G-wire-based self-quenched fluorescence probe combining with target-activated isothermal cascade amplification for ultrasensitive microRNA detection.

Herein, we reported the G-wire-based self-quenched fluorescence probe and its application in ultrasensitive microRNA (miRNA) detection by combining with target-activated isothermal cascade amplification. The terminal-single-fluorescein (FAM)-labeled G-rich oligonucletides self-assembled into G-wire nanostructures (G-wires) with K+ and Mg2+. Thereafter, the G-wires brought terminal-labeled FAM into close proximity, as a result, the self-quenched signal probe formed. Besides, when there was the target miRNA, target-activated isothermal cascade amplification converted miRNA into the copious trigger DNA. After hybridization between trigger DNA and the self-quenched probe, the G-wires were splited and forced the apart of proximate FAM, and then the self-quenched probe displayed an "on" mechanism. Therefore, the approach gave a limit of detection (LOM) of 0.82 aM to miRNA-21 and could be implemented within a wide linear range of 2 aM to 2 nM. This approach was able to distinguish the single-mismatched miRNA-21, which was selective and sensitive in detecting human spiked serum samples.

Label-free detection of microRNA: two-stage signal enhancement with hairpin assisted cascade isothermal amplification and light-up DNA-silver nanoclusters.

A method is described for the determination of microRNAs via two-stage signal enhancement. This is attained by combining hairpin (HP) assisted cascade isothermal amplification with light-up DNA-Ag nanoclusters. A rationally designed dual-functional HP is used, and microRNA-21 is chosen as a model analyte. At the first stage, upon the hybridization of the microRNA-21 with HP, microRNA recycling via polymerase-displacement reaction and a circulative nicking-replication process are achieved. This generates numerous G-abundant overhang DNA sequences. In the second stage, the above-released G-abundant overhang DNA sequences hybridize with the dark green Ag NCs, and this results in the appearance of bright red fluorescence. Thanks to the two signal enhancement processes, a linear dependence between the fluorescence intensity at 616 nm and the concentration of microRNA-21 is obtained in the range from 1 pM to 20 pM with a detection limit of 0.7 pM. The strategy clearly discriminates between perfectly-matched and mismatched targets. The method was applied to the determination of microRNA-21 in a spiked serum sample. Graphical abstractSchematic representation of microRNA detection by integrating hairpin assisted cascade isothermal amplification with light-up DNA Ag nanoclusters. With microRNA, G-abundant overhang DNA sequences from amplification reaction hybridize with dark green Ag nanoclusters to produce a concentration-dependent bright red fluorescence.

Graphene oxide-based fluorometric determination of microRNA-141 using rolling circle amplification and exonuclease III-aided recycling amplification.

A graphene oxide-based method has been developed for ultrasensitive and selective determination of microRNA-141 by means of rolling circle amplification (RCA) and exonuclease III (Exo III)-assisted recycling amplification. The method uses (a) a padlock probe with a hybrid sequence that is complementary to the target microRNA-141 at both the 5'- and the 3'-end, and (b) a long binding region of a signalling reporter strand. On addition of microRNA-141, it acts as the primer for triggering the RCA reaction following ligation. This results in the formation of a repeatedly concatenated sequence of the padlock probe. Subsequently, the RCA product hybridizes with the fluorescein-labelled signal strand to form the duplex DNA containing the blunt 3'-termini of signal strand. Addition of Exo III causes signal strand digestion and leads to RCA product recycling and liberation of fluorescein. Added graphene oxide does not absorb the fluorescein liberated because of the poor mutual interaction. Therefore, microRNA-141 can be quantified by measurement of the green fluorescence under excitation/emission wavelengths of 470/520 nm. The method has a 100 aM detection limit towards microRNA-141 and works in the wide range from 1 fM to 1 nM. It can discriminate even single-mismatched microRNA and shows good selectivity and sensitivity when applied to spiked human serum. Graphical abstract Schematic representation of a graphene oxide (GO)-based method for fluorometric determination of microRNA by using rolling circle amplification and exonuclease III (Exo III)-aided recycling amplification. With microRNA, the fluorescein-labelled signal strand becomes digested, and this genererates a fluorescent signal.

Spectroscopic investigation and in vitro cytotoxic activity toward HepG2 cells of a copper compound complexed with human serum albumin.

The human serum albumin (HSA) interaction of a mixed-ligand copper compound (1) with an imidazole and taurine Schiff base derived from salicylaldehyde and taurine was investigated using fluorescence spectroscopy, UV-vis spectroscopy, time-resolved fluorescence spectroscopy, circular dichroism (CD) spectroscopy, Fourier transform infrared (FT-IR) spectroscopy and a molecular docking technique. The results of fluorescence and time-resolved fluorescence spectroscopy indicated that 1 can effectively quench the HSA fluorescence by a static mechanism. Binding constants (K) and the number of binding sites (n ≈ 1) were calculated using modified Stern-Volmer equations. The thermodynamic parameters were calculated. UV-vis, CD and FT-IR spectroscopy measurements confirm the alterations in the HSA secondary structure induced by 1. The site marker competitive experiment confirms that 1 is located in subdomain IB of HSA. The combination of molecular docking results and fluorescence experimental results reveal that hydrophobic interaction and hydrogen bonds are the predominant intermolecular forces stabilizing the 1-HSA complex. The 1-HSA complex increases approximately three times its cytotoxicity in cancer cells but has no effect on normal cells in vitro. Compared with unbound 1, the 1-HSA complex promotes HepG2 cells apoptosis and also has a stronger capacity for cell cycle arrest at the S phase of HepG2 cells.