Grantee Research Project Results
Final Report: Rapid and Sensitive Electrochemical-Based Method for Improved Detection of Cryptosporidium parvum in Water
EPA Contract Number: 68D03036Title: Rapid and Sensitive Electrochemical-Based Method for Improved Detection of Cryptosporidium parvum in Water
Investigators: Aguilar, Zoraida P.
Small Business: Vegrandis Inc.
EPA Contact: Richards, April
Phase: I
Project Period: April 1, 2003 through September 1, 2003
Project Amount: $70,000
RFA: Small Business Innovation Research (SBIR) - Phase I (2003) RFA Text | Recipients Lists
Research Category: Watersheds , SBIR - Water and Wastewater , Small Business Innovation Research (SBIR)
Description:
This Phase I research project was designed to address an electrochemical sandwich-type immunosorbant assay approach for detection of Cryptosporidium parvum oocysts that would provide improvements for U.S. Environmental Protection Agency (EPA) Method 1623 in several respects on the macro- and microscales. The goals of this Phase I research project were to:
· Determine optimal conditions and limitations of capturing C. parvum oocysts using large, flat IgG antibody-modified gold surfaces (as compared to IgM-antibody modified immunomagnetic beads) after the filtration, concentration, and pellet stages of EPA Method 1623.
· Decrease the required skill level to perform analyses, compared to that for EPA Method 1623 (essentially eliminating the need to perform microscope inspections of immunofluorescently labeled oocysts).
· Identify general design parameters necessary to achieve good C. parvum detection limits using electrochemistry (Phase I target: 50 oocysts/L).
· Achieve short analyses time (Phase I target: less than 1 day).
· Determine whether there are natural field interferences from real sample matrices.
· Determine the extent of nonspecific adsorption on bare gold and future device materials.
The electrochemical sandwich-type immunosorbent assay involves assembly of mercaptoundecanol self-assembled monolayers to gold-coated silicon chips, followed by covalent immobilization (via carbodiimide coupling) of the primary antibody for an antigen on the coat of C. parvum oocysts. At this point, the chip's surface is ready for analysis of a sample. The chip then is exposed to a sample, which may contain the oocysts. This step is followed by assembly of a secondary antibody that is conjugated to an enzyme. If the enzyme is present, it then can convert a nonelectroactive precursor in a solution added at the very end of the assay to an electroactive one, which can be detected electrochemically (rinsing steps are used throughout).
C. parvum, a waterborne pathogen, is of particular interest because it is one of five major pathogens responsible for 5 million deaths each year worldwide. C. parvum invades gastrointestinal systems of hosts and does not respond to common drug treatments. This thick-walled oocyst resists typical water purification treatments and the infectious dose is low (e.g., 1-132 oocysts). Current EPA methods for detection in water, however, involve great expense ($400-$750 per sample), time (a few days to several weeks), highly skilled personnel, and poor accuracy and precision (approximately 100 oocysts/L detection limits). Thus, new methods are of interest that allow for more rapid detection and better detection limits, and require less skill from personnel.
Summary/Accomplishments (Outputs/Outcomes):
A series of studies using heat-shocked and gamma-irradiated C. parvum oocysts and a variety of assay conditions was performed to assess the lowest detectable oocyst quantities. Macrochip immunoassemblies and electrochemical detection with macroelectrodes and self-contained microcavity electrodes were investigated. Knowledge gained from these studies was used to perform a final assay on real environmental 10 L water samples that were filtered and concentrated using EPA Method 1623 and spiked with known amounts of live oocysts. Electrochemical results on the environmental samples were compared with analyses done by EPA Method 1623 (immunomagnetic bead recovery and immunofluorescent assay steps), in collaboration with a commercial laboratory.
The primary goals of this Phase I research project were achieved. Detection of 25 oocysts/L in buffer was demonstrated with minimal operator skills, better than the target of 50/L. Total assay times were diminished to less than the 1-day target. Real water matrices allow detection well above the background of 10 oocysts in a 2 mL filtered and centrifuged pellet of 10 L of lake sample (i.e., 1 oocyst/L in original sample, or 5,000 oocysts/L in a 2 mL pellet). The signal exhibits an increase with concentration of oocysts, and thus, quantitation is possible. Studies of the extent of nonspecific adsorption with oxidized silicon material, of interest in miniaturizing the method in Phase II, were performed. Microscale assays, to be developed in Phase II, are predicted to yield detectable signals within 25 minutes incubation time of enzyme substrate for concentrations of 100 oocysts/L of pelleted sample.
Conclusions:
The electrochemical immunoassay on macrochips using microelectrode detection has demonstrated ease-of-use, speed, and sensitivity advantages for EPA Method 1623. Although the detectable concentration and time-scale goals were achieved in Phase I, there is still ample room to improve detection limits and time of the assay by optimizing conditions and geometries, especially at the microscale. At the microscale, close proximity of immunoassembly site and detection electrodes will drastically enhance detection limits and time of assay, and significantly diminish size and volume (down to 16 pL). The experience and findings that were gained in Phase I poise Vegrandis, LLC, in an excellent position to perform Phase II work toward the automation of the method using integrated fluidics, integrating arrays for oocyst capture and electrochemical detection on the microscale, and establishing sampling features. The Phase II emphasis would lead to commercialization of a more portable, less expensive, and user-friendly device for not only timely quality assurance and control of water supplies, but also for studies of the fate of C. parvum oocysts in the environment from various sources to water intake sites as a function of weather and season. Such extensive and routine studies currently are not practical or affordable with current EPA methods of analyses.
Supplemental Keywords:
water, electrochemical sandwich-type immunosorbent assay, Cryptosporidium parvum, C. parvum detection, pathogen, antibody, antigen, gold, oocyst, electrochemistry, EPA Method 1623, detection electrodes, small business, SBIR., RFA, INTERNATIONAL COOPERATION, Water, Drinking Water, cryptosporidium parvum oocysts, pathogens, aquatic organisms, electrochemical detection, biopollution, early warning, analytical methods, cryptosporidium , water disinfection, drinking water contaminants, immunofluorescent assay, water treatmentThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.