Amplification-Free Strategy for miRNA Quantification in Human Serum Using Single Particle ICP-MS and Gold Nanoparticles as Labels.

ABSTRACT

MicroRNAs (miRNAs), which are short single-stranded RNA sequences between 18 and 24 nucleotides, are known to play a crucial role in gene expression. Changes in their expression are not only involved in many diseases but also as a response to physiological changes, such as physical exercise. In this work, a new analytical strategy for the sensitive and specific analysis of miRNA sequences in human plasma is presented. The developed strategy does not depend on any nucleic acid amplification process and can be obtained in direct correlation to the number of events obtained by using single-particle ICP-MS measurements. The high selectivity of the assay (up to single nucleotide polymorphisms) can be achieved by a double hybridization process of the target miRNA with a complementary capture oligonucleotide that is conjugated to a magnetic microparticle and simultaneously with a complementary reporter oligonucleotide conjugated to a gold nanoparticle. Thanks to the novel approach followed in this method, the stoichiometry of the oligonucleotide-nanoparticle conjugates does not need to be addressed for the quantification of the target miRNA, which also represents a big advantage over other similar methods. The optimized method is applied to the determination of a miRNA as a biomarker of physical exercise in non-spiked human serum samples, and the results are validated against rt-qPCR. The achieved sensitivity permits the direct differentiation among sedentary and sportive subjects. This general platform can be easily applied to any other sequence by only modifying the capture and reporter oligonucleotides, paving the way for multiple clinically interesting applications.