Abstract
We developed a simple procedure for the complete regeneration of silane-grafted gold surface. This method allowed the reuse of the regenerated gold chip for custom refunctionalization. In addition, we performed highly reproducible immunoassays using these chips over several cycles. The developed procedure was optimized and comprised of consecutive treatments of functionalized Au chip with 12 M HCl for 10 min and 29 W oxygen (O2)- plasma for 5 min. Monitoring and surface characterization of the developed methodology was performed with ellipsometry and Rutherford back scattering. The developed procedure was demonstrated on SPR-based Human Fetuin A (HFA) immunoassay, where the amino groups of APTES-functionalized Au chip were cross linked to the carboxyl groups of anti-HFA antibody using 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride and sulfo- N-hydroxy succinimide. The APTES-functionalization, anti-HFA antibody immobilization and HFA binding on the regenerated SPR Au chip were highly reproducible over 40 HFA immunoassay cycles.
Highlights
Regeneration of the surface immobilized with recognition biomolecules, such as antibody, after immunoassay is very important for expensive bioanalytical platforms such as Surface Plasmon Resonance (SPR), quartz crystal microbalance and micro-cantilevers
It was observed that strong acid treatment such as hydrochloric acid (HCl) can dissolve the siloxane bonds and remove upto the second aminopropyl triethoxysilane (APTES) layer as the first APTES layer on the Au surface was very strongly bound through Au-thiol interactions
During 40 consecutive Human Fetuin A (HFA) immunoassays, the areal density of APTES was highly-reproducible, which demonstrates that the developed regeneration procedure does not affect the surface properties of the Au layer.The developed Au surface regeneration procedure was optimized by determining the appropriate strength and treatment time of HCl and O2 plasma
Summary
Regeneration of the surface immobilized with recognition biomolecules, such as antibody, after immunoassay is very important for expensive bioanalytical platforms such as Surface Plasmon Resonance (SPR), quartz crystal microbalance and micro-cantilevers. The increase in cost-effectiveness of SPR Au chip was determined by highly-reproducible silanization, anti-HFA antibody binding, and HFA detection in 40 consecutive immunoassay cycles on the same SPR chip after regeneration. The thickness of APTES self-assembled monolayer (SAM) and the areal density of APTES on the bare, silanized and regenerated Au chip surfaces were determined by ellipsometry and RBS, respectively Figure 2.
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