Determination of surface concentrations of individual molecule-layers used in nanoscale biosensors by in situATR-FTIR spectroscopy

Abstract

For the development of nanowire sensors for chemical and medical detection purposes, the optimal functionalization of the surface is a mandatory component. Quantitative ATR-FTIR spectroscopy was used in situ to investigate the step-by-step layer formation of typical functionalization protocols and to determine the respective molecule surface concentrations. BSA, anti-TNF-α and anti-PSA antibodies were bound via 3-(trimethoxy)butylsilyl aldehyde linkers to silicon-oxide surfaces in order to investigate surface functionalization of nanowires. Maximum determined surface concentrations were 7.17 × 10⁻¹³ mol cm⁻² for BSA, 1.7 × 10⁻¹³ mol cm⁻² for anti-TNF-α antibody, 6.1 × 10⁻¹³ mol cm⁻² for anti-PSA antibody, 3.88 × 10⁻¹³ mol cm⁻² for TNF-α and 7.0 × 10⁻¹³ mol cm⁻² for PSA. Furthermore we performed antibody–antigen binding experiments and determined the specific binding ratios. The maximum possible ratio of 2 was obtained at bulk concentrations of the antigen in the μg ml⁻¹ range for TNF-α and PSA.