Use of a Branched Linker for Enhanced Biosensing Properties in IgG Detection from Mixed Chinese Hamster Ovary Cell Cultures

Tris(2-aminoethyl)-amine (TREN), a branched amine, was coupled to planar surfaces of alkanethiol self-assembled monolayers (SAMs) to increase the grafting density of IgG-binding peptide (HWRGWV or HWRGWVG) on gold surfaces. One of the three primary amine pendant groups of TREN anchors onto the SAM, while the other two are available for grafting with the C-termini of the peptide. The ellipsometric peptide density on the SAM-branched amine was 1.24 molecules nm ^-2 . The surfaces carrying the peptides were investigated via surface plasmon resonance (SPR) to quantify the adsorption of IgG and showed maximum binding capacity, Q _m of 4.45 mg m ^-2 , and dissociation constant, K _d of 8.7 × 10 ^-7 M. Real-time dynamic adsorption data was used to determine adsorption rate constants, k _a values, and the values were dependent on IgG concentration. IgG binding from complex mixtures of Chinese hamster ovary supernatant (CHO) was investigated and regeneration studies were carried out. Compared to the unbranched amine-based surfaces, the branched amines increased the overall sensitivity and selectivity for IgG adsorption from complex mixtures. Regeneration of the branched amine-based surfaces was achieved with 0.1 M NaOH, with less than 10% decline in peptide activity after 12 cycles of regeneration-binding.