A Novel DNA-Platinum Nanoparticle Conjugation Method for Attomolar Detection and Quantitation of Nucleic Acids Using the Microbubbling Digital Assay

Platinum nanoparticles (PtNPs) hold promise for developing novel point-of-care diagnostic tools. This study focuses on the synthesis of single-stranded DNA-platinum nanoparticle (DNA-PtNP) conjugates used as detection probes for nucleic acids in an ultrasensitive and amplification-free microbubbling digital assay. Although significant research exists for synthesizing DNA-gold nanoparticle (AuNP) conjugates, methods for synthesizing stable nucleic acid-PtNP conjugates, especially for larger PtNPs (>50 nm), remain unreported. The instability and slow ligand exchange rate of PtNP colloidal solutions make this synthesis challenging, as conventional methods such as salt aging cause PtNP aggregation and require prolonged durations. This study evaluates the effect of factors such as incubation time, DNA length, salt concentration, pH, and surfactants on DNA loading and hybridization on PtNPs. These findings led to a novel DNA-PtNP conjugation method featuring a 30 min pH-mediated conjugation followed by a 30 min freezing at −20 °C. This conjugation approach is rapid, efficient, and sonication-free, resulting in high DNA loading and hybridization efficiency and stable conjugates. Using these conjugates in the microbubbling digital assay enables the assay to detect and quantify nucleic acids in the attomolar range. Using HCV RNA as an example, the limit of detection of the microbubbling digital assay was 0.68 aM (408 copies/ml or 68 IU/ml). Our findings can be extended to other bio/nano systems, using oligonucleotide-loaded platinum nanoparticles to develop novel molecular diagnostics.