Multiplatform Protein Detection and Quantification Using Glutaraldehyde-Induced Fluorescence for 3D Systems

  • Mariana I. Neves
  • Marco Araújo
  • Cristina C. Barrias
  • Pedro L. Granja
  • Aureliana SousaEmail author


Glutaraldehyde (GTA) is a dialdehyde used as biological fixative and its interaction with proteins like bovine serum albumin (BSA) has been well described. Additionally, GTA is known to induce fluorescence when interacting with BSA molecules. In this work, it is developed a new sensitive and reproducible method for BSA quantification using GTA crosslinking to endow fluorescence to BSA molecules. This method can be used with standard lab equipment, providing a low cost, fast-tracking and straightforward approach for BSA quantification. Techniques such as confocal laser scanning microscopy (CLSM) and spectrofluorometry are applied for quantitative assessment, and widefield fluorescence microscopy for qualitative assessment. Qualitative and quantitative correlations between BSA content and GTA-induced fluorescence are verified. BSA concentrations as low as 62.5 μg/mL are detected using CLSM. This method can be highly advantageous for protein quantification in three-dimensional hydrogel systems, specially to evaluate protein loading/release in protein delivery or molecular imprinting systems.

Graphical Abstract

Preparation and analysis of glutaraldehyde-induced protein-fluorescence in 3D hydrogels. Alginate-methacrylate hydrogels containing varying amounts of bovine serum albumin (BSA) are prepared by photopolymerization and then incubated in glutaraldehyde solutions. Samples observation is performed using confocal laser scanning microscopy, spectrofluorometry and widefield fluorescence microscopy. Data is processed and retrieves a quantitative correlation between protein content and fluorescence levels.


Bovine serum albumin Biosensor Hydrogels Confocal laser scanning microscopy Spectrofluorometry 



The authors would like to acknowledge FEDER - Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020 - Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and Portuguese funds through FCT - Fundação para a Ciência e a Tecnologia/ Ministério da Ciência, Tecnologia e Ensino Superior in the framework of the Project PTDC/BBB-BIO/1889/2014, the doctoral grant SFRH/BD/129855/2017 to Mariana I Neves and the contract to Aureliana Sousa in the framework of the project “Institute for Research and innovation in Health Sciences” (POCI-01-0145-FEDER-007274). Marco Araújo gratefully acknowledges Interreg V-A Spain-Portugal (POCTEP) 2014-2020 and FEDER (0245_IBEROS_1_E) for the Postdoctoral grant.

The authors acknowledge the support of Ricardo Vidal from the Biointerfaces and Nanotechnology i3S Scientific Platform, the Bioimaging i3S Scientific Platform, member of the national infrastructure PPBI - Portuguese Platform of Bioimaging (PPBI-POCI-01-0145-FEDER-022122) and Daniela Silva from the Image, Microstructure and Microanalysis Unit (IMCROS) from the Materials Centre of the University of Porto (CEMUP). The authors thank Prof. Nuno Alves from the Centre for Rapid and Sustainable Product Development (CDRSP) of Politécnico de Leiria for the use of the photopolymerization system.

Supplementary material

10895_2019_2433_MOESM1_ESM.docx (378 kb)
ESM 1 Alginate methacrylation protocol, example of Mean Gray Value measurement, statistical analysis for CLSM and spectrofluorometry results, quantifications using protein assay commercial kits, spectrofluorometry preliminary assays and minimum protein levels measured by spectrofluorometry. (DOCX 377 kb)


  1. 1.
    Sabatini DD, Bensch K, Barrnett RJ (1963) J Cell Biol 17:19CrossRefGoogle Scholar
  2. 2.
    Migneault I, Dartiguenave C, Bertrand MJ, Waldron KC (2004) BioTechniques 37:790CrossRefGoogle Scholar
  3. 3.
    Shaimi R, Low Siew C (2016) Prolonged protein immobilization of biosensor by chemically cross-linked glutaraldehyde on mixed cellulose membrane. J Polym Eng 36:655Google Scholar
  4. 4.
    Akyilmaz E, Oyman G, Cınar E, Odabas G (2017) Prep Biochem Biotechnol 47:86CrossRefGoogle Scholar
  5. 5.
    Sargin İ, Arslan G (2016) Desalin Water Treat 57:10664CrossRefGoogle Scholar
  6. 6.
    Zhang M, Zhang Y, Helleur R (2015) Chem Eng J 264:56CrossRefGoogle Scholar
  7. 7.
    Aftab K, Akhtar K, Jabbar A (2014) Ecol Eng 73:319CrossRefGoogle Scholar
  8. 8.
    Lindén JB, Larsson M, Kaur S, Nosrati A, Nydén M (2016) J Appl Polym Sci 133 n/aGoogle Scholar
  9. 9.
    Wang W, Jin X, Zhu Y, Zhu C, Yang J, Wang H, Lin T (2016) Carbohydr Polym 140:356CrossRefGoogle Scholar
  10. 10.
    Spiller KL, Anfang RR, Spiller KJ, Ng J, Nakazawa KR, Daulton JW, Vunjak-Novakovic G (2014) Biomaterials 35:4477CrossRefGoogle Scholar
  11. 11.
    Tamimi E, Ardila DC, Haskett DG, Doetschman T, Slepian MJ, Kellar RS, Vande Geest JP (2015) J Biomech Eng 138:011001CrossRefGoogle Scholar
  12. 12.
    Lu W, Ma M, Xu H, Zhang B, Cao X, Guo Y (2015) Mater Lett 140:1CrossRefGoogle Scholar
  13. 13.
    Liu Y, An M, Wang L, Qiu H (2014) J Macromol Sci Part B: Phys 53:309CrossRefGoogle Scholar
  14. 14.
    Amadori S, Torricelli P, Rubini K, Fini M, Panzavolta S, Bigi A (2015) J Mater Sci Mater Med 26:69CrossRefGoogle Scholar
  15. 15.
    Gao S, Yuan Z, Guo W, Chen M, Liu S, Xi T, Guo Q (2017) Mater Sci Eng C 71:891CrossRefGoogle Scholar
  16. 16.
    Khalily MA, Goktas M, Guler MO (2015) Org Biomol Chem 13:1983CrossRefGoogle Scholar
  17. 17.
    Habeeb AFSA, Hiramoto R (1968) Arch Biochem Biophys 126:16CrossRefGoogle Scholar
  18. 18.
    Bowes JH, Cater CW (1966) J R Microsc Soc 85:193CrossRefGoogle Scholar
  19. 19.
    Hopwood D, Allen CR, McCabe M (1970) Histochem J 2:137CrossRefGoogle Scholar
  20. 20.
    Hardy PM, Nicholls AC, Rydon HN (1976) J Chem Soc Perkin Trans (1):958Google Scholar
  21. 21.
    Bowes JH, Cater CW (1968) Biochim Biophys Acta 168:341CrossRefGoogle Scholar
  22. 22.
    Melo RR, Alnoch RC, Vilela AFL, Souza EM, Krieger N, Ruller R, Sato HH, Mateo C (2017) Molecules 22Google Scholar
  23. 23.
    Rasmussen KE, Albrechtsen J (1974) Histochemistry 38:19CrossRefGoogle Scholar
  24. 24.
    Han Y, Duan Q, Li Y, Li Y, Tian J (2018) Adv Polym Technol 37:1214CrossRefGoogle Scholar
  25. 25.
    Gao J, Wu Y, Cui J, Wu X, Meng M, Li C, Yan L, Zhou S, Yang L, Yan Y (2018) J Taiwan Inst Chem Eng 91:468CrossRefGoogle Scholar
  26. 26.
    Majorek KA, Porebski PJ, Dayal A, Zimmerman MD, Jablonska K, Stewart AJ, Chruszcz M, Minor W (2012) Mol Immunol 52:174CrossRefGoogle Scholar
  27. 27.
    Ma X, Sun X, Hargrove D, Chen J, Song D, Dong Q, Lu X, Fan TH, Fu Y, Lei Y (2016) Sci Rep 6:19370CrossRefGoogle Scholar
  28. 28.
    Aston R, Sewell K, Klein T, Lawrie G, Grøndahl L (2016) Eur Polym J 82:1CrossRefGoogle Scholar
  29. 29.
    Tahtat D, Mahlous M, Benamer S, Khodja AN, Oussedik-Oumehdi H, Laraba-Djebari F (2013) Int J Biol Macromol 58:160CrossRefGoogle Scholar
  30. 30.
    Lu T, Xiang T, Huang X-L, Li C, Zhao W-F, Zhang Q, Zhao C-S (2015) Carbohydr Polym 133:587CrossRefGoogle Scholar
  31. 31.
    Chan AW, Whitney RA, Neufeld RJ (2008) Biomacromolecules 9:2536CrossRefGoogle Scholar
  32. 32.
    Yeom CK, Lee K-H (1998) J Appl Polym Sci 67:209CrossRefGoogle Scholar
  33. 33.
    Pawar SN, Edgar KJ (2012) Biomaterials 33:3279CrossRefGoogle Scholar
  34. 34.
    Bronze-Uhle ES, Costa BC, Ximenes VF, Lisboa-Filho PN (2016) Nanotechnol Sci Appl 10:11CrossRefGoogle Scholar
  35. 35.
    Yan J, Wang F, Chen J, Liu T, Zhang T (2016) Int J Police Sci Manag 2016:1Google Scholar
  36. 36.
    Taktak NEM, Awad OM, Elfiki SA, El-Ela NEA (2017) J Macromol Sci Part B: Phys 56:359CrossRefGoogle Scholar
  37. 37.
    Kiran Babu SN (2015) J Nanosci Nanotechnol 06Google Scholar
  38. 38.
    Kulkarni AR, Soppimath KS, Aminabhavi TM, Dave AM, Mehta MH (2000) J Control Release 63:97CrossRefGoogle Scholar
  39. 39.
    Bio-Rad, "DC™ Protein Assay", Accessed 20 June 2019
  40. 40.
    T. Scientific, "Pierce™ BCA Protein Assay Kit", Accessed 20 June 2019
  41. 41.
    Hou Q, Walsh MC, Freeman R, Barry JJ, Howdle SM, Shakesheff KM (2006) J Pharm Pharmacol 58:895CrossRefGoogle Scholar
  42. 42.
    Lima DS, Tenório-Neto ET, Lima-Tenório MK, Guilherme MR, Scariot DB, Nakamura CV, Muniz EC, Rubira AF (2018) J Mol Liq 262:29CrossRefGoogle Scholar
  43. 43.
    Wang H, Ying X, Liu J, Li X, Zhang W (2017) J Polym Res:24Google Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.i3S - Instituto de Investigação e Inovação em SaúdeUniversidade do PortoPortoPortugal
  2. 2.INEB - Instituto de Engenharia BiomédicaUniversidade do PortoPortoPortugal
  3. 3.FEUP- Faculdade de Engenharia da Universidade do PortoUniversidade do PortoPortoPortugal
  4. 4.ICBAS - Instituto de Ciências Biomédicas Abel SalazarUniversidade do PortoPortoPortugal

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