Fourier Analysis To Measure Diffusion Coefficients and Resolve Mixtures on a Continuous Electrophoresis Chip

• Published in: Analytical chemistry (Washington) Vol. 79; no. 21; pp. 8222 - 8231
• Main Authors: Estévez-Torres, A, Gosse, C, Le Saux, T, Allemand, J.-F, Croquette, V, Berthoumieux, H, Lemarchand, A, Jullien, L
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 01.11.2007
• Subjects: Acrylamides - chemistry; Analytical chemistry; Chemistry; Chromatographic methods and physical methods associated with chromatography; Diffusion; DNA - analysis; Electric fields; Electrophoresis, Microchip - instrumentation; Electrophoresis, Microchip - methods; Exact sciences and technology; Fluorescein - analysis; Fluorescent Dyes - chemistry; Fourier Analysis; Glass - chemistry; Molecular Weight; Oligonucleotides - analysis; Other chromatographic methods; Reproducibility of Results; Sensitivity and Specificity; Solution chemistry; Spectrometric and optical methods; Rhodamine; Fourier transform spectroscopy; Chemical analysis; Fourier analysis; Fluorescence spectrometry; Binary mixture; Electric field; Sample preparation; Simulation; Efficiency; Fluorescein; Electrophoresis; Oligonucleotide; DNA hybridization; Diffusion coefficient; Diffusion; Xanthene dye
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac070532z

We report a method to measure diffusion coefficients of fluorescent solutes in the 102−106 Da molecular mass range in a glass−PDMS chip. Upon applying a permanent electric field, the solute is introduced through a narrow channel into a wide analysis chamber where it migrates along the injection axis and diffuses in two dimensions. The diffusion coefficient is extracted after 1D Fourier transform of the resulting stationary concentration pattern. Analysis is straightforward, requiring no numerical integration or velocity field simulation. The diffusion coefficients measured for fluorescein, rhodamine green-labeled oligonucleotides, and YOYO-1-stained dsDNA fragments agree with the literature values and with our own fluorescence correlation spectroscopy measurements. As shown for 151 and 1257 base pair dsDNA mixtures, the present method allows us to rely on diffusion to quantitatively characterize the nature and the composition of binary mixtures. In particular, we implement a DNA hybridization assay to illustrate the efficiency of the proposed protocol for library screening.