European Biophysics Journal

, Volume 47, Issue 5, pp 573–581 | Cite as

Auto-fluorescence of a silk fibroin-based scaffold and its interference with fluorophores in labeled cells

  • Mehdi Amirikia
  • Seyed Mohammad Ali Shariatzadeh
  • Seyed Gholam Ali Jorsaraei
  • Malek Soleimani Mehranjani
Original Article


Silk fibroin is increasingly emerging as an important biomaterial for tissue engineering applications. The ability to fluorescently image silk matrices under a microscope would be helpful in differentiating embedded labeled cells from background signal, critical for the study of silk-based engineered tissues. In this study, we fabricated a scaffold using freeze drying and confirmed its structure by X-ray diffraction and Fourier transform infrared spectroscopy. We then examined the fluorescence of the silk fibroin scaffold using confocal microscopy, both before and after cell seeding and fluorescent labeling. We subsequently investigated the fluorescent signature of the silk fibroin scaffold chemically. Fluorophore-labeled cells seeded into the scaffold showed the same fluorescent color as the scaffold itself when excited by the same wavelength of light. UV–Vis and fluorescence spectroscopy of a silk fibroin solution indicated absorption and emission maxima at 277 and 345 nm, respectively, which is a typical protein-derived signal. HPLC and GC–MS were used to detect quercetin and quercetin derivatives, without success. We therefore conclude that unlike silk cocoons, the fluorescent behavior of silk fibroin scaffolds does not derive from quercetin and its derivatives but from the intrinsic fluorescence of fibroin protein. We also find that the fluorescent signals deriving from a scaffold and from labeled cells embedded in it can be distinguished when the different optical channels are merged.


Fluorescence Silk fibroin scaffold Fluorophore Cells Spectroscopy 



The authors would like to thank Babol University of Medical Sciences and Arak University for supporting this research. Also special thanks to Dr. Ebrahim Zabihi, head of the Cell and Molecular Research Centre, for his assistance in providing us with an optimal environment to carry out this research.


  1. Abdel-Fattah WI, Atwa N, Ali GW (2015) Influence of the protocol of fibroin extraction on the antibiotic activities of the constructed composites. Prog Biomater 4:77–88CrossRefPubMedPubMedCentralGoogle Scholar
  2. Andiappan M, Sundaramoorthy S, Panda N, Meiyazhaban G, Winfred SB, Venkataraman G, Krishna P (2013) Electrospun eri silk fibroin scaffold coated with hydroxyapatite for bone tissue engineering applications. Prog Biomater 2:1–11CrossRefGoogle Scholar
  3. Asakura T, Kuzuhara A, Tabeta R, Saito H (1985) Conformational characterization of Bombyx mori silk fibroin in the solid state by high-frequency carbon-13 cross polarization-magic angle spinning NMR, X-ray diffraction, and infrared spectroscopy. Macromolecules 18:1841–1845CrossRefGoogle Scholar
  4. Bhardwaj N, Devi D, Mandal BB (2015) Tissue-engineered cartilage: the crossroads of biomaterials, cells and stimulating factors. Macromol Biosci 15:153–182CrossRefPubMedGoogle Scholar
  5. Buer CS, Imin N, Djordjevic MA (2010) Flavonoids: new roles for old molecules. J Integr Plant Biol 52:98–111CrossRefPubMedGoogle Scholar
  6. Correia C, Bhumiratana S, Yan L-P, Oliveira AL, Gimble JM, Rockwood D, Kaplan DL, Sousa RA, Reis RL, Vunjak-Novakovic G (2012) Development of silk-based scaffolds for tissue engineering of bone from human adipose-derived stem cells. Acta Biomater 8:2483–2492CrossRefPubMedPubMedCentralGoogle Scholar
  7. Daimon T, Hirayama C, Kanai M, Ruike Y, Meng Y, Kosegawa E, Nakamura M, Tsujimoto G, Katsuma S, Shimada T (2010) The silkworm Green b locus encodes a quercetin 5-O-glucosyltransferase that produces green cocoons with UV-shielding properties. Proc Natl Acad Sci 107:11471–11476CrossRefPubMedPubMedCentralGoogle Scholar
  8. Formica J, Regelson W (1995) Review of the biology of quercetin and related bioflavonoids. Food Chem Toxicol 33:1061–1080CrossRefPubMedGoogle Scholar
  9. Georgakoudi I, Tsai I, Greiner C, Wong C, DeFelice J, Kaplan D (2007) Intrinsic fluorescence changes associated with the conformational state of silk fibroin in biomaterial matrices. Opt Express 15:1043–1053CrossRefPubMedGoogle Scholar
  10. Germain L, De Berdt P, Vanacker J, Leprince J, Diogenes A, Jacobs D, Vandermeulen G, Bouzin C, Préat V, Dupont-Gillain C (2015) Fibrin hydrogels to deliver dental stem cells of the apical papilla for regenerative medicine. Regen Med 10:153–167CrossRefPubMedGoogle Scholar
  11. Ghisaidoobe AB, Chung SJ (2014) Intrinsic tryptophan fluorescence in the detection and analysis of proteins: a focus on Förster resonance energy transfer techniques. Int J Mol Sci 15:22518–22538CrossRefPubMedPubMedCentralGoogle Scholar
  12. Harborne JB (1988) Flavonoids in the environment: structure-activity relationships. Prog Clin Biol Res 280:17PubMedGoogle Scholar
  13. Harizuka M (1953) Physiological genetics of the carotenoids in Bombyx mori, with special reference to the pink cocoon. Bull Seric Exp Stn Jpn 14:141–156Google Scholar
  14. Hodgkinson T, Yuan X-F, Bayat A (2014) Electrospun silk fibroin fiber diameter influences in vitro dermal fibroblast behavior and promotes healing of ex vivo wound models. J Tissue Eng 5:2041731414551661CrossRefPubMedPubMedCentralGoogle Scholar
  15. Huang GT-J, Sonoyama W, Liu Y, Liu H, Wang S, Shi S (2008) The hidden treasure in apical papilla: the potential role in pulp/dentin regeneration and bioroot engineering. J Endod 34:645–651CrossRefPubMedPubMedCentralGoogle Scholar
  16. Kato S, Kurata T, Fujimaki M (1971) Thermal degradation of aromatic amino acids. Agric Biol Chem 35:2106–2112CrossRefGoogle Scholar
  17. Kuboyama N, Kiba H, Arai K, Uchida R, Tanimoto Y, Bhawal UK, Abiko Y, Miyamoto S, Knight D, Asakura T (2013) Silk fibroin-based scaffolds for bone regeneration. J Biomed Mater Res B Appl Biomater 101:295–302CrossRefPubMedGoogle Scholar
  18. Kundu S, Kundu B, Talukdar S, Bano S, Nayak S, Kundu J, Mandal BB, Bhardwaj N, Botlagunta M, Dash BC (2012) Nonmulberry silk biopolymers. Biopolymers 97:455–467CrossRefPubMedGoogle Scholar
  19. Kusurkar TS, Tandon I, Sethy NK, Bhargava K, Sarkar S, Singh SK, Das M (2013) Fluorescent silk cocoon creating fluorescent diatom using a “water glass-fluorophore ferry”. Sci Rep 3:3290CrossRefPubMedPubMedCentralGoogle Scholar
  20. Lai G-J, Shalumon K, Chen J-P (2015) Response of human mesenchymal stem cells to intrafibrillar nanohydroxyapatite content and extrafibrillar nanohydroxyapatite in biomimetic chitosan/silk fibroin/nanohydroxyapatite nanofibrous membrane scaffolds. Int J Nanomed 10:567Google Scholar
  21. Li M, Lu S, Wu Z, Tan K, Minoura N, Kuga S (2002) Structure and properties of silk fibroin–poly (vinyl alcohol) gel. Int J Biol Macromol 30:89–94CrossRefPubMedGoogle Scholar
  22. Li X, You R, Luo Z, Chen G, Li M (2016) Silk fibroin scaffolds with a micro-/nano-fibrous architecture for dermal regeneration. J Mater Chem B 4:2903–2912CrossRefGoogle Scholar
  23. Liu J, Lawrence BD, Liu A, Schwab IR, Oliveira LA, Rosenblatt MI (2012) Silk fibroin as a biomaterial substrate for corneal epithelial cell sheet generation as a biomaterial substrate. Invest Ophthalmol Vis Sci 53:4130–4138CrossRefPubMedPubMedCentralGoogle Scholar
  24. Mandal BB, Kundu SC (2009) Cell proliferation and migration in silk fibroin 3D scaffolds. Biomaterials 30:2956–2965CrossRefPubMedGoogle Scholar
  25. Mobini S, Solati-Hashjin M, Hesaraki S, Gelinsky M (2012) Fabrication and characterization of regenerated silk/bioglass composites for bone tissue engineering. Modares J Med Sci Pathobiol 15:47–60Google Scholar
  26. Oku M (1934a) The chemical studies on the pigments in the cocoon filaments of Bombyx mori. Nippon Nogei Kaishi (in Japanese) 10:1014–1028CrossRefGoogle Scholar
  27. Oku M (1934b) Studies on cocoon pigment in the silkworm, Bombyx mori (VIII) quercetin glycosides in mulberry leaves. Nippon Nogei Kaishi 10:1029–1038CrossRefGoogle Scholar
  28. Razmara RS, Daneshfar A, Sahraei R (2010) Solubility of quercetin in water + methanol and water + ethanol from (292.8 to 333.8) K. J Chem Eng Data 55:3934–3936CrossRefGoogle Scholar
  29. Rice-Evans CA, Miller NJ, Paganga G (1996) Structure-antioxidant activity relationships of flavonoids and phenolic acids. Free Radical Biol Med 20:933–956CrossRefGoogle Scholar
  30. Rockwood DN, Preda RC, Yücel T, Wang X, Lovett ML, Kaplan DL (2011) Materials fabrication from Bombyx mori silk fibroin. Nat Protoc 6:1612–1631CrossRefPubMedGoogle Scholar
  31. Sah M, Pramanik K (2010) Regenerated silk fibroin from B. mori silkcocoon for tissue engineering applications. Int J Environ Sci Dev 1:404CrossRefGoogle Scholar
  32. Saha S, Kundu B, Kirkham J, Wood D, Kundu SC, Yang XB (2013) Osteochondral tissue engineering in vivo: a comparative study using layered silk fibroin scaffolds from mulberry and nonmulberry silkworms. PLoS One 8:e80004CrossRefPubMedPubMedCentralGoogle Scholar
  33. Sánchez-Rabaneda F, Jauregui O, Lamuela-Raventós RM, Viladomat F, Bastida J, Codina C (2004) Qualitative analysis of phenolic compounds in apple pomace using liquid chromatography coupled to mass spectrometry in tandem mode. Rapid Commun Mass Spectrom 18:553–563CrossRefPubMedGoogle Scholar
  34. Sionkowska A, Planecka A (2011) The influence of UV radiation on silk fibroin. Polym Degrad Stab 96:523–528CrossRefGoogle Scholar
  35. Sonoyama W, Liu Y, Yamaza T, Tuan RS, Wang S, Shi S, Huang GT-J (2008) Characterization of the apical papilla and its residing stem cells from human immature permanent teeth: a pilot study. J Endod 34:166–171CrossRefPubMedPubMedCentralGoogle Scholar
  36. Tamura Y, Nakajima K-I, Nagayasu K-I, Takabayashi C (2002) Flavonoid 5-glucosides from the cocoon shell of the silkworm, Bombyx mori. Phytochemistry 59:275–278CrossRefPubMedGoogle Scholar
  37. Tokuşoğlu Ö, Ünal M, Yıldırım Z (2003) HPLC-UV and GC-MS characterization of the flavonol aglycons quercetin, kaempferol, and myricetin in tomato pastes and other tomato-based products. Acta Chromatogr 13:196–207Google Scholar
  38. Um IC, Kweon H, Park YH, Hudson S (2001) Structural characteristics and properties of the regenerated silk fibroin prepared from formic acid. Int J Biol Macromol 29:91–97CrossRefPubMedGoogle Scholar
  39. Wang H-Y, Zhang Y-Q (2013) Effect of regeneration of liquid silk fibroin on its structure and characterization. Soft Matter 9:138–145CrossRefGoogle Scholar
  40. Weska RF, Vieira WC Jr, Nogueira GM, Beppu MM (2009) Effect of freezing methods on the properties of lyophilized porous silk fibroin membranes. Mater Res 12:233–237CrossRefGoogle Scholar
  41. Wray LS, Hu X, Gallego J, Georgakoudi I, Omenetto FG, Schmidt D, Kaplan DL (2011) Effect of processing on silk-based biomaterials: reproducibility and biocompatibility. J Biomed Mater Res B Appl Biomater 99:89–101CrossRefPubMedPubMedCentralGoogle Scholar
  42. Yamada H, Nakao H, Takasu Y, Tsubouchi K (2001) Preparation of undegraded native molecular fibroin solution from silkworm cocoons. Mater Sci Eng C 14:41–46CrossRefGoogle Scholar
  43. Zhang K, Zuo Y (2004) GC-MS determination of flavonoids and phenolic and benzoic acids in human plasma after consumption of cranberry juice. J Agric Food Chem 52:222–227CrossRefPubMedGoogle Scholar
  44. Zhang X, Baughman CB, Kaplan DL (2008) In vitro evaluation of electrospun silk fibroin scaffolds for vascular cell growth. Biomaterials 29:2217–2227CrossRefPubMedPubMedCentralGoogle Scholar
  45. Zhang Y, Fan W, Ma Z, Wu C, Fang W, Liu G, Xiao Y (2010) The effects of pore architecture in silk fibroin scaffolds on the growth and differentiation of mesenchymal stem cells expressing BMP7. Acta Biomater 6:3021–3028CrossRefPubMedGoogle Scholar
  46. Zhao B, Qi N, Zhang K-Q, Gong X (2016) Fabrication of freestanding silk fibroin films containing Ag nanowires/NaYF 4: Yb, Er nanocomposites with metal-enhanced fluorescence behavior. Phys Chem Chem Phys 18:15289–15294CrossRefPubMedGoogle Scholar
  47. Zuo B, Liu L, Wu Z (2007) Effect on properties of regenerated silk fibroin fiber coagulated with aqueous methanol/ethanol. J Appl Polym Sci 106:53–59CrossRefGoogle Scholar

Copyright information

© European Biophysical Societies' Association 2018

Authors and Affiliations

  • Mehdi Amirikia
    • 1
  • Seyed Mohammad Ali Shariatzadeh
    • 1
  • Seyed Gholam Ali Jorsaraei
    • 2
  • Malek Soleimani Mehranjani
    • 1
  1. 1.Department of Biology, Faculty of ScienceArak UniversityArakIran
  2. 2.Fatemeh Zahra Infertility and Reproductive Health Research Centre, Health Research InstituteBabol University of Medical SciencesBabolIran

Personalised recommendations