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

  • Andrea Sguassero
  • Álvaro Artiga
  • Carlo Morasso
  • Rafael Ramirez Jimenez
  • Rafael Martín Rapún
  • Roberta Mancuso
  • Simone Agostini
  • Ambra Hernis
  • Arturs Abols
  • Aija Linē
  • Alice Gualerzi
  • Silvia Picciolini
  • Marzia Bedoni
  • Marco Rovaris
  • Furio Gramatica
  • Jesus M. de la FuenteEmail author
  • Renzo VannaEmail author
Research Paper
Part of the following topical collections:
  1. Nanoparticles for Bioanalysis

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

SPR Nanoparticles Nanobiosensor miRNA Enhancement Multiplexing 

Abbreviations

Ab

Antibody

C12E5

Pentaethylene glycol monododecyl ether

CCD

Charge-coupled device

DEPC

Diethyl pyrocarbonate

EDC

1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide

GNSs

Gold nanospheres

ICP-AES

Inductively coupled plasma atomic emission spectroscopy

LOD

Limit of detection

miRNA

MicroRNA

MS

Multiple sclerosis

nGNSs

Neutravidin-coated gold nanospheres

NHS

N-Hydroxysuccinimide

NP

Nanoparticle

OD

Optical density

RT-PCR

Real-time PCR

SAM

Self-assembled monolayer

SPR

Surface plasmon resonance

SPRi

Surface plasmon resonance imaging

SSC3

Saline-sodium citrate

TCEP

Tris(2-carboxyethyl) phosphine

Notes

Acknowledgements

The authors thank Dr. Domenico Caputo for subjects’ recruitment and for his support for clinical aspects. They also thank Nicholas Corneli for his support for spotting and buffer optimisation. The authors gratefully acknowledge “The Advanced Microscopy Laboratory” (INA-Universidad de Zaragoza) for access to their instrumentation and expertise. They also thank J.C. Raposo of the Servicio Central de Análisis de Bizkaia from SGIker of Universidad del País Vasco (EHU) for the ICP technical support and J. Puertas of the Radioisotope Service of Universidad de Zaragoza for the radiolabelling technical support.

Funding information

Research funding was provided by the Italian Ministry of Health within the framework of the European EuroNanoMedII Project (Call 2015) entitled “NanoPlasmiRNA”; DGA-FSE (Diputación General de Aragón–Fondo Social Europeo); Ministerio de Educación, Cultura y Deportes of Spanish Government, FPU grant (FPU014/06249).

Compliance with ethical standards

Healthy control subjects were involved in the study. Three subjects have been enrolled at IRCCS Fondazione Don Carlo Gnocchi. These subjects gave written informed consent in accordance with the protocols approved by the ethics committee of the same institution and according to the principles of the Declaration of Helsinki.

Competing interests

The authors declare that they have no competing interests.

Supplementary material

216_2018_1331_MOESM1_ESM.pdf (839 kb)
ESM 1 (PDF 838 kb)

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Andrea Sguassero
    • 1
  • Álvaro Artiga
    • 2
    • 3
  • Carlo Morasso
    • 1
  • Rafael Ramirez Jimenez
    • 2
    • 3
  • Rafael Martín Rapún
    • 4
  • Roberta Mancuso
    • 5
  • Simone Agostini
    • 5
  • Ambra Hernis
    • 5
  • Arturs Abols
    • 6
  • Aija Linē
    • 6
  • Alice Gualerzi
    • 1
  • Silvia Picciolini
    • 1
    • 7
  • Marzia Bedoni
    • 1
  • Marco Rovaris
    • 8
  • Furio Gramatica
    • 1
  • Jesus M. de la Fuente
    • 2
    • 3
    Email author
  • Renzo Vanna
    • 1
    • 9
    Email author
  1. 1.Laboratory of Nanomedicine and Clinical Biophotonics (LABION)IRCCS Fondazione Don Carlo GnocchiMilanItaly
  2. 2.Instituto de Ciencia de Materiales de Aragon (CSIC-Universidad de Zaragoza)ZaragozaSpain
  3. 3.Centro de Investigacion Biomedica en Red in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN)MadridSpain
  4. 4.Instituto de Nanociencia de Aragón (INA)Universidad de ZaragozaZaragozaSpain
  5. 5.Laboratory of Molecular Medicine and BiotechnologiesIRCCS Fondazione Don Carlo GnocchiMilanItaly
  6. 6.Latvian Biomedical Research and Study CentreRigaLatvia
  7. 7.Nanomedicine Center NANOMIB, School of Medicine and SurgeryUniversity of Milano-BicoccaMonzaItaly
  8. 8.Multiple Sclerosis UnitIRCCS Fondazione Don Carlo GnocchiMilanItaly
  9. 9.Istituti Clinici Scientifici Maugeri IRCCSPaviaItaly

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