Photothermal PCR within 6 min based on pullulan-coated nanoplasmas for ultrafast nucleic acid analysis

Photothermal PCR based on specific nanomaterials has attracted attention due to their efficient photothermal conversion. However, these nanomaterials absorb polymerases, thereby inhibiting the PCR. Owing to their small size and highly active surface atoms, they are prone to aggregation and sedimentation, resulting in uneven heating within the reaction system. In addition, the fluorescent groups used for detection are susceptible to photobleaching under excitation light. Therefore, there is a need for stable photothermal nanomaterials and compatible photothermal PCR detection strategies.

This paper presents a coated-type nanoplasmas-based photothermal PCR method and platform for ultra-fast nucleic acid analysis. Pullulan-coated AuNRs were introduced, and pullulan-AuNRs nanoplasmas were prepared to prevent the aggregation of AuNRs and the adsorption of the polymerase. A photoelectric platform for ultrafast photothermal PCR was built. The photothermal effect of AuNRs was excited by an infrared laser, causing the PCR solution to rapidly heat up. By regulating the on-off cycle of the excitation light source and the fan through temperature feedback, the photothermal conversion of AuNRs was alternately excited, achieving ultra-fast photothermal PCR within 6 min. The CRISPR/Cas detection technology was coupled with the photothermal PCR to detect the amplified products and output fluorescence signals within 4 min, enabling ultra-fast detection of Salmonella DNA as low as 38 copies/reaction.

The pullulan-coated photothermal nanomaterials were introduced, which solved the problems of instability and incompatibility with the PCR system. The one-tube photothermal PCR-CRISPR assay maintains airtight conditions while avoiding interference between the excitation light source and the detection light source. This method achieves ultrafast detection within 10 min in a simple, rapid, accurate, and contamination-free manner.