Reversible Self-Assembled Monolayers (rSAMs): Adaptable Surfaces for Enhanced Multivalent Interactions and Ultrasensitive Virus Detection

Abstract

We report on the design of pH-switchable monolayers allowing a reversible and ordered introduction of affinity reagents on sensor surfaces. The principal layer building blocks consist of α-(4-amidinophenoxy)alkanes decorated at the ω-position with affinity ligands. These spontaneously self-assemble on top of carboxylic acid terminated SAMs to form reversible homo or mixed monolayers (rSAMs) that are tunable with respect to the nature of the head group, layer order and stability while featuring pH responsiveness and the dynamic nature of noncovalent build assemblies. We show that this results in a range of unique biosensor features. As a first example a sialic acid rSAM featuring strong lectin affinity is here used to sense hemagglutinin and influenza virus (H5N1) at the pM and fM level by in situ ellipsometry in a fully reversible fashion. We believe that the rSAM concept will find widespread use in surface chemistry and overall for boosting sensitivity in affinity biosensors.

Figure 1

(A) Synthetic pathway of OH-terminated amphiphile 1 and sialic acid terminated amphiphile 2 and (B) use of 1 and 2 to form an adaptable rSAM. Reagents and conditions in panel A: (a) 1,10-dibromodecane 3 10 equiv, K₂CO₃ 2 equiv, acetone, 80 °C, 24 h, 81%; (b) 4-(2-hydroxyethyl)phenol 6 2.0 equiv, K₂CO₃ 2.0 equiv, acetone, 80 °C, 24 h, ∼99%; (c) 2-chloroethyl ether 8 43 equiv, tetrabutylammonium hydrogen sulfate (THS) 2.0 equiv, NaOH solution (50% w/w), rt, 18 h, 56%; (d) HCl gas, MeOH, 0 °C → rt, 24 h, then NH₃ in MeOH, rt, 24 h, 82%; (e) NaN₃ 4.0 equiv, DMF, 90 °C, 24 h, 47%; (f) NaAsc 3.0 equiv, Cu(II)SO₄ 0.3 equiv, H₂O/2-butanol (1:2), rt, 4 h, 60%; (g) HCl gas, 1,4-dioxane, MeOH, 0 °C → rt, 24 h, then NH₃ in MeOH, rt, 24 h, 53%.

Figure 2

(A) Film thickness, d, and amount adsorbed, Γ, estimated by in situ ellipsometry, versus time during adsorption of 1 (blue trace), 2 (green trace), or a mixture of 1 and 2 (χ₂ = 0.2) (red trace) (50 μM in buffer) on MHA modified gold at pH 9. Thickness values after pH 9 adsorption, d_ads (Å), and after rinsing in pH 8 buffer, d_rinse (Å), are tabulated in Supporting Information Table 1. (B) Film thickness, d, and amount adsorbed, Γ, measured during the pH-driven self-assembly of 2 on MHA modified gold at pH 9 followed by cycling the pH between 9 and 3 in borate buffer (0.01 M). The desired pH was adjusted using 0.1 M NaOH or 0.1 M HCl solution in a discontinuous system.

Figure 3

(A–D) Baseline-corrected IR reflection–absorption (IRAS) spectra of (A) MHA on gold, (B) rSAM-1 (lower blue trace), (C) rSAM-1+2 (green trace), and (D) rSAM-2 (lower red trace). The black traces in panels B and D correspond to spectra of bulk 1 and 2 in their salt forms. (E–H) Topographical atomic force microscopy (AFM) images (1 μm × 1 μm) of (E) a SAM of MHA on a gold–mica surface, (F) rSAM-1, (G) rSAM-1+2, and (H) rSAM-2. The images were obtained in quantitative nanoscale mechanical (QNM) mode in air. The height differences between valley and peak are obtained from a section analysis as indicated by red arrows.

Figure 4

Film thickness, d, and adsorbed amount, Γ, estimated by ellipsometry for (A) a bare MHA-SAM on gold (orange bars), rSAM-1 (blue bars), or rSAM-2 (red bars) after exposure to solutions of HA, ConA, HSA, HA preincubated with mucin, or 0.005% (w/v) mucin until stable Δ and Ψ values were obtained or for a maximum duration of 5000 s (whichever came first); (B) rSAM-2 upon addition of incremental amounts of HA (red squares), ConA (green triangles), or HSA (blue circles); and (C) MHA-SAM (orange circles) or rSAM-2 (red squares) or SAM-14 (blue triangles) upon addition of deactivated influenza virus H5N1 (0.20–33 HAU) and rSAM 2 upon addition of deactivated influenza virus H5N1 preincubated with mucin (green triangles). Equilibrium dissociation constants, K_d, maximum specific binding, Γ_max, and the Hill slope, h, are tabulated in Supporting Information Table 7. (D) Surface topography of an rSAM of 2 on MHA modified gold after exposure to deactivated H5N1 (14 HAU) followed by rinsing with pH 8 HEPES buffer. Identified virus particles are indicated by arrows.

Figure 5

Hemagglutinin binding isotherms of rSAMs formed with varying density of EG4-sialic acid 16 (χ₁₆) in mixed rSAMs of 15 and 16.