Impact of surface roughness on Dielectrophoretically assisted concentration of microorganisms over PCB based platforms

This article presents a PCB based microfluidic platform for performing a dielectrophoretic capture of live microorganisms over inter-digitated electrodes buried under layers of different surface roughness values. Although dielectrophoresis has been extensively studied earlier over silicon and polymer surfaces with printed electrodes the issue of surface roughness particularly in case of buried electrodes has been seldom investigated. We have addressed this issue through a layer of spin coated PDMS (of various surface roughness) that is used to cover the printed electrodes over a printed circuit board. The roughness in the PDMS layer is generally defined by the roughness of the FR4 base which houses the printed electrodes as well as other structures. Possibilities arising out of COMSOL simulations have been well validated experimentally in this work.     

A rapid method for quantitation of antiviral antibodies

This paper examines the parameters necessary for the efficient measurement of anti-Theiler's murine encephalomyelitis virus (TMEV) antibodies in an affinity-dependent manner using a variation of a solid-phase particle concentration fluorescence immunoassay (PCFIA). By allowing antibody to react with fluorochrome-labelled virus in fluid phase and subsequently capturing the resulting virus-antibody complexes with anti-immunoglobulin coated polystyrene particles (fluid-phase PCFIA), the present assay allows for both greater sensitivity, specificity and preservation of conformational viral epitopes than do solid-phase immunoassays. Fluid-phase PCFIA proved to be a more rapid quantitative assay than ELISA and significantly diminished non-specific binding by both untreated and heat-inactivated normal mouse sera. This methodology also allowed us to perform competition assays and to determine the dissociation kinetics of anti-viral antibody preparations, investigations which cannot generally be performed as solid-phase immunoassays. Thus fluid-phase PCFIA is a rapid and efficient immunoassay with excellent reproducibility and great versatility.     

Effect of cryoprotectants and their concentration on post-thaw survival and development of rapid frozen-thawed pronudear stage mouse embryos

Experiments have been conducted to develop a simple rapid-freezingprotocol for pronudear stage mouse embryos. The effect of typeof cryoprotectant (dimethyl-sulphoxide (DMSO) or 1,2-propanediol(PROH)) and concentration (ranging from 3.5 to 8.0 mol/1 with0.5 mol/1 sucrose) on the post-thaw morphological survival rate,on cleavage rate and on development to the blastocyst stagewere studied. Further, in-vivo viability of embryos frozen usingthe most effective cryoprotectant concentration (PROH at 7.0mol/1) was compared with viability of non-frozen embryos. Thetype of cryoprotectant and its concentration influenced thesurvival and development of embryos to the blastocyst stagein vitro. The best development with PROH was achieved at 7.0mol/1 (66%, 128/193), whereas with DMSO the best developmentwas achieved at 6.0 mol/1 (42%, 71/171). The rates of survivaland cleavage did not differ between the two best cryoprotectantconcentrations (P > 0.01) but the proportion of embryos whichdeveloped to blastocyst was higher (P > 0.001) with PROHat 7.0 mol/1 compared with DMSO at 6.0 mol/1. The rates of survivaland development were higher (P < 0.001) with DMSO at 3.5and 6.0 mol/1 compared with similar concentrations of PROH.The cleavage and development, however, was higher (P < 0.001)at 7.0 mol/1 PROH compared with the same concentration of DMSO.At 8.0 mol/1 the survival and development was not different(P > 0.01) between DMSO and PROH. The rate of implantationand the percentage of live fetuses at autopsy of the recipientsreceiving non-frozen embryos was higher (63 and 41% respectively)than in those receiving frozen–thawed embryos (53 and37% respectively), but not significantly different. It may beconcluded that the concentration range of cryoprotectants whichallows acceptable embryo viability after freezing and thawingis very narrow. The rapid protocol using dehydration in 0.25mol/1 and 0.5 mol/1 sucrose followed by exposure to 7.0 mol/1PROH and 0.5 mol/1 sucrose for 45 s was the most effective forcryopreservation of pronudear stage mouse embryos.     

New method for concentration and quantitation of Mycobacterium leprae.

A new method of enumerating Mycobacterium leprae has been developed. Suspensions containing the organisms were filtered through a polycarbonate membrane filter (25-mm diameter, 0.4-micronm pore size, 10-micronm thick; Nucleopore) to concentrate the organisms. The membrane was then mounted on a glass slide and stained with a standard acid-fast stain. Finally, the membrane was treated with a small amount of chloroform to fix it to the slide and make it transparent. This method enabled us to detect M. leprae in quantities as small as 4.98 X 10(2) regardless of the total volume of the original material. Comparison with a standard method for enumerating M. leprae showed that both methods gave similar results when the organisms counted by the standard method were present in sufficient quantity for reproducibility. Because the least number of organisms that can be detected with the standard method is 10(4) ml and because the organisms detected with the new method could be concentrated on the polycarbonate filter from a large amount of infected fluid, a substantial number of suspensions were shown by the new method, but not by the standard method, to contain M. leprae.     

老化及年青红细胞电介质电泳的研究

本文介绍我们实验室自己试制的电介质电泳装置及测量方法,并用电介质电泳测定老化及年青红细胞的电介质电泳收集率(DCR)、细胞膜镶嵌蛋白分子水平扩散运动特征频率(Fc)及细胞在交流电场作用下的变形程度。并从测量结果分析老化及年青红细胞变形性及膜导电性、流动性、韧性、脆性等流变学性质。其中细胞变形性测定结果与衍射法进行了比较。     

Sudden origins: a general mechanism of evolution based on stress protein concentration and rapid environmental change

A major theme in Darwinian evolutionary theory is that novelty arises through a process in which organisms and their features are gradually transformed. Morgan provided Darwinism and the evolutionary synthesis with the idea that minor mutations produce the minuscule morphological variations on which natural selection then acts, and that, although mutation is random, once a process of gradual genetic modification begins, it becomes directional and leads to morphological, and consequently organismal, transformation. In contrast, studies on the role of cell membrane physical states in regulating the expression of stress proteins in response to environmental shifts indicate the existence of a downstream mechanism that prevents or corrects genetic change (i.e., maintains "DNA homeostasis"). However, episodic spikes in various kinds of environmental stress that exceed an organism's cells' thresholds for expression of proper amounts of stress proteins responsible for protein folding (including stochastically occurring DNA repair) may increase mutation rate and genetic change, which in turn will alter the pattern of gene expression during development. If severe stress disrupts DNA homeostasis during meiosis (gametogenesis), this could allow for the appearance of significant mutational events that would otherwise be corrected or suppressed. In evolutionary terms, extreme spikes in environmental stress make possible the emergence of new genetic and consequent developmental and epigenetic networks, and thus also the emergence of potentially new morphological traits, without invoking geographic or other isolating mechanisms.     

A rapid, automated approach for quantitation of rotavirus and reovirus infectivity

Current microscopy-based approaches for immunofluorescence detection of viral infectivity are time consuming and labor intensive and can yield variable results subject to observer bias. To circumvent these problems, we developed a rapid and automated infrared immunofluorescence imager-based infectivity assay for both rotavirus and reovirus that can be used to quantify viral infectivity and infectivity inhibition. For rotavirus, monolayers of MA104 cells were infected with simian strain SA-11 or SA-11 preincubated with rotavirus-specific human IgA. For reovirus, monolayers of either HeLa S3 cells or L929 cells were infected with strains type 1 Lang (T1L), type 3 Dearing (T3D), or either virus preincubated with a serotype-specific neutralizing monoclonal antibody (mAb). Infected cells were fixed and incubated with virus-specific polyclonal antiserum, followed by an infrared fluorescence-conjugated secondary antibody. Well-to-well variation in cell number was normalized using fluorescent reagents that stain fixed cells. Virus-infected cells were detected by scanning plates using an infrared imager, and results were obtained as a percent response of fluorescence intensity relative to a virus-specific standard. An expected dose-dependent inhibition of both SA-11 infectivity with rotavirus-specific human IgA and reovirus infectivity with T1L-specific mAb 5C6 and T3D-specific mAb 9BG5 was observed, confirming the utility of this assay for quantification of viral infectivity and infectivity blockade. The imager-based viral infectivity assay fully automates data collection and provides an important advance in technology for applications such as screening for novel modulators of viral infectivity. This basic platform can be adapted for use with multiple viruses and cell types.     

Anthracyclines: recent developments in their separation and quantitation

Anthracyclines are among the most widely used anticancer agents. Notwithstanding the large efforts to develop new drugs with a better pharmaceutical profile, daunorubicin, doxorubicin, epirubicin and idarubicin are still the most used in clinical practice. Many efforts are now ongoing to reduce the side effects by using pharmaceutical formulations able to release the drug in the most appropriate way and monitoring the quantity of anthracyclines and their metabolites in the body fluids or tissues frequently and in every patient to maintain the drug concentration within the expected range. This review describes the most recent developments in the separation and quantitation of the above clinically useful drugs, together with their principal metabolites. Some less widely used derivatives will also be considered.     

A rapid method for the quantitation of haemoglobin A2.

A rapid accurate and inexpensive technique for the measurement of haemoglobin A2 has been devised. It uses a small plastic device which embodies a miniature chromatographic column and reservoirs for the eluting buffer and eluates. The device is charged with whole blood, pH 8.25 buffer and centrifuged. The eluate is removed and the centrifugation repeated using pH 7.0 buffer to remove the remaining haemoglobin. The haemoglobin concentration in the 2 volumes of eluates is measured in a spectrophotometer at 414 nm and the percentage of HbA2 calculated. Results are similar to those obtained by standard methods.     

Computer assisted quantitation of terminal hepatic vein connective tissue in the rat.

Computer assisted image analysis has been used to quantify the cellular and extracellular connective tissue component of rat liver terminal hepatic venules, in control animals and those exposed to 40% ethanol in drinking water. A significant relationship existed between the size of the terminal hepatic venule and the amount of connective tissue it contained in 14 of 15 controls and 17 of 18 ethanol exposed rats. Thickening of the terminal hepatic vein wall assessed to be present in ethanol treated rats by direct observation was confirmed by image analysis in all cases (p < 0.01). Significant differences between treated and control livers (p < 0.05) were detected by image analysis when not apparent to human observers. Sensitive quantitative assessment of terminal hepatic vein wall thickening was thus achieved by computerized analysis of liver sections.     

Quantifying continuous-flow dielectrophoretic trapping of cells and micro-particles on micro-electrode array

An interdigitated electrode array embedded within a micro-channel with forced flow is shown to enable dielectrophoretic (DEP) characterization of particles and/or cells based on measurements of their trapping percentage over a continuous frequency range. A simplified model of the trapping percentage, using spatial averaging of the convective and DEP force, linearly correlated it to the effective DEP force (in its positive mode). Thus, the Clausius–Mossotti factor was fitted to the experimental data, yielding effective electrical characteristics of the particles and/or cells. Also, the generated trapping percentage curve response over a continuous range of frequencies facilitates sorting and detection based on differences other than just the cross-over frequencies.     

A rapid rosette technique for quantitation and separation of mononuclear cell subsets using monoclonal antibodies

We present a rapid and simple method for simultaneous quantitation and separation of mononuclear cell (MNC) subsets. When lymphoid cells are sensitized with monoclonal antibodies of the OK and Leu series, they rapidly form rosettes with ox erythrocytes (ORBC) coated with affinity-purified rabbit IgG against mouse IgG. Rosette-forming cells (RFC) may then be counted and separated from non-rosetting MNC by Isopaque-Ficoll gradient centrifugation. The yield and viability are close to 100% after ORBC lysis. Adherent cells do not interfere. Isolated T8⁺ and Leu3a⁺ cells were further tested: the purity was 97–99%, and the cells were functionally intact with respect to their modulating activity on the generation of immunoglobulin-secreting cells by MNC after stimulation with pokeweed mitogen.     

Measurement of immunoperoxidase activity. A rapid and reproducible immunoassay for quantitation of cellular antigens.

A procedure is described making it possible to obtain quantitative data with the immunohistochemical peroxidase anti--peroxidase (PAP) technique. After applying PAP on fixed fibroblast preparations, peroxidase enzymic activity is measured on solubilized immune-complexes. The results show this technique to be sensitive and reproducible. Differences in the amounts of the same antigen (alpha2-macroglobulin) are detected in differently treated fibroblast preparations. The value of the technique for the demonstration of cellular antigens is discussed.