Contact pin-printing of albumin-fungicide conjugate for silicon nitride-based sensors biofunctionalization: Multi-technique surface analysis for optimum immunoassay performance

Protein adsorption/desorption and antibody binding stoichiometry on silicon interferometric biosensors examined with TOF-SIMS

“…3 and 5). Also, partial protein desorption during primary immunoreaction, concluded in [20], is shown here to depend on the probe spotting concentration, confirming the generality of this recently reported phenomenon [25]. Third, the molecular orientation of immunoadsorbed primary (side-on) and secondary antibody (headon) [20] corresponds well to their binding stoichiometry in the immune complexes formed onto the MZI transducer (with the ratio of ~1.3 for primary antibody to probe, and ~2.5 for secondary to primary antibody), evaluated here with TOF-SIMS more accurately than estimated from sensor response.…”

“…reaching a value of about 1.1 mg/m 2 for C = 200 µg/mL. As discussed in Section 3.2.1 a similar phenomenon was observed for BSA adsorbed on APTES modified substrates[25].…”

“…Therefore, protein desorption initiated by subsequent immobilization of another macromolecule is to some extent expected. Such a behavior has been also observed on silicon surfaces modified with aminosilane at certain protein surface density values [13,25]. evidence of reduced surface density of the OTA-OVA conjugate after the first immunoreaction step due to partial desorption (see Fig.…”

“…A similar analysis has been previously performed on planar substrates imitating the MZI transducer [20], and the new findings are discussed in relation to those. To optimize the biofunctionalization procedure for efficient detection of analyte, the on-chip sensors where the analyte-protein conjugate (probe) has been immobilized from solutions with different concentration were examined following a strategy introduced recently [25]. Although TOF-SIMS provides a valuable information about molecular composition of multi-component layers on surfaces, it is not able to quantify directly the molecules surface density and for this reason it is complemented by ellipsometric analysis of plain surfaces prepared following the same procedure applied for the chips.…”

Controlling orientation, conformation, and biorecognition of proteins on silane monolayers, conjugate polymers, and thermo-responsive polymer brushes: investigations using TOF-SIMS and principal component analysis

“…Silicon surfaces are activated with silane selfassembled monolayers prior to their biofunctionalization [97]. Therefore, surface analysis methods including TOF-SIMS have been applied to examine the antibodies, antigens, and blocking proteins adsorbed and covalently attached to silicon surfaces functionalized with organo-silanes for silicon-based biosensors [34,74,[98][99][100]. In the following section, we discuss the TOF-SIMS examination of the factors affecting antibodies orientation, with focus on the surfaces modified with self-assembled monolayers, in particular the organo-silanes on silicon surfaces.…”