Biomedical subjects

Tsuyoshi Sonehara

Publications and source records attributed to Tsuyoshi Sonehara.

5 recordsLinked to original sources

PubMed2006

Improvement of biomolecule quantification precision and use of a single-element aspheric objective lens in fluorescence correlation spectroscopy.

We found a way to increase the precision with which biomolecules present at concentrations below 10(-10) M can be quantified by fluorescence correlation spectroscopy (FCS). The effectiveness of the way was demonstrated experimentally by using a single-element aspheric objective lens, which was newly developed to reduce the cost of FCS instruments. In the first part of this paper, the relative standard deviation (RSD) of FCS-based concentration measurements is estimated theoretically by an analytical approximation assuming the detection volume profiles in FCS setups to be Gaussian and by molecular simulations in which more realistic profiles are calculated from physical parameters of the measurement setups. In a limit of infinitely bright molecules and zero background emission, the analytical approximation predicts that the RSD at a concentration is minimized when the mean number of molecules in a detection volume is approximately 0.5. A detection volume of the order of 10(-13) L thus gives smaller RSD values for concentrations from 10(-11) to 10(-10) M than does one of the order of 10(-15) L, which is widely used in FCS. This prediction is supported by the molecular simulations, taking into account the finite molecule brightness and background emission. In the second part of the paper, the RSD is evaluated experimentally with an FCS setup with a detection volume of 1.1 x 10(-13) L. The newly developed objective lens, serving as the bottom of the sample cell in this setup, has a large numerical aperture (0.9) without using immersion liquid. When a calibration line was made by 30-s FCS measurements of Cy3-labeled, 112-mer single-stranded DNA solutions, the RSD roughly agreed with the simulation result and was less than 0.1 for DNA concentrations from 2 x 10(-11) to 10(-10) M.

Algorithms↗

PubMed2006

Ultra-slim laminated capillary array for high-speed DNA separation.

We developed a new kind of capillary array for electrophoresis by using the numerical-control (NC) wiring technique conventionally used to produce printed-circuit boards. Laminating two polyimide sheets after laying cylindrical capillaries between them according to designed geometries, we fabricated a 16-lane laminated capillary array (LCA) 9.9 cm long, 7.2 cm wide, and 0.5 mm thick in which the effective length of all capillaries was only 10.9 cm. This compact LCA thus had separation columns as short as those in capillary array electrophoresis chips fabricated by lithography techniques. Like conventional capillary arrays, it also enabled pipetting-less direct injection of analytes from sample preparation plates. Using the LCA with LIF detection and a replaceable fluid sieving matrix, we demonstrated high-speed ssDNA fragment separations. At an electric field strength of 316 V/cm, 15 fragments ranging from 50 to 500 bases were completely separated within 5.8 min in all lanes. The lane-to-lane CV of migration time was only 0.38%, and the fragment size for which the resolution per base was 0.59 was 258 +/- 15 bases (average +/-SD).

DNA, Single-Stranded↗

PubMed2004

High resolution for single-strand conformation polymorphism analysis by capillary electrophoresis.

Since the successful completion of the Human Genome Project, increasing concern is being directed toward the polymorphic aspect of the genome and its clinical relevance. A form of single-strand DNA-conformation polymorphism analysis (SSCP) employing nondenaturing slab-gel electrophoresis (SGE) is applicable to the genetic diagnosis of bladder cancer from urine samples. To bring this technique into routine clinical practice, the use of capillary electrophoresis (CE) is naturally favorable in terms of speed and automation. However, the resolving power of SSCP, a prerequisite basis for reliability required in diagnostics, remains as a challenge for CE systems. We thus focused on this topic and conducted studies on CE instruments equipped with a single capillary or an array of multiple capillaries, using the resolution (Rs) as a quantitative scale for the resolving power. Polymer concentration and buffer are shown to be the decisive parameters. High Rs values of >2.5 are achieved for representative SNPs markers under the optimized conditions, without sacrificing such intrinsic advantages of CE over SGE as the 10-fold quicker migration time and operation that is reproducible, continuous, and automatic. The strategies presented broaden the limits of CE in both the current and related applications.

Buffers↗

PubMed2004

High-speed DNA sequencing by tube-based capillary electrophoresis.

We assessed the feasibility of high-speed DNA sequencing by tube-based capillary electrophoresis (TCE) with electrokinetic sample injections. We developed a water-circulated TCE system to control the capillary temperature precisely. Using this system and a ready-made sieving matrix at 50 degrees C, single-stranded DNA size marker fragments were separated at various pairs of the electric field strength, E, of 128-480 V/cm and the capillary effective length, L, of 100-360 mm. Assuming the read length (RL) is the fragment size at which the peak width equals the peak interval per base in obtained electropherograms, we estimated the values of RL (E, L), the RL at the pair (E, L). The points in ELz-space, (E, L, RL(E, L)), form a curved surface expressed by z = RL(E, L). Analyzing the contour lines of this curved surface, we determined the pairs of E and L providing target RLs of 300-500 bases within a minimum time. At a pair optimized for a 500-base RL (330 V/cm, 200 mm), one-color sequencing fragments were successfully separated up to 529 bases within 9.6 min. These results demonstrate that high-speed DNA sequencing comparable with that obtained by microfabricated chip-based capillary electrophoresis (MCE) can be achieved with TCE, which is more suitable in automation than MCE.

Electrophoresis, Capillary↗

PubMed2002

Fluorescence correlation spectroscopy excited with a stationary interference pattern for capillary electrophoresis.

Using a combination of capillary electrophoresis (CE) and patterned fluorescence correlation spectroscopy (patterned FCS), we have developed a new technique for performing electrophoretic analysis independently of the initial length of injected analyte plugs. In t histechnique, which is abbreviated as CE/patterned FCS, fluorescent analyte molecules dispersed continuously in a capillary migrate through a stationary interference pattern created by two intersecting excitation laser beams, and their fluorescence emission is monitored. We prove theoretically that the power spectrum of fluctuations in the fluorescence intensity gives a virtual electropherogram. The profile of the electropherogram and the number of theoretical plates are in general obtained by using analytical methods. Characterizing the capillary length within the excitation beams as the effective length, we compare CE/ patterned FCS with conventional CE. Numerical simulations on capillary gel electrophoresis of DNA predict that the optimized CE/patterned FCS is superior to conventional CE when the effective length is shorter than 1 cm. The experimental feasibility of this technique is demonstrated in the fluorometry of TOTO-1-stained DNA. For an effective length of 740 microm, a maximum number of plates of 7400, and a resolution of 1.0 were obtained with a one-component injection of pUC18 DNA and a two-component injection of pUC 18 DNA and lambda DNA, respectively.

Journal Article↗