Claire K. Sanders

Separation of Escherichia coli bacteria from peripheral blood mononuclear cells using standing surface acoustic waves.

A microfluidic device was developed to separate heterogeneous particle or cell mixtures in a continuous flow using acoustophoresis. In this device, two identical surface acoustic waves (SAWs) generated by interdigital transducers (IDTs) propagated toward a microchannel, which accordingly built up a standing surface acoustic wave (SSAW) field across the channel. A numerical model, coupling a piezoelectric effect in the solid substrate and acoustic pressure in the fluid, was developed to provide a better understanding of SSAW-based particle manipulation. It was found that the pressure nodes across the channel were individual planes perpendicular to the solid substrate. In the separation experiments, two side sheath flows hydrodynamically focused the injected particle or cell mixtures into a very narrow stream along the centerline. Particles flowing through the SSAW field experienced an acoustic radiation force that highly depends on the particle properties. As a result, dissimilar particles or cells were laterally attracted toward the pressure nodes at different magnitudes, and were eventually switched to different outlets. Two types of fluorescent microspheres with different sizes were successfully separated using the developed device. In addition, Escherichia coli bacteria premixed in peripheral blood mononuclear cells (PBMCs) were also efficiently isolated using the SSAW-base separation technique. Flow cytometric analysis on the collected samples found that the purity of separated E. coli bacteria was 95.65%.

Correlating light scattering with internal cellular structures.

The origins of side scattering from a fibroblast and cervical cell line were determined by comparing side-scatter images with images stained for lysosomes, nuclei, and mitochondria on a cell by cell basis. Lysosomes or nuclei are the most efficient type of scatterer depending on the cell type and incident light polarization. The relative scattering efficiencies of lysosomes and mitochondria were the same for both cell lines, while the scattering efficiencies of the nuclei differed. The percent of 90° scattering from the nucleus, mitochondria, and lysosomes as well as the group of other internal cellular objects was estimated. The nucleus was the largest contributor to side scatter in the cervical carcinoma cells. The contributions of lysosomes, mitochondria, the nucleus, and particles unstained by either Hoechst, LysoSensor or MitoTracker ranges from ∼20% to ∼30% in fibroblast cells. The contribution of lysosomes to side scatter was much stronger when the incident light was polarized perpendicular to the scattering plane than when the polarization of the side scatter laser was parallel to the scattering plane. This dependence on side scatter polarization indicates that lysosomes contain scattering structures that are much smaller than the wavelength of light used in the measurements (785 nm). In conclusion, mitochondria were not found to be either the most efficient scatterer or to have the largest contribution to scattering in either cell line, in contrast to previous reports. Rather lysosomes, nuclei and unknown particles all have significant contributions to 90° scattering and the contributions of some of these particles can be modulated by changing the polarization of the incident light.

Evaluation of ionic liquids on phototrophic microbes and their use in biofuel extraction and isolation

Multiple ionic liquids (ILs) were assessed for their ability to extract branched, unsaturated hydrocarbons from an aqueous medium. In addition, IL cytotoxicity studies were performed on two phototrophic microbes, Synechocystis sp. PCC6803 and Botryococcus braunii var Showa. The optimum IL for use in an isoprenoid hydrocarbon extraction may vary based on the biological source of the isoprenoids. Our results suggest that ionic liquids have the potential to serve as novel biocompatible milking agents for extracting high-value chemicals from the microbes, with toxicity to both species minimized by considerations of ionic liquid structure and hydrophobicity.

A yEGFP-based reporter system for high-throughput yeast two-hybrid assay by flow cytometry.

The yeast two‐hybrid (Y2H) assay is a widely used method to study protein–protein interactions, a major objective in postgenome research. Despite the tremendous utility of the Y2H assay, several issues including accuracy, speed, automation, and cost‐effectiveness limit its application in high‐throughput analysis. We have created an improved Y2H assay reporter system by integrating the codon‐optimized yeast enhanced green fluorescent protein (yEGFP) gene into the ADE2 locus of the AH109 yeast strain and evaluated reporter expression using the strong and weak triggers via flow cytometry. We also performed flow cytometry‐based Y2H assays in liquid cultures as well as in a cDNA library screening. We have shown that yEGFP, but not EGFP, is a sensitive Y2H reporter for flow cytometric detection. We also show that this yEGFP‐based Y2H could be easily adapted to high‐throughput format without conventional agar plating. Moreover, our data demonstrate that this system can be used for cDNA library screening. We have developed sensitive and efficient flow cytometry‐based Y2H assay system that is well suited for large‐scale protein–protein interaction identification and characterization. When compared with the conventional plate and filter membrane‐based nutrient and colorimetric analysis, our flow cytometric assay offers convenient, quantitative, and faster reporter analysis compatible with existing liquid handling robots.

Quantitative analysis of the effect of cell type and cellular differentiation on protective antigen binding to human target cells.

We quantitatively measured protective antigen (PA) binding to human cells targeted by anthrax lethal toxin (LT). Affinities were less than 50 nM for all cells, but differentiated cells (macrophages and neutrophils) had significantly increased PA binding and endothelial cells demonstrated the most binding. Combined with the function of such cells, this suggests that PA receptors interact with the extracellular matrix and that differentiation increases the number of PA‐specific receptors, which supports previously observed differentiation‐induced LT susceptibility. Our results quantifiably confirm that the generality of PA binding will complicate its use as a tumor targeting agent.

Advantages of full spectrum flow cytometry

Abstract. A charge coupled device-based flow-cytometer for the measurement of full spectra was implemented and characterized. The spectral resolution was better than 1.5 nm and the coefficient of variation for fluorescence from flow check beads was 5% or better. Both cell and bead data were analyzed by fitting to measured component spectra. Separation of flow check and align flow beads, which have similar spectra, was nearly identical whether using a spectral analysis or a scatter analysis. After mixing, cells stained with ethidium bromide or propidium iodide were measured at different timepoints. The contribution of these 12 nm separated emission spectra could be separately quantified and the kinetic process of the samples becoming homogeneous due to fluorophor dissociation and rebinding was observed. Principle component analysis was used to reduce noise and alternating least squares (ALS) was used to analyze one set of noise-reduced cell data without knowledge of the component spectra. The component spectra obtained via ALS are very similar to the measured component spectra. The contributions of ethidium bromide and propidium iodide to the individual spectra are also similar to those obtained via the spectral fitting procedure.

Binding and cell intoxication studies of anthrax lethal toxin.

Anthrax lethal toxin (LT) is a major virulence factor of Bacillus anthracis. The vast majority of the anthrax toxin-related literature describes the assembly of LT as a cell-dependent process. However, some reports have provided evidence for the existence of a fully assembled LT, either in vitro or in the bloodstream of anthrax-infected animals. To follow up on this work, we present studies on fully-assembled LT. We first demonstrate facile and cell-free assembly and purification of LT. We then show that fully assembled LT binds an anthrax toxin receptor with almost 100-fold higher affinity than the protective antigen (PA) alone. Quantitative cell intoxication assays were used to determine the LD50 (lethal dose 50) for LT. The cell-binding studies revealed that LT binds mammalian cells using a different mode from PA. Even when PA-specific receptors were blocked, fully assembled LT was able to bind the cell surface. Our studies support the existing evidence that LT fully assembles in the blood stream and can bind and intoxicate mammalian cells with very high affinity and efficacy. More importantly, the data presented here invoke the possibility that LT may bind cells in a receptor-independent fashion, or recognize receptors that do not interact with PA. Hence, blood borne LT may emerge as a novel therapeutic target for combating anthrax.

Effects of acetic acid on light scattering from cells

Acetic acid has been used for decades as an aid for the detection of precancerous cervical lesions, and the use of acetic acid is being investigated in several other tissues. Nonetheless, the mechanism of acetowhitening is unclear. This work tests some of the hypotheses in the literature and measures changes in light scattering specific to the nucleus and the cytoplasm. Wide angle side scattering from both the nucleus and the cytoplasm increases with acetic application to tumorigenic cells, with the increase in nuclear scattering being greater. In one cell line, the changes in nuclear scattering are likely due to an increase in number or scattering efficiency of scattering centers smaller than the wavelength of excitation light. There are likely several cellular changes that cause acetowhitening and the cellular changes may differ with cell type. These results should lead to a better understanding of acetowhitening and potentially the development of adjunct techniques to improve the utility of acetic acid application. For the well-studied case of cervical tissue, acetowhitening has been shown to be sensitive, but not specific for oncogenic changes needing treatment.

Acoustic lysis of vegetative bacterial cells: Method and device development

A critical problem of many pathogen detection assays is the availability of intracellular protein and deoxyribonucleic acid (DNA). Acoustic lysis of suspended vegetative bacterial cells in a microfluidic system offers several advantages over conventional lysis techniques. The intracellular proteins and DNA are released and available for detection. A novel acoustic lysing alternative technique to the existing lysing methods for sample preparation and lysis step is proposed. We report here an efficient lysis device that uses acoustic excitation for performing lysis of Gram-positive and Gram-negative vegetative cells and has a high yield in a short amount of time. We also verified the condition of released protein since one of the major uses of vegetative cells lysis is for protein expression studies. Fluorimetry and flow cytometry were used to assess the degree of damage induced on the cells by the actual lysis method. The acoustic device allows the delivery of proteins in a non-denatured form, without adding chemicals, particles or other substances (e.g. enzymes) that could complicate the process or the detection procedure. The lysis device operates at low power (50–400 mW) and short time (3 min) and has high efficiency in comparison to current lysis standards (>85% vs. 12–50%).

The contribution of specific organelles to side scatter

Knowledge of which cellular structures scatter light is needed to fully utilize the information available from light scattering measurements of cells and tissues. To determine how specific organelles contribute to light scattering, wide angle side scattering was imaged simultaneously with fluorescence from specific organelles for thousands of cells using flow cytometry. Images were obtained with different depth of field conditions and analyzed with different assumptions. Both sets of data demonstrated that mitochondria and lysosomes, contribute similarly to side scatter. The nucleus contributes as much or more light scatter than either the mitochondria or the lysosomes.