Ultrasensitive detection of nonfluorescent molecules in microspace

Increasingly, tiny chemical laboratories are finding their way into analytical applications. Small device size and reduced sample and reagent volumes have dramatically reduced the time required for analysis. Moreover, integration of chemical procedures on microchips has enabled novel functions. Chip-based processes such as mixing, reaction, separation, and detection are advancing rapidly. Practical applications for analytical systems include clinical diagnosis, environmental monitoring, food quality control, and bioanalysis. These systems are also very promising for high-throughput production of medicines and fine chemicals. Yet, to date, there exist no sufficiently-sensitive and universal detection methods suited to the vastly constrained space and miniscule volumes of these applications. Additional specialty functions also await development, including detection of optically active compounds (important in drug discovery), individual molecule counting, imaging, and in situ flow velocity sensing.

One method popular with researchers is fluorescence detection following light absorption by target molecules. This method currently represents the most sensitive of the optical approaches, and under the right conditions, even individual molecules can be detected. But fluorescence detection is limited because most molecules release light energy as heat to the surrounding medium. Thus a method of detecting heat at the microscale would be a welcome improvement.

For the first time, our group has developed a coaxial thermal lens microscope (TLM).¹ TLM is a kind of absorption photometry based on photothermal phenomena. The basic principle is illustrated in Figure 1. The TLM consists of a laser beam, which is tuned to the absorption wavelength of a sample, used for excitation, and a probe laser beam used for detection. These two beams are coaxially introduced to an objective lens and focused on the sample. The sample absorbs the excitation beam, and the light energy is converted to heat. The intensity distribution of the excitation beam shows a Gaussian profile, and the resultant temperature distribution is likewise proportional to a Gaussian distribution. The rise in temperature decreases the refractive index of the solvent. Consequently, the refractive index distribution works as a concave lens. We call this the thermal lens (TL) effect. When the probe beam is focused at a longer distance than the excitation beam, the TL acts as a converging lens. We can detect the TL effect by measuring the power transmitted by the probe beam through a pinhole. The change in transmittance is proportional to the degree of thermal lensing, which is proportional to the concentration of the sample. Thus, the TLM can detect the concentration via the TL effect in a small space. The most important element in achieving sensitive TL detection is the focus difference (Δf) between the excitation and probe beams. Our original microscope was quite old (made half a century ago) and had adequate chromatic aberration (∼2μm), which just happened to enable sensitive TL detection.

So far, we have demonstrated that the TLM can sense single-molecule concentrations in detection volumes of approximatelya femtoliter (1fl=1μm³).¹ The excitation wavelength currently rangesfrom the deep-UV to the near-IR thanks to a specialized optical design for UV laser beams.² In addition to the concentration determination function, the TLM can sensitively and selectively detect optical activity of molecules by phase modulation of the excitation beam.³ The sensitivity is a million orders higher than that of the best conventional detector (the circular dichroism spectrometer). The TLM can be also be used to determine in situ flow velocity by tuning the wavelength of the excitation beam to the IR(1480nm) region and detecting the light absorption of downstream water molecules via the TL effect. A small rod lens and optical fiber technology have reduced the size of the TLM so that it now fits in the palm of the hand.⁴

In summary, we have developed a functional TLM for analysis at the microscale. Its high sensitivity and broad applicability make this instrument suitable for a number of microchip-based analytical and chemical-synthesis systems. Additional capabilities are being developed to enable rapid imaging, individual molecule detection, and time-resolved measurements. TLMs can be expected to become powerful tools for both basic science and practical applications using micro- and nano-chip technologies.