Study on the dielectrophoretic characteristics of malaria-infected red blood cells.

Malaria is a tropical disease caused by parasites in the genus Plasmodium, which still presents 241 million cases and nearly 627,000 deaths recently. In this work, we used the dielectrophoresis (DEP) to characterize red blood cells in a microchannel. The purpose of this work is to determine the difference between the normal and the malaria-infected cells based on the DEP characteristics. The samples were infected cells and normal red blood cells, which were either prepared in culture or obtained from volunteers. Diamond-shaped and curved micropillars were used to create different degrees of DEP in the gap between them. The DEP crossover frequencies were observed with the diamond-shaped micropillars. The cell velocity under negative dielectrophoresis (nDEP) at a low frequency was examined with the curved micropillars. The measured lower crossover frequencies were remarkably different between the malaria-infected cells and the normal cells, whereas the higher crossover frequencies were similar among the samples. The velocity under nDEP was lower for the infected cells than the normal cells. The results imply that the malaria infection significantly decreases the capacitance but increases the conductance of the cell membrane, whereas a change in cytoplasmic conductivity may occur in a later stage of infection.

Characterization of Extra-Cellular Vesicle Dielectrophoresis and Estimation of Its Electric Properties.

Dielectrophoresis (DEP) refers to a type of electrical motion of dielectric particles. Because DEP is caused by particle polarization, it has been utilized to characterize particles. This study investigated the DEP of three types of exosomes, namely bovine milk, human breast milk, and human breast cancer exosomes. Exosomes are kinds of extracellular vesicles. The crossover frequencies of the exosomes were determined by direct observation of their DEPs. Consequently, bovine and human milk exosomes showed similar DEP properties, whereas the cancer exosomes were significantly different from the others. The membrane capacitance and conductivity of the exosomes were estimated using determined values. A significant difference was observed between bovine and human milk exosomes on their membrane capacitance. It was revealed that the membrane capacitances of human breast milk and human breast cancer exosomes were almost identical to those of their host cells and the conductivity of the exosomes were much lower than that of the host cell. Based on these results, DEP separation of the human breast milk and cancer exosomes was demonstrated. These results imply that DEP can be utilized to separate and identify cancer exosomes rapidly. Additionally, our method can be utilized to estimate the electric property of other types of extracellular vesicles.

Rapid and low-cost amplicon visualization for nucleic acid amplification tests using magnetic microbeads.

This study presents a rapid and low-cost amplicon detection method in which amplicons are attached to magnetic microbeads, suspended in deionized water, and subjected to a magnetic field on a hydrophilic surface resulting in the circular agglomeration of amplicon-conjugated microbeads, visible to the naked eye.

Simple microfluidic device for detecting the negative dielectrophoresis of DNA labeled microbeads.

We propose a new microfluidic device that can be used to determine the change in the negative dielectrophoresis (n-DEP) of dielectric microbeads when a small amount of DNA is attached to them. We previously proposed a DNA detection method based on changes in the DEP of microbeads induced by the attachment of DNA. When target DNA is attached to the microbeads having n-DEP property, the DEP changes from negative to positive. This occurs because electric charges of the DNA increase the surface conductance of the microbeads. Thus, only the DNA-labeled microbeads are attracted to a microelectrode by positive DEP. The trapped DNA-labeled microbeads can be counted by dielectrophoretic impedance measurements. A large amount of DNA (approximately 105 DNA molecules) is required to change the DEP from negative to positive. Even though this method can be combined with DNA amplification, reducing the amount of DNA required can help us to shorten the reaction time. In this study, we aimed to detect DNA less than 105 DNA molecules by determining the change in the n-DEP change. To achieve this, we proposed a simple microfluidic device consisting of a single microchannel and a single pair of microelectrodes. Numerical simulations revealed that the device can identify the slight change in the n-DEP of the microbeads corresponding to the attachment of a small amount of DNA. In practical experiments, the fabricated device distinguished 10-1000 DNA molecules per microbead. This method represents a fast and easy method of DNA detection when combined with DNA amplification techniques.

Development of the School Teachers Job Stressor Scale (STJSS).

AIM: Japanese teachers are not only responsible for students but also for tasks outside the classroom, including engagement with parents and the community, and maintaining safety. They work longer hours and have lower self-efficacy than teachers in other countries. Thus, we aimed to develop an assessment scale for job stress in teachers and to evaluate its psychometric properties. METHODS: We developed the "School Teachers Job Stressor Scale (STJSS) Draft" comprising 45 items, based on previous anonymous self-report questionnaires collected from 98 teachers in four elementary and middle schools in Miyazaki City, Japan. Subsequently, the scale draft and the previously validated Brief Job Stress Questionnaire (23-item abridged version) were distributed to 2276 teachers from 73 elementary and middle schools in Miyazaki City. Finally, we analyzed data from 1300 participants. After excluding inappropriate data based on ceiling and floor effect analysis, we carried out a good-poor, item-total correlation, and exploratory factor analyses. We then verified construct validity, criterion-related validity, and reliability using correlation analysis, confirmatory factor analysis, and Cronbach's alpha, respectively. RESULTS: After item-total correlation analysis, five items were excluded. Exploratory factor analysis extracted five factors: "Time spent outside of work," "Self-assessment of one's ability as a teacher," "Relationship with other teachers," "Social interactions outside of teaching," and "Duties outside of teaching." The final version of the STJSS comprised 23 items and five factors. CONCLUSION: The 23-item STJSS developed to measure specific stressors in Japanese teachers to improve their mental health care could provide an accurate assessment tool with adequate reliability and validity.

Fast and sensitive isothermal DNA assay using microbead dielectrophoresis for detection of anti-microbial resistance genes.

Antimicrobial resistant pathogens are a growing worldwide threat to human health. This study describes a novel method for rapid and sensitive detection of antimicrobial resistance (AMR) genes, specifically blaCTX-M-15 which encodes for the enzyme that offers resistance to extended spectrum ß-lactam antibiotics. The method combines isothermal DNA amplification by recombinase polymerase amplification (RPA), with microbead dielectrophoresis (DEP)-based DNA detection. The RPA amplicon is captured onto dielectric microbeads, and the amount of amplicon determined by dielectrophoretic impedance measurement (DEPIM) of the microbeads. Amplicon-labeled microbeads were prepared by either a two-step or one-step method. A purified recombinant plasmid containing blaCTX-M-15 and genomic DNA (with plasmid) extracted from an AMR bacteria (Escherichia coli NCTC 13441) were used as target samples. A one-step method in which RPA and DNA immobilization on the microbeads is carried out simultaneously, has a detection limit of 2 copies/reaction for pure plasmid and 50 copies/reaction for genomic DNA. The assays are quantitative with a dynamic range up to 105 copies/reaction, with a total detection time of 26 min. Both methods are easy, rapid, and unlike lateral flow detection are quantitative.

Comparison of Sensitivity and Quantitation between Microbead Dielectrophoresis-Based DNA Detection and Real-Time PCR.

In this study, we describe a microbead-based method using dielectrophoresis (DEP) for the fast detection of DNA amplified by polymerase chain reaction (PCR). This electrical method measures the change in impedance caused by DEP-trapped microbeads to which biotinylated target DNA molecules are chemically attached. Using this method, measurements can be obtained within 20 min. Currently, real-time PCR is among the most sensitive methods available for the detection of target DNA, and is often used in the diagnosis of infectious diseases. We therefore compared the quantitation and sensitivity achieved by our method to those achieved with real-time PCR. We found that the microbead DEP-based method exhibited the same detection limit as real-time PCR, although its quantitative detection range was slightly narrower at 10-105 copies/reaction compared with 10-107 copies/reaction for real-time PCR. Whereas real-time PCR requires expensive and complex instruments, as well as expertise in primer design and experimental principles, our novel method is simple to use, inexpensive, and rapid. This method could potentially detect viral and other DNAs efficiently in combination with conventional PCR.

Bacterial detection based on polymerase chain reaction and microbead dielectrophoresis characteristics.

In this study, an electrical DNA detection method was applied to bacterial detection. DNA was extracted from bacteria and amplified by polymerase chain reaction. The microbeads were labelled with amplicons, altering their surface conductance and therefore their dielectrophoresis characteristics. Amplicon-labelled microbeads could thus be trapped within a high-strength electric field, where they formed a pearl chain between the electrodes, resulting in an increased conductance between the electrodes. This method reduces the amplicon detection time from 1-2 h to 15 min, compared with the conventional method. The presented method realised quantitative detection of specific bacteria at concentrations above 1 × 105 and 2.4 × 104 CFU/ml for bacterial solutions with and without other bacterial presence, respectively.

Development of an anti-norovirus single-chain Fv for immunochromatographic test kit.

It was developed that an anti-norovirus single-chain Fv (scFv) antibody for immunochromathographic test kit by using the phage displayed technique and biopanning. To obtain the scFv having wide reactivity for several norovirus genotypes, a mixture of recombinant norovirus capsid proteins, including 11 norovirus genotypes, was used for biopanning. Then, one anti-norovirus scFv antibody that recognized both of norovirus genogroups GI and GII was identified. An immunochromatographic test strip was constructed by using the scFv and demonstrated it to detect norovirus in stool samples. The immunochromatographic strip showed similar sensitivity to a commercially available one on which several monoclonal antibodies are included.

Real-time direct observation of single-molecule DNA hydrolysis by exonuclease III.

Single-molecule DNA digestion by exonuclease III, which has 3' to 5' exonuclease activity, was analyzed using a micro-channel with two-layer laminar flow. First, a DNA-bead complex was optically trapped in one layer in the absence of exonuclease III permitted the DNA to be stretched by the laminar flow. The exonuclease III reaction was initiated by moving the trapped DNA-bead complex to another layer of flow, which contained exonuclease III. As the reaction proceeded, the fluorescently-stained DNA was observed to shorten. The process was photographed; examination of the photographs showed that the DNA molecule shortened in a linear fashion with respect to the reaction time. The digestion rate obtained from the single-molecule experiment was compared to that measured from a bulk experiment and was found to be ca. 28 times higher than the bulk digestion rate.

Electrospinning of poly(lactic acid) stereocomplex nanofibers.

The electrospinning of stereocomplex nanofibers of high-molecular-weight poly(L-lactic acid) (PLLA)/poly(D-lactic acid) (PDLA) (PLLA/PDLA = 1:1) was carried out with chloroform as the spinning solvent. The stereocomplex nanofibers with diameters of 830-1400 and 400-970 nm were successfully obtained at voltages of -12 and -25 kV, respectively. Wide-angle X-ray scattering indicated that with an increasing absolute value of voltage from 0 to 25 kV the crystallinity of homo-crystallites composed of either PLLA or PDLA decreased from 5% to 1%, whereas the crystallinity of stereocomplex crystallites increased slightly from 16% to 20%. The obtained results reveal that electrospinning is an effective method to prepare stereocomplex nanofibers with a negligibly small amount of homo-crystallites, even when high-molecular-weight PLLA and PDLA are used, and that the orientation caused by high voltage (or electrically induced high shearing force) during electrospinning enhances the formation and growth of stereocomplex crystallites and suppresses the formation of homo-crystallites.

Recovery of intact yeast chromosomal DNA from agarose gel plugs using coil-globule transition.

In the current studies, we designed a new approach for sizing and isolating chromosomal DNA using coil-globule transition, which avoids fragmentation of giant DNA due to mechanical stress. Although coil-globule transition is reversible and globular DNA is tolerant to mechanical stress, globular DNA cannot be manipulated by an electric field because of the loss of its negative charges. In our system, however, DNA is extracted from an agarose gel in a coiled state into a solution of PEG, and coil-globule transition is induced by cations generated at the anode. This method achieves buffer exchange without stirring, which is the main cause of mechanical stress. Real-time analysis of T4dc viral DNA molecules revealed that they change immediately from a coiled to a globular form when the cation concentration is sufficiently high. This method was used to prepare yeast chromosomal DNA in a globular state without fragmentation.

Single-molecule reverse transcription polymerase chain reaction using water-in-oil emulsion.

A new method based on a combination of reverse transcription polymerase chain reaction (RT-PCR) and a water-in-oil (W/O) emulsion was developed. Reverse transcription and initial thermal cycles were carried out in droplets of the W/O emulsion. Then, the droplets were united, followed by remaining secondary PCR cycles. This two-step method succeeded in detecting a single RNA molecule.

Adaptor polymerase chain reaction for single molecule amplification.

The adaptor polymerase chain reaction (PCR) permits the amplification of DNA fragments with arbitrary sequences. In this paper, we describe the successful amplification of plasmid-derived single molecule DNAs digested by a restriction enzyme. By using adaptors made of short and long oligonucleotides, nonspecific interactions during PCR were suppressed. The method will be applicable to the detection of single molecule DNA fragments even if their sequence is unknown.

Single-molecule PCR using water-in-oil emulsion.

Polymerase chain reaction (PCR) using a single molecule of DNA is very useful for analysis, detection and cloning of the desired DNA fragment. We developed a simple PCR method utilizing a water-in-oil (W/O) emulsion that included numerous droplets of reaction mixture in bulk oil phase. These droplets, which were stable even at high temperatures, functioned as micro-reactors. This allows the effective concentration of template DNA to be increased, even for low concentrations of template DNA. The present method consists of a two-step thermal cycle. The first step was carried out using the W/O emulsion. During this step, the template DNA was amplified in the limited volume of the droplets in the W/O emulsion. The W/O emulsion was broken and the second PCR step was carried out. This method can be easily applied to amplify a single DNA molecule.

One-end immobilization of individual DNA molecules on a functional hydrophobic glass surface.

We demonstrate an effective method for DNA immobilization on a hydrophobic glass surface. The new DNA immobilizing technique is extremely simple compared with conventional techniques that require heterobifunctional crosslinking reagent between DNA and substrate surface that are both modified chemically. In the first process, a coverslip was treated with dichlorodimethylsilane resulting in hydrophobic surface. lambda DNA molecules were ligated with 3'-terminus disulfide-modified 14 mer oligonucleotides at one cohesive end. After reduction of the disulfide to sulfhydryl (thiol) groups the resulting thiol-modified lambda DNA molecules were reacted on silanized coverslip. Fluorescent observation showed that the thiol-modified lambda DNA molecules were anchored specifically to the hydrophobic surface at one terminus, although non-specific binding of the DNA molecules was suppressed. It was observed that the one-end-attached DNA molecule was bound firmly to the surface and stretched reversibly in one direction when a d.c. electric field was applied.