UV excitation thermal lens microscope for sensitive and nonlabeled detection of nonfluorescent molecules

An ultrasensitive and nonlabeled detection method of nonfluorescent molecules on a microchip was developed by realizing a thermal lens microscope (TLM) with a 266-nm UV pulsed laser as an excitation light source (UV-TLM). Pulsed laser sources have advantages over continuous-wave laser sources in more compact size and better wavelength tuning, which are important for microchip-based analytical systems. Their disadvantage is difficulty in applying a lock-in amplifier due to the high (>10⁴) duty ratio of pulse oscillation. To overcome this problem, we realized a quasi-continuous-wave excitation by modulating the pulse trains at ∼1 kHz and detecting the synchronous signal with a lock-in amplifier. The optimum pulse repetition frequency was obtained at 80 kHz, which was reasonable considering thermal equilibrium time. Furthermore, a permissible flow velocity in the range of 6.6-19.8 mm/s was found to avoid sensitivity decrease due to photochemical reactions and thermal energy dissipation. Under these conditions, we detected adenine aqueous solutions on a fused-silica microchip without labeling and obtained a sensitivity that was 350 times higher than that in a spectrophotometric method. The sensitivity was enough for detection on a microchip with an optical path length that was 2-3 orders shorter than that in conventional cuvettes. Finally, the UV-TLM method was applied to liquid chromatography detection. Fluorene and pyrene were separated in a microcolumn and detected in a capillary (50-μm inner diameter) with 150 times higher sensitivity than a spectrophotometric method. Our method provides highly sensitive and widely applicable detections for various analytical procedures and chemical syntheses on microchips.