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United States Department of Agriculture

Agricultural Research Service

Research Project: PATHOGEN FATE AND TRANSPORT IN IRRIGATION WATERS

Location: Environmental Microbial and Food Safety Laboratory

Title: Ultra-sensitive detection using integrated waveguide technologies

Authors
item Li, Shuhong
item Kiefer, Lynda
item Zhu, Piexuan
item Shelton, Daniel
item Amstutz, Pete
item Tang, Cha-Mei

Submitted to: Book Chapter
Publication Type: Book / Chapter
Publication Acceptance Date: April 15, 2011
Publication Date: July 25, 2011
Citation: Li, S., Kiefer, L., Zhu, P., Shelton, D.R., Amstutz, P., Tang, C. 2011. Ultra-sensitive detection using integrated waveguide technologies. In: Somerset, V. Environmental Biosensors. Rijeka, Croatia: InTech. p.305-313.

Interpretive Summary: Scientists from ARS and Creatv MicroTech have collaboratively developed a new fluorescence detection method based on integrating waveguide technology (IWT). IWT achieves high sensitivity by maximizing the signal while minimizing background noise. IWT assays can be conducted either in a solid-phase or liquid-phase format. In the solid-phase IWT format, analytes are first captured on the inner surface of the capillary tube or cuvette; while in the liquid-phase IWT format, the analyte is in solution inside of the cuvette. Assays have been developed for both formats. For example, solid-phase IWT assay have been published for both Escherichia coli O157 cells Bacillus anthracis spores, where cell/spores were captured on the inner surface of an open ended capillary tube and analyzed using an instrument called SignalyteTM; dection limits were 10 cells and 1000 spores, respectively. More recently, liquid-phase assays have been developed for E. coli O157 cells using the SignalyteTM-II. Assays were developed using two different formats. One was based on immunomagnetic separation (IMS) techniques with a sensitivity of 10 cells in a 1 mL sample. The other format included filtration of cell-antibody complexes, followed by washing and release of the antibody with a low pH buffer. The sensitivity of this assay was 1000 cells; but since large sample volumes can be analyzed, the sensitivity is between 10-100 cells per mL. Other applications are also presented. This new method should be useful to laboratories analyzing water, manure and/or produce for the presence of pathogens.

Technical Abstract: There is a pressing need to detect analytes at very low concentrations, such as food- and water-borne pathogens (e.g. E. coli O157:H7) and biothreat agents (e.g., anthrax, toxins). Common fluorescence detection methods, such as 96 well plate readers, are not sufficiently sensitive for low concentrations. We describe here a novel detection principle---integrating waveguide technology (IWT)---that allows for greater sensitivity. The ultra-sensitive fluorescence detection instrument, SignalyteTM-II, is based on Integrating Waveguide Technology. The sensitivity is achieved by maximizing the signal while minimizing background noise. A very sensitive E. coli O157:H7 detection assay based on immunomagnetic separation (IMS) techniques was developed and fluorescent signal was tested on SignalyteTM-II. Testing data demonstrate that as few as 10 cfu/ml of E. coli in a 1 ml sample is detectable. Another application of Integrating Waveguide Technology is in flow-through format. We demonstrate a real time fluorescence detection system using Cy5 dye. The flow through format can also be adapted to luminescence detection. We developed an in-line detection system of treated waste water using ATP assay. Testing result shows that 100 cells of E. coli in a 100 mL sample of reclaimed waste water is detectable.

Last Modified: 10/21/2014