DNA Linkers and Diluents for Ultrastable Gold Nanoparticle Bioconjugates in Multiplexed Assay Development

Abstract

A novel bioconjugation strategy leading to ultrastable gold nanoparticles (AuNPs), utilizing DNA linkers and diluents in place of traditional self-assembled monolayers, is reported. The protective capacity of DNA confers straightforward biomolecular attachment and multistep derivatization capabilities to these nanoparticles and, more significantly, substantially enhances their stability in demanding and complex sensing environments. The DNA/AuNPs were assembled through pH-assisted thiol-gold bonding of single stranded DNA and salt aging, with preconjugated biotin moieties facing outward from the gold surface. These nanoparticles remain a stable colloidal suspension under a wide range of buffers and ionic strengths and can endure multiple rounds of lyophilization while retaining high biological activity. Furthermore, the high stability of the DNA/AuNPs allows for multiple reactions and conjugations to be performed within the colloidal suspensions (i.e., Protein A and antibody binding) for tailored and specific recognition to take place. We have demonstrated the applications of the DNA/AuNPs for colorimetric assays and ELISA feasibility; additionally, SPR imaging analysis of a supported membrane microarray shows excellent results with DNA/AuNPs as the enhancing agent. Together, the properties imparted by this interface render the material suitable for clinical and point-of-care applications where stability, throughput, and extended shelf lives are needed.

Synthesis of Ultrastable Gold Nanoparticles as a New Drug Delivery System

“…In recent decades, intensive research has been undertaken to develop new synthesis leading to ultrastable GNP. An exhaustive review of highly-stable and ultrastable GNPs led to only a dozen publications describing new synthesis conditions and resistance against different criteria [11,12,13,14,15,16,17,18,19,20,21,22]. However, most of the time, GNPs presented ultrastable properties only under limited conditions and were not water-soluble.…”

“…For instance, numerous GNPs have been reported to be stable for more than six months at room temperature without any alteration [18,23], but this feature should not be sufficient to define them as ultrastable [12,16,20]. The notion of time must be taken into account and the stability kinetics explained when the aggregation is observed after a few minutes following salt exposure [11,13,16,17,19,20]. Nonetheless, some studies have suggested a degree of stability against freeze drying, salts resistance [11,13,15,16,17] and/or autoclaving [24].…”

Plasmonic Sensing with 3D Printed Optics

“…Binding patterns for cholera toxin are strong and specific, with signal intensities comparable to established SPR sensors in the literature. 25,26 …”

Advancements in DNA-assisted Immunosensors

“…Cheng's group developed a very stable conjugate using DNA as a linker on the surface of gold nanoparticles, applied to the sandwich assay to detect cholera toxin (Figure 7a) 46 . In the proposed method, DNA linker on the surface of gold nanoparticles replaced the alkanethiol/PEG linker used in the synthesis of gold nanoparticle conjugates, which helps the conjugate be stable in a complex sensing environment, improving the detected signal.…”