Comparison of Physical Adsorption and Covalent Coupling Methods for Surface Density-Dependent Orientation of Antibody on Silicon

Abstract

The orientation of antibodies, employed as capture molecules on biosensors, determines biorecognition efficiency and bioassay performance. In a previous publication we demonstrated for antibodies attached covalently to silicon that an increase in their surface amount Γ, evaluated with ellipsometry, induces changes in their orientation, which is traced directly using Time-of-Flight Secondary Ion Mass Spectroscopy combined with Principal Component Analysis. Here, we extend the above studies to antibodies adsorbed physically on a 3-aminopropyltriethoxysilane (APTES) monolayer. Antibodies physisorbed on APTES (0 ≤ Γ ≤ 3.5 mg/m2) reveal the Γ ranges for flat-on, side-on, and vertical orientation consistent with random molecular packing. The relation between orientation and Γ is juxtaposed for silicon functionalized with APTES, APTES modified with glutaraldehyde (APTES/GA) and N-hydroxysuccinimide-silane (NHS-silane). Antibody reorientation occurs at lower Γ values when physisorption (APTES) is involved rather than chemisorption (APTES/GA, NHS-silane). At high Γ values, comparable proportions of molecules adapting head-on and tail-on vertical alignment are concluded for APTES and the NHS-silane monolayer, and they are related to intermolecular dipole–dipole interactions. Intermolecular forces seem to be less decisive than covalent binding for antibodies on the APTES/GA surface, with dominant head-on orientation. Independently, the impact of glutaraldehyde activation of APTES on vertical orientation is confirmed by separate TOF-SIMS measurements.

Preparation of Temperature-Responsive Antibody–Nanoparticles by RAFT-Mediated Grafting from Polymerization

“…Furthermore, APC technologies enable the tethering of antibodies to hydrophobic drugs [ 12 , 13 ], contrast agents [ 14 ], and phosphors [ 15 ]. Antibodies have also been immobilized on the surface of substrates [ 16 , 17 , 18 , 19 ] and particles [ 20 ]. Particularly, nanoparticulation is considered to improve the payload selection and the amount of drug introduced [ 21 ].…”

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

“…The Langmuir model can provide a useful reference to the observed adsorption isotherms (Figure ) with a binding capacity equal to (Γ ind Θ ∝ ), reflecting the (maximum) mass loading Γ ind of the individual IgG molecule. , The inner structure of immobilized IgG molecules (Figure ) depends on surface density and immobilization scheme. A decrease in the surface area accessible to each molecule forces a more vertical IgG orientation with the Γ ind values critical for each specific orientation provided by geometric considerations. , The transition from flat-on to side-on and up to vertical alignment is expected every time the surface density Γ reaches a critical (Γ ind Θ ∝ ) value (rescaled to d Γ ∼1 and 1.7 nm , ) and consistent with other reports, e.g., ref and . For vertical alignment, the immobilization scheme determines the proportions of IgG molecules that adapt the coexisting orientations of tail-on and head-on. , …”

“…For vertical alignment, the immobilization scheme determines the proportions of IgG molecules that adapt the coexisting orientations of tail-on and head-on. 7,8 The adsorption isotherms for in-flow immobilization of IgG antibodies (Figures 6a and S5) indicate that the surface binding capacity obtained by covalent coupling on APTES/GA is approximately 15% larger than that achieved employing physical adsorption to APTES. This is consistent with previous studies on antibody immobilization under static conditions, performed by droplet deposition of the protein solution, which reported a slightly higher surface amount of IgG molecules immobilized on glutaraldehyde activated APTES surfaces compared to those modified only with APTES.…”

“…In the case of immunosensors, immobilization of antibodies, which act as capture molecules, requires special attention due to the antigen binding efficiency that depends on the orientation adopted by surface immobilized antibodies. − Although the immobilization scheme controls the molecular orientation, − the latter is also determined by surface density, with a more vertical arrangement forced by a decreasing surface area accessible to each molecule . Recently, antibody immobilization described by random sequential adsorption, forming random rather than close-packed molecular arrangement, was shown to describe the relationship between antibody orientation and its surface density. , The stability of the immobilization of the capture protein could be hindered by partial desorption or exchange with other molecules that may occur during subsequent steps of biofunctionalization and assay. The observation of these complex phenomena, which involve protein–surface and protein–protein interactions, , is difficult due to the question of protein composition in formed multi-molecular adlayers. − For biofunctionalization and assay protocols, molecular desorption and exchange have been reported mainly for immobilization based on the physical adsorption approach, − however, they can also occur for covalent immobilization schemes under certain conditions .…”

“…Immobilization of biomolecules generally requires modification of the biosensor surface to provide the chemical properties that promote physical adsorption or allow covalent coupling. , For this purpose, the application of self-assembled monolayers involving alkanethiols and alkoxysilanes is a robust and versatile approach for modification of gold and silicon-based surfaces, respectively. , For silicon-based biosensors, surface modification with amino-terminated silane, 3-aminopropyltriethoxysilane (APTES), is a primary strategy for the physical adsorption of proteins, while subsequent activation of the APTES layer with glutaraldehyde is a common procedure for the covalent coupling of proteins. , Although glutaraldehyde activation is considered to prevent protein desorption, the impact of this procedure on the stability of the immobilization has not been explicitly examined. Previous comparative analyses between physical adsorption and covalent attachment have focused their effect in assay efficiency , or antibody orientation …”