Hiki Shinichiro, Mawatari Kazuma, Hibara Akihide, Tokeshi Manabu, Kitamori Takehiko
Institute of Microchemical Technology, KSP East207, 3-2-1 Sakado, Takatsu, Kawasaki, Kanagawa 213-0012, Japan.
Anal Chem. 2006 Apr 15;78(8):2859-63. doi: 10.1021/ac051967u.
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.
通过利用266纳米紫外脉冲激光作为激发光源实现热透镜显微镜（UV-TLM），开发了一种用于在微芯片上超灵敏且无标记检测非荧光分子的方法。脉冲激光源相较于连续波激光源具有尺寸更紧凑和波长调谐更好的优势，这对于基于微芯片的分析系统很重要。其缺点是由于脉冲振荡的占空比高（>10⁴），难以应用锁相放大器。为克服此问题，我们通过以约1千赫兹调制脉冲序列并使用锁相放大器检测同步信号来实现准连续波激发。在80千赫兹获得了最佳脉冲重复频率，考虑到热平衡时间这是合理的。此外，发现允许流速在6.6 - 19.8毫米/秒范围内可避免由于光化学反应和热能耗散导致的灵敏度降低。在这些条件下，我们在熔融石英微芯片上检测腺嘌呤水溶液而无需标记，获得的灵敏度比分光光度法高350倍。该灵敏度足以在光程长度比传统比色皿短2 - 3个数量级的微芯片上进行检测。最后，UV-TLM方法应用于液相色谱检测。芴和芘在微柱中分离，并在内径50微米的毛细管中检测，灵敏度比分光光度法高150倍。我们的方法为微芯片上的各种分析程序和化学合成提供了高灵敏度且广泛适用的检测。
