Rapid quantitative PCR equipment using photothermal conversion of Au nanoparticles

Woongkyu Park¹, Sun-Hee Ahn¹, and Jae Sung Ahn^2,*

¹Medical&Bio Photonics Research Center, Korea Photonics Technology Institute, Gwangju, 61007, Republic of Korea

²Pukyong National University, Pusan, 48513, Republic of Korea

*E-mail address: jsahn@pknu.ac.kr

The worldwide epidemic of infectious diseases has created an urgent need for rapid on-site diagnosis, leading to the development of various molecular diagnostic technologies, such as Loop Mediated Isothermal Amplification (LAMP), microfluidic PCR, and photothermal PCR. Among these methods, photothermal PCR has attracted significant attention due to its ability to deliver fast and precise results by converting absorbed light energy into thermal energy, enabling faster thermal cycling through plasmonic resonance.

We utilized a collimated laser beam (λ = 808 nm) to heat Au nanoshells for photothermal PCR, with temperature monitored by a K-type thermocouple and pyrometer. The maximum optical power of the laser used in the heating process was 2.5 W. Thermal cycling was controlled through the LabVIEW program using the laser, cooling fan, and thermocouple. PCR mixture consisted of 10 μL template λ-DNA, Au nanoshell, 10× Z-Taq Buffer, Takara Z-TaqTM DNA polymerase, dNTP, forward and backward primers, bovine serum albumin, and distilled water. To ensure thermal stability, the surface of the Au nanoshell was coated with silica and the concentration used was 150 μg/mL. Thermal cycling was performed for 40 cycles using a two-step procedure between 55 degrees (annealing and extension) and 96 degrees (denaturation), with PID feedback for each step to maintain the temperature of the PCR sample for 10 seconds. Electrophoretic imaging confirmed the DNA amplification results and showed bands of comparable intensity to the results of PCR experiments performed under the same conditions using benchtop PCR equipment.

For photothermal qPCR experiment, PCR mixture consisted of 10 μL Human gingival fibroblast DNA, Au nanoshell, 20× Taqman probe, 2× Master Mix, bovine serum albumin, and distilled water. Thermal cycling was performed between 55 (for 60 seconds) and 96 degrees (for 15 seconds). Fluorescence signal from the PCR mix was excited by 465 ~ 495 nm light and detected by a CMOS camera followed by a 515 ~ 555 nm bandpass filter. PCR amplification plot was constructed by recording averaged pixel intensity of the fluorescence signal at the end of extension step of each cycle. Even cycle threshold value (Ct) from the photothemal PCR was slightly increased comparing the Ct value from benchtop qPCR equipment, we could verify the DNA amplification result from PCR amplification plot and electrophoretic imaging. Through the integration of the photothermal qPCR technology, the heating and cooling rates were significantly increased to 4 ℃/s, permitting the reduction of the amplification time. As a consequence, the photothermal qPCR might facilitate the execution of point-of-care qPCR.