Design of Bi-functional Bioconjugated Gold Nanoparticles by Pulsed Laser Ablation with Minimized Degradation

Femtosecond-pulsed laser ablation of gold is investigated in electron donor moiety containing biomolecule solutions, resulting in the controlled design of biocompatible, partially charged and electrostatic-stabilized gold nanoconjugates by in situ surface functionalization. The cell-penetrating peptide penetratin and a targeting miniStrep aptamer sequence are conjugated simultaneously to a single nanoparticle for the first time, producing highly stable gold nanoparticle bi-conjugates with defined degrees of surface coverages for each biomolecule. Ablating gold by 100 J laser pulses in presence of 1 M penetratin and 4.5 M miniStrep yields an average bioconjugate size of 10 nm and results in 61 pmol cm -2 surface coverage for the aptamer and 30 pmol cm -2 for the penetratin. Whereat, degree of surface coverage shows direct correlation to process parameters as well as to biomolecule size, concentration and steric dimension of the biomolecules used. To investigate the influence of time-delayed bioconjugation in view of more efficient up-scaling process, a fast ex situ conjugation is presented in a continuous flow chamber secondary, allowing highly controlled bioconjugation without irradiating the photo-and thermal-sensitive biomolecules and permitting the use of nanosecond laser pulses.