Focal Volume Confinement by Submicrometer-Sized Fluidic Channels

Abstract

Microfluidic channels with two lateral dimensions smaller than 1 microm were fabricated in fused silica for high-sensitivity single-molecule detection and fluorescence correlation spectroscopy. The effective observation volumes created by these channels are approximately 100 times smaller than observation volumes 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. Depending on the channel geometries, the effective dimensionality of diffusion is reduced, which is taken into account by simple solutions to diffusion models with boundaries. Driven by electrokinetic forces, analytes could be flowed rapidly through the observation volume, drastically increasing the rate of detection events and reducing data acquisition times. The statistical accuracy of single-molecule characterization is improved because all molecules are counted and contribute to the analysis. Velocities as high as 0.1 m/s were reached, corresponding to average molecular residence times in the observation volume as short as 10 micros. Applications of these nanofabricated devices for high-throughput, single-molecule detection in drug screening and genomic analysis are discussed.

Nanofluidic structures for single biomolecule fluorescent detection

“…Submicrometer-sized fluidic channels allow for this balance while maintaining focal volume restriction. 28 Such channels can be fabricated on fused silica wafers using metal oxide semiconductor compatible processes, allowing for manufacturing scalability and permitting visible wavelength spectroscopy with low autofluorescence as compared to borosilicate or pyrex glass. A focused laser beam aligned to the channel results in an effective probe size with a length defined by the diffraction limit, but a width and height defined by the channel walls ( Figure 4).…”

Fluorescence Fluctuation Spectroscopy in Subdiffraction Focal Volumes

“…Diffraction has, indeed, imposed a maximum molecular concentration that can be handled by far-field optical FFS. All efforts to reduce the detection volume any further implied the use of evanescent fields and mechanical constraints such as near-field optical tips [7], interfaces providing total internal reflection [8,9], and waveguide structures [10,11]. However, mechanical confinement hampers noninvasive intracellular investigations, and the interaction with the probe is likely to affect the dynamics to be probed.…”

Design of a confocal microfluidic particle sorter using fluorescent photon burst detection

“…Furthermore, the twodimensional particle focusing mechanism that is applied successfully for m-sized beads is not working for these phages due to their small size. Possible solutions could be found in reduction of the channel dimensions, 11,12 rapid scanning of the laser beam 35,36 or by using waveguides. 37,38 At present, the speed of detection and sorting is too low for practical use.…”

“…Single molecules have been detected by means of fluorescence in relatively large capillaries, 4-6 micrometer 7-10 and even submicrometer sized channels. [11][12][13][14][15] Previous work in our laboratories reported on the detection of flowing fluorescent particles like bacteria and microspheres in a microcapillary mounted on a confocal fluorescence microscopy setup. 16 These results led to the design of a microfluidic biochip aimed at detection and sorting of relatively small particles in real time using the same confocal fluorescence microscope.…”