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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

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.

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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

References

  1. 1.

    Kappel A, Keller A. miRNA assays in the clinical laboratory: workflow, detection technologies and automation aspects. Clin Chem Lab Med. 2016; https://doi.org/10.1515/cclm-2016-0467.

  2. 2.

    Pritchard CC, Cheng HH, Tewari M. MicroRNA profiling: approaches and considerations. Nat Rev Genet. 2012;13:358–69. https://doi.org/10.1038/nrg3198.

  3. 3.

    Graybill RM, Bailey RC. Emerging biosensing approaches for microRNA analysis. Anal Chem. 2016;88:431–50. https://doi.org/10.1021/acs.analchem.5b04679.

  4. 4.

    Degliangeli F, Kshirsagar P, Brunetti V, Pompa PP, Fiammengo R. Absolute and direct microRNA quantification using DNA–gold nanoparticle probes. J Am Chem Soc. 2014;136:2264–7. https://doi.org/10.1021/ja412152x.

  5. 5.

    Chamorro-Garcia A, Merkoçi A. Nanobiosensors in diagnostics. Nanobiomedicine. 2016;3:1849543516663574. https://doi.org/10.1177/1849543516663574.

  6. 6.

    Kalogianni DP, Kalligosfyri PM, Kyriakou IK, Christopoulos TK. Advances in microRNA analysis. Anal Bioanal Chem. 2018;410:695–713. https://doi.org/10.1007/s00216-017-0632-z.

  7. 7.

    Carrascosa LG, Huertas CS, Lechuga LM. Prospects of optical biosensors for emerging label-free RNA analysis. TrAC Trends Anal Chem. 2016;80:177–89. https://doi.org/10.1016/j.trac.2016.02.018.

  8. 8.

    Scarano S, Mascini M, Turner APF, Minunni M. Surface plasmon resonance imaging for affinity-based biosensors. Biosens Bioelectron. 2010;25:957–66. https://doi.org/10.1016/j.bios.2009.08.039.

  9. 9.

    Szunerits S, Spadavecchia J, Boukherroub R. Surface plasmon resonance: signal amplification using colloidal gold nanoparticles for enhanced sensitivity. Rev Anal Chem. 2014;33:153–64. https://doi.org/10.1515/revac-2014-0011.

  10. 10.

    Fang S, Lee HJ, Wark AW, Corn RM. Attomole microarray detection of microRNAs by nanoparticle-amplified SPR imaging measurements of surface polyadenylation reactions. J Am Chem Soc. 2006;128:14044–6. https://doi.org/10.1021/ja065223p.

  11. 11.

    Vaisocherová H, Šípová H, Víšová I, Bocková M, Špringer T, Ermini ML, et al. Rapid and sensitive detection of multiple microRNAs in cell lysate by low-fouling surface plasmon resonance biosensor. Biosens Bioelectron. 2015;70:226–31. https://doi.org/10.1016/j.bios.2015.03.038.

  12. 12.

    Wang Q, Liu R, Yang X, Wang K, Zhu J, He L, et al. Surface plasmon resonance biosensor for enzyme-free amplified microRNA detection based on gold nanoparticles and DNA supersandwich. Sensors Actuators B Chem. 2016;223:613–20. https://doi.org/10.1016/j.snb.2015.09.152.

  13. 13.

    Wang Q, Li Q, Yang X, Wang K, Du S, Zhang H, et al. Graphene oxide–gold nanoparticles hybrids-based surface plasmon resonance for sensitive detection of microRNA. Biosens Bioelectron. 2016;77:1001–7. https://doi.org/10.1016/j.bios.2015.10.091.

  14. 14.

    Zhang D, Yan Y, Cheng W, Zhang W, Li Y, Ju H, et al. Streptavidin-enhanced surface plasmon resonance biosensor for highly sensitive and specific detection of microRNA. Microchim Acta. 2013;180:397–403. https://doi.org/10.1007/s00604-013-0945-3.

  15. 15.

    Liu R, Wang Q, Li Q, Yang X, Wang K, Nie W. Surface plasmon resonance biosensor for sensitive detection of microRNA and cancer cell using multiple signal amplification strategy. Biosens Bioelectron. 2017;87:433–8. https://doi.org/10.1016/j.bios.2016.08.090.

  16. 16.

    Šípová H, Zhang S, Dudley AM, Galas D, Wang K, Homola J. Surface plasmon resonance biosensor for rapid label-free detection of microribonucleic acid at subfemtomole level. Anal Chem. 2010;82:10110–5. https://doi.org/10.1021/ac102131s.

  17. 17.

    Nasheri N, Cheng J, Singaravelu R, Wu P, McDermott MT, Pezacki JP. An enzyme-linked assay for the rapid quantification of microRNAs based on the viral suppressor of RNA silencing protein p19. Anal Biochem. 2011;412:165–72. https://doi.org/10.1016/j.ab.2011.01.030.

  18. 18.

    Ding X, Yan Y, Li S, Zhang Y, Cheng W, Cheng Q, et al. Surface plasmon resonance biosensor for highly sensitive detection of microRNA based on DNA super-sandwich assemblies and streptavidin signal amplification. Anal Chim Acta. 2015;874:59–65. https://doi.org/10.1016/j.aca.2015.03.021.

  19. 19.

    Zhou W-J, Chen Y, Corn RM. Ultrasensitive microarray detection of short RNA sequences with enzymatically modified nanoparticles and surface plasmon resonance imaging measurements. Anal Chem. 2011;83:3897–902. https://doi.org/10.1021/ac200422u.

  20. 20.

    Qiu X, Liu X, Zhang W, Zhang H, Jiang T, Fan D, et al. Dynamic monitoring of microRNA-DNA hybridization using DNAase-triggered signal amplification. Anal Chem. 2015;87:6303–10. https://doi.org/10.1021/acs.analchem.5b01159.

  21. 21.

    Hu Z, Zhang A, Storz G, Gottesman S, Leppla SH. An antibody-based microarray assay for small RNA detection. Nucleic Acids Res. 2006;34:e52. https://doi.org/10.1093/nar/gkl142.

  22. 22.

    Tran HV, Piro B, Reisberg S, Duc HT, Pham MC. Antibodies directed to RNA/DNA hybrids: an electrochemical immunosensor for microRNAs detection using graphene-composite electrodes. Anal Chem. 2013;85:8469–74. https://doi.org/10.1021/ac402154z.

  23. 23.

    Goldenberg MM. Multiple sclerosis review. Pharmacol Ther. 2012;37:175–84.

  24. 24.

    Lublin FD, Reingold SC, Cohen JA, Cutter GR, Sørensen PS, Thompson AJ, et al. Defining the clinical course of multiple sclerosis. Neurology. 2014;83:278–86. https://doi.org/10.1212/WNL.0000000000000560.

  25. 25.

    Teunissen CE, Malekzadeh A, Leurs C, Bridel C, Killestein J. Body fluid biomarkers for multiple sclerosis—the long road to clinical application. Nat Rev Neurol. 2015;11:585–96. https://doi.org/10.1038/nrneurol.2015.173.

  26. 26.

    Keller A. Multiple sclerosis: microRNA expression profiles accurately differentiate patients with relapsing-remitting disease from healthy controls. PLoS One. 2009;4:e7440. https://doi.org/10.1371/journal.pone.0007440.

  27. 27.

    Cox MB, Cairns MJ, Gandhi KS, Carroll AP, Moscovis S, Stewart GJ, et al. MicroRNAs miR-17 and miR-20a inhibit T cell activation genes and are under-expressed in MS whole blood. PLoS One. 2010;5:e12132. https://doi.org/10.1371/journal.pone.0012132.

  28. 28.

    De Santis G, Ferracin M, Biondani A, Caniatti L, Rosaria Tola M, Castellazzi M, et al. Altered miRNA expression in T regulatory cells in course of multiple sclerosis. J Neuroimmunol. 2010;226:165–71. https://doi.org/10.1016/j.jneuroim.2010.06.009.

  29. 29.

    Fenoglio C, Ridolfi E, Cantoni C, De Riz M, Bonsi R, Serpente M, et al. Decreased circulating miRNA levels in patients with primary progressive multiple sclerosis. Mult Scler Houndmills Basingstoke Engl. 2013;19:1938–42. https://doi.org/10.1177/1352458513485654.

  30. 30.

    Mancuso R, Hernis A, Agostini S, Rovaris M, Caputo D, Clerici M. MicroRNA-572 expression in multiple sclerosis patients with different patterns of clinical progression. J Transl Med. 2015;13:148. https://doi.org/10.1186/s12967-015-0504-2.

  31. 31.

    Guerau-de-Arellano M, Alder H, Ozer HG, Lovett-Racke A, Racke MK. miRNA profiling for biomarker discovery in multiple sclerosis: from microarray to deep sequencing. J Neuroimmunol. 2012;248:32–9. https://doi.org/10.1016/j.jneuroim.2011.10.006.

  32. 32.

    Junker A, Hohlfeld R, Meinl E. The emerging role of microRNAs in multiple sclerosis. Nat Rev Neurol. 2011;7:56–9. https://doi.org/10.1038/nrneurol.2010.179.

  33. 33.

    Ojea-Jiménez I, Bastús NG, Puntes V. Influence of the sequence of the reagents addition in the citrate-mediated synthesis of gold nanoparticles. J Phys Chem C. 2011;115:15752–7. https://doi.org/10.1021/jp2017242.

  34. 34.

    Conde J, Ambrosone A, Sanz V, Hernandez Y, Marchesano V, Tian F, et al. Design of multifunctional gold nanoparticles for in vitro and in vivo gene silencing. ACS Nano. 2012;6:8316–24. https://doi.org/10.1021/nn3030223.

  35. 35.

    Puertas S, de Gracia Villa M, Mendoza E, Jiménez-Jorquera C, de la Fuente JM, Fernández-Sánchez C, et al. Improving immunosensor performance through oriented immobilization of antibodies on carbon nanotube composite surfaces. Biosens Bioelectron. 2013;43:274–80. https://doi.org/10.1016/j.bios.2012.12.010.

  36. 36.

    Sola L, Damin F, Cretich M, Chiari M. Novel polymeric coatings with tailored hydrophobicity to control spot size and morphology in DNA microarray. Sensors Actuators B Chem. 2016;231:412–22. https://doi.org/10.1016/j.snb.2016.03.049.

  37. 37.

    Ermini ML, Mariani S, Scarano S, Minunni M. Direct detection of genomic DNA by surface plasmon resonance imaging: an optimized approach. Biosens Bioelectron. 2013;40:193–9. https://doi.org/10.1016/j.bios.2012.07.018.

  38. 38.

    Simon L, Lautner G, Gyurcsányi RE. Reliable microspotting methodology for peptide-nucleic acid layers with high hybridization efficiency on gold SPR imaging chips. Anal Methods. 2015;7:6077–82. https://doi.org/10.1039/C5AY01239B.

  39. 39.

    Fernández F, Sánchez-Baeza F, Marco M-P. Nanogold probe enhanced surface plasmon resonance immunosensor for improved detection of antibiotic residues. Biosens Bioelectron. 2012;34:151–8. https://doi.org/10.1016/j.bios.2012.01.036.

  40. 40.

    Špringer T, Chadtová S, Ermini ML, Lamačová J, Homola J. Functional gold nanoparticles for optical affinity biosensing. Anal Bioanal Chem. 2017;409:4087–97. https://doi.org/10.1007/s00216-017-0355-1.

  41. 41.

    Junker A. MicroRNA profiling of multiple sclerosis lesions identifies modulators of the regulatory protein CD47. Brain. 2009;132:3342–52. https://doi.org/10.1093/brain/awp300.

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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

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).

Author information

Correspondence to Jesus M. de la Fuente or Renzo Vanna.

Ethics declarations

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.

Additional information

Published in the topical collection Nanoparticles for Bioanalysis with guest editors María Carmen Blanco-López and Montserrat Rivas.

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Cite this article

Sguassero, A., Artiga, Á., Morasso, C. et al. A simple and universal enzyme-free approach for the detection of multiple microRNAs using a single nanostructured enhancer of surface plasmon resonance imaging. Anal Bioanal Chem 411, 1873–1885 (2019). https://doi.org/10.1007/s00216-018-1331-0

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Keywords

  • SPR
  • Nanoparticles
  • Nanobiosensor
  • miRNA
  • Enhancement
  • Multiplexing