Final Report: Nanoparticle-Enhanced Immunoassay for Monitoring Organic PollutantsEPA Contract Number: EPD04055
Title: Nanoparticle-Enhanced Immunoassay for Monitoring Organic Pollutants
Investigators: Bastiaans, Glenn J.
Small Business: Intelligent Optical Systems Inc.
EPA Contact: Manager, SBIR Program
Project Period: April 1, 2004 through June 30, 2005
Project Amount: $224,996
RFA: Small Business Innovation Research (SBIR) - Phase II (2004) Recipients Lists
Research Category: Small Business Innovation Research (SBIR) , Nanotechnology , SBIR - Nanotechnology
Intelligent Optical Systems, Inc. (IOS) has demonstrated the feasibility of achieving highly effective assays for organic pollutants on a continuous basis by adapting known principles of immunoassays in an innovative way. This achievement will make it possible to develop field-deployable monitoring systems to guard against the presence and effects of organic pollutants and other harmful substances in water. Immunoassays allow the highly selective detection of a wide variety of substances of organic and biological origin using a very sensitive approach. IOS is developing a technology to conduct these selective and sensitive assays in a continuous or periodic manner.
Phase I demonstrated the feasibility of detecting polyaromatic hydrocarbons down to concentrations of 10 parts per billion or less using this new technique. Improved limits of detection may be expected through refinement of spectroscopic labeling techniques. Objectives of the Phase II research included expanding the number of classes of compounds that can be assayed and improving the Phase I immunoassay design.
Following the success of Phase I, improved methods of developing the immunoassay, the bead substrate, and the instrument were sought in Phase II to expand the system beyond detection of only phenanthrene (Phen). Atrazine (ATZ) was chosen as a second target analyte for assay development. Although ATZ was a more desirable target for detection, it proved to possess characteristics that challenged the parameters established for the Phen assay. A different strategy to attach surrogate antigens to quantum dots (QDs) had to be demonstrated.
The major objectives of this Phase II work were to refine the displacement immunoassay technology to the point where prototypes of contaminant monitors could be field tested in collaboration with industrial partners. In Phase II, refinement of the microsphere solid substrate used for immobilization of the displacement immunoassay reagents was done by evaluating a variety of bead materials. Beads were used in a solid phase so they could be sequestered onto porous polymer membranes and placed in flow cells where the displacement immunoassay reagents could be continuously exposed to a flowing water sample in a filtration arrangement. This design allows the continuous immunodetection of targeted organic pollutants present in the flowing liquid sample. Decreases in levels of the optical luminescence due to the displacement of the QD-labeled surrogate antigen portion of the immunoassay reagent (indicative of the amount of pollutants present) were detected in a very sensitive manner.
The Phase I immunoassay for polyaromatic hydrocarbons demonstrated the detection of pyrene. In Phase II, an improved displacement immunoassay was demonstrated for the detection of phenanthrene using the same monoclonal antibody. Secondly, a displacement immunoassay for atrazine was developed using a different monoclonal antibody. Additional surrogate antigens were developed for the atrazine assay. The pesticide atrazine is the most commonly detected contaminant that occurs above the U.S. Environmental Protection Agency-mandated maximum concentration limit in monitored water systems. The chemical name of atrazine is 2-chloro-4-ethylamino-6-isopropylamino-s-triazine. A surrogate antigen for atrazine that can be readily coupled to QDs through a primary amine group is one of its degradation products, 2-amino-4-chloro-6-ethylamino-s-triazine. In both cases, however, fluorescent dye labeling of the surrogate antigens was required to obtain immunoassays of moderate sensitivity. Much was learned about QD labeling of the surrogate antigens, but a functional assay with QD labeling was not brought to common practice.
Several variations of a porous membrane flow cell were developed. QD-labeled and dye-labeled microspheres were loaded successfully on the porous membranes within the cells, and fluorescence was observed from the trapped microspheres. One variation of the flow cell, comprised of thin flow channels laser machined into thin plastic sheets, was co-developed by a subcontractor. This flow cell design subsequently was developed into a commercial product by the subcontractor.
Several variations of a bench level optical fluorescence detection system were developed. These prototypes used light emitting diodes or arrays of diodes as the light source and a photodiode or commercial photodiode array spectrometer as the light detector. All the prototypes relied on the use of a bifurcated optical fiber bundle to conduct excitation light to the sample and to collect fluorescent emission from the sample. A field deployable prototype instrument was not fabricated in this work. Complications in the development of QD-labeled surrogate antigens resulted in the use of extra resources in an attempt to resolve the research problem. Resources originally planned for the fabrication of a field deployable unit, as well as corporate internal research and development funds, were used to pursue additional work with QD labels.
Because of the unsuitability of commercial QD labels for immunoassay labeling applications, minimal progress was made in refining the displacement immunoassay technology to the point where prototypes of contaminant monitors could be field tested in collaboration with industrial partners. As a result, the pursuit of the commercial opportunities established in the Phase I work was postponed. The development of the displacement immunoassay, however, has been significantly advanced during the Phase II work. Reduction of this assay to a field-ready technique and development of a field deployable instrument is expected to be achieved with additional funding obtained for a related, non-overlapping application. Thus, the commercialization of this monitoring technology is still quite possible.