Microfluidic Droplet Stabilization via SPAAC Promoted Antibody Conjugation at the Water/Oil Interface.

Droplet-based microfluidics is leading the development of miniaturized, rapid, and sensitive version of enzyme-linked immunosorbent assays (ELISAs), a central method for protein detection. These assays involve the use of a functionalized surface able to selectively capture the desired analyte. Using the droplet's oil water interface as a capture surface requires designing custom-perfluorinated fluorosurfactants bearing azide-containing polar groups, which spontaneously react when forming the droplet with strain-alkyne-functionalized antibodies solubilized in the aqueous phase. In this article, we present our research on the influence of the structure of surfactant's hydrophilic heads on the efficiency of SPAAC functionalization and on the effect of this antibody grafting process on droplet stability. We have shown that while short linkers lead to high grafting efficiency, long linkers lead to high stability, and that an intermediate size is required to balance both parameters. In the described family of surfactants, the optimal structure proved to be a PEG4 linker connecting a polar di-azide head and a per-fluoropolyether tail (Krytox). We also found that grafting an increasing amount of antibody, thus increasing interface coverage, increases droplet stability. It thus appears that such a bi-partite system with a reactive fluoro-surfactant in the oil phase and reactive antibody counterpart in the aqueous phase gives access in situ to novel surfactant construct providing unexplored interface structures and droplet functionality.

Droplet Surface Immunoassay by Relocation (D-SIRe) for High-Throughput Analysis of Cytosolic Proteins at the Single-Cell Level.

Enzyme-linked immunosorbent assay (ELISA) is a central analytic method in biological science for the detection of proteins. Introduction of droplet-based microfluidics allowed the development of miniaturized, less-consuming, and more sensitive ELISA assays by coencapsulating the biological sample and antibody-functionalized particles. We report herein an alternative in-droplet immunoassay format, which avoids the use of particles. It exploits the oil/aqueous-phase interface as a protein capture and detection surface. This is achieved using tailored perfluorinated surfactants bearing azide-functionalized PEG-based polar headgroups, which spontaneously react when meeting at the droplet formation site, with strained alkyne-functionalized antibodies solubilized in the water phase. The resulting antibody-functionalized inner surface can then be used to capture a target protein. This surface capture process leads to concomitant relocation at the surface of a labeled detection antibody and in turn to a drastic change in the shape of the fluorescence signal from a convex shape (not captured) to a characteristic concave shape (captured). This novel droplet surface immunoassay by fluorescence relocation (D-SIRe) proved to be fast and sensitive at 2.3 attomoles of analyte per droplet. It was further demonstrated to allow detection of cytosolic proteins at the single bacteria level.

Plasma induced acceleration and selectivity in strain-promoted azide-alkyne cycloadditions.

Strain-promoted azide-alkyne cycloaddition (SPAAC) is an important member of the bioorthogonal reaction family. Over the past decade, much work has been dedicated to the generation of new strained alkynes with improved reactivity. While kinetics studies of SPAAC are often conducted in organic solvents, buffered solutions or mixtures, these media do not reflect the complexity of in vivo systems. In this work, we show that performing SPAAC in human plasma leads to intriguing kinetics and selectivity effects. In particular, we observed that reactions in plasma could be accelerated up to 70-fold compared to those in methanol, and that selective couplings between a pair of reagents could be possible in competition experiments. These findings highlight the value of evaluating bioorthogonal reactions in such a complex medium, especially when in vivo applications are planned, as unsuspected behaviour can be observed, disrupting the usual rules governing the reactivity in simple solvent systems.

Bicyclo[6.1.0]nonyne carboxylic acid for the production of stable molecular probes.

Bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN alcohol) is the most prominent strained-alkyne scaffold in chemical biology. Described herein is the synthesis of an oxidized analogue - BCN acid - whose facile functionalization via amide bond formation yields more stable derivatives than the classically encountered carbamates.

Expedient synthesis of trifunctional oligoethyleneglycol-amine linkers and their use in the preparation of PEG-based branched platforms.

We designed a convergent synthesis pathway that provides access to trifunctional oligoethyleneglycol-amine (OEG-amine) linkers. By applying the reductive coupling of a primary azide to bifunctional OEG-azide precursors, the corresponding symmetrical dialkylamine bearing two homo-functional end chain groups and a central nitrogen was obtained. These building blocks bear minimal structural perturbation compared to the native OEG backbone which makes them attractive for biomedical applications. The NMR investigations of the mechanism process reveal the formation of nitrile and imine intermediates which can react with the reduced free amine form. Additionally, these trifunctional OEG-amine linkers were employed in a coupling reaction to afford branched multifunctional PEG dendrons which are molecularly defined. These discrete PEG-based dendrons (n = 16, 18 and 36) could be useful for numerous applications where multivalency is required.

Structural investigation of cyclo-dioxo maleimide cross-linkers for acid and serum stability.

The biochemical characteristics of hetero-bifunctional cross-linkers used in bioconjugates are of essential importance to the desired features of the final adduct (i.e. antibody-drug conjugates). These include stability in biological media, chemical and biological reactivities, cleavability under defined conditions, and solubility. In our previous work, we introduced a new amino-to-thiol linker, maleimidomethyl dioxane (MD), as an alternative to classical maleimide conjugation, with increased hydrophilicity and serum stability due to succinimidyl ring-opening. In this work, we investigate the generality of linkers containing a dioxo-ring with regard to their ability to self-hydrolyze and their surprising stability at a low pH. We synthesized four FRET probes which allowed us to address the stability of the dioxo-ring and to study the maleimide ring-opening and the thiol-exchange processes by means of detecting and measuring the generation of fluorescence. It was found that the ring expansion (from a 5- to a 6-membered ring) improved the stability of the probes in aqueous media, and the increase of the chain length between the dioxo-ring and the succinimide ring (from methylene to ethylene) decreased the rate of succinimidyl ring-opening.

From solution to in-cell study of the chemical reactivity of acid sensitive functional groups: a rational approach towards improved cleavable linkers for biospecific endosomal release.

pH-Sensitive linkers designed to undergo selective hydrolysis at acidic pH compared to physiological pH can be used for the selective release of therapeutics at their site of action. In this paper, the hydrolytic cleavage of a wide variety of molecular structures that have been reported for their use in pH-sensitive delivery systems was examined. A wide variety of hydrolytic stability profiles were found among the panel of tested chemical functionalities. Even within a structural family, a slight modification of the substitution pattern has an unsuspected outcome on the hydrolysis stability. This work led us to establish a first classification of these groups based on their reactivities at pH 5.5 and their relative hydrolysis at pH 5.5 vs. pH 7.4. From this classification, four representative chemical functions were selected and studied in-vitro. The results revealed that only the most reactive functions underwent significant lysosomal cleavage, according to flow cytometry measurements. These last results question the acid-based mechanism of action of known drug release systems and advocate for the importance of an in-depth structure-reactivity study, using a tailored methodology, for the rational design and development of bio-responsive linkers.

Novel chiral derivatizing isothiocyanate-based agent for the enantiomeric excess determination of amines.

The expedient synthesis of a novel chiral (1S,2S)-N-[(2-isothiocyanato)cyclohexyl] trifluoromethanesulfonamide and its use as a derivatizing auxiliary for the straightforward determination of optical purity of chiral amines are reported.