It has been shown before that serum albumin clusters are formed upon adsorption of this protein onto sound surfaces, especially at high protein concentrations that favor proteinCprotein interactions.39,40 Whether the homogeneous distribution of streptavidin is a result of specific binding rather than nonspecific binding remains elusive. Open in a separate window Figure 6 Confocal images of nonmodified, pSB-coated, and pSB-biotin beads incubated with BSA-AF488 (0.5 mg/mL) or Strep-PE (50 g/mL) or a mixture of the two. equipped with acknowledgement elements of choice, to enable the specific binding of target molecules. First, we present a proof of theory 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. Circulation 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. polymerization via atom transfer radical polymerization (ATRP),20 that is, growing polymer brushes from a surface, has become the method of choice for the preparation of zwitterionic coatings. It yields highly antifouling, densely packed coatings with tunable thicknesses.21 Carboxybetaines (CBs) and sulfobetaines (SBs) have become the zwitterionic monomers of choice because of their commercial availability, straightforward synthesis, and outstanding performance.22,23 Although antifouling coatings have been widely investigated on flat surfaces, their use in nano- and micron-sized particles remains largely unexplored. Apart from a small number of studies describing the use of PEG-derived materials9,24 or zwitterionic coatings prepared by a method with altered poly(acrylic acid),8,25 only two preliminary studies by Jia et al.6 and Yang et al.7 describe ATRP-prepared zwitterionic CB polymer-coated nanoparticles for sensing purposes. However, both these studies Vegfb lack not only a sophisticated biological read-out system that allows for the discrimination between specific and nonspecific protein binding, but also the ability to perform analysis on single beads rather than on the bulk. We anticipated, therefore, that a sensing platform based on ATRP-prepared antifouling beads that can be analyzed by circulation cytometry would be highly valuable. Circulation cytometry is usually indispensable in biomedical research and is Morusin routinely used in clinical diagnostics, for example, to monitor the course and treatment of HIV infections and to determine the prognoses and optimal treatment for several types of cancers.26?28 Moreover, flow cytometry allows for the simultaneous and multiparametric analysis of hundreds to thousands of cells or micron-sized particles per second,26 which makes it a very powerful and frequently used read-out system. Herein, we demonstrate that circulation cytometry is also an excellent technique to study specific versus nonspecific binding on micron-sized particles. Here, we develop a route toward generically antifouling zwitterionic SB polymer (pSB)-coated beads that still can bind a specific protein of interest (see Figure ?Physique11). With this aim, magnetic amine-functionalized beads (Dynabeads; 2.8 m diameter) were selected as the starting material. These beads are compatible with flow cytometry devices, stable in both aqueous and organic solvents, and can be very easily separated from solvents/reactants because of their magnetic core. Top functionalization of the pSB-coated beads with biotin or mannose allowed for the specific binding of streptavidin or concanavalin A (ConA), respectively, from complex biological media. These pSB-coated beads showed excellent antifouling properties combined with a selective binding of the protein of interest. This platform, therefore, shows great potential for the development of a range of bioassays that require ultralow biofouling conditions. Open in a separate window Physique 1 Schematic representation of (A) a nonmodified bead with a significant amount of nonspecifically bound proteins, (B) an antifouling zwitterionic polymer-coated bead that repels all biomolecules (before functionalization with a biorecognition unit), and (C) an antifouling polymer-coated bead equipped with a acknowledgement unit that specifically binds its target while still being able to repel all unwanted proteins. Materials and Methods Materials All chemicals and solvents were used without further purification. -Bromoisobutyryl bromide (98%), trimethylamine (BioUltra, 99.5%), copper(I) chloride (99%), copper(II) chloride (97%), sodium azide (BioUltra, 99.5%), dimethyl sulfoxide (DMSO) (anhydrous, 99.9%), acetone semiconductor grade (VLSI PURANAL Honeywell 17617), tris(3-hydroxypropyltriazolylmethyl)amine (THPTA; 95%), copper(II) sulfate pentahydrate (98%), and (+)-sodium l-ascorbate (BioXtra, 99.0%) were purchased from Sigma-Aldrich. Dimethylformamide (DMF) for peptide synthesis (99.8%) and monopropargylamine (99%) were obtained from Acros Organics, 2,2-bipyridine (98%) from Alfa Aesar, isopropanol (high performance liquid chromatography) from BioSolve, 2,2,3,3,4,4,4-heptafluorobutylamine (97%) from Fisher Scientific, dichloromethane (DCM) from VWR International S.A.S., and 8-(+)-biotinylamino-3,6-dioxa-octyl(1at 4 C. The obtained concentrated labeled serum had a final concentration of 21.2 mg/mL. From this, a 10% serum-HLF488 answer was reconstituted with Morusin a concentration of 6 mg/mL. Serum Biotinylation The same pooled cows serum that was utilized for the HiLyte Fluor 488 serum labeling was also utilized for biotinylation. Serum proteins were biotinylated using an. Morusin