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Nano- and microparticles are of increasing importance for a wide range of applications in the material and life sciences. Examples are their use as carriers for dye molecules and drugs, multichromophoric reporters for signal enhancement strategies in optical assays, targeted probes for bioimaging, and biosensors. All these applications require surface functionalization of the particles with e.g., ligands (to tune the dispersibility and prevent unspecific interactions), crosslinkers, sensor dyes, or analyte recognition moieties like biomolecules, and subsequently, the knowledge of the chemical nature and total number of surface groups as well as the number of groups accessible for coupling reactions. Particularly attractive for the latter are sensitive and fast photometric or fluorometric assays, which can be read out with simple, inexpensive instrumentation. Here, we present a novel family of cleavable photometric and multimodal reporters for the quantification of conjugatable amino and carboxyl surface groups on nano- and microparticles. These probes allow for the determination of particle-bound labels, unbound reporters in the supernatant, and reporters cleaved off from the particle surface as well as the remaining thiol groups on the particles by photometry and inductively coupled optical emission spectrometry (32S ICP-OES).3 Comparison of the performance of these cleavable reporters with conventional labels, utilizing changes in intensity and/or colour of absorption and/or emission, underlines the analytical potential of this versatile concept which elegantly circumvents signal distortions by light scattering and encoding dyes, and enables straightforward validation by method comparison.
Polymer nanoparticles (NPs) are of increasing importance for a wide range of applications in the material and life sciences. This includes their use as carriers for dye molecules and drugs, multichromophoric reporters for signal enhancement strategies in optical assays, targeted probes in bioimaging studies, and nanosensors.[1] Application-relevant properties of such NPs include their size, morphology, colloidal stability, and ease of surface functionalization with e.g., sensor molecules and targeting ligands. Many of these features as well as the interaction of NPs with their microenvironment are closely linked to the knowledge of the chemical nature and total number of surface groups as well as the number of surface functionalities accessible for subsequent coupling reactions of differently sized ligands, biomolecules or reporter molecules. This underlines the importance of simple, robust, reliable, and validated methods, which can be employed for the characterization of a broad variety of particle systems independent of their optical properties, i.e., scattering or the presence of encoding dyes.[2]
In this respect, we assessed a variety of conventional and newly developed colorimetric or fluorometric labels for the optical surface group analysis, utilizing e.g., changes in intensity and/or color for signal generation.[3] Moreover, novel cleavable and multimodal reporters were developed which consist of a reactive group, a cleavable linker, and an optically active moiety, chosen to contain also heteroatoms for straightforward method validation by elemental analysis, ICP-OES, ICP-MS or NMR. In contrast to conventional labels measured bound at the particle surface, which can favor signal distortions by scattering and encoding dyes, cleavable reporters can be detected colorimetrically or fluorometrically both attached at the particle surface and after quantitative cleavage of the linker in the transparent supernatant after particle removal e.g., by centrifugation. Here, we present first results obtained for the optical quantification of carboxylic and amino groups on a series of self-made NPs with different types of labels and compare their potential and drawbacks for surface group analysis.
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.
Nanometer- and micrometer-sized particles are of increasing importance for a wide range of applications in the material and life sciences. This includes carriers for, e.g., drugs as well as dye molecules for use as multichromophoric reporters for signal enhancement in optical assays, platforms for DNA sequencing, and the fabrication of nanosensors and targeted probes for bioimaging studies.
Application-relevant properties of such particles include their size (and size distribution), shape, colloidal stability, biocompatibility, and ease of subsequent functionalization, e.g., with linkers, sensor molecules, and targeting ligands. The latter requires knowledge of the number of groups effectively accessible for subsequent coupling reactions and hence, selective and sensitive methods of analysis, which can be ideally employed for the characterization of a broad variety of particle systems independent of their optical properties, i.e., scattering or the presence of encoding dyes. For product control, robust, reliable and fast methods performable with inexpensive equipment are prefered.
In this respect, we studied a variety of conventional labels for optical readout, utilizing changes in intensity and/or color of absorption and/or emission. In addition, we developed a platform of cleavable and multimodal labels for optical assays which consist of a cleavable linker and an optically active reporter moiety. In contrast to conventional reporters measured directly at the particle surface, which are prone to signal distortions by scattering and encoding dyes, these cleavable labels can be detected colorimetrically or fluorometrically both bound at the particle surface and after quantitative cleavage of the linker in the transparent supernatant. Moreover, they enable straightforward validation by method comparison with elemental analysis, ICP-OES or ICP-MS.
Here, we present representative examples of newly synthesized cleavable labels and their application for the quantification of amino, thiol and carboxy surface groups on different nanomaterials and compare these results with measurements using conventional optical labels.
The amount of grafted poly(acrylic acid) on poly(methyl methacrylate) micro- and nanoparticles was quantified by conductometry, 13C solid-state NMR, fluorophore labeling, a supramolecular assay based on high-affinity binding of cucurbit[7]uril, and two colorimetric assays based on toluidine blue and nickel complexation by pyrocatechol violet. The methods were thoroughly validated and compared with respect to reproducibility, sensitivity, and ease of use. The results demonstrate that only a small but constant fraction of the surface functional groups is accessible to covalent surface derivatization independently of the total number of surface functional groups, and different contributing factors are discussed that determine the number of probe molecules which can be bound to the polymer surface. The fluorophore labeling approach was modified to exclude artifacts due to fluorescence quenching, but absolute quantum yield measurements still indicate a major uncertainty in routine fluorescence-based surface group quantifications, which is directly relevant for biochemical assays and medical diagnostics. Comparison with results from protein labeling with streptavidin suggests a porous network of poly(acrylic acid) chains on the particle surface, which allows diffusion of small molecules (cutoff between 1.6 and 6.5 nm) into the network.
In order to develop simple and versatile procedures for the preparation of red emissive particles, various one-step swelling procedures for the loading of fluorophores into nanometer- and micrometer-sized polystyrene particles were systematically assessed. Parameters studied for model dyes from common dye classes include the composition of the swelling medium, dye charge and polarity, dye concentration, and particle surface chemistry. The dye loading procedures were compared based upon the efficiency of dye incorporation, fluorescence intensity, and colloidal stability of the resulting particles as well as the absence of dye leaking as determined by absorption and fluorescence spectroscopy, flow cytometry, and measurements of zeta potentials. In addition, for the first time, the influence of the amount of incorporated dye on the absolute fluorescence quantum yield and brightness of the fluorescent particles was investigated for selected chromophores in differently sized particles using a custom-made calibrated integrating sphere setup. Our results demonstrate the general suitability of these one-step loading procedures for efficient particle staining with neutral, zwitterionic, and charged fluorophores like oxazines, coumarines, squaraines, xanthenes, and cyanines emitting in the visible and near infrared. Dye polarity was identified as a suitable tool to estimate the loading efficiency of fluorophores into these polymer particles.