Highly Specific Binding on Antifouling Zwitterionic Polymer-Coated Microbeads as Measured by Flow Cytometry

Abstract

Micron- and nano-sized particles are extensively used in various biomedical applications. However, their performance is often drastically hampered by the nonspecific adsorption of biomolecules, a process called biofouling, which can cause false-positive and false-negative outcomes in diagnostic tests. Although antifouling coatings have been extensively studied on flat surfaces, their use on micro- and nanoparticles remains largely unexplored, despite the widespread experimental (specifically, clinical) uncertainties that arise because of biofouling. Here, we describe the preparation of magnetic micron-sized beads coated with zwitterionic sulfobetaine polymer brushes that display strong antifouling characteristics. These coated beads can then be equipped with recognition elements of choice, to enable the specific binding of target molecules. First, we present a proof of principle with biotin-functionalized beads that are able to specifically bind fluorescently labeled streptavidin from a complex mixture of serum proteins. Moreover, we show the versatility of the method by demonstrating that it is also possible to functionalize the beads with mannose moieties to specifically bind the carbohydrate-binding protein concanavalin A. Flow cytometry was used to show that thus-modified beads only bind specifically targeted proteins, with minimal/near-zero nonspecific protein adsorption from other proteins that are present. These antifouling zwitterionic polymer-coated beads, therefore, provide a significant advancement for the many bead-based diagnostic and other biosensing applications that require stringent antifouling conditions.

Atomistic Study of Zwitterionic Peptoid Antifouling Brushes

“…16,23 Tightly controlled polymers pre-formed with a surface anchor group may also be grafted to a surface, but these brushes may be limited in molecular weight to enable efficient surface anchoring and hence require relatively high grafting densities to be effective. 18 Although recent work has continued to expand the potential applications of zwitterionic surface brushes from biomedical to membrane filtration and marine antifouling, [24][25][26][27][28] a clear advantage compared to other polymer designs is not always observed. 24,[29][30][31] The excellent antifouling properties of zwitterionic polymer brushes are frequently attributed to their high hydration by charge groups.…”

PLL–Poly(HPMA) Bottlebrush-Based Antifouling Coatings: Three Grafting Routes

“… 7 Antifouling coatings frequently consist of poly(ethylene glycol) (PEG) 8 , 9 or zwitterionic polymers. 10 − 15 More recently, however, also poly( N -(2-hydroxypropyl)methacrylamide) [poly(HPMA)] brushes grown by controlled radical polymerizations have been reported to result in stable and highly antifouling coatings, on par with and in some cases outperforming zwitterionic coatings. 7 , 13 , 16 − 19 Although the antifouling properties of poly(HPMA) brushes are not entirely understood, the reported fouling levels are extremely low.…”

Chain End‐Functionalized Polymer Brushes with Switchable Fluorescence Response

“…These include converting alkyl bromide end groups into amines before reacting with isothiocyanate‐functionalized dye as well as nitroxide radical exchange during polymerization . A straightforward alternative to these synthetic routes could be “click chemistry,” which was previously used to modify chain‐ends of polymer brushes with, for example, recognition elements, DNA, and polyethylene glycol (PEG) …”

“…[1][2][3] For example, fluorescent dye-labeled polymers have been used to study polymer chain dynamics in solution, [4][5][6] local glass transition temperature of block copolymers, [7] and structures of block copolymer micelles. [8] Localization of the dye at the chain-end of the polymer backbone can offer advantages including minimally affected intrinsic polymer properties (such as solubility and conformation), minimum self-quenching between closely located dyes, and the binding possibility of recognition molecules in chain-ends of polymer brushes with, for example, recognition elements, [34][35][36][37] DNA, [38] and polyethylene glycol (PEG). [39] Here we present switchable fluorescent response of chain end dye-functionalized poly(methyl methacrylate) (PMMA) polymer brushes utilizing co-solvency effect displayed by PMMA in alcohol-water mixtures.…”