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

Immunosensors thatcombine planar transducers with microfluidicsto achieve in-flow biofunctionalization and assay were analyzed hereregarding surface binding capacity, immobilization stability, bindingstoichiometry, 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), followedby blocking with bovine serum albumin (BSA) and streptavidin (STR)capture, are monitored with white light reflectance spectroscopy (WLRS)sensors as thickness d (& UGamma;) of the adlayerformed on top of aminosilanized silicon chips. Multi-protein surfacecomposition (IgG, BSA, and STR) is determined by time of flight secondaryion mass spectrometry (TOF-SIMS) combined with principal componentanalysis (applying barycentric coordinates to the score plot). In-flowimmobilization shows at least 1.7 times higher surface binding capacitythan static adsorption. In contrast to physical immobilization, whichis unstable during blocking with BSA, chemisorbed antibodies desorb(reducing d (& UGamma;)) only when the bilayeris formed. Also, TOF-SIMS data show that IgG molecules are partiallyexchanged with BSA on APTES but not on APTES/GA modified chips. Thisis confirmed by the WLRS data that show different binding stoichiometrybetween the two immobilization schemes for the direct binding IgG/anti-IgGassay. The identical binding stoichiometry for STR capture resultsfrom partial replacement with BSA of vertically aligned antibodieson APTES, with fraction of exposed Fab domains higher than on APTES/GA.

Automated summary

Immunosensors that combine planar transducers with microfluidics were analyzed for their surface binding capacity, immobilization stability, and binding stoichiometry. Two IgG immobilization schemes, physical adsorption and glutaraldehyde covalent coupling, were compared and monitored using white light reflectance spectroscopy sensors. The results showed that in-flow immobilization had higher surface binding capacity compared to static adsorption, and chemisorbed antibodies were more stable during blocking with bovine serum albumin. The data also revealed that IgG molecules were partially exchanged with BSA on APTES chips but not on APTES/GA chips.