Mari Tabuchi

Nanospheres for DNA separation chips

We report here a technology to carry out separations of a wide range of DNA fragments with high speed and high resolution. The approach uses a nanoparticle medium, core-shell type nanospheres, in conjunction with a pressurization technique during microchip electrophoresis. DNA fragments up to 15 kilobase pairs (kbp) were successfully analyzed within 100 s without observing any saturation in migration rates. DNA fragments migrate in the medium while maintaining their characteristic molecular structure. To guarantee effective DNA loading and electrofocusing in the nanosphere solution, we developed a double pressurization technique. Optimal pressure conditions and concentrations of packed nanospheres are critical to achieve improved DNA separations.

A 15-s protein separation employing hydrodynamic force on a microchip

We report here a novel pressurization technique for microchip electrophoresis that enables 15-s separation of protein mixtures extracted from biological samples. Although pressure-driven flow is usually parabolic flow, pressurization prior to electrophoresis separation produced a plug flow and achieved a dramatic migration time reduction without compromising resolution. Sample plugs were pushed forward by pressurization after loading the sample but before electrophoresis separation, in the absence of an electric potential. Higher pressures enabled higher speed separation; furthermore, the resolution could be easily controlled using an optimal pressure. In addition, the slow medium-pressurization technique enabled 2-D separation in only a single channel on a microchip. Utilizing this technique, 12 samples of complex protein mixture extracted from a human T lymphoblastic cell line, Jurkat cells, were separated within 15 s in a single run using a 12-microchannel array. In addition, target proteins from Jurkat cells were detected within this time. This novel pressurization technique on a microchip will offer enormous advantages for proteome analysis over commonly used 2-D electrophoresis.

Cell-free protein synthesis on a microchip

We evaluated the expression of various proteins by using a cell‐free protein synthesis system with a rapid translation system, a microtube, and a microchip technique. Protein expression was successfully achieved with a microfabricated reaction chamber on a plastic chip. Proteins were expressed effectively by use of expression vectors for T7 RNA polymerase (pET) instead of a plasmid for in vitro expression vector, which is recommended by the manufacture of the rapid translation system. Expression of the proteins depended on the type of proteins chosen. Two mammalian proteins were synthesized simultaneously with two expression vectors of pET species. Effective application of the cell‐free protein synthesis system will enable miniaturization of protein synthesis and mediation between the transcriptome and proteome.

Bio-sensing on a chip with compact discs and nanofibers

This paper describes a novel, sensitive detection system for biomolecules (DNA and proteins etc.) that is integrated in a lab-on-a-chip utilizing optical compact discs (CDs) and bio-nanofibers. The new method comprises a microchannel containing CD grating that confines fragments of unique bacterial cellulose fibrils (BC), which have nanometre scale fibers and holes. A maximum of six times higher sensitivity to detect DNA was obtained with this CD and BC system compared to a conventional method. We also demonstrate an effective light-confining effect for biological application with the new method.

Applicability of bacterial cellulose as an alternative to paper points in endodontic treatment

Dental root canal treatment is required when dental caries progress to infection of the dental pulp. A major goal of this treatment is to provide complete decontamination of the dental root canal system. However, the morphology of dental root canal systems is complex, and many human dental roots have inaccessible areas. In addition, dental reinfection is fairly common. In conventional treatment, a cotton pellet and paper point made from plant cellulose is used to dry and sterilize the dental root canal. Such sterilization requires a treatment material with high absorbency to remove any residue, the ability to improve the efficacy of intracanal medication and high biocompatibility. Bacterial cellulose (BC) is produced by certain strains of bacteria. In this study, we developed BC in a pointed form and evaluated its applicability as a novel material for dental canal treatment with regard to solution absorption, expansion, tensile strength, drug release and biocompatibility. We found that BC has excellent material and biological characteristics compared with conventional materials, such as paper points (plant cellulose). BC showed noticeably higher absorption and expansion than paper points, and maintained a high tensile strength even when wet. The cumulative release of a model drug was significantly greater from BC than from paper points, and BC showed greater compatibility than paper points. Taken together, BC has great potential for use in dental root canal treatment.

Rapid measurement of deoxyribonuclease I activity with the use of microchip electrophoresis based on DNA degradation

Deoxyribonuclease I (DNase I) activity in serum has been shown to be a novel diagnostic marker for the early detection of acute myocardial infarction (AMI). However, the conventional method to measure DNase I activity is time-consuming. In the current study, to develop a rapid assay method for DNase I activity for clinical purposes, a microchip electrophoresis device was used to measure DNase I activity. Because DNase I is an endonuclease that degrades double-stranded DNA endo-nucleolytically to produce oligonucleotides, degradation of the DNA standard caused by DNase I action was detected using microchip electrophoresis. We detected DNase I activity within 10 min. This is the first study to apply microchip electrophoresis for the detection of DNase I activity; furthermore, it seems plausible that reduction of analysis time for DNase I activity could make this novel assay method using microchip electrophoresis applicable in clinical use.

A novel injection method for high-speed proteome analysis by capillary electrophoresis.

We have developed a new sample injection method for capillary electrophoresis (CE) that reduces the required migration time. We demonstrated a pressurization technique that was performed with buffer in the outlet after the electrokinetic sample injection with no buffer in the outlet. To reduce the migration time, the sample injection had to be performed with no buffer in the outlet; water should be pressurized while the buffer is in the outlet. Though the resolution was slightly decreased using this method, the addition of a separation carrier (curdlan) to the run buffer restored the resolution without delaying the migration time. The use of our new sample injection method combined with our high‐quality separation carrier will enable us to improve the efficiency of the high‐throughput screening (HTS) system for proteome analysis.

A separation carrier in high-speed proteome analysis by capillary electrophoresis.

We obtained a high‐efficiency separation carrier for proteome analysis by capillary electrophoresis. The addition of curdlan or laminaran to the run buffer hastened the migration time without any degradation in resolution. We propose that for the development of the separation carrier it is necessary to synthetically analyze each of the following mobility factors of electroosmotic flow: buffer ionic strength, additional disturbance and adsorption. The total analysis for buffer and additive will be useful for designing high‐throughput screening (HTS) systems for proteome analysis without annoying adsorption.

Direct and simple fluorescence detection method for oxidized lipoproteins.

The quantification of low-density lipoprotein (LDL) and high-density lipoprotein (HDL) is currently one of the most important clinical measurements for characterizing metabolic syndrome. However, recent studies have revealed additional factors that may be more strongly associated with the coronary heart disease than simple measurement of LDL or HDL levels, such as small dense (sd) LDL particles and oxidized LDL or HDL particles. Although several methods using enzyme-antibody detection systems or fluorescent probes have been devised to characterize these factors, such methods are expensive to implement for clinical measurements. Here, we present a straightforward analytical method for direct quantitation of oxidized lipoproteins by fluorescence spectrometry, with excitation in the UV (365 +/- 10 nm) or visible (470 +/- 10 nm) range and emission detected at 450 +/- 30 nm or 535 +/- 15 nm. This method can be readily applied for clinical measurement in patients with dyslipidemia using only 1 microL of 1 mg/mL of lipoprotein and without the need for any expensive detection antibodies. Using this new technique, biological samples from patients with dyslipidemia showed higher fluorescence intensities than samples from normal subjects when detecting oxidized LDL and light HDL (d = 1.063-1.125 g/mL), whereas samples from patients with dyslipidemia showed lower fluorescence intensities than samples from normal subjects when measuring oxidized heavy HDL (d = 1.125-1.210 g/mL) levels.

Geometrical Separation Method for Lipoproteins Using Bioformulated-Fiber Matrix Electrophoresis: Size of High-Density Lipoprotein Does Not Reflect Its Density

The increasing number of patients with metabolic syndrome is a critical global problem. In this study, we describe a novel geometrical electrophoretic separation method using a bioformulated-fiber matrix to analyze high-density lipoprotein (HDL) particles. HDL particles are generally considered to be a beneficial component of the cholesterol fraction. Conventional electrophoresis is widely used but is not necessarily suitable for analyzing HDL particles. Furthermore, a higher HDL density is generally believed to correlate with a smaller particle size. Here, we use a novel geometrical separation technique incorporating recently developed nanotechnology (Nata de Coco) to contradict this belief. A dyslipidemia patient given a 1-month treatment of fenofibrate showed an inverse relationship between HDL density and size. Direct microscopic observation and morphological observation of fractionated HDL particles confirmed a lack of relationship between particle density and size. This new technique may improve diagnostic accuracy and medical treatment for lipid related diseases.