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


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.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6





Pentaethylene glycol monododecyl ether


Charge-coupled device


Diethyl pyrocarbonate




Gold nanospheres


Inductively coupled plasma atomic emission spectroscopy


Limit of detection




Multiple sclerosis


Neutravidin-coated gold nanospheres






Optical density


Real-time PCR


Self-assembled monolayer


Surface plasmon resonance


Surface plasmon resonance imaging


Saline-sodium citrate


Tris(2-carboxyethyl) phosphine


  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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  5. 5.

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

    Article  PubMed  PubMed Central  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  23. 23.

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

    Google Scholar 

  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.

    Article  PubMed  PubMed Central  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  PubMed  Google Scholar 

Download references


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.


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



Corresponding authors

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.

Electronic supplementary material


(PDF 838 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

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

Download citation


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