TY - CONF A1 - Rühle, Bastian A1 - Roloff, Alexander A1 - Nirmalananthan-Budau, Nithiya A1 - Borcherding, H. A1 - Thiele, T. A1 - Schedler, U. A1 - Resch-Genger, Ute T1 - Quantification of Aldehydes on Polymeric Microbead Surfaces via Catch and Release of Reporter Chromophores N2 - 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 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. T2 - Anakon CY - Münster, Germany DA - 25.04.2019 KW - Optical quantification KW - Polymer particles KW - CHO functions KW - Catch and release assay PY - 2019 AN - OPUS4-49781 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Nirmalananthan-Budau, Nithiya A1 - Roloff, Alexander A1 - Rühle, Bastian A1 - Borcherding, H. A1 - Thiele, T. A1 - Schedler, U. A1 - Resch-Genger, Ute T1 - Quantification of Aldehydes via Catch and Release of Reporter Chromophores on Polymeric Microbeads N2 - 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 the surface of 2D- and 3D-supports. We present here a versatile concept to quantify the amount of accessible aldehyde moieties on the surface of PMMA particles through the specific binding and subsequent release of small reporter molecules such as absorbing and fluorescent dyes utilizing acylhydrazone formation as a reversible covalent labeling strategy. Unbound reporter molecules can be easily removed by washing steps, eliminating inaccuracies caused by unspecific adsorption to hydrophobic surfaces. Cleavage of the hydrazones at acidic pH assisted by a carbonyl trap releases the optical reporters rapidly and quantitatively and allows for their optical detection at low concentration. Importantly, this strategy separates the signal-generating molecules from the bead surface, thereby circumventing common pitfalls of optical assays associated with light scattering and signal distortions due to binding-induced changes in reporter fluorescence and quenching dye-dye interactions on crowded material surfaces. The potential of this catch-and-release strategy for surface group quantification is representatively demonstrated for a set of microparticles functionalized with different aldehyde densities. This concept is validated by a colorimetric assay with a different optical probe, which contains a reductively cleavable disulfide bond and a reporter that can be quantified photometrically in solution after its release. The excellent match of the results of both optical assays confirms their suitability for the rapid and sensitive quantification of aldehydes on microbead surfaces. These simple catch-and-release assays are excellent tools for process control during bead fabrication and the comparison of different bead batches. Their potential for predicting biomolecule coupling efficiencies in bioconjugation reactions is currently assessed in reductive amination reactions with streptavidin. T2 - Tag der Chemie CY - Berlin, Germany DA - 11.07.2019 KW - Optical quantification KW - Catch and release assay KW - CHO functions KW - Polymer particles PY - 2019 AN - OPUS4-49783 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Nirmalananthan-Budau, Nithiya A1 - Roloff, Alexander A1 - Rühle, Bastian A1 - Borcherding, H. A1 - Thiele, T. A1 - Schedler, U. A1 - Resch-Genger, Ute T1 - Quantification of Aldehyde Functional Groups on Polymeric Microbeads via Catch and Release of Reporters N2 - Aldehyde-functionalized materials have found broad use in bioconjugation applications. For example, coupling of aldehyde surface groups with to proteins or amine-functionalized oligonucleotides can readily produce biomolecule-covered chip microarray and bead surfaces for multiplex analyses. Additionally, aldehyde-modified nanoparticles can possess bioadhesive properties that can extend their retention time in biological compartments. These emerging novel bioanalytical applications call for reliable tools and methods to detect and quantify accessible aldehyde functionalities. We present here a straightforward concept to quantify the amount of accessible aldehyde moieties on the surface of polymethylmethacrylate (PMMA) particles through the specific binding and subsequent release of small reporter molecules such as absorbing and fluorescent dyes utilizing hydrazone formation as a reversible covalent labeling strategy. Unbound reporter molecules can be easily removed by washing steps, eliminating inaccuracies caused by unspecific adsorption to hydrophobic surfaces. Cleavage of the hydrazones at acidic pH assisted by a carbonyl trap releases the optical reporters rapidly and quantitatively and allows for their optical detection at low concentration. Importantly, this strategy separates the signal-generating molecules from the bead surface, thereby circumventing light scattering and signal distortions due to binding-induced changes in reporter fluorescence and quenching dye-dye interactions on crowded material surfaces. The potential of this catch-and-release strategy for surface group quantification is representatively demonstrated for a set of microparticles functionalized with different aldehyde densities. This concept is validated by a colorimetric assay with a different optical probe, which contains a reductively cleavable disulfide bond and a reporter that can be quantified photometrically in solution after its cleavage. The excellent match of the results of both optical assays confirms their suitability for the rapid and sensitive quantification of aldehydes on microbead surfaces. These simple catch-and-release assays are excellent tools for process control during bead fabrication and the comparison of different bead batches. T2 - 29th Annual Conference of the German Society for Cytometry CY - Berlin, Germany DA - 25.09.2019 KW - Optical quantification KW - Catch and release assay KW - CHO functions KW - Polymer particles PY - 2019 AN - OPUS4-49784 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER -