Multimodal Cleavable Reporters for Quantifying Carboxy and Amino Groups on Organic and Inorganic Nanoparticles

Abstract

Organic and inorganic nanoparticles (NPs) are increasingly used as drug carriers, fluorescent sensors, and multimodal labels in the life and material sciences. These applications require knowledge of the chemical nature, total number of surface groups, and the number of groups accessible for subsequent coupling of e.g., antifouling ligands, targeting bioligands, or sensor molecules. To establish the concept of catch-and-release assays, cleavable probes were rationally designed from a quantitatively cleavable disulfide moiety and the optically detectable reporter 2-thiopyridone (2-TP). For quantifying surface groups on nanomaterials, first, a set of monodisperse carboxy-and amino-functionalized, 100 nm-sized polymer and silica NPs with different surface group densities was synthesized. Subsequently, the accessible functional groups (FGs) were quantified via optical spectroscopy of the cleaved off reporter after its release in solution. Method validation was done with inductively coupled plasma optical emission spectroscopy (ICP-OES) utilizing the sulfur atom of the cleavable probe. This comparison underlined the reliability and versatility of our probes, which can be used for surface group quantification on all types of transparent, scattering, absorbing and/or fluorescent particles. The correlation between the total and accessible number of FGs quantified by conductometric titration, qNMR, and with our cleavable probes, together with the comparison to results of conjugation studies with differently sized biomolecules reveal the potential of catch-and-release reporters for surface analysis. Our findings also underline the importance of quantifying particularly the accessible amount of FGs for many applications of NPs in the life sciences.

Analyzing the surface of functional nanomaterials—how to quantify the total and derivatizable number of functional groups and ligands

“…The quantification of FG and ligands on NM surfaces can be carried out by solution phase NMR techniques and by solid-state NMR. It typically involves the addition of an internal standard of known concentration and known, high purity, that is chosen to reveal NMR signals (chemical shift) well separated from the NMR signals originating from the FG or ligands of interest and the matrix [ 51 , 89 ]. Particularly for solution NMR, it must also be considered that the NMR signals of the FG or ligands bound to a NM surface can significantly change compared to the signals of the free molecule or FG in solution.…”

“…However, both methods can also be applied to detect elements that directly correspond to ligands and FG native on the NM surface, and thus, the total amount of FG, or to detect specific labels conjugated or associated to the FG, and hence, the number of derivatizable FG. Examples for the latter case present the quantification of elements such as sulfur present in certain dyes or cleavable probes [64,89,127], or the use of metal ion containing reporters [173]. ICP-OES has been used to quantify FG on various NM such as carbon-based NM, noble metal NP, polymer beads, and lanthanide-based NP [127,[174][175][176][177].…”

“…In a conductometric titration, the conductivity of a sample is measured as a function of the added amount of acid or base. Typical examples present the quantification of the total amount of amino and carboxy groups on polystyrene (PS) particles [ 64 , 89 , 90 ]. The suitability of conductometry for carboxy group quantification on polymeric particles has been validated for polymethylmethacrylate (PMMA) particles grafted with polyacrylic acid (PAA) by comparison with quantitative NMR spectroscopy (qNMR) [ 63 ] and for PS particles by comparison with Zeta potential measurements [ 90 ].…”

“…As summarized in Fig. 4 , this includes (i) conventional (“always ON”) dyes, (ii) chromogenic/fluorogenic (“chameleon”-type) dyes and activatable (“turn-ON”) dye reporters that change either the spectral position of their absorption and/or emission bands upon reaction with the respective FG or become absorptive (colored) or emissive upon the binding event [ 64 , 114 , 115 ], and (iii) cleavable probes that can be quantitatively cleaved off from the NM surface and subsequently quantified in solution [ 64 , 89 ]. In addition, (iv) adsorption/desorption assays relying on negatively or positively charged reporters and electrostatic interactions with oppositely charged FG are utilized [ 116 ].…”

“…These cleavable probes are also suited for quantifying amino and carboxy groups at various FG densities and even on absorbing and fluorescent NM like dye-stained fluorescent particles. [ 89 ]. Moreover, this design principle can be easily adapted to other FG.…”