Final Report: Nanoparticle-Enhanced Immunoassay for Monitoring Organic PollutantsEPA Contract Number: 68D03026
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, 2003 through September 1, 2003
Project Amount: $69,996
RFA: Small Business Innovation Research (SBIR) - Phase I (2003) RFA Text | Recipients Lists
Research Category: Nanotechnology , SBIR - Nanotechnology , Small Business Innovation Research (SBIR)
This Phase I research project successfully demonstrated, for the first time, the feasibility of developing a continuous monitor for environmental contaminants in water using immunoassay methodology. Relatively large polystyrene microspheres were used as the solid phase in the nanoparticle-labeled heterogeneous displacement immunoassay. These microspheres were obtained in three variations with three different proteins immobilized on their surfaces to allow different methods of antibody attachment to their surfaces. Methods were developed to image the microspheres in both their initial nonfluorescent state and after fluorescent labels had been attached to their surface. The methods also were developed to quantitatively measure the fluorescence of the labeled microspheres in an aqueous suspension.
Intelligent Optical Systems, Inc. (IOS), demonstrated the feasibility of achieving highly effective assays for organic pollutants on a continuous basis by adapting known immunoassay principles in an innovative approach. A displacement immunoassay was demonstrated that could detect as little as 14 parts per billion (ppb) of phenanthrene, an organic water contaminant. To enhance the sensitivity and robustness of the assay, quantum dot (QD) nanoparticles were employed as luminescent labels to achieve highly sensitive detection. A second innovation was to use two complementary sets of solid-phase microspheres to: (1) serve as a site for displacement immunoassay to occur; and (2) collect and concentrate displaced, labeled surrogate antigen to achieve highly sensitive pollutant detection. The use of two sets of microspheres, in turn, allowed the implementation of an innovative method for achieving continuous or periodic monitoring.
A third innovation, which will be included in the Phase II research project, is to embed the two sets of microspheres into two different porous polymer membranes. These membranes will be placed in a flow cell and continuously exposed to a flowing water sample in a filter arrangement. This design will allow for the continuous immunodetection of targeted organic pollutants present in the flowing water sample. The proposed innovative arrangement will result in the design of an elegant field-deployable system to monitor organic pollutants in water.
To achieve the goals for this Phase I research project, an initialization task and the following seven tasks were planned and executed: (1) prepare and coat QDs, (2) bioconjugate surrogate antigen to luminescent QDs, (3) immobilize capture antibody on polymer beads, (4) demonstrate displacement immunoassay for polyaromatic hydrocarbons (PAHs), (5) plan the Phase II research project, (6) explore the commercial potential, and (7) prepare and submit the report.
Through the performance of these tasks, all goals were achieved and are summarized in the following sections.
Fluorescence Measurement Systems. As part of the project initialization task, protocols for the measurement of fluorescence from microspheres and from QD solutions were developed. For quantitative measurements, a commercial Spex fluorolog spectrometer was used. To visualize fluorescence from individual microspheres, an Olympus fluorescence microscope was used. To validate the quantitative fluorescence measurement system, solutions containing commercial fluorescent microspheres (Magsphere) were placed in a sample cell, and excitation and emission spectra were obtained. These spectra displayed good signal-to-noise ratios and were free from any interference. To validate the fluorescence imaging process using the fluorescent microscope, polymer beads coated with strepavidin were imaged before and after labeling with dye particles. The detection of individual labeled beads was easily achieved.
Prepare and Coat QDs. The QDs used in this work were obtained commercially from Evident Technologies (EviTags).
Bioconjugate Surrogate Antigen to Luminescent QDs. To implement a displacement immunoassay, a labeled surrogate antigen is required. This surrogate antigen is initially complexed to an immobilized antibody. The surrogate antigen for the PAH displacement immunoassay developed in this project was 1-aminopyrene (1-AP). This compound was covalently bonded to the QDs. To confirm that 1-AP coupled to the QDs, measurements of the luminescence from the QDs were made before and after coupling to 1-AP. This procedure provided labeled surrogate antigen for use in the displacement immunoassay.
Immobilize Capture Antibody on Polymer Beads. The displacement immunoassay implemented in this project requires a solid phase for immobilization of the antibody, so that free and bound antigens can be separated. Polystyrene microspheres were used as solid phases in the experimental work. An antibody to PAHs was immobilized to the surfaces of the microspheres. Spectroscopic examination of the complex formed between the immobilized antibodies and the labeled surrogate antigen revealed a shift in the QD fluorescence spectrum. The spectral changes, however, did not prevent demonstration of the displacement immunoassay.
Demonstrate Displacement Immunoassay for PAHs. The critical feasibility test of the proposed displacement immunoassay technology was the demonstration that a different PAH, in this case phenanthrene, could displace the labeled surrogate antigen (1-AP-QD) from its immunocomplex with the immobilized antibody x-PAH. This displacement was demonstrated using sample solutions of phenanthrene having concentrations over the range of 14 to 140 ppb. Observation of fluorescence spectra provided a clear indication that a displacement immunoassay to detect low concentrations of PAHs in water is feasible. A phenanthrene concentration as low as 14 ppb generated an easily detectable fluorescence signal with a high signal-to-noise ratio.
Plan Phase II Studies. Significant planning was carried out to advance the immunoassay system approach to demonstrate feasibility in a field-testable prototype. An important part of the Phase II research project is the use of membranes. Two manufacturers were contacted to discuss membrane product specifications and ability. Sample membranes from both manufacturers have been obtained to aid planning. Preliminary drawings of a flow cell design have been made. IOS already has designed and fabricated optical systems similar to the system that will be needed for the Phase II prototype. These designs and planning will be used to develop field-deployable prototypes in the proposed Phase II research project.
Explore Commercial Potential. IOS started exploring the commercial potential of this technology via collaboration with Foresight Technology, who performed a Technology Niche Analysis that provided IOS with a good start toward researching the market potential of the continuous immunoassay technology for water contaminant monitoring. Based on this Technology Niche Analysis, IOS reached preliminary agreements with two companies that IOS can collaborate with in the field-testing of Phase II prototype instruments. IOS also conducted additional marketing studies, extending the work done by Foresight.
Realization of Phase I Goals. All of the Phase I goals were achieved in this research project through the successful completion of the tasks described.
By meeting the goals of this Phase I research project and demonstrating that phenanthrene could be detected at a concentration as low as 14 ppb via the proposed displacement immunoassay procedure, the technical feasibility of the proposed technology has been proven. The successful use of QD nanoparticles as fluorescent labels also proved the technical feasibility of applying nanotechnology to environmental monitoring needs.
Market studies and preliminary contacts with companies in the water monitoring market have led IOS to conclude that the commercial prospects for the proposed continuous water pollution monitor are quite promising. Given technical feasibility and commercial promise, IOS has decided to propose the development of field-deployable prototypes for monitoring three classes of water contaminants. The prototypes will be tested by industrial collaborators as part of the Phase II research project.