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Fluorogenic atom transfer radical polymerization (ATRP) directly detects initiator-dependent polymer formation, as initially non-fluorescent polycyclic aromatic probe monomers reveal visible fluorescence upon polymerization in real time. Advancement of this initial proof-of-concept toward biodetection applications requires both a more detailed mechanistic understanding of probe fluorescence activation, and the ability to initiate fluorogenic polymerization directly from a biomolecule surface. Here, we show that simple monomer hydrogenation, independent of polymerization, reveals probe fluorescence, supporting the critical role of covalent enone attachment in fluorogenic probe quenching and subsequent fluorescence activation. We next demonstrate bioorthogonal, protein-initiated fluorogenic ATRP by the surface conjugation and characterization of protein–initiator conjugates of a model protein, bovine serum albumin (BSA). Fluorogenic ATRP from initiator-modified protein allows for real-time visualization of polymer formation with negligible background fluorescence from unmodified BSA controls. We further probe the bioorthogonality of this fluorogenic ATRP assay by assessing polymer formation in a complex biological environment, spiked with fetal bovine serum. Taken together, we demonstrate the potential of aqueous fluorogenic ATRP as a robust, bioorthogonal method for biomolecular-initiated polymerization by real-time fluorescence activation. 

The development of novel approaches to signal amplification in aqueous media could enable new diagnostic platforms for the detection of water-soluble analytes, including biomolecules. This paper describes a fluorogenic polymerization approach to amplify initiator signal by the detection of visible fluorescence upon polymerization in real-time. Fluorogenic monomers were synthesized and co-polymerized by atom transfer radical polymerization (ATRP) in water to reveal increasing polymer fluorescence as a function of both reaction time and initiator concentration. Optimization of the fluorogenic ATRP reaction conditions allowed for the quantitative detection of a small-molecule initiator as a model analyte over a broad linear concentration range (pM to mM). Raising the reaction temperature from 30 °C to 60 °C facilitated sensitive initiator detection at sub-picomolar concentrations in as little as 1 h of polymerization. This method was then applied to the detection of streptavidin as a model biological analyte by fluorogenic polymerization from a designed biotinylated ATRP initiator. Taken together, these studies represent the first example of a fluorogenic ATRP reaction and establish fluorogenic polymerization as a promising approach for the direct detection of aqueous analytes and biomolecular recognition events.