the MWCNTs@Au NCs, resulting in the fluorescence recovery of Atto-425. DSN can cleave the probe and result in the release of miR-92a-3p. The released miR-92a-3p can hybridize with other probes to form a signal amplification cycle. The fluorescence of MWCNTs@Au NCs remains stable and constitutes a ratiometric fluorescence system with that of Atto-425. A detection concentration interval of 0.1-10 pM and a limit of detection of 31 fM was obtained under optimized measurement conditions. In addition, the accuracy of the biosensor was validated by detecting the concentration of miR-92a-3p extracted from clinical exosome samples.

Keywords: Au nanoclusters; biosensor; duplex-specific nuclease; exosomal miRNA; multi-walled carbon nanotubes; ratiometric fluorescence.

Figure 1

(a) Schematic illustration of the synthesis of MWCNTs@Au NCs. TEM images of (b) MWCNTs, (c) Au NCs (the inset depicts the size distribution of Au NCs) and (d,e) MWCNTs@Au NCs.

Figure 2

(a) FTIR spectra and (b) zeta potentials of MWCNTs and MWCNTs@Au NCs. (c) Fluorescence excitation and emission spectra of MWCNTs@Au NCs (the inset shows the optical photographs of MWCNTs@Au NCs under sunlight (left) and 360 nm UV light (right)). (d) UV–vis absorption spectra of MWCNTs and MWCNTs@Au NCs.

Figure 3

(a) Schematic diagram of fluorescence detection of miR-92a-3p based on MWCNTs@Au NCs and DSN-assisted signal amplification. (b) Fluorescence spectra of probe, MWCNTs@Au NCs, probe + MWCNTs, probe + MWCNTs@Au NCs, probe + MWCNTs@Au NCs + miR-92a-3p, and probe + MWCNTs@Au NCs + miR-92a-3p + DSN.

Figure 4

(a) The effect of the amount of MWCNTs@Au NCs on the fluorescence signals of the biosensing system. (b) The variation of the fluorescence of Atto-425 with the mixing time of the probe and MWCNTs@Au NCs. (c) The effect of incubation temperature on the fluorescence signals (n = 3, mean ± s.d.).

Figure 5

(a) Fluorescence spectra of the biosensor under different concentrations of miR-92a-3p. (b) The calibration line of the Δprobe/MWCNTs@Au NCs fluorescence ratio against the concentration of miR-92a-3p (n = 3, mean ± s.d.). (c) Comparison of the fluorescence signals produced by miR-92a-3p and mismatched sequences (n = 3, mean ± s.d.).

Figure 6

(a) TEM image of exosomes. (b) Western blotting of exosome-enriched (En.) and exosome-unenriched (Unen.) samples. C1~C3 represent CRC patients; H1~H3 represent healthy controls. (c) Comparison of the concentration of exosomal miR-92a-3p of three CRC patients and three healthy controls detected by RT-qPCR and the proposed biosensor (n = 3, mean ± s.d.).