Project Research Results
Grantee Research Project Results
Final Report: Online Water Monitoring Utilizing an Automated Microarray Biosensor InstrumentEPA Contract Number: EPD09016
Title: Online Water Monitoring Utilizing an Automated Microarray Biosensor Instrument
Investigators: Trindade, Theresa
Small Business: Constellation Technology Corporation
EPA Contact: Manager, SBIR Program
Project Period: February 1, 2009 through July 31, 2009
Project Amount: $70,000
RFA: Small Business Innovation Research (SBIR) - Phase I (2009)
Research Category: Small Business Innovation Research (SBIR)
Three organisms were chosen for concentration from water using an automated concentration system and immunoassay development on the ABS 400 instrument. Those organisms included Cryptosporidium parvum, Hepatitis A, and Escherichia coli (E. coli) O157:H7. Fifteen liter volumes of water were spiked with E. coli O157:H7 and concentrated manually using a Fresenius 80K molecular weight cutoff filter. A volume of 250 milliliters or less was then further concentrated using a Microza hollow fiber module with a 50K molecular weight cutoff. Samples were plated to calculate the recovery efficiency of each of the filters. Following concentration, samples from both filtrations were tested for detection on the ABS 400 instrument. A new next generation version of the Automated Concentration System (ACS) developed at Constellation Technology was built for concentrating water samples in the latter half of the contract period. The same procedures used in manual concentration were used in the automated system.Summary/Accomplishments (Outputs/Outcomes):
Multiple antibodies were tested for assay development on the ABS 400. Antibodies with great enough affinity to C. parvum or Hepatitis A virus for development of a sensitive and specific assay were not identified. Due to the cost of antibodies, these assays were not developed any further. A polyclonal goat anti-E. coli O157:H7 antibody was utilized to develop an immunoassay on the ABS 400. Assay sensitivity was determined to be 105 cfu/ml. Fifteen liter volumes of sterile deionized water were spiked with a known concentration of E. coli O157:H7 and concentrated using both filters described above. Each filter also was found to be reusable a minimum of three times. Overall recovery with a final sample eluted from the Microza filter never exceeded 48 percent in the manual process. A one-filter system (only the Fresenius filter) tested manually gave a mean recovery of 73 percent. The automated concentrator incorporating only a large scale filtration step was used to process a 15 liter volume of water in 1 hour. The sample generated was tested on the ABS 400 instrument. Concentration and subsequent detection were performed in combination in less than 90 minutes using the ACS-ABS 400 system. Recovery efficiency could not be assessed using the automated concentrator. Positive detection on the ABS 400 of E. coli O157:H7 was observed following processing on the ACS.Conclusions:
The purpose of this SBIR project was to develop a rapid test method for pathogens of interest in water utilizing a combined concentration and detection system. The advantages to this system include low consumable costs due to reusability of the cartridges and filters and also rapid processing and detection time. Three assays were proposed for development in Phase I of this contract, Hepatitis A, Cryptospordium parvum, and E. coli O157:H7. The former two assays were not developed due to lack of antibodies with affinity great enough for sensitive detection on the ABS 400 instrument. The limit of detection of the E. coli O157:H7 assay was 105 cfu/ml.
A one-filter system provided better recovery of the organisms spiked into the large volumes of water to be concentrated. A final concentration procedure developed proved successful at processing samples with a mean recovery of 73 percent. Recovery using the automated concentrator was not assessed due to a lack of data from viable counts performed. This was attributed to mechanical stress that the system put on the filtered organisms that may have lysed the cells or rendered them unculturable. Staining methods and ABS 400 test results verified the presence of E. coli O157:H7 in the eluted sample. Overall results showed that the combined system can concentrate a large volume of water and generate a sample suitable for testing and detection on the ABS 400. The entire procedure can be performed in less than 90 minutes. This combined system can be used for testing larger volumes of water and each assay cartridge for the ABS 400 can be developed for testing up to 10 different water pathogens at a time. This system is a rapid, specific, and versatile instrument that would be an asset for municipal water systems to have online for prevention of public health illness.
The system proposed has a number of water analysis applications. These applications include measurement of contaminants in groundwater, surface water, effluent, and drinking water. The current proposal addressed applications in the measurement of drinking water.
Without the automated in-line analysis afforded by the proposed system, water samples must be transported to the laboratory for analysis or reagents must be mixed in the field to be used with screening instruments. Typically, these screening instruments do not have the sensitivity required to meet the detection limits required by the primary and secondary drinking water standards. With the proposed system and method, operation is fully automated and requires only occasional end-user action, such as when consumables need replacing. The water collection, testing, and analysis will be fully automated and verifiable. The proposed system bridges the gap between available screening instruments and laboratory measurements.
Another key advantage to this system is the reusability of test cartridges and the filter module. Unlike other single-use consumables, the ABS cartridge and the hollow fiber filter in the concentrator can be reused. Current data suggest that a single channel on the cartridge can be reused at least three times given negative test results, and it is expected that it can be used up to 10 times or until a positive result occurs. Therefore, it is possible that a single ABS cartridge can be used for up to 20 total tests, which would significantly lower the overall cost of operation. The filtration module can be reused three times or more.Supplemental Keywords:
small business, SBIR, EPA, microarray biosensor, water supply monitoring, detection system, recovery system, pathogen, bacteria, virus, parasite, toxin, water supply, pathogen detection, ultrafiltration, immunoassay, E. coli, Crypotsporidium parvum, hepatitis A, sensitive, specific, cost-effective, water monitoring, measurement and monitoring
Progress and Final Reports: