DNA-gold nanoparticle probes are implemented in a simple strategy for direct microRNA (miRNA) quantification. Fluorescently labeled DNA-probe strands are immobilized on PEGylated gold nanoparticles (AuNPs). In the presence of target miRNA, DNA-RNA heteroduplexes are formed and become substrate for the endonuclease DSN (duplex-specific nuclease). Enzymatic hydrolysis of the DNA strands yields a fluorescence signal due to diffusion of the fluorophores away from the gold surface. We show that the molecular design of our DNA-AuNP probes, with the DNA strands immobilized on top of the PEG-based passivation layer, results in nearly unaltered enzymatic activity toward immobilized heteroduplexes compared to substrates free in solution. The assay, developed in a real-time format, allows absolute quantification of as little as 0.2 fmol of miR-203. We also show the application of the assay for direct quantification of cancer-related miR-203 and miR-21 in samples of extracted total RNA from cell cultures. The possibility of direct and absolute quantification may significantly advance the use of microRNAs as biomarkers in the clinical praxis.

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MicroRNAs (miRNAs) are important regulators of gene expression, and many pathological conditions, including cancer, are characterized by altered miRNA expression levels. Therefore, accurate and sensitive quantification of miRNAs may result in correct disease diagnosis establishing these small noncoding RNA transcripts as valuable biomarkers. Aiming at overcoming some limitations of conventional quantification strategies, nanotechnology is currently providing numerous significant alternatives to miRNA sensing. In this review an up-to-date account of nanotechnology-based strategies for miRNA detection and quantification is given. The topics covered are: nanoparticle-based approaches in solution, sensing based on nanostructured surfaces, combined nanoparticle/surface sensing approaches, and single-molecule approaches.