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Synthesis and Optical Quantification of Surface Groups on Organic and Inorganic Particle-Carriers
(2020)
Differently sized organic and inorganic particles are of great interest in the life and material sciences, as they can be used e.g. as drug carriers, fluorescent sensors, and multimodal labels in bioanalytical assays and imaging applications.1 Particle performance in such applications depends mainly on the sum of their intrinsic physicochemical properties. Here, the surface chemistry, i.e., the total number of surface functional groups (FG) and the number of FG accessible for subsequent modification with ligands and/or biomolecules, is one of the key parameters. Moreover, the surface chemistry of these materials controls the behavior and fate of the particles when released to the environment or taken up by cells. Nevertheless, it is still relatively rare that FG are quantified in particle safety studies. Methods for FG quantification should be simple, robust, reliable, fast, and inexpensive, and allow for the characterization of a broad variety of materials differing in size, chemical composition, and optical properties.
Aiming at the development of simple, versatile, and multimodal tools for the quantification of bioanalytically relevant FG such as amine2,3, carboxy2,3, thiol, and aldehyde4 functionalities, we designed a catch-and-release assay utilizing cleavable probes that enable the quantification of the cleaved-off reporters in the supernatant after particle separation, and thus, circumvent interferences resulting from particle light scattering and sample-inherent absorption or emission.2 The potential of our cleavable probes for the quantification of carboxy and amino groups was demonstrated for commercial and custom-made polymer and silica particles of varying FG densities, underlining the benefit of the catch-and-release assays as a versatile method for the FG quantification on all types of transparent, scattering, absorbing and/or fluorescent particles.2,3 In the future, our cleavable probe strategy can be easily adapted to other analytical techniques requiring different reporters, or to different types of linkers that can be cleaved thermally, photochemically, or by pH, utilizing well-established chemistry, e.g. from drug delivery systems. It can contribute to establish multi-method characterization strategies for particles to provide a more detailed picture of the structure-properties relationship and thus can support the design of sustainable and safe(r) materials.
Quantification of Aldehydes via Catch and Release of Reporter Chromophores on Polymeric Microbeads
(2019)
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.
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.