Covalent and Non-covalent In-Flow Biofunctionalization for Capture Assays on Silicon Chips: White Light Reflectance Spectroscopy Immunosensor Combined with TOF-SIMS Resolves Immobilization Stability and Binding Stoichiometry

Abstract

Immunosensors that combine planar transducers with microfluidics to achieve in-flow biofunctionalization and assay were analyzed here regarding surface binding capacity, immobilization stability, binding stoichiometry, and amount and orientation of surface-bound IgG antibodies. Two IgG immobilization schemes, by physical adsorption [3-aminopropyltriethoxysilane (APTES)] and glutaraldehyde covalent coupling (APTES/GA), followed by blocking with bovine serum albumin (BSA) and streptavidin (STR) capture, are monitored with white light reflectance spectroscopy (WLRS) sensors as thickness d Γ of the adlayer formed on top of aminosilanized silicon chips. Multi-protein surface composition (IgG, BSA, and STR) is determined by time of flight secondary ion mass spectrometry (TOF-SIMS) combined with principal component analysis (applying barycentric coordinates to the score plot). In-flow immobilization shows at least 1.7 times higher surface binding capacity than static adsorption. In contrast to physical immobilization, which is unstable during blocking with BSA, chemisorbed antibodies desorb (reducing d Γ ) only when the bilayer is formed. Also, TOF-SIMS data show that IgG molecules are partially exchanged with BSA on APTES but not on APTES/ GA modified chips. This is confirmed by the WLRS data that show different binding stoichiometry between the two immobilization schemes for the direct binding IgG/anti-IgG assay. The identical binding stoichiometry for STR capture results from partial replacement with BSA of vertically aligned antibodies on APTES, with fraction of exposed Fab domains higher than on APTES/GA.

4-(Triethoxysilyl)butanoic acid as a self-assembled monolayer for surface modification of titanium dioxide

“…16 Thus, the probability of the exposure of the Fab region of the antibody would increase. 15 Then, the capture antibody could recognize the antigen more effectively by using APTES followed by GA as the SAM. Thus, the modification and detection effects achieved by using APTES followed by GA as the SAM were better than those achieved by using APTES as the SAM.…”