Focal Volume Confinement by Submicrometer-Sized Fluidic Channels

Mathieu Foquet, Jonas Korlach, Warren Zipfel, Watt Webb, H.G.Craighead

Applied and Engineering Physics, Cornell University, Ithaca, NY

Microfluidic channels (Figure 1) with two lateral dimensions smaller than 1 m are fabricated in fused silica for high sensitivity single molecule detection and Fluorescence Correlation Spectroscopy (FCS) (Figure 2). The channels are fabricated using the Sacrificial Layer Technique (Figure 3). The effective observation volumes (Figure 4) created by these channels are about 100 times smaller than observation volumes (Figure 5) using conventional confocal optics and thus enable single fluorophore detection at higher concentrations. Increased signal-to-noise ratios are also attained because the molecules are restricted to diffuse through the central regions of the excitation volume, where the laser intensity is maximal. Depending on the channel geometries, the effective dimensionality of diffusion (Figure 6) is reduced which is taken into account by simple solutions to diffusion models with boundaries. A direct electrokinetic flow can be established in the capillaries. The flow speed (Figure 7) can be characterized using Fluorescence correlation spectroscopy as well. An extensive study of the effect of the alignment of the channels (Figure 8) with the fluorescence optics is done. Finally, single fluorophore detection (Figure 9) was realized in the channels, under various flow conditions. The number of photon (Figure 10) collected in function of both the laser illumination intensity and the flow condition is plotted and analyzed. Such channels are ideal for high concentration FCS, detection of rare species such as dimmers with a low association rate, and other single molecule techniques.

Publication Reference:

M. Foquet, J. Korlach, W. Zipfel, W. Webb, H. G. Craighead, "Focal Volume Confinement by Submicrometer-Sized Nanofabricated Channels", Analytical Chemistry, In Press (2004).

Figure 1: Pictures of Microcapillaries fabricated by the sacrificial layer process. (A) Top view showing a wide channel narrowing in to a 1 micrometer wide channel and several of the side irrigation channels. (B) Cross section of a 250 nm tall microchannel. (C) Top view in an optical microscope of an microchannel.

Figure 2: Explanatory slide of the underlying principles to fluorescence correlation spectroscopy. It relies on the measurement of the correlation function of the fluorescence signal, whose expression is given at the top. A typical intensity curve and autocorrelation curve are shown under this definition. Among the most important parameters one can quickly identify from a correlation curve is the value at its origin, inversely proportional to the number of molecules in the focal volume and the time window of the fluctuation of fluorescence intensity.

Figure 3: The different steps of the sacrificial layer process. Step 1: Deposition of the sacrificial material. Step2: Patterning of the sacrificial layer using either photolithography or electron beam lithography. Step 3: Covering of the sacrificial layer by the capillary wall material. Step 4: Opening of irrigation holes at regular intervals on the devides, these wholes will give access to the sacrificial layer material. Step 5: Wet etching of the sacrificial material, leaving behind the cavity defining the microfluidics system. Step 6: Deposition of additional wall material to close the irrigation holes. Step 7: Opening of access ports at the extremeties of the device.

Figure 4: Example of a Correlation curve in bulk solution (dark blue) and of a correlation curve in a nanocapillary (cyan). Even though the concentration in bulk is a hundred fold lower than in the channel, the abscise at origin of the correlation function is lower, indicated a greater number of molecule in the focal volume. Hence the channels have brought about a 100-fold improvement in the concentrations possibly studied.

Figure 5: The relevance to pursue single molecule detection at concentration higher than those conventionally achievable using confocal optics is illustrated here by the wide gap between the existing technologies and the concentrations of interest to biologist. One possible way to realize these goals is the use of micro and nanostructures..

Figure 6: The effect of dimensionality on the observed shape of correlation curve. The experimental curve is shown in dark gray, the theoretical curve is shown in orange. (A) Correlation curve in an open confocal volume and the corresponding model. (B) Correlation curve in a small section of a conventional confocal volume and the corresponding model. If the slice taken is thin enough, one can see that a simple two dimension model is sufficient. This requires to remove one of the terms of the conventional theoretical form. (C) An example of a correlation in a thin and narrow channel. Here, not only are two dimensions restricted, but because of the relatively closeness of the laser focal spot and the channel width, a corrective function g^*(t) must be introduced.

Figure 7: Fluorescence correlation spectroscopy curves in a 0.5 micrometer wide by 0.25 tall channel, under various electrokinetic flow conditions. As the flow speed is increased, the residency time of the molecules is shortened. These curves can be fitted to a mixed diffusion-flow model of FCS.

Figure 8: Study of the effect of alignment of the capillary with the observed Signal and correlation. (A) Study of the z-focus effect on a flat and wide channel. The parameter w_x is an estimate of the width of the laser beam spot deduced from the correlation curve. (B) A similar study on a narrow channel. (C) a study of the effect of lateral alignment on the direct intensity of the signal. One notices that the position needs to be controlled within only a couple hundred nanometers.

Figure 9: Measured signal intensity indicating the passage of single molecules in the focal volume. Each data point is the number of photons detected in a 5 microseconds time window. (A) pure diffusion, no flow applied. (B) Flow corresponding to a field of 7.5kV/m. (C) Flow at 75 kV/m. (D) Flow at 750 kV/M

Figure 10: Plot of the number of detected photons per molecule in function of both the flow speed and the laser intensity. As expected, the number of photons is initially stationary with the flow speed (speed lower than 10kV/m), indicating the number of photons detected is diffusion limited. It then starts decreasing linearly with increasing speed as the systems enters the flow dominated regime. The number of photons increases with increasing laser power, but the systems starts saturating at power above 100 microWatt.