Fluorescence-enhanced p19 proteins-conjugated single quantum dot with multiplex antenna for one-step, specific and sensitive miRNAs detection.

Ideal methods for miRNA detection should be rapid and amplification-free in the mix-and-measure format. Here we report a novel FRET strategy based on fluorescence-enhanced p19 proteins-conjugated single quantum dot (QD) with multiplex antenna for one-step, specific and sensitive miRNAs quantitative analysis. It is simple in design and operation, and possesses high sensitivity and selectivity. In the strategy, RNA-binding viral p19 proteins-conjugated quantum dot (p19-QD) with high fluorescence and multiplex antenna was designed, and exploited for the capture recognition element and the donor. In the presence of target miRNAs, the p19-QD would tightly capture the miRNA-21-antimiRNA-21 duplex (dsRNA) formed in solution by hybridization of the specific antimiRNA-21-Cy3 probe (receptor) to the single-stranded miRNA-21 target. The FRET detection system between QD and Cy3 was constructed, and the signal read-out was measured. Conversely, in the absence of target miRNA-21, the p19-QD refuse to capture the free single-stranded antimiRNA-21-Cy3 probe (receptor), the FRET between quantum dot and Cy3 is deterred, thereby providing a low-background signal to improve sensitivity. Benefiting from the high affinity and specificity of p19 protein for duplex RNA and the fluorescence enhancement of donor p19-QD by the passivation and protection from p19 protein, a detection limit as low as 0.6 fM was achieved without employing any amplification techniques and pre-concentration or purification steps. The performance can be achieved by only one-step incubation without additional reagents and washing steps, thus greatly reducing the operating difficulty and saving time. Moreover, the fluorescence-enhanced p19-QD conjugate-based FRET strategy avoids the specific design in the number of the bases for interval in the DNA-QD conjugate-based FRET system using DNA strands as the FRET linker, and improves the FRET efficiency. Furthermore, it has been successfully applied to analyze the content of miRNA-21 in breast cancer cell. The results indicated that, the strategy will become a sensitive and reliable miRNAs quantification method in biomedical research and early clinical diagnostics.