Ultra-high multiplex PCR uniformity is enhanced by carbon nanotube-dendrimer-encapsulated gold nanoparticles

Abstract

Polymerase chain reaction (PCR) remains the gold standard in molecular diagnostics. However, under highly multiplexed conditions, PCR often suffers from poor amplification uniformity, which limits its high-throughput potential and clinical applicability. To address this limitation, we developed a novel nanocomposite—carbon nanotube-dendrimer-encapsulated gold nanoparticles (CNT-G3-Au), synthesized by covalently conjugating third-generation dendrimer-coated gold nanoparticles to carboxylated carbon nanotubes (CNT–COOH) via EDC·HCl/NHS coupling chemistry. The resulting point–line–surface nanostructure integrates the high thermal conductivity of CNT–COOH and gold nanoparticles with the electrostatic DNA enrichment capability of dendrimer assemblies, enabling efficient heat transfer and electrostatic enrichment of DNA during amplification. When applied to 120-plex ultra-high multiplex PCR and 40-plex methylation-specific PCR systems, CNT-G3-Au significantly enhanced amplification uniformity. Quantitative polymerase chain reaction analysis revealed cycle threshold variation of less than 4 among amplicons, while high-throughput sequencing revealed a read uniformity of 83.6%, along with increased mapping rates and improved genomic coverage depth. Mechanistic studies, including zeta potential analysis, thermal conductivity testing and finite element modeling, confirmed the synergistic effects of thermal conduction (1.503 W m⁻¹ K⁻¹) and electrostatic enrichment. Furthermore, CNT-G3-Au exhibited excellent specificity, structural stability and compatibility under highly multiplexed conditions. These results highlight CNT-G3-Au as a promising and broadly applicable PCR enhancer, offering a new strategy for improving amplification performance in complex systems and enabling more robust and accurate nucleic acid detection in both clinical and research settings.