Microfabricated flow cytometers can detect, count, and analyze cells or particles using microfluidics and electronics to give impedance-based characterization. Such systems are being developed to provide simple, low-cost, label-free, and portable solutions for cell analysis. Recent work using microfabricated systems has demonstrated the capability to analyze micro-organisms, erythrocytes, leukocytes, and animal and human cell lines. Multifrequency impedance measurements can give multiparametric, high-content data that can be used to distinguish cell types. New combinations of microfluidic sample handling design and microscale flow phenomena have been used to focus and position cells within the channel for improved sensitivity. Robust designs will enable focusing at high flowrates while reducing requirements for control over multiple sample and sheath flows. Although microfluidic impedance-based flow cytometers have not yet or may never reach the extremely high throughput of conventional flow cytometers, the advantages of portability, simplicity, and ability to analyze single cells in small populations are, nevertheless, where chip-based cytometry can make a large impact. ' 2010International Society for Advancement of Cytometry Key terms microfluidics; impedance characterization; label free; single cell analysis; hydrodynamic focusing; sorting MICROFLUIDIC flow cytometers can bring many advantages to the field of flow cytometry (FCM). Compared to typical flow cytometry channel sizes, the miniaturized dimensions permit microfluidic systems to analyze single cells, to identify cellular variability in gene expression, or drug response within a cell population. Chipbased cytometers can have lower size and costs than conventional benchtop instruments, and may be portable. Today, the developmental aim for microfluidic systems is to reach the same sensitivity and capability for multiparametric analyses as delivered by conventional flow cytometers. Many efforts have been made to improve existing devices and to create new miniaturized high-end instruments. Microfluidic chips can incorporate on-chip cell preparation, cell culture, lysis, and modules for optical, electrophoretic, or genomic analysis (1). They are also suitable for analysis of cells in suspension as well as adherent cells.Miniaturized cytometry devices will have high impact in the development of point-of-care devices in developing countries. Accurate CD4 1 T-cell counts are used to monitor human immunodeficiency virus (HIV)-infected patients, and various thresholds of the number of CD4 1 T lymphocytes per ll of whole blood are used to start antiretroviral therapy (2-5). A simple, single-purpose CD4 cell counting device, which does not require standard laboratory equipment or trained laboratory personnel, could help some of the 33 million HIV-infected people worldwide monitor the stage of infection (6)(7)(8). In this application, increased analysis throughput is a secondary concern compared to increased sensitivity and specificity. Portable, miniatu...

Lipid bilayers in nanopores are stable for days. The stability depends on the nature of the lipid as well on the pore diameter as investigated by impedance spectroscopy. Bilayers formed of palmitoyl‐oleoyl‐phosphatidylcholine are stable up to 4 days in 200 nm pores regularly arrayed in a 300 nm thin silicon nitride support. The transport of K+ across bilayers by valinomycin demonstrates that this support material has a high potential to monitor functions of membrane proteins.

IntroductionAn efficient and reliable method to estimate plant cell viability, especially of pollen, is important for plant breeding research and plant production processes. Pollen quality is determined by classical methods, like staining techniques or in vitro pollen germination, each having disadvantages with respect to reliability, analysis speed, and species dependency. Analysing single cells based on their dielectric properties by impedance flow cytometry (IFC) has developed into a common method for cellular characterisation in microbiology and medicine during the last decade. The aim of this study is to demonstrate the potential of IFC in plant cell analysis with the focus on pollen.MethodDeveloping and mature pollen grains were analysed during their passage through a microfluidic chip to which radio frequencies of 0.5 to 12 MHz were applied. The acquired data provided information about the developmental stage, viability, and germination capacity. The biological relevance of the acquired IFC data was confirmed by classical staining methods, inactivation controls, as well as pollen germination assays.ResultsDifferent stages of developing pollen, dead, viable and germinating pollen populations could be detected and quantified by IFC. Pollen viability analysis by classical FDA staining showed a high correlation with IFC data. In parallel, pollen with active germination potential could be discriminated from the dead and the viable but non-germinating population.ConclusionThe presented data demonstrate that IFC is an efficient, label-free, reliable and non-destructive technique to analyse pollen quality in a species-independent manner.

Using conserved fingerprints in the glycosyltransferase (GTase) domain of high-molecular-weight penicillin-binding proteins (PBP), a gene (mgt) encoding a putative monofunctional glycosyltransferase has been identified in Haemophilus influenzae and in other bacterial species. Here we report the cloning of the homologous Escherichia coli gene and show that the solubilised membrane fraction of E. coil cells overexpressing the mgt gene contain a significantly increased peptidoglycan synthesis activity. In contrast to the high-molecular-weight PBPs, this activity is not inhibited by Flavomycin.

We report here a new mode of coupling between the chemical and vectorial reaction explored for the oxaloacetate decarboxylase Na+ pump from Klebsiella pneumoniae. The membrane‐bound beta‐subunit is responsible for the decarboxylation of carboxybiotin and the coupled translocation of Na+ ions across the membrane. The biotin prosthetic group which is attached to the alpha‐subunit becomes carboxylated by carboxyltransfer from oxaloacetate. The two conserved aspartic acid residues within putative membrane‐spanning domains of the beta‐subunit (Asp149 and Asp203) were exchanged by site‐directed mutagenesis. Mutants D149Q and D149E retained oxaloacetate decarboxylase and Na+ transport activities. Mutants D203N and D203E, however, had lost these two activities, but retained the ability to form the carboxybiotin enzyme. Direct participation of Asp203 in the catalysis of the decarboxylation reaction is therefore indicated. In addition, all previous and present data on the enzyme support a model in which the same aspartic acid residue provides a binding site for the metal ion catalysing its movement across the membrane. The model predicts that asp203 in its dissociated form binds Na+ and promotes its translocation, while the protonated residue transfers the proton to the acid‐labile carboxybiotin which initiates its decarboxylation. Strong support for the model comes from the observation that Na+ transport by oxaloacetate decarboxylation is accompanied by H+ transport in the opposite direction. The inhibition of oxaloacetate decarboxylation by high Na+ concentrations in a pH‐dependent manner is also in agreement with the model.