Bright
signal outputs are needed for fluorescence detection of
biomolecules at their native expression levels. Increasing the number
of labels on a probe often results in crowding-induced self-quenching
of chromophores, and maintaining the function of the targeting moiety
(e.g., an antibody) is a concern. Here we demonstrate a simple method
to accommodate thousands of fluorescent dye molecules on a single
antibody probe while avoiding the negative effects of self-quenching.
We use a bottlebrush polymer from which extend hundreds of duplex
DNA strands that can accommodate hundreds of covalently attached and/or
thousands of noncovalently intercalated fluorescent dyes. This polymer–DNA
assembly sequesters the intercalated fluorophores against dissociation
and can be tethered through DNA hybridization to an IgG antibody.
The resulting fluorescent nanotag can detect protein targets in flow
cytometry, confocal fluorescence microscopy, and dot blots with an
exceptionally bright signal that compares favorably to commercially
available antibodies labeled with organic dyes or quantum dots.
Molecular bottlebrushes with hydrophobic poly(n-butyl acrylate) or polystyrene and hydrophilic poly(di-(ethylene glycol) ethyl ether acrylate)) side chains were successfully synthesized by grafting from a poly(2-(2bromoisobutyryloxy)ethyl methacrylate) macroinitiator using iron-based atom transfer radical polymerization (ATRP). Iron(II) bromide, iron(III) bromide, and tetrabutylammonium bromide catalyst was employed for an ATRP grafting-from reaction, resulting in brush macromolecules with a narrow molecular weight distribution (M w /M n = 1.18−1.28). Molecular weights measured by multiangle laser light scattering correlates well with the theoretical values for all bottlebrushes. Imaging of individual bottlebrushes by atomic force microscopy exhibited a wormlike conformation. Initiation efficiencies were calculated by cleaving the side chains by alcoholysis and then injecting to gel permeation chromatography. The initiation efficiencies were ca. 80−95%, showing relatively high values for a grafting from polymerization with an iron catalyst. These results indicate that iron-catalyzed ATRP allows well-controlled polymerization even when targeting dense grafting from procedures. The 0.1% (w/ w) of water-soluble molecular bottlebrushes with poly(di(ethylene glycol) ethyl ether acrylate)) side chains displayed a lower critical solution temperature behavior in distilled water, and the average particle size started to increase above 8 °C due to intermolecular aggregation of the bottlebrushes. The slight decrease of the size in highly diluted solution (0.005% w/w) of the bottlebrush was observed as the temperature was increased, suggesting that intramolecular collapse of the individual molecules.
Phototunable supersoft elastomers were prepared by photo-cross-linking molecular bottlebrushes with poly(n-butyl acrylate) (PBA) side chains. Bottlebrushes were synthesized by atom transfer radical polymerization (ATRP) and Br-chain end functionalities were replaced by photo-crosslinkable coumarin units. Photo-cross-linking and photoscission of molecular bottlebrushes resulted in tunable elastomeric behavior quantified by dynamic mechanical analysis. The cell− substrate interactions, tuned by UV-light, depended on surface morphology and softness that could be as low as several kilopascals.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.