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Carboxy, amino, and thiol groups play a critical role in a variety of physiological and biological processes and are frequently used for bioconjugation reactions. Moreover, they enable size control and tuning of the surface during the synthesis of particle systems. Especially, thiols have a high binding affinity to noble metals and semiconductors (SC). Thus, simple, inexpensive, robust, and fast methods for the quantification of surface groups and the monitoring of reactions involving ligands are of considerable importance for the characterization of modified or stabilized nanomaterials including polymers.
We studied the potential of the Ellman’s assay, recently used for the quantification of thiol ligands on SC nanocrystals by us1 and the 4-aldrithiol assay for the determination of thiol groups in molecular systems and on polymeric, noble and SC nanomaterials. The results were validated with ICP-OES and reaction mechanisms of both methods were studied photometrically and with ESI-TOF-MS.
The investigation of the reaction mechanisms of both methods revealed the influence of different thiols on the stoichiometry of the reactions2, yielding different mixed disulfides and the thiol-specific products spectroscopically detected. The used methods can quantify freely accessible surface groups on nanoparticles, e.g., modified polystyrene nanoparticles. For thiol ligands coordinatively bound to surface atoms of, e.g., noble or SC nanomaterials, depending on the strength of the thiol-surface bonds, particle dissolution prior to assay performance can be necessary.
We could demonstrate the reliability of the Ellman’s and aldrithiol assay for the quantification of surface groups on nanomaterials by ICP-OES and derived assay-specific requirements and limitations. Generally, it is strongly recommended to carefully control assay performance for new samples, components, and sample ingredients to timely identify possible interferences distorting quantification.
Engineered nanomaterials (NM) with their unique size-dependent properties are of increasing relevance for current and future developments in various fields such as medical and pharmaceutical industry, computing and electronics or food and consumer products. The performance and safety of NM are determined by the sum of their intrinsic physicochemical properties. Especially, the particle surface chemistry, which is largely controlled by the chemical nature and density of functional groups and ligands, is an important key driver for the performance, stability, and processibility of NM, as well as their interaction with the environment. Thus, methods for functional group quantification can foster the sustainable development of functional and safe(r) NM.
Aiming at the development of simple, versatile and multimodal tools for the quantification of common bioanalytically relevant functional groups, we designed a catch-and-release assay based on cleavable probes that enable the quantification of the cleaved-off reporters in the supernatant after particle separation. Thus, the approach circumvents interferences resulting from particle light scattering and sample-inherent absorption or emission. To study the potential of the assay, commercially available and in-house synthesized aminated and carboxylated polymer and silica nanoparticles of different functional group densities were tested.
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 variation of pH, utilizing well-established chemistry. In addition, it can contribute to the development of multi-method characterization strategies to provide a more detailed picture of the intrinsic physicochemical property - performance/safety relationships and thus can support the design of tailored nanomaterials with better controlled properties.
Quantification of the Total and Accessible Number of Functional Groups and Ligands on Nanomaterials
(2022)
Surface-functionalized organic and inorganic nanoparticles (NP) 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. NP performance in such applications depends not only on particle size, size distribution, and morphology, but also on surface chemistry, i.e. the total number of surface functional groups (FG) and the number of FG accessible for subsequent functionalization with ligands or biomolecules, which in turn determines surface charge, colloidal stability, biocompatibility, and toxicity. Methods for FG quantification should be simple, robust, reliable, fast, and inexpensive, and allow for the characteriza-tion of a broad variety of nanomaterials differing in size, chemical composition, and optical properties.
Aiming at the development of simple, versatile, and multimodal tools for the quantification of many bioanalytically relevant FG such as amine, carboxy, thiol and aldehyde functionalities, we investigated and compared various analytical methods commonly used for functional group quantification. This includes electrochemical titration methods, dye-based optical assays, and other instrumental analytical techniques such as nuclear magnetic resonance, mass spectrometry, and thermal analysis methods.
Nowadays amorphous silica nanoparticles (SiO2-NP) are one of the most abundant engineered nanomaterials, that are highly stable and can be easily produced on a large scale at low cost. Surface functionalized SiO2-NP 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. Their performance in such applications depends not only on particle size, size distribution, and morphology, but also on surface chemistry, i.e. the total number of surface functional groups (FG)
and the number of FG accessible for subsequent functionalization with ligands or biomolecules, which in turn determines surface charge, colloidal stability, biocompatibility, and toxicity. Aiming at the development of simple, versatile, and multimodal tools for the quantification of many bioanalytically relevant FG and ligands, we investigated and compared various analytical methods commonly used for FG quantification. This includes electrochemical titration methods, dye-based optical assays, and other instrumental analytical techniques such as nuclear magnetic resonance and thermal analysis methods.
The potential of our multimodal approach for FG quantification was demonstrated for commercial and custom-made silica particles of varying FG, showing not only an influence of the synthesis methods on the number of FG but also on the performance. In the future, our strategy can contribute to establish multi-method characterization strategies to provide a more detailed picture of the structure-properties relationship.