Oriented Immobilization of Antibodies through Recombinant Protein-G on Assembled Gold Nanorods for Label-Free Biosensing Applications

Aguero Villarreal, Victor Efrain
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Conjugation of biomolecules on gold nanorod(GNR) surfaces is the basis for successful biosensing applications. Current functionalization methods are problematic as they are inefficient, they modify the antibody native structure and they do not orient the antibody on the sensor surface. Instead of the intensive surface modification of GNRs and random immobilization approach, a facile oriented immobilization method using recombinant protein-G(RPG) to functionalize any type of antibody on the GNR surface was developed.

GNRs synthesized using the bi-surfactant seed-mediated growth method were covalently immobilized to the glass substrate using 10% MPTMS solution. A 21 x 18 x 9.7 mm PLA disposable custom-made chamber was implemented to serve as incubation site for all biomolecular reactions. A layer-by-layer approach was utilized to deposit the protein layers on the GNR surface. Anti-human IgG and anti-rabbit IgG were successfully oriented and immobilized on the GNR surface using a combination of thiol-PEG-biotin, streptavidin and biotinylated RPG molecules. The spectral response of the RPG biochip to 600 nM anti-human IgG was 12 times larger than covalent-modified biosensors. Fluorescence microscopy and fluorescence intensity confirmed antibody binding on the GNR surface. The RPG biochip increased the surface mass density of antibody by ~80% in comparison to random immobilized techniques. The RPG biochip decreased the development cost by 307% with a net sensor worth of $3.21 dollars. Concentrations from 20 to 320 nM of human IgG were detected and the sensor surface was successfully regenerated. Developed immobilization technique clearly surpassed the antibody efficiency deposition of random methods.

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Antibody, Biosensing, Gold Nanorod, Label free, Oriented Immobilization, Protein G
Biomedical Engineering