Magnetic separation-assisted cluster-amplified versatile fluorescent aptasensors for the sensitive detection of target biomolecules.

A sensitive and versatile platform for detecting diverse target biomolecules was developed by combining a magnetic separation module and a fluorescence amplification module in a plug-and-play manner. The magnetic separation module was constructed using magnetic beads (MBs), whose surfaces were modified with aptamer-blocked captor DNAs. The fluorescence amplification module was constructed by loading the fluorescent dye rhodamine 6G (Rh6G) into the pores of mesoporous silica nanoparticles (MSNs). The MSN surfaces were modified with prey DNAs, of which the MSN-near ends hybridized with complementary DNAs (sealing DNAs) to form duplexes to seal the pores, and the free ends were designed to be single-stranded that were complementary to the captor DNAs. Upon binding of targets to their aptamers, the captor DNAs were unblocked and thus were able to hybridize with the prey DNAs, to capture Rh6G-laden MSNs, forming MB-MSN clusters. The clusters were isolated by magnetic separation and heated to dissociate the DNA duplexes, to unseal the MSN pores and release the inner Rh6G; thus a target was converted into a cluster of Rh6G dyes. By simply changing the target aptamers and related DNA connectors, this strategy detected ATP, thrombin, and platelet-derived growth factor BB with detection limits of 2.1 nM, 4.1 pM, and 2.4 pM, respectively. A wide range of targets, high amplification efficiency and universal functional modules endow the aptasensors with good potential as versatile platforms for detecting target molecules in vitro and in medical research.

MnO2 nanosheet-mediated target-binding-induced FRET strategy for multiplexed microRNAs detection and imaging in living cells.

In the regulatory network, miRNAs play a regulatory role in a cooperative or antagonistic manner. Simultaneous accurate detection and imaging of multiplexed miRNAs in living cells are of great significance for miRNA-associated biological research and disease diagnosis and treatment. Herein, a MnO2 nanosheet-mediated target-binding-induced fluorescence resonance energy transfer (FRET) strategy was developed for detection and imaging of multiplexed miRNAs in living cells. Two pairs of DNA probes (P1-AF 488/P1'-Cy3 and P2-AF 488/P2'-AF 594) contained the complementary sequence to target miRNAs (miRNA-373 and miRNA-96) and labelled with different fluorescence dyes were designed. They were adsorbed onto MnO2 nanosheets by physisorption to form DNA/MnO2 nanocomposite probes. When the DNA/MnO2 nanocomposite probes were taken up by cells, the MnO2 nanosheets were reduced by intracellular glutathione, accompanying the release of DNA probe pairs. Then the DNA probe pairs specifically recognized and combined with miRNA-373 and miRNA-96 to form stable duplexes, respectively, bringing labelled fluorophores into close proximity to occur FRET. Based on this, the simultaneous imaging of miRNA-373 and miRNA-96 in MDA-MB-231 and L02 cells was successfully implemented. The results displayed a higher expression level of target miRNAs in MDA-MB-231 cells compared to L02 cells. The changes in expression levels of miRNA-96 induced by anti-miRNA-96 or mimics in MDA-MB-231 cells could also be monitored. In addition, the ratiometric detections of multiplexed miRNAs were achieved by utilizing the DNA probe pairs. The proposed strategy provides an alternative method for simultaneous accurate detection and imaging of multiplexed miRNAs and has potential application in biomedical applications.