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.

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.

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.

Conformational separation of monosaccharides of glycoproteins labeled with 2-aminoacrydone using microchip electrophoresis.

The conformational separation of monosaccharides labeled with fluorescent 2-aminoacrydone (AMAC) was performed by electrophoresis on a plastic microchip with light-emitting diode confocal fluorescence detection. The AMAC-labeled five neutral monosaccharide mixture (D-glucose (Glc), D-mannose, D-galactose, L-fucose, and D-xylose) or two amino monosaccharide mixture (N-acetyl-D-glucosamine and N-acetyl-D-galactosamine) were well separated at pH 8.5 and 0.5% w/v methylcellulose of 200 mM borate buffer conditions using microchip electrophoresis. The separation was successfully performed considering the difference in stability of the complex between the hydroxyl residue of the monosaccharide and borate ions, and we found that 200 mM and pH 8.5 of borate buffer conditions were critical. High-speed separation for the neutral monosaccharides (50 s) and for amino monosaccharides (70 s) was attained at a 400 V/cm of electric field condition, showing all peak resolutions were greater than 0.9% and RSD of mobility were less than 1.9%. The detection limits of 0.86 microM for Glc and <1 microM for all other monosaccharides were enhanced with the addition of 0.5% w/v methylcellulose to the buffer. These attainments are fully compatible with conventional CE. The analysis of the subtle differences in the conformational stability and the value of the hydroxyl residue of the borate complex allowed the development of an efficient prospective tool for attaining high-resolution separation of monosaccharide mixtures having complicated and analogous conformations.

On-line microdevice for stress proteomics.

The handling of the cells or tissues is essential for proteomics research or drug screening, where labor is not avoidable. The steps of cell wash, protein extraction, protein denaturing are complicated procedures in conventional method using centrifugation and pipetting in the laboratory. This is the bottle-neck for proteome research. To solve these problems, we propose to utilize the nanotechnology, which will improve the proteomics methodology. Utilizing the nanotechnology, we developed a novel microseparation system, where centrifugation and pipetting are needless. This system has a nanostructured microdevice, by which the cell handling, protein extraction, and antibody assay can be performed. Since cell transfer is needless, all cells are corrected without any loss during the cell-pretreatment procedures, which allowed high reproducibility and enabled the detection of low amount of protein expression. Utilizing the microdevice, we analyzed the stress induced proteins. We further succeeded the screening of food that was useful for immunity and found that an extraction from seaweed promoted the apoptosis of T-lymphoblastic cells. Here, we present an on-line microdevice for stress proteomics.

Design for DNA separation medium using bacterial cellulose fibrils.

In this paper, we present a novel DNA separation medium using bacterial cellulose fibrils. Bacterial cellulose has an intrinsic three-dimensional micrometer- to nanometer-scale network structure. Addition of this material to a low-concentration polymer solution (<5 cP) enables high-resolution electrophoretic separation of DNA, even for fragments of 10-100-bp or single-nucleotide polymorphism. The newly designed medium consists of a double mesh: a 10-nm flexible mesh derived from a conventional polymer medium containing 10-nm to 1-microm rigid pores made up of 10-microm bacterial cellulose fragments.

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.

High-speed separation of proteins by microchip electrophoresis using a polyethylene glycol-coated plastic chip with a sodium dodecyl sulfate-linear polyacrylamide solution.

In this paper, we describe a method for size-based electrophoretic separation of sodium dodecyl sulfate (SDS)-protein complexes on a polymethyl methacrylate (PMMA) microchip, using a separation buffer solution containing SDS and linear polyacrylamide as a sieving matrix. We developed optimum conditions under which protein separations can be performed, using polyethylene glycol (PEG)-coated polymer microchips and electrokinetic sample injection. We studied the performance of protein separations on the PEG-coated PMMA microchip. The electrophoretic separation of proteins (21.5-116.0 kDa) was completed with separation lengths of 3 mm, achieved within 8 s on the PEG-coated microchip. This high-speed method may be applied to protein separations over a large range of molecular weight, making the PEG-coated microchip approach applicable to high-speed proteome analysis systems.

Microchip electrophoretic protein separation using electroosmotic flow induced by dynamic sodium dodecyl sulfate-coating of uncoated plastic chips.

Separation of sodium dodecyl sulfate (SDS)-protein complexes is difficult on plastic microchips due to protein adsorption onto the wall. In this paper, we elucidated the reasons for the difficulties in separating SDS-protein complexes on plastic microchips, and we then demonstrated an effective method for separating proteins using polymethyl methacrylate (PMMA) microchips. Separation difficulties were found to be dependent on adsorption of SDS onto the hydrophobic surface of the channel, by which cathodic electroosmotic flow (EOF; reversed flow) was generated. Our developed method effectively utilized the reversed flow from this cathodic EOF as a driving force for sample proteins using permanently uncoated but dynamic SDS-coated PMMA microchips. High-speed (6 s) separation of proteins and peptides up to 116 kDa was successfully achieved using this system.

A design of nanosized PEGylated-latex mixed polymer solution for microchip electrophoresis.

We report here advanced microchip electrophoresis using a nanoparticle doped polymer solution that enables greater separation of DNA. The proposed system is simple and effective without any new apparatus or complicated procedures. Various amounts and sizes (80 nm, 110 nm, and 193 nm) of polymer nanoparticle solutions (PEGylated-latex) were mixed with a conventional polymer solution for microchip electrophoresis. When a 0.49 wt% hydroxyl propyl methyl cellulose (HPMC) buffer solution was mixed with a 2.25 wt% 80 nm-PEGylated-latex a higher separation efficiency and a higher mobility of a wider molecular range of dsDNA (10 bp to 2 kbp) was achieved under low viscosity conditions (<5.5 cP) than in conventional 0.7% HPMC. The separation performance was dependent on the particle size and concentration. Furthermore, the effectiveness of the larger PEGylated-latex (193 nm) was not as high as the smaller one (80 to 110 nm). The observed separation improvement by polymer solution with latex-nanoparticles seems to derive from the balance between wider polymer mesh size and the structural obstacles of particles in the buffer.

A triple-injection method for microchip electrophoresis.

We report here a novel triple injection method for microchip electrophoresis (micro-CE) that results in a higher intensity of DNA peaks. This new method includes a triple-repeated process of a combination of a sample loading voltage and a separation voltage in each interval, namely (loading time) + (separation time) + (loading time) + (separation time) + (loading time), prior to electrophoretic separation. All these injections were electrokinetically controlled by a software. Although the usual sample injection, which included the process of one 60 s electrokinetically application, was limited by the amount of sample, peaks of 40% higher intensity were obtained using the new method within half of the conventional injection time compared to the conventional method. Maximum peak intensity was successfully achieved with integration of the intensities of the triple-repeated peaks by adjusting the application period of the separation voltage. Repetition of the sample loading voltage for an adjusted period with a further adjusted period of separation voltage in each interval may be an effective method for injection of samples that results in peaks with higher intensity.

Self-contained on-chip cell culture and pretreatment system.

In this study, we describe a simple on-chip cell culture and pretreatment system that requires no external machines. Conventional cell culture utilizes culture dishes or microtiter plates, where pipetting and centrifugation are indispensable for washing cells and changing media. However, our microdevice requires no external centrifugation or pump. Utilizing this microdevice, we attained dramatically shorter total analytical time with a high-throughput screening system for proteomic analysis (1 min per 12 samples; one eightieth of the conventional time). Protein expression of Jurkat cells during stress-shock induced apoptosis was readily analyzed using this system. We found that a seaweed extraction effectively induced apoptosis of Jurkat cells.

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.

Rapid subpicogram protein detection on a microchip without denaturing.

Protein size separation based on sodium dodecyl sulfate-gel electrophoresis (SDS-GE) requires denaturing, but we propose that denaturing is unnecessary for analysis by microchip electrophoresis (micro-CE). By omitting the protein denaturing process, we achieved not only shortened total analysis time, but also dramatically improved sensitivity without compromising size determination. The detection limit was improved to 0.1 ng/microL under conditions without denaturing and 600 pg (9.0 femtomol) of bovine serum albumin was detectable, which equals levels detectable by Silver stain, although a routine method by microchips in the Coomassie Blue detection level.

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.