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

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.

Tracking soluble and nanoparticulated titanium released in vivo from metal dental implant debris using (single-particle)-ICP-MS.

BACKGROUND: This work studies the presence of the Ti, Al and V metal ions and Ti nanoparticles released from the debris produced by the implantoplasty, a surgical procedure used in the clinic, in rat organs. METHODS: The sample preparation for total Ti determination was carefully optimized using microsampling inserts to minimize the dilution during the acid attack of the lyophilized tissues by a microwave-assisted acid digestion method. An enzymatic digestion method was optimized and applied to the different tissue samples in order to extract the titanium nanoparticles for the single-particle ICP-MS analysis. RESULTS: A statistically significant increase was found for Ti concentrations from control to experimental groups for several of the studied tissues, being and particularly significant in the case of brain and spleen. Al and V concentrations were detected in all tissues but they were not different when comparing control and experimental animals, except for V in brain. The possible presence of Ti-containing nanoparticles mobilized from the implantoplasty debris was tested using enzymatic digestions and SP-ICP-MS. The presence of Ti-containing nanoparticles was observed in all the analyzed tissues, however, differences on the Ti mass per particle were found between the blanks and the digested tissue and between control and experimental animals in some organs. CONCLUSION: The developed methodologies, both for ionic and nanoparticulated metal contents in rat organs, have shown the possible increase in the levels of Ti both as ions and nanoparticles in rats subjected to implantoplasty.