Quantification of Aldehydes on Polymeric Microbead Surfaces via Catch and Release of Reporter Chromophores
- Aldehyde-functionalized materials have found widespread use in bioconjugation applications. For example, coupling of aldehyde surface groups with proteins, peptides or amine-functionalized oligonucleotides can readily produce biomolecule-decorated chip and bead surfaces for multiplex analyses. Furthermore, aldehyde-modified nanoparticles can possess bioadhesive properties that can prolong their retention time in biological compartments. These emerging novel bioanalytical and biomedical applications call for reliable tools and methods to detect and quantify accessible aldehyde functionalities on material surfaces. We report on a versatile concept to quantify the accessible aldehyde moieties on particle surfaces through the specific binding and subsequent release of small reporter molecules such as fluorescent dyes and non-fluorescent chromophores utilizing acylhydrazone formation as a reversible covalent labeling strategy. This is representatively demonstrated for a set of polymerAldehyde-functionalized materials have found widespread use in bioconjugation applications. For example, coupling of aldehyde surface groups with proteins, peptides or amine-functionalized oligonucleotides can readily produce biomolecule-decorated chip and bead surfaces for multiplex analyses. Furthermore, aldehyde-modified nanoparticles can possess bioadhesive properties that can prolong their retention time in biological compartments. These emerging novel bioanalytical and biomedical applications call for reliable tools and methods to detect and quantify accessible aldehyde functionalities on material surfaces. We report on a versatile concept to quantify the accessible aldehyde moieties on particle surfaces through the specific binding and subsequent release of small reporter molecules such as fluorescent dyes and non-fluorescent chromophores utilizing acylhydrazone formation as a reversible covalent labeling strategy. This is representatively demonstrated for a set of polymer microparticles with different aldehyde labeling densities. Excess reporter molecules can be easily removed by washing, eliminating inaccuracies caused by unspecific adsorption to hydrophobic surfaces. Cleavage of hydrazones at acidic pH assisted by a carbonyl trap releases the fluorescent reporters rapidly and quantitatively and allows for their fluorometric detection at low concentration. Importantly, this strategy separates the signal-generating molecule from the bead surface, thereby circumventing common issues associated with light scattering and signal distortions due to binding-induced changes in reporter fluorescence as well as quenching dye-dye interactions on crowded material surfaces. In addition, we demonstrate that the release of a non-fluorescent chromophore via disulfide cleavage and subsequent quantification by absorption spectroscopy gives comparable results, verifying that both assays are capable of rapid and sensitive quantification of aldehydes on microbead surfaces. These strategies enable a quantitative comparison of bead batches with different functionalization densities, and a qualitative prediction of their coupling efficiencies in bioconjugations, as demonstrated in reductive amination reactions with Streptavidin.…
Autor*innen: | Bastian RühleORCiD |
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Koautor*innen: | Alexander Roloff, Nithiya Nirmalananthan-Budau, H. Borcherding, T. Thiele, U. Schedler, Ute Resch-GengerORCiD |
Dokumenttyp: | Posterpräsentation |
Veröffentlichungsform: | Präsentation |
Sprache: | Englisch |
Jahr der Erstveröffentlichung: | 2019 |
Organisationseinheit der BAM: | 1 Analytische Chemie; Referenzmaterialien |
1 Analytische Chemie; Referenzmaterialien / 1.2 Biophotonik | |
DDC-Klassifikation: | Naturwissenschaften und Mathematik / Chemie / Analytische Chemie |
Freie Schlagwörter: | CHO functions; Catch and release assay; Optical quantification; Polymer particles |
Themenfelder/Aktivitätsfelder der BAM: | Chemie und Prozesstechnik |
Veranstaltung: | Anakon |
Veranstaltungsort: | Münster, Germany |
Beginndatum der Veranstaltung: | 25.04.2019 |
Enddatum der Veranstaltung: | 28.04.2019 |
Verfügbarkeit des Dokuments: | Datei im Netzwerk der BAM verfügbar ("Closed Access") |
Datum der Freischaltung: | 28.11.2019 |
Referierte Publikation: | Nein |