Immunochemical Micro Imaging Analyses for the Detection of Proteins in Artworks

  • Giorgia Sciutto
  • Martina Zangheri
  • Silvia Prati
  • Massimo Guardigli
  • Mara Mirasoli
  • Rocco Mazzeo
  • Aldo Roda
Part of the following topical collections:
  1. Analytical Chemistry for Cultural Heritage


The present review is aimed at reporting on the most advanced and recent applications of immunochemical imaging techniques for the localization of proteins within complex and multilayered paint stratigraphies. Indeed, a paint sample is usually constituted by the superimposition of different layers whose characterization is fundamental in the evaluation of the state of conservation and for addressing proper restoration interventions. Immunochemical methods, which are based on the high selectivity of antigen–antibody reactions, were proposed some years ago in the field of cultural heritage. In addition to enzyme-linked immunosorbent assays for protein identification, immunochemical imaging methods have also been explored in the last decades, thanks to the possibility to localize the target analytes, thus increasing the amount of information obtained and thereby reducing the number of samples and/or analyses needed for a comprehensive characterization of the sample. In this review, chemiluminescent, spectroscopic and electrochemical imaging detection methods are discussed to illustrate potentialities and limits of advanced immunochemical imaging systems for the analysis of paint cross-sections.


Paint cross-section Immunoassay Imaging Proteins Chemiluminescence Immuno-SERS Immuno-SECM 


  1. 1.
    Wild D (2013) The immunoassay handbook, 4th edn. Elsevier, AmsterdamGoogle Scholar
  2. 2.
    Cartechini L, Vagnini M, Palmieri M, Pitzurra L, Mello T, Mazurek J, Chiari G (2010) Immunodetection of proteins in ancient paint media. Acc Chem Res 43:867CrossRefGoogle Scholar
  3. 3.
    Cartechini L, Palmieri M, Vagnini M, Pitzurra L (2016) Immunochemical methods applied to art-historical materials: identification and localization of proteins by ELISA and IFM. Top Curr Chem. doi: 10.1007/s41061-015-0006-y Google Scholar
  4. 4.
    Palmieri M, Vagnini M, Pitzurra L, Rocchi P, Brunetti BG, Sgamellotti A, Cartechini L (2011) Development of an analytical protocol for a fast, sensitive and specific protein recognition in paintings by enzyme-linked immunosorbent assay (ELISA). Anal Bioanal Chem 399:3011CrossRefGoogle Scholar
  5. 5.
    Palmieri M, Vagnini M, Pitzurra L, Brunetti BG, Cartechini L (2013) Identification of animal glue and hen-egg yolk in paintings by use of enzyme-linked immunosorbent assay (ELISA). Anal Bioanal Chem 405:6365CrossRefGoogle Scholar
  6. 6.
    Zangheri M, Sciutto G, Mirasoli M, Prati S, Mazzeo R, Roda A, Guardigli M (2016) A portable device for on site detection of chicken ovalbumin in artworks by chemiluminescent immunochemical contact imaging. Microchem J 124:247CrossRefGoogle Scholar
  7. 7.
    Dolci LS, Sciutto G, Guardigli M, Rizzoli M, Prati S, Mazzeo R, Roda A (2008) Ultrasensitive chemiluminescent immunochemical identification and localization of protein components in painting cross-sections by microscope low-light imaging. Anal Bioanal Chem 392:29CrossRefGoogle Scholar
  8. 8.
    Sciutto G, Dolci LS, Buragina A, Prati S, Guardigli M, Mazzeo R, Roda A (2011) Development and optimization of a multiplex chemiluminescent immunochemical technique for the simultaneous detection of different proteins in paint micro cross-sections. Anal Bioanal Chem 399:2889CrossRefGoogle Scholar
  9. 9.
    Sciutto G, Dolci LS, Guardigli M, Zangheri M, Prati S, Mazzeo R, Roda A (2013) Single and multiplexed immunoassays for the chemiluminescent imaging detection of animal glues in historical paint cross-sections. Anal Bioanal Chem 405:933CrossRefGoogle Scholar
  10. 10.
    Sciutto G, Prati S, Mazzeo R, Zangheri M, Roda A, Bardini L, Valenti G, Rapino S, Marcaccio M (2014) Localization of proteins in paint cross-sections by scanning electrochemical microscopy as an alternative immunochemical detection technique. Anal Chim Acta 831:31CrossRefGoogle Scholar
  11. 11.
    Arslanoglu J, Zaleski S, Loike J (2011) An improved method of protein localization in artworks through SERS nanotag-complexed antibodies. Anal Bioanal Chem 399:2997CrossRefGoogle Scholar
  12. 12.
    Sciutto G, Litti L, Lofrumento C, Prati S, Ricci M, Gobbo M, Roda A, Castellucci E, Meneghetti M, Mazzeo R (2013) Alternative SERRS probes for the immunochemical localization of ovalbumin in paintings: an advanced mapping detection approach. Analyst 138:4532CrossRefGoogle Scholar
  13. 13.
    Perets EA, Indrasekara ASDS, Kurmis A, Atlasevich N, Fabrisb L, Arslanoglu J (2015) Carboxy-terminated immuno-SERS tags overcome non-specific aggregation for the robust detection and localization of organic media in artworks. Analyst 140:5971CrossRefGoogle Scholar
  14. 14.
    Sandu I, Schäfer S, Magrini D, Bracci S, Roque C (2012) Cross-section and staining-based techniques for investigating organic materials in painted and polychrome works of art: a review. Microsc Microanal 18:860CrossRefGoogle Scholar
  15. 15.
    Magrini D, Bracci S, Sandu ICA (2013) Fluorescence of organic binders in painting cross-sections. Proc Chem 8:194CrossRefGoogle Scholar
  16. 16.
    Kockaert L, Gausset P, Dubi-Rucquoy M (1989) Detection of ovalbumin in paint media by immuno-fluorescence. Stud Conserv 34:183Google Scholar
  17. 17.
    Raminez-Barat B, de la Vinã S (2001) Characterization of proteins in paint media by immunofluorescence: a note on methodological aspects. Stud Conserv 46:282Google Scholar
  18. 18.
    Heginbotham A, Millay V, Quick M (2004) The use of immuno-fluorescence microscopy (IFM) and enzyme-linked immunosorbent assay (ELISA) as complementary techniques for protein identification in artists’ materials. J Am Inst Conserv 45:89CrossRefGoogle Scholar
  19. 19.
    Gosling JP (1990) A decade of development in immunoassay methodology. Clin Chem 36:1408Google Scholar
  20. 20.
    DeLuca MA (1978) Methods in enzymology. Academic Press, New YorkGoogle Scholar
  21. 21.
    Kricka LJ, Stanley PE, Thorpe GHG, Whitehead TP (1984) Analytical applications of bioluminescence and chemiluminescence. Academic Press, London, New YorkGoogle Scholar
  22. 22.
    Scott D, Dikici E, Ensor M, Daunert S (2011) Bioluminescence and its impact on bioanalysis. Ann Rev Anal Chem 4:297–319CrossRefGoogle Scholar
  23. 23.
    Kamnev AA, Tugarova AV, Selivanova MA, Tarantilis PA, Polissiou MG, Kudryasheva NS (2013) Effects of americium-241 and humic substances on photobacterium phosphoreum: bioluminescence and diffuse reflectance FTIR spectroscopic studies. Spectrochim. Acta Part A Mol Biomol Spectrosc 100:171–175CrossRefGoogle Scholar
  24. 24.
    Mirasoli M, Michelini E (2014) Analytical bioluminescence and chemiluminescence. Anal Bioanal Chem 406:5529–5530CrossRefGoogle Scholar
  25. 25.
    Alieva RR, Belogurova NV, Petrova AS, Kudryasheva NS (2014) Effects of alcohols on fluorescence intensity and color of a discharged-obelin-based biomarker. Anal Bioanal Chem 406:2965–2974CrossRefGoogle Scholar
  26. 26.
    Burakova LP, Kudryavtsev AN, Stepanyuk GA, Baykov IK, Morozova VV, Tikunova NV, Dubova MA, Lyapustin VN, Yakimenko VV, Frank LA (2015) Bioluminescent detection probe for tick-borne encephalitis virus immunoassay. Anal Bioanal Chem 407:5417–5423CrossRefGoogle Scholar
  27. 27.
    Marzocchi E, Grilli S, Della Ciana L, Prodi L, Mirasoli M, Roda A (2008) Chemiluminescent detection systems of horseradish peroxidase employing nucleophilic acylation catalysts. Anal Biochem 377:189CrossRefGoogle Scholar
  28. 28.
    Zomer J (2011) The nature of chemiluminescent reactions, chemiluminescence and bioluminescence past present and future, 1st edn. Royal Society Chemistry, Cambridge, pp 54–90Google Scholar
  29. 29.
    Ximenes VF, Campa A, Baader WJ, Catalani LH (1999) Facile chemiluminescent method for alkaline phosphatase determination. Anal Chim Acta 402:99CrossRefGoogle Scholar
  30. 30.
    Mirasoli M, Venturoli S, Guardigli M, Dolci LS, Simoni P, Musiani M, Roda A (2011) Ultrasensitive bioanalytical imaging, chemiluminescence and bioluminescence: past, present and future. Royal Society of Chemistry, Cambridge, pp 398–424Google Scholar
  31. 31.
    Roda A, Pasini P, Musiani M, Baraldini M, Mirasoli M, Guardigli M, Russo C (2001) Bioanalytical applications of chemiluminescent imaging. In: GarcÍa-Campaña AM, Baeyens W (eds) Chemiluminescence in analytical chemistry. Marcel Dekker, New York, pp 473–495Google Scholar
  32. 32.
    Roda A, Guardigli M, Pasini P, Musiani M, Baraldini M (2002) Luminescence biotechnology: instruments and applications. CRC Press, FloridaGoogle Scholar
  33. 33.
    Roda A, Guardigli M, Michelini E, Pasini P, Mirasoli M (2003) Peer reviewed: analytical bioluminescence and chemiluminescence. Anal Chem 75:462ACrossRefGoogle Scholar
  34. 34.
    Roda A, Guardigli M, Michelini E, Mirasoli M (2009) Bioluminescence in analytical chemistry and in vivo imaging. Trends Anal Chem 28:307CrossRefGoogle Scholar
  35. 35.
    Creton R, Lionel FJ (2001) Chemiluminescence microscopy as a tool in biomedical research. Biotechniques 31:1098Google Scholar
  36. 36.
    Zollner H (1999) Handbook of enzyme inhibitors, 3rd edn. Wiley, WeinheimCrossRefGoogle Scholar
  37. 37.
    Price D, Worsfold PJ, Mantoura RFC (1994) Determination of hydrogen peroxide in seawater by flow injection analysis with chemiluminescence detection. Anal Chim Acta 298:121CrossRefGoogle Scholar
  38. 38.
    Yuan J, Shiller AM (1999) Determination of subnanomolar levels of hydrogen peroxide in seawater by reagent-injection chemiluminescence detection. Anal Chem 71:1975CrossRefGoogle Scholar
  39. 39.
    Rej R, Bretaudiere J-P (1980) Effects of metal ions on the measurement of alkaline phosphatase activity. Clin Chem 26:423Google Scholar
  40. 40.
    Camden JP, Dieringer JA, Wang Y, Masiello DJ, Marks LD, Schatz GC, Van Duyne RP (2008) Probing the structure of single-molecule surface-enhanced raman scattering. J Am Chem Soc 130:12616CrossRefGoogle Scholar
  41. 41.
    Henry A-I, Bingham JM, Ringe E, Marks LD, Schatz GC, Van Duyne RP (2011) Correlated structure and optical property studies of plasmonic nanoparticles. J Phys Chem 115:9291CrossRefGoogle Scholar
  42. 42.
    Amendola V, Meneghetti M (2012) Exploring how to increase the brightness of surface-enhanced raman spectroscopy nanolabels: the effect of the Raman-active molecules and of the label size. Adv Funct Mater 22:353–360CrossRefGoogle Scholar
  43. 43.
    Lutz BR, Dentinger CE, Nguyen LN, Sun L, Zhang J, Allen AN, Chan S, Knudsen BS (2008) Spectral analysis of multiplex Raman probe signatures. ACS Nano 2:2306CrossRefGoogle Scholar
  44. 44.
    Lofrumento C, Ricci M, Platania E, Becucci M, Castellucci E (2013) SERS detection of red organic dyes in Ag-agar gel. J Raman Spectrosc 44:47CrossRefGoogle Scholar
  45. 45.
    Leona M, Decuzzi P, Kubic TA, Gates G, Lombardi JR (2011) Nondestructive identification of natural and synthetic organic colorants in works of art by surface enhanced Raman scattering. Anal Chem 83:3990CrossRefGoogle Scholar
  46. 46.
    Murcia-Mascarós S, Domingo C, Sanchez-Cortes S, Cañamares MV, Garcia-Ramos JV (2005) Spectroscopic identification of alizarin in a mixture of organic red dyes by incorporation in Zr-Ormosil. J Raman Spectrosc 36:420CrossRefGoogle Scholar
  47. 47.
    Doherty B, Brunetti BG, Sgamellotti A, Miliani C (2011) A detachable SERS active cellulose film: a minimally invasive approach to the study of painting lakes. J Raman Spectrosc 42:1932CrossRefGoogle Scholar
  48. 48.
    Casadio F, Leona M, Lombardi JR, Van Duyne R (2010) Identification of organic colorants in fibers, paints, and glazes by surface enhanced Raman spectroscopy. Acc Chem Res 43:7827CrossRefGoogle Scholar
  49. 49.
    Samanta A, Maiti KK, Soh KS, Liao X, Vendrell M, Dinish US, Yun SW, Bhuvaneswari R, Kim H, Rautela S, Chung J, Olivo M, Chang YT (2011) Ultrasensitive near-infrared Raman reporters for SERS-based in vivo cancer detection. Angew Chem 50:6089CrossRefGoogle Scholar
  50. 50.
    Küstner B, Gellner M, Schütz M, Schöppler F, Marx A, Ströbel P, Adam P, Schmuck C, Schlücker S (2009) SERS labels for red laser excitation: silica-encapsulated SAMs on tunable gold/silver nanoshells. Angew Chem 48:1950CrossRefGoogle Scholar
  51. 51.
    Nie S, Emory SR (1997) Probing Single Molecules and Single Nanoparticles by Surface-Enhanced Raman Scattering. Science 275:1102CrossRefGoogle Scholar
  52. 52.
    Aroca R (2006) Surface-enhanced vibrational spectroscopy. Wiley, West SussexCrossRefGoogle Scholar
  53. 53.
    Le Ru EC, Etchgoin PG (2009) Principles of surface enhanced Raman spectroscopy and related plasmonic effects. Elsevier, AmsterdamGoogle Scholar
  54. 54.
    Moskovits M (2005) Surface-enhanced Raman spectroscopy: a brief retrospective. J Raman Spectrosc 36:485CrossRefGoogle Scholar
  55. 55.
    Lewis IR, Edwards H (2001) Handbook of Raman spectroscopy: from the research laboratory to the process line, 1st edn. CRC Press, New YorkGoogle Scholar
  56. 56.
    Lombardi JR, Birke RL, Lu T, Xu J (1986) Charge-transfer theory of surface enhanced Raman spectroscopy: herzberg-Teller contributions. J Chem Phys 84:4174CrossRefGoogle Scholar
  57. 57.
    Lombardi JR, Birke RL (2008) A unified approach to surface-enhanced Raman spectroscopy. J Phys Chem 112:5605Google Scholar
  58. 58.
    Maiti KK, Dinish US, Fu CY, Lee JJ, Soh KS, Yun SW, Bhuvaneswari R, Olivo M, Chang YT (2010) Development of biocompatible SERS nanotag with increased stability by chemisorptions of reporter molecule for in vivo cancer detection. Biosens Bioelectron 26:398CrossRefGoogle Scholar
  59. 59.
    Dykman L, Khlebtsov N (2012) Gold nanoparticles in biomedical applications: recent advances and perspectives. Chem Soc Rev 41(6):2256–2282CrossRefGoogle Scholar
  60. 60.
    Khlebtsov N, Bogatyrev V, Dykman L, Khlebtsov B, Staroverov S, Shirokov A, Matora L, Khanadeev V, Pylaev T, Tsyganova N, Terentyuk G (2013) Analytical and theranostic applications of gold nanoparticles and multifunctional nanocomposites. Theranostics 3(3):167–180CrossRefGoogle Scholar
  61. 61.
    Fojtik A, Henglein A (1993) Laser ablation of films and suspended particles in a solvent: formation of cluster and colloid solutions. Ber Bunsenges Phys Chem 97:252CrossRefGoogle Scholar
  62. 62.
    Amendola V, Meneghetti M (2009) Laser ablation synthesis in solution and size manipulation of noble metal nanoparticles. PCCP 11:3805CrossRefGoogle Scholar
  63. 63.
    Amendola V, Meneghetti M (2009) Size evaluation of gold nanoparticles by UV–vis spectroscopy. J Phys Chem 113:4277Google Scholar
  64. 64.
    Amendola V, Meneghetti M (2013) What controls the composition and the structure of nanomaterials generated by laser ablation in liquid solution. Phys Chem Chem Phys 15:3027CrossRefGoogle Scholar
  65. 65.
    Meneghetti M, Scarsi A, Litti L, Marcolongo G, Amendola V, Gobbo M, Di Chio M, Boscaini A, Fracasso G, Colombatti M (2012) Plasmonic nanostructures for SERRS multiplexed identification of tumor-associated antigens. Small 8:3733CrossRefGoogle Scholar
  66. 66.
    Kleinman SL, Frontiera RR, Henry AI, Dieringer JA, Van Duyne RP (2013) Creating, characterizing, and controlling chemistry with SERS hot spots. Phys Chem Chem Phys 15:21CrossRefGoogle Scholar
  67. 67.
    Bottari F, Oliveri P, Ugo P (2014) Electrochemical immunosensor based on ensemble of nanoelectrodes for immunoglobulin IgY detection: application to identify hen’s egg yolk in tempera paintings. Biosen Bioelectron 52:403CrossRefGoogle Scholar
  68. 68.
    Guadagnini L, Chiavari C, Martini C, Bernardi E, Morselli L, Tonelli D (2011) The use of scanning electrochemical microscopy for the characterisation of patinas on copper alloys. Electrochim Acta 56:6598CrossRefGoogle Scholar
  69. 69.
    Fan FF, Demail C (2012) Preparation of tips for scanning electrochemical microscopy, scanning electrochemical microscopy. In: Marcel D (ed) New York, pp 25–52Google Scholar
  70. 70.
    Roberts WS, Lonsdale DJ, Griffiths J, Higson SPJ (2007) Advances in the application of scanning electrochemical microscopy to bioanalytical systems. Biosens Bioelectron 23:301–318CrossRefGoogle Scholar
  71. 71.
    Carano M, Lion N, Girault HH (2007) Detection of proteins on membranes and in microchannels using copper staining combined with scanning electrochemical microscopy. J Electroanal Chem 599:349CrossRefGoogle Scholar
  72. 72.
    Stagni S, Palazzi A, Zacchini S, Ballarin B, Bruno C, Paolucci F, Marcaccio M, Carano C, Bard AJ (2006) New family of ruthenium (II) polypyridine complexes bearing 5-aryltetrazolate ligands as systems for electrochemiluminescent devices. Inorg Chem 45:695CrossRefGoogle Scholar
  73. 73.
    Zanarini S, Bard AJ, Marcaccio M, Palazzi A, Paolucci F, Stagni S (2006) Ruthenium(II) complexes containing tetrazolate group: electrochemiluminescence in solution and solid state. J Phys Chem B 110:22551CrossRefGoogle Scholar
  74. 74.
    Rapino S, Valenti G, Marcu R, Giorgio M, Marcaccio M, Paolucci F (2010) Microdrawing and highlighting a reactive surface. J Mater Chem 20:7272CrossRefGoogle Scholar
  75. 75.
    Valenti G, Bardini L, Bonazzi D, Rapino S, Marcaccio M, Paolucci F (2010) Creation of reactive micro patterns on silicon by scanning electrochemical microscopy. J Phys Chem C 114:22165CrossRefGoogle Scholar
  76. 76.
    Sun P, Laforge FO, Mirkin MV (2007) Scanning electrochemical microscopy in the 21st century. Phys Chem Chem Phys 9:802–823CrossRefGoogle Scholar
  77. 77.
    Wittstock G, Wilhelm T, Bahrs S, Steinrücke P (2001) SECM feedback imaging of enzymatic activity on agglomerated microbeads. Electroanal 13:669CrossRefGoogle Scholar
  78. 78.
    Shiku H, Matsue T, Uchida I (1996) Detection of microspotted carcinoembryonic antigen on a glass substrate by scanning electrochemical microscopy. Anal Chem 68:1276CrossRefGoogle Scholar
  79. 79.
    Zhang X, Peng X, Jin W (2006) Scanning electrochemical microscopy with enzyme immunoassay of the cancer-related antigen CA15-3. Anal Chim Acta 558:110CrossRefGoogle Scholar
  80. 80.
    Yasukawa T, Hirano Y, Motochi N, Shiku H, Matsue T (2007) Enzyme immunosensing of pepsinogens 1 and 2 by scanning electrochemical microscopy. Biosens Bioelectron 22:3099CrossRefGoogle Scholar
  81. 81.
    Kasai S, Yokota A, Zhou H, Nishizawa M, Niwa K, Onouchi T, Matsue T (2000) Immunoassay of the MRSA-related toxic protein, leukocidin, with scanning electrochemical microscopy. Anal Chem 72:5761CrossRefGoogle Scholar

Copyright information

© Springer International Publishing Switzerland 2016

Authors and Affiliations

  • Giorgia Sciutto
    • 1
  • Martina Zangheri
    • 2
  • Silvia Prati
    • 1
  • Massimo Guardigli
    • 2
  • Mara Mirasoli
    • 2
  • Rocco Mazzeo
    • 1
  • Aldo Roda
    • 2
  1. 1.Department of Chemistry “G. Ciamician”, Microchemistry and Microscopy Art Diagnostic Laboratory (M2ADL)University of BolognaRavennaItaly
  2. 2.Department of Chemistry “G. Ciamician”University of BolognaBolognaItaly

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