Bioconjugation of Ultrabright Semiconducting Polymer Dots for Specific Cellular Targeting

Abstract

Semiconducting polymer dots (Pdots) represent a new class of ultrabright fluorescent probes for biological imaging. They exhibit several important characteristics for experimentally demanding in vitro and in vivo fluorescence studies, such as their high brightness, fast emission rate, excellent photostability, non-blinking, and non-toxic feature. However, controlling the surface chemistry and bioconjugation of Pdots has been a challenging problem that prevented their widespread applications in biological studies. Here, we report a facile yet powerful conjugation method that overcomes this challenge. Our strategy for Pdot functionalization is based on entrapping heterogeneous polymer chains into a single dot, driven by hydrophobic interactions during nanoparticle formation. A small amount of amphiphilic polymer bearing functional groups is cocondensed with the majority of semiconducting polymers to modify and functionalize the nanoparticle surface for subsequent covalent conjugation to biomolecules, such as streptavidin and immunoglobulin G (IgG). The Pdot bioconjugates can effectively and specifically label cellular targets, such as cell surface marker in human breast cancer cells, without any detectable nonspecific binding. Single-particle imaging, cellular imaging, and flow cytometry experiments indicate a much higher fluorescence brightness of Pdots compared to those of Alexa dye and quantum dot probes. The successful bioconjugation of these ultrabright nanoparticles presents a novel opportunity to apply versatile semiconducting polymers to various fluorescence measurements in modern biology and biomedicine.

Light‐Induced PEGylation and Functionalization of Semiconductor Polymer Dots

“…[33] In at ypical bioconjugation reaction, 60 mLo fp olyethylene glycol (PEG, 5% w/ v) and an equal amount of concentrated HEPES buffer (1 m)w ere added to 3mLo faPdot solution (50 mgmL ,i nd eionized water) was added and the resulting mixture was left on ar otary shaker for 4h at room temperature. The resulting Pdot-streptavidin bioconjugates were separated from free biomolecules by gel filtration using Sephacryl HR-300 gel media.…”

Fluorescent nanoparticles from PEGylated polyfluorenes

“…15 potential for use in applications ranging from biological imaging to consumer electronics. [1][2][3][4][5][6][7] CPNs can be considered .as alternatives to the use of Quantum Dots (QDs) as they are small (ca. 10 -200 nm), bright, exhibit photostable fluorescence that can be tuned across the visible spectrum and can be isolated as 20 stable dispersions in water.…”

“…PEG groups have been incorporated to reduce non-specific binding of biomolecules and carboxy groups have been utilised for attachment of biomolecules using this approach. 3,4 Given that 65 the factors that control the size and microstructure of CPNs formed by nanoprecipitation are not well understood, we feel that development of a more straightforward and reproducible approach to providing surface functionality is desirable. Our chosen approach is to synthesise bespoke conjugated polymers 70 with the desired functional groups covalently attached to the polymer backbone and to develop an understanding of how these modifications influence the nanoprecipitation process and the properties of the resultant nanoparticles.…”

Size-dependent optical properties of conjugated polymer nanoparticles

“…[5][6][7][8][9] Among numerous species of uorescent particles, conjugated polymer nanoparticles (CPNs), arouse great interest in terms of following charming characteristics: ultrahigh brightness, structural diversity, functional designability, excellent biocompatibility, good photostability and water dispersion stability, 10 thus efficiently avoiding the shortcomings of conventional uorescent dyes and Qdots. Although CPNs have been proposed and applied in biological eld for only a few years, in virtue of the excellent optical properties and water dispersion stability, conjugated polymer nanoparticles have become a research hotspot as a candidate for labelling materials, such as cellular labelling, [11][12][13] in vivo imaging, [14][15][16][17][18] biosensing, 19 drug delivery 20 and photodynamic cancer therapy.…”