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 (>10(4)) duty ratio of pulse oscillation. To overcome this problem, we realized a quasi-continuous-wave excitation by modulating the pulse trains at approximately 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-microm 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.
Hiki
Shinichiro
S
Institute of Microchemical Technology, KSP East207, 3-2-1 Sakado, Takatsu, Kawasaki, Kanagawa 213-0012, Japan.
Mawatari
Kazuma
K
Hibara
Akihide
A
Tokeshi
Manabu
M
Kitamori
Takehiko
T
D000225:Adenine / Q000032:analysis*; D005449:Fluorenes / Q000032:analysis*; D007834:Lasers; D007909:Lenses; D046208:Microchip Analytical Procedures; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods*; D011721:Pyrenes / Q000032:analysis*; D012680:Sensitivity and Specificity; D012822:Silicon Dioxide / Q000737:chemistry*; D013696:Temperature; D013997:Time Factors; D014466:Ultraviolet Rays
D005449:Fluorenes; D011721:Pyrenes; C041509:fluorene; D012822:Silicon Dioxide; C030984:pyrene; D000225:Adenine