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 (R2 = 0.991), while PNA@AuNRs-qPCR reached 10 copies/μL with excellent linearity (R2 = 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.