Total Bound Nitrogen Analysis for the Quantification of Immobilized Peptides on Dynabeads

According to the most recent listing reported by the European Commission, rare-earth elements (REEs) are the critical raw materials with the highest supply risk, whereas their recycling rates remain very low in the European Union [1]. End-of-life fluorescent lamps are a promising secondary source of REEs, but their recycling requires innovative separation processes [2,3]. By using phage surface display, Lederer and co-workers identified selectively surface-binding peptides that specifically bind to fluorescent lamp phosphors [4]. In a following study, Schrader et al. immobilized these peptides on coated well plates to investigate their binding to various REE phosphors [5]. The immobilization was facilitated by an activation with benzotriazole-1-yl-oxytripyrrolidinophosphonium-hexafluorophosphate (PyBOP) in the aprotic solvent N-Methyl-2-pyrrolidone (NMP) in the presence of the sterically hindered base diisopropylethylamine (DiPEA), a coupling reaction commonly used for chemical peptide synthesis. Recently, we investigated the immobilization method presented by Schrader et al. for the functionalization of Dynabeads [6]. Dynabeads are highly spherical and monodisperse composite magnetic beads, consisting of superparamagnetic iron oxide nanoparticles dispersed in a polystyrene matrix. They are commercially available with various surface coatings. The functionalization of amine coated Dynabeads with phosphor binding peptides, immobilized with the coupling reaction described above, did not change the Dynabeads’ zeta potential and had no significant effect on the interaction with REE phosphors [6]. On the other hand, we found that the immobilization onto carboxylic acid coated Dynabeads changed the Dynabeads’ zeta potential and isoelectric point.
We also observed that this immobilization had a detrimental effect on the interaction of the beads with the targeted phosphor particles and suggested that this may be an indication of polymerization of the peptides on the Dynabeads’ surfaces. In this work, we present a quantitative analysis of the total bound nitrogen (TNb) for the quantification of the immobilized peptides on the Dynabeads.

References
1. European Commission, Study on the EU’s list of Critical Raw Materials - Final Report (2020).
2. Patil, A.B., Paetzel, V., Struis, R.P.W.J., Ludwig, C. Separations 9 (2022), https://doi.org/10.3390/separations9030056
3. Binnemans, K., Jones, P. Journal of Rare Earths 32, 195-200 (2014), https://doi.org/10.1016/S1002-0721(14)60051-X
4. Lederer, F., Curtis, S., Bachmann, S., Dunbar, S., MacGillivray, R. Biotechnol. Bioeng. 114, (2016), https://doi.org/10.1002/bit.26240
5. Schrader, M., Bobeth, C., Lederer, F. ACS Omega XXXX, (2021), https://doi.org/10.1021/acsomega.1c04343
6. Boelens, P., Bobeth, C., Hinman, N., Weiss, S., Zhou, S., Vogel, M., Drobot, B., Azzam, S.S.A., Pollmann, K., Lederer, F. J. Magn. Magn. Mater. 169956 (2022), https://doi.org/10.1016/j.jmmm.2022.169956