Microwave-Assisted Synthesis of PNA@AuNRs with Temperature Gating for Precise Amplification of Nucleic Acids

Nucleic acid amplification tests are widely used for molecular diagnostics. However, primer-dimer formation and nonspecific amplification may compromise analytical accuracy and increase the risk of false-positive results. Here, temperature-gated PNA@AuNRs (peptide nucleic acid-modified gold nanorods) were introduced to improve amplification specificity. A microwave-assisted modification method efficiently immobilized PNA onto AuNRs, enabling directional primer capture at low temperature and controlled release near polymerase-optimal conditions. PNA@AuNRs-LAMP achieved a limit of quantification (LOQ) of 10² copies/μL with strong linearity (<i>R</i>² = 0.991), while PNA@AuNRs-qPCR reached 10 copies/μL with excellent linearity (<i>R</i>² = 0.998). Mechanistic analysis demonstrated strong PNA-primer affinity and a distinct thermal-start effect at 60 °C, absent in traditional LAMP. In spiked equine serum and nasal samples, PNA@AuNRs effectively eliminated nonspecific amplification and improved detection accuracy. These results highlight PNA@AuNRs-gated amplification as a promising strategy for high-specificity molecular diagnostics.