Nanofluidic Devices for Single Molecule Spectroscopy
Benjamin R. Cipriany
Micrometer and sub-micrometer fluidic devices are used in a variety of applications for analytical biochemistry. Previous work describes the benefits of performing single molecule spectroscopy in small engineered focal volumes with regard to uniform illumination intensity and controllable analyte flow . The goal of this project is to further reduce the engineered focal volume with sub-100nm fluidic channel dimensions. Reduction of the focal volume will reduce the probability of multiple molecule occupancy and could lead to spectroscopy with higher accuracy. Biophysical interaction at the channel-fluid interface becomes increasingly dominant at smaller dimensions and therefore warrants comparison of the practical benefits of each design .
A two-step lithographic process was used to create these nanofluidic devices. Electron beam lithography was used to pattern the nanofluidic channel region and an anisotropic reactive ion etch of the fused silica formed channels of dimension 100nm by 45nm, as shown in Figure 1 and 2. A photolithography step defined the reservoirs and analyte supply channels to the nanofluidic channel region. Channels were sealed in a direct bonding process using a fused silica cover wafer, to reduce autofluorescence and allow standard coverslip-corrected microscope objectives to be used.
To provide a comparison with nanofluidic devices, submicrometer fluidic channels were created with a similar process involving a single photolithograpy step. Submicrometer channels were patterned with projection photolithography and then created with an anistroptic reactive ion etch resulting in channel dimensions of 500nm by 250nm, as shown in Figure 3. Direct bonding with a fused silica cover wafer is used again .
Fluidic channels have been used in experiments involving fluorescent dye labeled DNA and proteins. In particular, a relatively-new molecular engineered probe, the molecular beacon , is under investigation for use with fluidic channels in single molecule spectroscopy.
Submicrometer (500nm x 250nm) and nanometer (100nm x 25nm) scale fluidic channels were fabricated in fused silica to conduct single molecule spectroscopy. A nanometer-scale engineered focal volume permits micromolar range analyte sample concentrations, while increased biophysical interactions at the channel-fluid interface warrant comparative study with submicrometer channels.
Investigation was conducted using molecular beacons in fluidic channels to provide more robust single molecule spectroscopy, with possibilities for the analysis of heterogeneous analyte solutions. The highly sequence-specific nature of molecular beacons, combined with rapid fluidic channel analysis, presents new possible opportunities for early detection of disease.
Figure 1: Optical micrograph of electron beam lithography fabricated nanofluidic channels.
Figure 2: Atomic force microscope image of a single nanofluidic channel profile (approximately 100nm wide x 45 nm deep).
Figure 3: Optical micrograph of an array of submicrometer fluidic channels. Spectroscopy is
performed in the narrow region (500nm wide x 250nm deep) of each channel near the numbers.
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