Skip to main content
SpringerLink
Log in

Quantification of fractional and absolute functionalization of gelatin hydrogels by optimized ninhydrin assay and 1H NMR

  • Paper in Forefront
  • Published:
Analytical and Bioanalytical Chemistry Aims and scope Submit manuscript

Abstract

3D cell culture in protein-based hydrogels often begins with chemical functionalization of proteins with cross-linking agents such as methacryloyl or norbornene. An important and variable characteristic of these materials is the degree of functionalization (DoF), which controls the reactivity of the protein for cross-linking and therefore impacts the mechanical properties and stability of the hydrogel. Although 1H NMR has emerged as the most accurate technique for quantifying absolute DoF of chemically modified proteins, colorimetric techniques still dominate in actual use and may be more useful for quantifying fractional or percent DoF. In this work, we sought to develop an optimized colorimetric assay for DoF of common gelatin-based biomaterials and validate it versus NMR; along the way, we developed a set of best practices for both methods and considerations for their most appropriate use. First, the amine-reactive ninhydrin assay was optimized in terms of solvent properties, temperature, ninhydrin concentration, and range of gelatin standards. The optimized assay produced a linear response to protein concentration in a convenient, 96-well plate format and yielded a fractional DoF similar to NMR in most cases. In comparing with NMR, we identified that DoF can be expressed as fractional or absolute, and that fractional DoF can be inaccurate if the amino acid content of the parent protein is not properly accounted for. In summary, the fractional DoF of methacryloyl- and norbornene-functionalized gelatins was quantified by an optimized colorimetric ninhydrin assay and orthogonally by 1H NMR. These methods will be valuable for quality control analysis of protein-based hydrogels and 3D cell culture biomaterials.

Graphical abstract

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

Data availability

Raw data is available upon request.

References

  1. Choi JR, Yong KW, Choi JY, Cowie AC. Recent advances in photo-crosslinkable hydrogels for biomedical applications. BioTechniques. 2019;66:40–53.

    Article  CAS  Google Scholar 

  2. Annabi N, Tamayol A, Uquillas JA, Akbari M, Bertassoni LE, Cha C, et al. 25th anniversary article: rational design and applications of hydrogels in regenerative medicine. Adv Mater. 2014;26:85–124.

    Article  CAS  Google Scholar 

  3. Ifkovits JL, Burdick JA. Review: photopolymerizable and degradable biomaterials for tissue engineering applications. Tissue Eng. 2007;13:2369–85.

    Article  CAS  Google Scholar 

  4. Li Y, Rodrigues J, Tomás H. Injectable and biodegradable hydrogels: gelation, biodegradation and biomedical applications. Chem Soc Rev. 2012;41:2193–221.

    Article  CAS  Google Scholar 

  5. Yue K, Trujillo-de Santiago G, Alvarez MM, Tamayol A, Annabi N, Khademhosseini A. Synthesis, properties, and biomedical applications of gelatin methacryloyl (GelMA) hydrogels. Biomaterials. 2015;73:254–71.

    Article  CAS  Google Scholar 

  6. Mũnoz Z, Shih H, Lin C-C. Gelatin hydrogels formed by orthogonal thiol–norbornene photochemistry for cell encapsulation. Biomater Sci. 2014;2:1063–72.

    Article  Google Scholar 

  7. Hoorick JV, Gruber P, Markovic M, et al. Highly reactive thiol-norbornene photo-click hydrogels: toward improved processability. Macromol Rapid Commun. 2018;39:1800181.

    Article  Google Scholar 

  8. Claaßen C, Claaßen MH, Truffault V, Sewald L, Tovar GEM, Borchers K, et al. Quantification of substitution of gelatin methacryloyl: best practice and current pitfalls. Biomacromolecules. 2018;19:42–52.

    Article  Google Scholar 

  9. Yue K, Li X, Schrobback K, et al. Structural analysis of photocrosslinkable methacryloyl-modified protein derivatives. Biomaterials. 2017;139:163–71.

    Article  CAS  Google Scholar 

  10. Van Den Bulcke AI, Bogdanov B, De Rooze N, Schacht EH, Cornelissen M, Berghmans H. Structural and rheological properties of methacrylamide modified gelatin hydrogels. Biomacromolecules. 2000;1:31–8.

    Article  Google Scholar 

  11. Zhu M, Wang Y, Ferracci G, Zheng J, Cho N-J, Lee BH. Gelatin methacryloyl and its hydrogels with an exceptional degree of controllability and batch-to-batch consistency. Sci Rep. 2019;9:1–13.

    Article  Google Scholar 

  12. Vlierberghe SV, Fritzinger B, Martins JC, Dubruel P. Hydrogel network formation revised: high-resolution magic angle spinning nuclear magnetic resonance as a powerful tool for measuring absolute hydrogel cross-link efficiencies. Appl Spectrosc. 2010;64:1176–80.

    Article  Google Scholar 

  13. Hafidz R, Yaakob CM, Amin I, Noorfaizan A. Chemical and functional properties of bovine and porcine skin gelatin. Int Food Res J. 2011;18:813–7.

    CAS  Google Scholar 

  14. Karim AA, Bhat R. Fish gelatin: properties, challenges, and prospects as an alternative to mammalian gelatins. Food Hydrocoll. 2009;23:563–76.

    Article  CAS  Google Scholar 

  15. Loessner D, Meinert C, Kaemmerer E, Martine LC, Yue K, Levett PA, et al. Functionalization, preparation and use of cell-laden gelatin methacryloyl–based hydrogels as modular tissue culture platforms. Nat Protoc. 2016;11:727–46.

    Article  CAS  Google Scholar 

  16. Shirahama H, Lee BH, Tan LP, Cho N-J. Precise tuning of facile one-pot gelatin methacryloyl (GelMA) synthesis. Sci Rep. 2016;6:1–11.

    Article  Google Scholar 

  17. Aldana AA, Malatto L, Rehman MAU, Boccaccini AR, Abraham GA. Fabrication of gelatin methacrylate (GelMA) scaffolds with nano- and micro-topographical and morphological features. Nanomaterials. 2019;9:120.

    Article  Google Scholar 

  18. Ma Z, Gao C, Gong Y, Shen J. Paraffin spheres as porogen to fabricate poly(L-lactic acid) scaffolds with improved cytocompatibility for cartilage tissue engineering. J Biomed Mater Res B Appl Biomater. 2003;67B:610–7.

    Article  CAS  Google Scholar 

  19. Mario Perera M, Ayres N. Gelatin based dynamic hydrogels via thiol–norbornene reactions. Polym Chem. 2017;8:6741–9.

    Article  Google Scholar 

  20. Friedman M. Applications of the ninhydrin reaction for analysis of amino acids, peptides, and proteins to agricultural and biomedical sciences. J Agric Food Chem. 2004;52:385–406.

    Article  CAS  Google Scholar 

  21. Nayuni NK, Cloutman-Green E, Hollis M, Hartley J, Martin S, Perrett D. Critical evaluation of ninhydrin for monitoring surgical instrument decontamination. J Hosp Infect. 2013;84:97–102.

    Article  CAS  Google Scholar 

  22. Sewald L, Claaßen C, Götz T, Claaßen MH, Truffault V, Tovar GEM, et al. Beyond the modification degree: impact of raw material on physicochemical properties of gelatin type A and type B methacryloyls. Macromol Biosci. 2018;18:1800168.

    Article  Google Scholar 

  23. McCormack T, O’Keeffe G, Craith BM, O’Kennedy R. Assessment of the effect of increased fluorophore labelling on the binding ability of an antibody. Anal Lett. 1996;29:953–68.

    Article  CAS  Google Scholar 

  24. Vira S, Mekhedov E, Humphrey G, Blank PS. Fluorescent-labeled antibodies: balancing functionality and degree of labeling. Anal Biochem. 2010;402:146–50.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

We acknowledge Andrew Kinman for testing various versions of the ninhydrin assay and Parastoo Anbaei for aiding in the synthesis of GelNB-NHS. NMR was performed at the University of Virginia Biomolecular Magnetic Resonance facility under the guidance of Dr. Jeffrey Ellena. We thank the faculty of Chemical Engineering, especially the Lampe, Caliari, and Letteri laboratories, for generous access to their lyophilization equipment.

Funding

This work was supported by the National Institute of Biomedical Imaging and Bioengineering (NIBIB) under Award Number U01EB029127 through the National Institutes of Health (NIH), with co-funding from the National Center for Advancing Translational Sciences (NCATS). JMZ was supported in part by the Graduate Research Fellowship Program through the National Science Foundation. ANM was supported in part by a summer undergraduate research award through the Institute for Nanoscale and Quantum Scientific Advanced Research (nanoSTAR) at the University of Virginia. JOC was supported in part by the NIH-funded T32 Biotechnology Training Program at the University of Virginia.

Author information

Authors and Affiliations

  1. Department of Chemistry, University of Virginia, Charlottesville, VA, 22904, USA

    Jonathan M. Zatorski, Alyssa N. Montalbine, Jennifer E. Ortiz-Cárdenas & Rebecca R. Pompano

Authors
  1. Jonathan M. Zatorski
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Alyssa N. Montalbine
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jennifer E. Ortiz-Cárdenas
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Rebecca R. Pompano
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

JM Zatorski, AN Montalbine, J Ortiz-Cárdenas, and RR Pompano designed the experimental approach, interpreted the results, and wrote and edited the manuscript. In addition, JM Zatorski and AN Montalbine optimized the ninhydrin assay conditions, JM Zatorski designed and performed the NMR experiments, and J Ortiz-Cárdenas synthesized GelNB-NHS.

Corresponding author

Correspondence to Rebecca R. Pompano.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Disclaimer

The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Published in the topical collection featuring Female Role Models in Analytical Chemistry.

Electronic supplementary material

ESM 1

(PDF 294 kb).

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zatorski, J.M., Montalbine, A.N., Ortiz-Cárdenas, J.E. et al. Quantification of fractional and absolute functionalization of gelatin hydrogels by optimized ninhydrin assay and 1H NMR. Anal Bioanal Chem 412, 6211–6220 (2020). https://doi.org/10.1007/s00216-020-02792-5

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-020-02792-5

Keywords

Search

Navigation