Spectroscopic and electrophoresis study of substitution on the surface of gold nanoparticles by different mercaptoalkyl carboxylic acids and bioconjugation with bovine serum albumin

  • Raisa L. Silveira
  • Mónica B. Mamián-López
  • Joel C. Rubim
  • Marcia L. A. Temperini
  • Paola Corio
  • Jonnatan J. SantosEmail author
Research Paper


To develop bioconjugated materials, it is necessary to understand how the various elements present in a conjugate interact with one another. To gain insights into nanoparticle–capping agent–protein interactions, gold nanoparticles (AuNPs) measuring 30 nm in diameter were coated with different molecules bearing a thiol group: 3-mercaptopropionic acid, 6-mercaptohexanoic acid, and 11-mercaptoundecanoic acid. The covalent conjugation of AuNPs to the protein bovine serum albumin (BSA) via a cross-linker reaction with N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide was systematically investigated under different reaction conditions with variation of the concentrations of the mercaptoalkyl carboxylic acid (MA) and BSA. All the products were analyzed by UV–vis spectroscopy, gel electrophoresis, and Raman spectroscopy in every modification step. From analysis of the UV–vis results, it is possible at low concentrations of MA to see strong coupling among AuNPs, observed when they are aggregated by KCl, which does not happen at higher concentration of MA, indicating an AuNP-to-MA ratio of 1:130,000 is best for bioconjugation purposes. Agarose gel electrophoresis, a classic technique for biomolecule characterization, indicated that BSA is capable of altering the mobility of AuNPs when it modifies completely the surface of AuNPs because of its high molecular mass (around 66 kDa). Principal component analysis of surface-enhanced Raman spectroscopy data was successfully used as a chemometric tool to assist the characterization of the nanoparticle modification with linker molecules in the absence and presence of different BSA concentrations, making it possible to clearly evaluate the gradual substitution/modification of AuNPs (1:13,000 < 1:65,000 < 1:130,000 AuNP-to-MA ratio) and the conjugation with BSA, which is homogenous at a concentration of 0.01 g L-1.

Graphical abstract


Gold nanoparticles Mercaptoalkyl carboxylic acid Bioconjugation Bovine serum albumin Surface-enhanced Raman spectroscopy Principal component analysis 



The authors thank the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for financial support. This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Brazil, Finance Code 001.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Supplementary material

216_2019_1758_MOESM1_ESM.pdf (963 kb)
ESM 1 (PDF 962 kb)


  1. 1.
    Lane LA, Qian X, Nie S. SERS nanoparticles in medicine: from label-free detection to spectroscopic tagging. Chem Rev. 2015;115(19):10489–529.Google Scholar
  2. 2.
    Choi N, Lee J, Ko J, Jeon JH, Rhie GE, deMello AJ, et al. Integrated SERS-based microdroplet platform for the automated immunoassay of F1 antigens in Yersinia pestis. Anal Chem. 2017;89(16):8413–20.Google Scholar
  3. 3.
    Shi W, Paproski RJ, Moore R, Zemp R. Detection of circulating tumor cells using targeted surface-enhanced Raman scattering nanoparticles and magnetic enrichment. J Biomed Opt. 2014;19(5):056014.Google Scholar
  4. 4.
    Alkilany AM, Thompson LB, Boulos SP, Sisco PN, Murphy CJ. Gold nanorods: their potential for photothermal therapeutics and drug delivery, tempered by the complexity of their biological interactions. Adv Drug Deliv Rev. 2012;64(2):190–9.Google Scholar
  5. 5.
    Thakor AS, Jokerst J, Zavaleta C, Massoud TF, Gambhir SS. Gold nanoparticles: a revival in precious metal administration to patients. Nano Lett. 2011;11(10):4029–36.Google Scholar
  6. 6.
    Bhamidipati M, Fabris L. Multiparametric assessment of gold nanoparticle cytotoxicity in cancerous and healthy cells: the role of size, shape, and surface chemistry. Bioconjug Chem. 2017;28(2):449–60.Google Scholar
  7. 7.
    Bastús NG, Comenge J, Puntes V. Kinetically controlled seeded growth synthesis of citrate-stabilized gold nanoparticles of up to 200 nm: size focusing versus Ostwald ripening. Langmuir. 2011;27(17):11098–105.Google Scholar
  8. 8.
    Cañaveras F, Madueño R, Sevilla JM, Blázquez M, Pineda T. Role of the functionalization of the gold nanoparticle surface on the formation of bioconjugates with human serum albumin. J Phys Chem C. 2012;116(18):10430–7.Google Scholar
  9. 9.
    Caporusso AM, Aronica LA, Schiavi E, Martra G, Vitulli G, Salvadori P. Hydrosilylation of 1-hexyne promoted by acetone solvated gold atoms derived catalysts. J Organomet Chem. 2005;690(4):1063–6.Google Scholar
  10. 10.
    Jiang W, Mardyani S, Fischer H, Chan WCW. Design and characterization of lysine cross-linked mercapto-acid biocompatible quantum dots. Chem Mater. 2006;18(4):872–8.Google Scholar
  11. 11.
    Kairdolf BA, Qian X, Nie S. Bioconjugated nanoparticles for biosensing, in vivo Imaging, and medical diagnostics. Anal Chem. 2017;89(2):1015–31.Google Scholar
  12. 12.
    Mamian-Lopez MB, Corio P, Temperini ML. Cooperative hydrogen-bonding of the adenine-thymine pair as a strategy for lowering the limit of detection of thymine by surface-enhanced Raman spectroscopy. Analyst. 2016;141(11):3428–36.Google Scholar
  13. 13.
    Fan M, Andrade GFS, Brolo AG. A review on the fabrication of substrates for surface enhanced Raman spectroscopy and their applications in analytical chemistry. Anal Chim Acta. 2011;693(1):7–25.Google Scholar
  14. 14.
    Mieszawska AJ, Mulder WJ, Fayad ZA, Cormode DP. Multifunctional gold nanoparticles for diagnosis and therapy of disease. Mol Pharm. 2013;10(3):831–47.Google Scholar
  15. 15.
    Bhamidipati M, Cho H-Y, Lee K-B, Fabris L. SERS-based quantification of biomarker expression at the single cell level enabled by gold nanostars and truncated aptamers. Bioconjug Chem. 2018;29(9):2970–81.Google Scholar
  16. 16.
    Narayan S, Rajagopalan A, Reddy JS, Chadha A. BSA binding to silica capped gold nanostructures: effect of surface cap and conjugation design on nanostructure-BSA interface. RSC Adv. 2014;4(3):1412–20.Google Scholar
  17. 17.
    Selva Sharma A, Ilanchelian M. Comprehensive multispectroscopic analysis on the interaction and corona formation of human serum albumin with gold/silver alloy nanoparticles. J Phys Chem B. 2015;119(30):9461–76.Google Scholar
  18. 18.
    Baptista PV, Doria G, Quaresma P, Cavadas M, Neves CS, Gomes I, et al. Nanoparticles in molecular diagnostics. Prog Mol Biol Transl Sci. 2011;104:427–88.Google Scholar
  19. 19.
    Di Marco M, Shamsuddin S, Razak KA, Aziz AA, Devaux C, Borghi E, et al. Overview of the main methods used to combine proteins with nanosystems: absorption, bioconjugation, and encapsulation. Int J Nanomedicine. 2010;5:37–49.Google Scholar
  20. 20.
    Moore CJ, Monton H, O'Kennedy R, Williams DE, Nogues C, Crean C, et al. Controlling colloidal stability of silica nanoparticles during bioconjugation reactions with proteins and improving their longer-term stability, handling and storage. J Mater Chem B. 2015;3(10):2043–55.Google Scholar
  21. 21.
    Chan WCW. Nanomedicine 2.0. Acc Chem Res. 2017;50(3):627–32.Google Scholar
  22. 22.
    Santos JJ, Leal J, Dias LAP, Toma SH, Corio P, Genezini FA, et al. Bovine serum albumin conjugated gold-198 nanoparticles as model to evaluate damage caused by ionizing radiation to biomolecules. ACS Appl Nano Mater. 2018;1(9):5062–70.Google Scholar
  23. 23.
    Shinzawa H, Awa K, Kanematsu W, Ozaki Y. Multivariate data analysis for Raman spectroscopic imaging. J Raman Spectrosc. 2009;40(12):1720–5.Google Scholar
  24. 24.
    Das G, Gentile F, Coluccio ML, Perri AM, Nicastri A, Mecarini F, et al. Principal component analysis based methodology to distinguish protein SERS spectra. J Mol Struct. 2011;993(1):500–5.Google Scholar
  25. 25.
    Wold S, Esbensen K, Geladi P. Principal component analysis. Chemom Intell Lab Syst. 1987;2(1–3):37–52.Google Scholar
  26. 26.
    Bro R, Smilde AK. Principal component analysis. Anal Methods. 2014;6(9):2812–31.Google Scholar
  27. 27.
    Kaminska A, Winkler K, Kowalska A, Witkowska E, Szymborski T, Janeczek A, et al. SERS-based immunoassay in a microfluidic system for the multiplexed recognition of interleukins from blood plasma: towards picogram detection. Sci Rep. 2017;7:11.Google Scholar
  28. 28.
    Kaminska A, Kowalska A, Albrycht P, Witkowska E, Waluk J. ABO blood groups' antigen-antibody interactions studied using SERS spectroscopy: towards blood typing. Anal Methods. 2016;8(7):1463–72.Google Scholar
  29. 29.
    Lee C, Yang W, Parr RG. Development of the Colle-Salvetti correlation-energy formula into a functional of the electron density. Phys Rev B. 1988;37(2):785–9.Google Scholar
  30. 30.
    Becke AD. Density-functional thermochemistry. III. The role of exact exchange. J Chem Phys. 1993;98(7):5648–52.Google Scholar
  31. 31.
    Stephens PJ, Devlin FJ, Chabalowski CF, Frisch MJ. Ab initio calculation of vibrational absorption and circular dichroism spectra using density functional force fields. J Phys Chem. 1994;98(45):11623–7.Google Scholar
  32. 32.
    Kim K, Lee HB, Yoon JK, Shin D, Shin KS. Ag nanoparticle-mediated Raman scattering of 4-aminobenzenethiol on a Pt substrate. J Phys Chem C. 2010;114(32):13589–95.Google Scholar
  33. 33.
    Frens G. Controlled nucleation for regulation of particle-size in monodisperse gold suspensions. Nat Phys Sci. 1973;241(105):20–2.Google Scholar
  34. 34.
    Turkevich J, Stevenson PC, Hillier J. A study of the nucleation and growth processes in the synthesis of colloidal gold. Discuss Faraday Soc. 1951;11:55–75.Google Scholar
  35. 35.
    Lewis DJ, Day TM, MacPherson JV, Pikramenou Z. Luminescent nanobeads: attachment of surface reactive Eu(III) complexes to gold nanoparticles. Chem Commun. 2006;13:1433–5.Google Scholar
  36. 36.
    Toma SH, Santos JJ, Araki K, Toma HE. Pushing the surface-enhanced Raman scattering analyses sensitivity by magnetic concentration: a simple non core-shell approach. Anal Chim Acta. 2015;855:70–5.Google Scholar
  37. 37.
    Santos JJ, Toma SH, Corio P, Araki K. Key role of surface concentration on reproducibility and optimization of SERS sensitivity. J Raman Spectrosc. 2017;48(9):1190–5.Google Scholar
  38. 38.
    Cavadas MAS, Monopoli MP, Cunha CSE, Prudencio M, Pereira E, Lynch I, et al. Unravelling malaria antigen binding to antibody-gold nanoparticle conjugates. Part Part Syst Charact. 2016;33(12):906–15.Google Scholar
  39. 39.
    Fang T, Ma KG, Ma LL, Bai JY, Li X, Song HH, et al. 3-Mercaptobutyric acid as an effective capping agent for highly luminescent CdTe quantum dots: new insight into the selection of mercapto acids. J Phys Chem C. 2012;116(22):12346–52.Google Scholar
  40. 40.
    Kalia J, Raines RT. Advances in bioconjugation. Curr Org Chem. 2010;14(2):138–47.Google Scholar
  41. 41.
    Xia N, Xing Y, Wang GF, Feng QQ, Chen QQ, Feng HM, et al. Probing of EDC/NHSS-mediated covalent coupling reaction by the immobilization of electrochemically active biomolecules. Int J Electrochem Soc. 2013;8(2):2459–67.Google Scholar
  42. 42.
    Laaksonen T, Ahonen P, Johans C, Kontturi K. Stability and electrostatics of mercaptoundecanoic acid-capped gold nanoparticles with varying counterion size. Chemphyschem. 2006;7(10):2143–9.Google Scholar
  43. 43.
    Totaro KA, Liao X, Bhattacharya K, Finneman JI, Sperry JB, Massa MA, et al. Systematic investigation of EDC/sNHS-mediated bioconjugation reactions for carboxylated peptide substrates. Bioconjug Chem. 2016;27(4):994–1004.Google Scholar
  44. 44.
    Yu CJ, Tseng WL. Colorimetric detection of mercury(II) in a high-salinity solution using gold nanoparticles capped with 3-mercaptopropionate acid and adenosine monophosphate. Langmuir. 2008;24(21):12717–22.Google Scholar
  45. 45.
    Halas NJ, Lal S, Chang WS, Link S, Nordlander P. Plasmons in strongly coupled metallic nanostructures. Chem Rev. 2011;111(6):3913–61.Google Scholar
  46. 46.
    Toma SH, Santos JJ, Araki K, Toma HE. Supramolecular approach to gold nanoparticle/triruthenium cluster hybrid materials and interfaces. Eur J Inorg Chem. 2011;10:1640–8.Google Scholar
  47. 47.
    Sendroiu IE, Mertens SFL, Schiffrin DJ. Plasmon interactions between gold nanoparticles in aqueous solution with controlled spatial separation. Phys Chem Chem Phys. 2006;8(12):1430–6.Google Scholar
  48. 48.
    Ikeda S, Nishinari K. Intermolecular forces in bovine serum albumin solutions exhibiting solidlike mechanical behaviors. Biomacromolecules. 2000;1(4):757–63.Google Scholar
  49. 49.
    Flecha FLG, Levi V. Determination of the molecular size of BSA by fluorescence anisotropy. Biochem Mol Biol Educ. 2003;31(5):319–22.Google Scholar
  50. 50.
    Frens G, Overbeek JT. Repeptization and theory of electrocratic colloids. J Colloid Interface Sci. 1972;38(2):376–87.Google Scholar
  51. 51.
    Hanauer M, Pierrat S, Zins I, Lotz A, Sonnichsen C. Separation of nanoparticles by gel electrophoresis according to size and shape. Nano Lett. 2007;7(9):2881–5.Google Scholar
  52. 52.
    Park S, Brown KA, Hamad-Schifferli K. Changes in oligonucleotide conformation on nanoparticle surfaces by modification with mercaptohexanol. Nano Lett. 2004;4(10):1925–9.Google Scholar
  53. 53.
    Wang A, Wu CJ, Chen SH. Gold nanoparticle-assisted protein enrichment and electroelution for biological samples containing low protein concentrations - a prelude of gel electrophoresis. J Proteome Res. 2006;5(6):1488–92.Google Scholar
  54. 54.
    Sheehan D. Physical biochemistry: principles and applications. Chichester: Wiley; 2000. p. 153–213.Google Scholar
  55. 55.
    Lopez-Lorente AI, Simonet BM, Valcarcel M. Electrophoretic methods for the analysis of nanoparticles. Trends Anal Chem. 2011;30(1):58–71.Google Scholar
  56. 56.
    Férard G. Quantities and units for electrophoresis in the clinical laboratory (IUPAC recommendations 1994). Pure Appl Chem. 1994;66(4):891–6.Google Scholar
  57. 57.
    Zanchet D, Micheel CM, Parak WJ, Gerion D, Williams SC, Alivisatos AP. Electrophoretic and structural studies of DNA-directed Au nanoparticle groupings. J Phys Chem B. 2002;106(45):11758–63.Google Scholar
  58. 58.
    Ibii T, Kaieda M, Hatakeyama S, Shiotsuka H, Watanabe H, Umetsu M, et al. Direct immobilization of gold-binding antibody fragments for immunosensor applications. Anal Chem. 2010;82(10):4229–35.Google Scholar
  59. 59.
    Castro JL, Lopez-Ramirez MR, Arenas JF, Otero JC. Surface-enhanced Raman scattering of 3-mercaptopropionic acid adsorbed on a colloidal silver surface. J Raman Spectrosc. 2004;35(11):997–1000.Google Scholar
  60. 60.
    Iosin M, Toderas F, Baldeck PL, Astilean S. Study of protein-gold nanoparticle conjugates by fluorescence and surface-enhanced Raman scattering. J Mol Struct. 2009;924:196–200.Google Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.University of Sao PauloSão PauloBrazil
  2. 2.Federal University of ABCSanto AndréBrazil
  3. 3.University of BrasiliaBrasíliaBrazil

Personalised recommendations