Quantification of Aldehydes on Polymeric Microbead Surfaces via Catch and Release of Reporter Chromophores

Abstract

Aldehyde moieties on 2D-supports or microand nanoparticles can function as anchor groups for the attachment of biomolecules or as reversible binding sites for proteins on cell surfaces. The use of aldehyde-based materials in bioanalytical and medical settings calls for reliable methods to detect and quantify this functionality. We report here 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 nonfluorescent 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 quasi-quantitatively and allows for their fluorometric detection at low concentration. Importantly, this strategy separates the signal-generating molecules from the bead surface. This circumvents common issues associated with light scattering and signal distortions that are caused by binding-induced changes in reporter fluorescence as well as quenching dye− dye interactions on crowded particle surfaces. In addition, we demonstrate that the release of a nonfluorescent 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.

Fabrication of a “Selenium Signature” Chemical Probe-Modified Paper Substrate for Simultaneous and Efficient Determination of Biothiols by Paper Spray Mass Spectrometry

“…In the process, the Ebselen selenenyl amide (Se–N) bond was cleaved and combined with a thiol (−SH) group in Cys, and a selenenylsulfide (Se–S) bond was formed. When an acid spray solvent was applied, a Schiff-base linkage (Scheme ) between the cellulose on paper and the chemical probe was cleaved; consequently, the ion peak m / z 412 of aimed product [Ebselen-NH 2 -Cys + H] + was obtained by PS-MS. Other biothiols (Hcy, Nac, CysGly, GSH) have a similar derivatization behavior like Cys, and their paper derivative products were illustrated (Figures S8a–c and c). The ion peaks m / z 426, m / z 454, m / z 469, and m / z 598 of aimed products [Ebselen-NH 2 -Hcy + H] + , [Ebselen-NH 2 -Nac + H] + , [Ebselen-NH 2 -CysGly + H] + , and [Ebselen-NH 2 -GSH + H] + were obtained, respectively.…”

“…In the process, the Ebselen selenenyl amide (Se−N) bond was cleaved and combined with a thiol (−SH) group in Cys, and a selenenylsulfide (Se−S) bond was formed. When an acid spray solvent was applied, a Schiff-base linkage (Scheme 2) between the cellulose on paper and the chemical probe was cleaved; 45 To obtain the best analytical performance, a tandem mass spectrometry mode in PS-MS is usually performed to reduce the effect of the matrix ion suppression. 46 In this respect, analyte ions or derivatized ions within the mixture should be subjected to collisional activation, and an efficient generation of fragment ion product is expected.…”

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

“…Moreover, this design principle can be easily adapted to other FG. For example, we developed the SPDP derivative 3-(2-pyridyldithio)propionyl hydrazide (PDPH) bearing a reactive hydrazide group for the quantification of aldehyde groups on a set of PMMA microparticles [ 140 ]. Validation was done by comparison with another catch-and-release assay utilizing a hydrazide-functionalized fluorescent BODIPY dye (BDP-hzd) as reporter that proved to be even more sensitive due to the fluorometric readout.…”

Influence of Nanoparticle Encapsulation and Encoding on the Surface Chemistry of Polymer Carrier Beads

“…Therefore, we evaluated and compared both fabrication methods in terms of PSMP size, surface charge, amount of total and accessible FG, and luminescence properties. Based upon previously examined and validated protocols for FG quanti cation, [37][38][39][40] the total amount of (de)protonable COOH groups was determined by a conductometric titration. In addition, Fourier transform infrared (FTIR) spectroscopy was used as a semiquantitative method for the determination of the number of total COOH acid groups.…”