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Anal Bioanal Chem. 2018 Aug 28. doi: 10.1007/s00216-018-1331-0. [Epub ahead of print]

A simple and universal enzyme-free approach for the detection of multiple microRNAs using a single nanostructured enhancer of surface plasmon resonance imaging.

Author information

1
Laboratory of Nanomedicine and Clinical Biophotonics (LABION), IRCCS Fondazione Don Carlo Gnocchi, 20121, Milan, Italy.
2
Instituto de Ciencia de Materiales de Aragon (CSIC-Universidad de Zaragoza), 50009, Zaragoza, Spain.
3
Centro de Investigacion Biomedica en Red in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), 28029, Madrid, Spain.
4
Instituto de Nanociencia de Aragón (INA), Universidad de Zaragoza, Campus Rio Ebro, Edificio I+D, INA, C/Mariano Esquillor s/n, 50018, Zaragoza, Spain.
5
Laboratory of Molecular Medicine and Biotechnologies, IRCCS Fondazione Don Carlo Gnocchi, 20121, Milan, Italy.
6
Latvian Biomedical Research and Study Centre, Riga, 1067, Latvia.
7
Nanomedicine Center NANOMIB, School of Medicine and Surgery, University of Milano-Bicocca, 20900, Monza, Italy.
8
Multiple Sclerosis Unit, IRCCS Fondazione Don Carlo Gnocchi, 20121, Milan, Italy.
9
Instituto de Ciencia de Materiales de Aragon (CSIC-Universidad de Zaragoza), 50009, Zaragoza, Spain. jmfuente@unizar.es.
10
Centro de Investigacion Biomedica en Red in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), 28029, Madrid, Spain. jmfuente@unizar.es.
11
Laboratory of Nanomedicine and Clinical Biophotonics (LABION), IRCCS Fondazione Don Carlo Gnocchi, 20121, Milan, Italy. renzo.vanna@icsmaugeri.it.
12
Istituti Clinici Scientifici Maugeri IRCCS, Via Maugeri 10, 27100, Pavia, Italy. renzo.vanna@icsmaugeri.it.

Abstract

Here we describe a simple approach for the simultaneous detection of multiple microRNAs (miRNAs) using a single nanostructured reagent as surface plasmon resonance imaging (SPRi) enhancer and without using enzymatic reactions, sequence specific enhancers or multiple enhancing steps as normally reported in similar studies. The strategy involves the preparation and optimisation of neutravidin-coated gold nanospheres (nGNSs) functionalised with a previously biotinylated antibody (Ab) against DNA/RNA hybrids. The Ab guarantees the recognition of any miRNA sequence adsorbed on a surface properly functionalised with different DNA probes; at the same time, gold nanoparticles permit to detect this interaction, thus producing enough SPRi signal even at a low ligand concentration. After a careful optimisation of the nanoenhancer and after its characterisation, the final assay allowed the simultaneous detection of four miRNAs with a limit of detection (LOD) of up to 0.5 pM (equal to 275 attomoles in 500 μL) by performing a single enhancing injection. The proposed strategy shows good signal specificity and permits to discriminate wild-type, single- and triple-mutated sequences much better than non-enhanced SPRi. Finally, the method works properly in complex samples (total RNA extracted from blood) as demonstrated by the detection of four miRNAs potentially related to multiple sclerosis used as case study. This proof-of-concept study confirms that the approach provides the possibility to detect a theoretically unlimited number of miRNAs using a simple protocol and an easily prepared enhancing reagent, and may further facilitate the development of affordable multiplexing miRNA screening for clinical purposes.

KEYWORDS:

Enhancement; Multiplexing; Nanobiosensor; Nanoparticles; SPR; miRNA

PMID:
30155701
DOI:
10.1007/s00216-018-1331-0

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