Development of 1480 nm Photothermal High-Speed Real-Time Polymerase Chain Reaction System for Rapid Nucleotide Recognition

Abstract

The polymerase chain reaction (PCR) is a key technology used in genome-based biological analysis; however, requests have been made to shorten the operation time for emergency tests such as medical diagnostics, and countermeasures against bioterrorism. We have developed a novel rapid real-time PCR system using the direct absorption of an IR laser beam by water droplets as the heating device. The advantage of this system is that only the target water droplet was heated photothermally without transmitting any heat to the surroundings, which is important for the production of fast thermal cycle intervals. The system consists of a fluorescent microscope, an oil chamber with a set of water droplets lined up at the bottom, a 1480 nm IR laser unit, which is absorbed by water and can be focused on the droplets on the stage of the microscope, and an image intensifier to quantify the PCR reaction within a water droplet by measuring the change of fluorescent intensity. Using the system, we examined the PCR procedure under the following conditions: initial heating to 95 °C, maintaining this temperature for 10 s, and the suggested here and in similar places throughout 50 cycles of 1 s at 95 °C for denaturation and 3 s at 60 °C for annealing/extension. The temperature increase and decrease between the two temperatures 95 and 60 °C, were within 1 and 0.8 s respectively, i.e., 32 K/s, which is 1.5 times faster than the conventional heat conduction-based system. Rapid PCR amplification was observed successfully by the rise change in the sigmoidal curvature of fluorescent intensity, and the procedure was accomplished within 3.5 min, including the initial heating and complete 50 PCR cycles. The results indicate that the direct absorption-based heating of water droplets photothermally could give us a faster temperature chnage than the conventional heat-conduction-based systems such as Peltier heating/cooling.