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.

Keywords: antifouling; biosensing; flow cytometry; microbead; sulfobetaine; zwitterionic polymer.

Figure 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 recognition unit that specifically binds its target while still being able to repel all unwanted proteins.

Figure 2

(A) XPS wide scans of nonmodified beads, initiator-functionalized and pSB polymer-coated beads. (B) XPS N 1s and (C) C 1s narrow scans of pSB-coated beads. The spectra show the successful growth of zwitterionic polymer brushes from the beads.

Scheme 1. Overview of Chemical Modifications on Amine-Terminated Beads to Yield Functionalized Antifouling pSB-Coated Beads

The reaction conditions represented by the arrows are as follows: (a) 2.4 M α-bromoisobutyryl bromide, DCM, Et₃N, RT, 3 h. (b) SB, Cu(I)/Cu(II) (9/1), bipyridine, isopropanol/Milli-Q (1/4), RT, 1 min. (c1) 2 M heptafluorobutylamine, DMF, 65 °C, 16 h. (c2) 0.5 M NaN₃, PBS, RT, 16 h. (c3) 2 M propargylamine, PBS, RT, 16 h. (d1) SPAAC, 20 mM BCN-R₁, DMSO/Milli-Q (1/1), RT, 16 h. (d2) CuAAC, 10 mM F₉-alkyne, 0.25 mM CuSO₄, 5 mM Na ascorbate, 1.25 mM THPTA, DMSO/Milli-Q (1/1), RT, 16 h. (d3) CuAAC, 500 μM mannose-C₁₁-azide, 100 μM CuSO₄, 2.5 mM sodium ascorbate, 500 μM THPTA, DMSO/Milli-Q (1/1), RT, 16 h.

Figure 3

Representative flow cytometry data of (A) nonmodified and (B) pSB-coated beads incubated in either PBS or a protein solution containing BSA-AF488 (0.5 mg/mL) or serum-HLF488 (∼6 mg/mL).

Figure 4

Representative flow cytometry data summarizing the MFI of nonmodified and pSB-coated beads incubated with PBS, BSA-AF488 (0.5 mg/mL), serum-HLF488 (10%, ∼6 mg/mL), Strep-PE (50 μg/mL), or ConA-AF635 (50 μg/mL). MFI values are corrected for the autofluorescence of the beads by subtracting MFI values of their corresponding PBS samples; see the Supporting Information Table S1 for noncorrected values.

Figure 5

F 1s peaks from XPS wide scans obtained from pSB beads substituted with a heptafluorinated amine, pSB-azide beads functionalized with BCN-CF₃ via SPAAC, and pSB-azide beads functionalized with F₉-alkyne via CuAAC. The appearance of F 1s peaks in these XPS spectra indicates the successful functionalization of the polymer-coated beads with fluorinated model compounds.

Figure 6

Confocal images of nonmodified, pSB-coated, and pSB-biotin beads incubated with BSA-AF488 (0.5 mg/mL) or Strep-PE (50 μg/mL) or a mixture of the two. The green channel shows the fluorescence as measured with a 488-laser line and a 500-531 nm band-pass filter, and the red channel shows the fluorescence as measured with a 561-laser line with a 570-610 nm band-pass filter.

Figure 7

Flow cytometry data of pSB-biotin beads incubated with PBS or a mixture of Strep-PE (50 μg/mL) and either BSA-AF4888 (0.5 mg/mL) or serum-HLF488 (10%, ∼6 mg/mL). (A) 488 channel showing BSA-AF488 and serum-HLF488 binding; PBS (blue) and BSA-AF488 (dark green) completely overlap, (B) PE channel showing Strep-PE binding; Strep-PE mixed with BSA-AF488 (orange) completely overlaps with Strep-PE mixed with serum-HLF488 (magenta).