Protein adsorption on poly(N-isopropylacrylamide) brushes: dependence on grafting density and chain collapse

Abstract

The protein resistance of poly(N-isopropylacrylamide) brushes grafted from silicon wafers was investigated as a function of the chain molecular weight, grafting density, and temperature. Above the lower critical solution temperature (LCST) of 32 °C, the collapse of the water-swollen chains, determined by ellipsometry, depends on the grafting density and molecular weight. Ellipsometry, radio assay, and fluorescence imaging demonstrated that, below the lower critical solution temperature, the brushes repel protein as effectively as oligoethylene oxide-terminated monolayers. Above 32 °C, very low levels of protein adsorb on densely grafted brushes, and the amounts of adsorbed protein increase with decreasing brush-grafting-densities. Brushes that do not exhibit a collapse transition also bind protein, even though the chains remain extended above the LCST. These findings suggest possible mechanisms underlying protein interactions with end-grafted poly(N-isopropyl acrylamide) brushes.

Figure 1

XPS spectra of a self-assembled monolayer of initiator silane on a silicon wafer before (a) and after (b) the polymerization of PNIPAM from the monolayer.

Figure 2

Advancing water contact angle on dry PNIPAM brushes at 23°C and 37°C. The numbers below the graph indicate the estimated chain grafting densities and corresponding molecular weights. Here * indicates a significant difference between the values measured at 23°C and 37°C for each sample (p < 0.001), and ** indicates a significant difference between the water contact angle at 37°C on the low density brush (0.08 chains/nm² at 44kD or 0.09 chains/nm² at 48kD) relative to the other samples (p < 0.01).

Figure 3

Measured changes in the ellipsometric thickness of PNIPAM brushes in pure water as a function of temperature. The curves correspond to different brush preparations: black squares (0.08/nm², 44kDa); open squares (0.09/nm², 48kDa); open circles (0.09/nm², 85kDa); black circles (0.11/nm², 126kDa); open triangles (0.21/nm², 101kDa).

Figure 4

Difference in ellipsometric angles ψ (a) and Δ (b) determined with incident light at 546 nm, before and after immersion in a 1 mg/mL lysozyme solution. The dashed lines correspond to the simulated values for the adsorbed amount of 1 mg/m² on PNIPAM. The dotted line indicates the simulated shift in Δ for 0.1 mg/m² adsorbed protein on the OEG monolayer.

Figure 5

¹²⁵[I]-BSA adsorption on PNIPAM brushes at 23°C and at 37°C. The polymer grafting density and molecular weight are indicated below the bars. Adsorption on the OEG-terminated alkylsilane monolayer is shown for comparison. Statistically significant differences (p < 0.05) are indicated.

Figure 6

Fluorescence images of adsorbed FITC-BSA (1mg/mL in 0.01M PBS buffer) on different micropatterned surfaces at two different temperatures. (a) Microcontact-printed OEG silane (dark zigzag region) on a clean glass substrate at 37 °C and (b) at 23°C; (c) Microcontact-printed PNIPAM brush (dark zigzag region) on a clean glass substrate at 37 °C and (d) at 23°C; (e) Microcontact-printed OTS on clean glass substrate (bright zigzag), which was backfilled with OEG silane (dark region) at 37 °C and (f) at 23°C.