An antigen-targeting assay for Lyme disease: Combining aptamers and SERS to detect the OspA protein.

Lyme disease is the fastest growing vector-borne disease in the United States. However, current testing modalities are ill suited to detection of Lyme disease, leading to the diagnosis of many cases after treatment is effective. We present an improved, direct method Lyme disease diagnosis, where the Lyme specific biomarker Outer Surface Protein A (OspA) in clinical serum samples is identified using a diagnostic platform combining surface enhanced Raman scattering (SERS) and aptamers. Employing orthogonal projections to latent structures discriminant analysis, the system accurately identified 91% of serum samples from Lyme patients, and 96% of serum samples from symptomatic controls. In addition, the OspA limit-of-detection, determined to be 1 × 10-4 ng/mL, is greater than four orders of magnitude lower than that found in serum samples from early Lyme disease patients. The application of this platform to detect this difficult-to-diagnose disease suggests its potential for detecting other diseases that present similar difficulties.

Improved cell sensitivity and longevity in a rapid impedance-based toxicity sensor.

A number of toxicity sensors for testing field water using a range of eukaryotic cell types have been proposed, but it has been difficult to identify sensors with both appropriate sensitivity to toxicants and the potential for long-term viability. Assessment of bovine pulmonary artery endothelial cell (BPAEC) monolayer electrical impedance with electric cell-substrate impedance sensing (ECIS) showed promise in a previous systematic evaluation of toxicity sensor technologies. The goal of the study reported here was to improve toxicant responsiveness and field portability of this cell-based toxicity sensor. A variety of human cells, non-human mammalian cells, and non-mammalian vertebrate cells were screened for sensitivity to 12 waterborne industrial chemicals. The results of this assessment show that bovine lung microvessel endothelial cell (BLMVEC) monolayers and iguana heart (IgH-2) cell monolayers could detect nine out of the 12 waterborne industrial chemicals, an improvement over the seven chemicals previously detected using BPAEC monolayers. Both the BLMVEC and IgH-2 cell monolayers were tested for their ability for long-term survival on the ECIS test chips in a laboratory environment. Both cell lines were able to maintain high impedance readings on the ECIS electrodes for 37 days, a key trait in developing a field-portable toxicity sensor for water. Cell line optimization has greatly contributed to the on-going development of a field-portable cell-based biosensor that detects with sensitivity a wide range of waterborne toxicants.

Prolonged stochastic single ion channel recordings in S-layer protein stabilized lipid bilayer membranes.

S-layer proteins are commonly found in bacteria and archaea as two-dimensional monomolecular crystalline arrays as the outermost cell membrane component. These proteins have the unique property that following disruption by chemical agents, monomers of the protein can re-assemble to their original lattice structure. This unique property makes S-layers interesting for utilization in bio-nanotechnological applications. Here, we show that the addition of S-layer proteins to bilayer lipid membranes increases the lifetime and the stability of the bilayer. M2delta ion channels were functionally incorporated into these S-layer stabilized membranes and we were able to record their activity for up to 20 h. Transmission electron microscopy (TEM) was used to visualize the 2D crystalline pattern of the S-layer and the M2delta ion channel characteristics in bilayer lipid membrane's were compared in the presence and absence of S-layers.

Development of a mast cell-based biosensor.

A mast cell-based biosensor has been developed to enable the use of these cells in numerous applications including pharmaceutical screening, environmental monitoring, clinical diagnosis and homeland security. Rat basophilic leukemia (RBL) mast cells offer excellent potential for biosensor applications because they are robust and undergo a dramatic exocytotic response within minutes of antigen addition. To monitor mast cell activation, fluorescent dyes were loaded into the cells and used as indicators of alkalinization of secretory granules, calcium fluxes or generation of reactive oxygen species. These fluorescence assays efficiently measure activation of antigen-stimulated RBL mast cells, detecting the antigen with picomolar sensitivity. To demonstrate the utility of this mast cell-based biosensor for detection of microbial pathogens, an IgE chimeric protein was created by fusing the Fc region of the IgE antibody to CD14, a receptor for lipopolysaccharide. This chimeric protein has the capacity to bind to Escherichia coli and Listeria monocytogenes and also to IgE receptors on the mast cells, thereby stimulating a signaling response to bacteria. RBL mast cells labeled with the calcium indicator Fluo-4 are shown to be responsive to E. coli, only when sensitized with the chimeric protein, thus demonstrating a highly versatile biosensor for bacterial contamination.

Detection of single bacterial pathogens with semiconductor quantum dots.

Semiconductor quantum dots (QDs) have been used in a simple fluorometric assay to detect single cells of the pathogenic Escherichia coli O157:H7 serotype. Composed of CdSe/ZnS core/shell QDs conjugated to streptavidin, this system exhibits 2 orders of magnitude more sensitivity than a similar assay using a common organic dye. Selectivity for this pathogenic bacterial strain over a common lab strain (E. coli DH5alpha), which is gained from the use of specific biotinylated antibodies, is also demonstrated for QD labeling. Under continuous excitation, these QDs retain high fluorescence intensities for hours, whereas a typical organic dye bleaches within seconds, allowing for more rapid and accurate identification of E. coli O157:H7 in single-cell fluorescence-based assays. This indirect QD labeling method, based on antibody-antigen and streptavidin-biotin interactions, is flexible enough to expand to other systems and has great potential for use in simultaneous multicolor detection schemes.

In situ measurement of degranulation as a biosensor based on RBL-2H3 mast cells.

A direct degranulation assay has been developed to enable the use of RBL mast cells as a biosensor for screening chemical libraries for drug discovery and environmental toxicity evaluation. Release of beta-hexosaminidase into the extracellular milleu is widely used to characterize cellular components and mechanisms involved in stimulated exocytosis, including those initiated by crosslinking of IgE receptors on mast cells. To adapt this versatile assay for high throughput screening, we developed a direct, in situ method in which beta-hexosaminidase detection is carried out in a single step, convenient for multi-sample processing and thus for biosensor applications. This direct assay is efficient for measuring exocytosis in antigen-stimulated RBL mast cells, detecting antigen concentrations as low as 1 pM. We also demonstrate its utility in detecting inhibition of degranulation by a known pharmacologic inhibitor that blocks Syk tyrosine kinase activity critical for cell activation.