A plasmonic MNAzyme signal amplification strategy for quantification of miRNA-4739 breast cancer biomarker.

A major challenge in the 21st century is the development of point-of-care diagnostic tools capable to detect and quantify disease biomarkers in a straightforward, affordable, sensitive, and specific manner. The remarkable plasmonic properties of gold nanoparticles (AuNPs) have promoted their use for development of simple methodologies for nucleic acid detection in combination with a variety of oligonucleotides amplification techniques. Here, assemblies of AuNPs with Multicomponent Nucleic Acid enzymes (MNAzymes) has been successfully used in the design of a highly sensitive and simple bioassay for rapid spectroscopic detection and quantification of miRNA-4739 in blood samples. The miRNA selected is a doxorubicin chemoresistant biomarker in breast cancer which overexpression promotes the proliferation, progression, and survival of cancer cells. In this work, two alternatives experimental designs, based on use of MNAzymes and AuNPs, have been optimized and applied for sensitive miRNA-4739 quantification: one based on a traditional direct measurement of wavelength shift and a second non-conventional simple approach based on isolation and measurement of free nanoparticles absorbance. Improvement in sensitivity and, higher measurement accuracy and precision were achieved with the second approach. The developed bioassay provides a detection limit as low as 7 pmolL-1 for miRNA-4739 quantification and performed satisfactory selectivity and well practical applicability by analysis of the miRNA-4739 in blood, demonstrating that the proposed strategy is a promising and suitable spectroscopic method for breast cancer diagnosis thought liquid biopsy of circulating tumoral cells.

AF4-UV/VIS-MALS-ICPMS/MS for the characterization of the different nanoparticulated species present in oligonucleotide-gold nanoparticle conjugates.

In-depth characterization of functionalized nanomaterials is still a remaining challenge in nanobioanalytical chemistry. In this work, we propose the online coupling of Asymmetric Flow Field-Flow Fractionation (AF4) with UV/Vis, Multiangle Light Scattering (MALS) and Inductively Coupled Plasma-Tandem Mass Spectrometry (ICP-MS/MS) detectors to carry out, in less than 10 min and directly in the functionalization reaction mixture, the complete characterization of gold nanoparticles (AuNPs) functionalized with oligonucleotides and surface-modified with polyethylene glycol (PEG). AF4 separation provided full separation of the bioconjugates from the original AuNPs while P/Au and S/Au ICP-MS/MS ratios in the bioconjugate fractographic peaks could be used to compute the corresponding stoichiometries, oligonucleotide/AuNP and PEG/AuNPs. MALS detection clearly showed the coexistence of two distinct nanoparticulated populations in the bioconjugation mixture, which were demonstrated to be different not only in size but in functionality as well. The major bioconjugate population showed lower hydrodynamic ratios (18 nm) with higher and steadier oligonucleotides/AuNPs (92) and PEG/AuNPs (2350) stoichiometries, in comparison to the minor abundant population (54 nm, 51 and 1877, respectively). Moreover, the ratio between the absorbance signals measured at 520 nm and 650 nm reflects a lower AuNP aggregation in the major (10.5) than in the minor (4.5) population. Results obtained prove the benefits of a detailed characterization to find out if subsequent purification of functionalized AuNP-oligonucleotides is required to design more efficiently their final bioanalytical application.

Signal amplification strategies for clinical biomarker quantification using elemental mass spectrometry.

The current trends in modern medicine towards early diagnosis, or even prognosis, of different diseases have brought about the need for the corresponding biomarker detection at ever lower levels in really complex matrices. To do so, it is necessary to use proper extremely sensitive detection techniques such as elemental mass spectrometry. However, target labelling with metals for subsequent sensitive ICP-MS detection falls short nowadays even if resorting to inorganic nanoparticles containing a high number of detectable elements. Thus, new amplification strategies are being proposed to face this analytical challenge that will be critically discussed in this paper. Fundamentals of different novel strategies developed to achieve signal amplification and sensitive elemental mass spectrometry detection are here discussed. Some representative examples of relevant clinical applications are highlighted, along with future prospects and challenges.