Conjugation Efficiency of Laser-Based Bioconjugation of Gold Nanoparticles with Nucleic Acids

Ultrashort pulsed laser ablation represents a powerful tool for the generation of pure gold nanoparticles avoiding chemical precursors, reducing agents, and stabilizing ligands. The bare surface of the charged nanoparticles makes them highly available for functionalization and as a result especially interesting for biomedical applications. The functionalization with oligonucleotides is achievable simultaneously (in situ) or after (ex situ) nanoparticle generation by adding the conjugative agents to the ablation media prior or after laser ablation, respectively. In order to obtain information on the conjugation efficiency and resulting surface coverages, we applied two approaches modified from an established fluorescence-based method for the quantification of oligonucleotides after displacement from the surfaces of the gold nanoparticles. Surface saturation values up to 165 pmol cm−2 were observed for the in situ and ex situ laser-based conjugation. The observed conjugation efficiency in terms of the percentage of total oligonucleotides conjugated to the gold nanoparticles is about four times higher for the in situ conjugation. Nanoparticles with well-defined diameters were already obtained performing an in situ conjugation with an oligonucleotide to the nanoparticle ratio of 20. At this minimum required concentration, more than 90% of added oligonucleotides are conjugated to the surfaces of the nanoparticles, and a surface coverage of 29 pmol cm−2, being in the range of reported values in the literature, was observed. In conclusion, the laser-based bioconjugation seems to be an alternative approach to gold nanoparticle bioconjugates with defined surface coverage. Additionally, observed high conjugation efficiencies are especially interesting for precious biomolecules.