Final Report: Nanoparticle-Based Lateral Flow Microarray Test Strip AssayEPA Contract Number: EPD07031
Title: Nanoparticle-Based Lateral Flow Microarray Test Strip Assay
Investigators: Venkatasubbarao, Srivatsa
Small Business: Intelligent Optical Systems Inc.
EPA Contact: Manager, SBIR Program
Project Period: March 1, 2007 through August 31, 2007
Project Amount: $70,000
RFA: Small Business Innovation Research (SBIR) - Phase I (2007) RFA Text | Recipients Lists
Research Category: Nanotechnology , SBIR - Nanotechnology , Small Business Innovation Research (SBIR)
Nanotechnology is a transformational technology that can benefit many different disciplines of science and commerce. Nanomaterials have dimensions of less than 100 nm, and exhibit unique optical, chemical, and physical properties. This project describes using nanomaterials to develop a multiplexed assay kit for detecting pathogens in water samples. The assay format chosen here incorporates commercially validated and user-friendly lateral flow test strips. A microarray is incorporated as part of the lateral flow test strips to advance the multiplexed detection of pathogens. The choice of nanomaterials used in the assay development improves the sensitivity of detection and makes quantitative and/or semi-quantitative measurements feasible.
Pathogens such as E. coli, Clostridium, Pseudomonas, and others that are present in water can cause disease and, in some cases, can result in fatalities. Therefore, there is an important need to detect these organisms in water samples. Traditional methods, such as culture-based detection, are time consuming and can take several days before positive results are achieved. During this period of testing, this potentially contaminated water can pose a risk to the user. Therefore, rapid detection methods are needed to minimize this risk, and improve the monitoring for hazardous pathogens. High false-positive rates and low sensitivities currently undermine the rapid detection methods that have been developed for the detection of pathogens.
In this project, the assay detection sensitivity was improved over traditional lateral flow assays by using nanoparticles as labels. The specificity also was improved by developing an ability to incorporate multiple antibodies, thereby providing a broader detection profile. The multiplex capability, increased specificity, and quantification capabilities were improved because of the use of nanoparticles as detection labels. The incorporation of the microarray as part of the lateral flow test strip design ensures multiplex capability and the potential for improved specificity.
This user-friendly assay format can be used easily by semi-skilled personnel to conduct tests from water samples. The user simply applies a small volume of water on the sample application region of the test strip. The pathogens, if present in the sample, interact with the detector reagents on the test strip and migrate across the lateral-flow membrane, and the pathogens and detector agents are captured on immobilized microarray spots. The signal from the microarray spots is measured using a simple, portable reader. The signal then is related to the presence and concentration of pathogens in the samples.
During the course of the Phase I project, test strips were designed to measure E. coli 0157 and Pseudomonas aeruginosa. These two water pathogens were selected for this work because the detection of these organisms is a U.S. Environmental Protection Agency (EPA) priority, and also because easy-to-use and reliable test kits for these pathogens are commercially attractive. The protocols to fabricate microarray test strips were established. Several different biochemical reagents were tested to determine suitable candidates for the assay development. Protocols to conduct tests were developed, and the tests for E. coli O157 and Pseudomonas were conducted on the fabricated microarray test strips. The nanoparticle concentrations on the microarray spots were measured using a portable reader.
The results obtained in this Phase I project show that an easy-to-use lateral flow microarray test strip can be fabricated to measure waterborne pathogens. The E. coli O157 assay has a sensitivity to measure 1,000 organisms present in a sample and 10,000 Pseudomonas organisms from the sample. Based on the tests conducted so far, the false-positive and false-negative values are zero. The presence of Clostridium and E. coli O157 had no bearing on the results obtained for Pseudomonas. Similarly, the E. coli O157 measurements were not affected by the presence of Clostridium or Pseudomonas in the water samples. The portable reader developed for this work allows for very sensitive and reproducible measurements of the spots on the test strip.
In this project, Intelligent Optical Systems, Inc. (IOS) demonstrated the feasibility of using nanomaterial labeled reagents on a lateral flow microarray test strip to provide the quantitative and multiplexed detection of pathogens in water samples. This work has laid a solid foundation for development of easy-to-use and reliable test kits for environmental monitoring applications. The protocols for fabricating microarray test strips, conjugating the biomolecules to nanomaterials, and identifying the antibodies to be used in the assay development have been established. Furthermore, IOS also has determined the detector antibody concentrations that need to be used for developing these assays and the procedures to optimize the nanomaterials signal. The reader developed for this purpose allows for the accurate and reproducible measurements of the nanomaterials present on the microarray.
The developed test strips are likely to find applications both in drinking water and beach water monitoring applications. The end users will be small and large water processing facilities, beach water quality monitors, and regulatory agencies. The platform technology could be applicable to most medical and monitoring applications. IOS has received significant interest from end users, as well as other commercial companies, regarding the exploration of future collaboration and additional testing on this project. IOS is very optimistic about the possibility of transitioning this technology into a commercial product. The EPA regulatory approval of the technology will be a key event toward the commercialization of this assay format.