Grantee Research Project Results

Final Report: Neurotoxin and Cytotoxin Detection in Water Supplies During Sample Collection

EPA Contract Number: EPD04067
Title: Neurotoxin and Cytotoxin Detection in Water Supplies During Sample Collection
Investigators: Spencer, Kevin M.
Small Business: EIC Laboratories Inc.
EPA Contact: Richards, April
Phase: II
Project Period: April 1, 2004 through June 30, 2005
Project Amount: $225,000
RFA: Small Business Innovation Research (SBIR) - Phase II (2004) Recipients Lists
Research Category: SBIR - Water and Wastewater , Small Business Innovation Research (SBIR) , Drinking Water

Description:

There has been an alarming increase in toxic cyanobacteria over the past two decades, with numerous poisonings reported from Australia to the United States. The increased toxic risks led the U.S. Environmental Protection Agency (EPA) to include cyanotoxins on the 1998 Contaminant Candidate List. Most cyanobacterial blooms, however, are not toxic. Furthermore, bloom toxicity will change over time. Therefore, cyanobacterial identification is not enough; toxin presence must be confirmed. There are many biological/toxicological methods available to detect these toxins. The main drawback of these biological techniques is that they are time-consuming, laboratory-based and require significant technical expertise. With dynamic systems such as cyanobacterial blooms, detection and identification of toxins should be conducted as quickly as possible, preferably in the field.

The goal of this research project was to develop a field-portable automated sensor, based on surface-enhanced Raman spectroscopy (SERS), which can be used by nonspecialists. The entire system will weigh approximately 10 pounds and will detect the different toxins through a simple dipstick arrangement. SERS, which directly measures chemical bonding, theoretically would allow direct determination of all analytes; however, practical identification of the desired toxins in the presence of nontoxic bacteria and other chemical constituents in the water supply could become unwieldy. There are methods by which SERS substrates can be made specific for certain analytes, using chemical modifications. These modifications can alter the surface chemistry to attract chemicals with a certain electronegativity, pH or chemical reactivity. EIC Laboratories Inc. (EIC) will use the latter and develop SERS substrates highly specific for the toxins of interest.

Phase I demonstrated the potential for SERS as a cyanotoxin sensor, in which cylindrospermopsin, microcystins, anatoxin-a, saxitoxins and domoic acid all were detected. Rapid detection of high femtogram toxin quantities was demonstrated with the SERS sensor. The purpose of the Phase II project was to advance the Phase I results in an effort to optimize the sensors for rapid detection at desired sensitivity levels, with the need for minimal sampling volumes. The preliminary Phase I coating research was expanded to facilitate increased selectivity and sensitivity toward each cyanotoxin. The key to the eventual integration of the SERS technique into an EPA-approved protocol is the reproducibility and analytical precision to ensure measurement validity. Therefore, the majority of the Phase II program emphasized the optimization of the SERS-sensing elements and the entire fabrication process.

Summary/Accomplishments (Outputs/Outcomes):

During the Phase II project, the main thrust of the research was to develop a SERS-sensing element that enabled detection of trace levels of cyanotoxins reproducibly. With the proof of principle demonstrated, a mass-production method had to be developed that would ensure that any element used would be reliable and quantitative. These requirements led to a new fabrication procedure for the SERS-sensing elements based on sputtering thick metal films, followed by electrochemical roughening procedures. With the change in the base material, the following parameters had to be optimized: roughening time, cycling limits, electrolyte, carbonate concentration, surface oxidation, surface acidity, surface stability and the need for protective overcoatings. EIC optimized each of these parameters to produce a final SERS-sensing element that appears to have the desired reproducibility and sensitivity. This SERS-sensing element was produced by electrochemically roughening sputtered gold using KCl electrolyte, with carbonate included during the electrochemical roughening, and a 30-minute post-roughening, plasma-cleaning procedure. With this sensor, high-quality reproducible spectra of anatoxin-a were demonstrated.

Conclusions:

During the Phase II project, the effort was transitioned from proof of principle to a final device that could be used reliably for field operations. The sensing elements required an improved fabrication process to facilitate cheaper SERS-sensing element cost, increased mass production capabilities and, most significantly, increased measurement precision through reduced batch-to-batch variability. Through a detailed analysis of the various parameters that can affect the fabrication of the SERS-sensing elements, EIC developed a vapor-deposited, gold SERS-sensing element that has improved in-batch reproducibility (50 to 100 can now be fabricated in a batch) by a factor of two, and has stymied the large batch-to-batch variations that EIC would occasionally observe for the electrochemically roughened foils used in the Phase I project. The sensitivity towards the cyanotoxins appears to have also increased.

The Phase II results are encouraging, although the sensing-element optimization process did require the brunt of the research. These results point to the possibility for a fieldable instrument that can rapidly identify and quantify toxic blooms in surface water or for an online instrument that can monitor toxins in treated water. This sensor can streamline sampling at the source and could be used in an environmental laboratory as a rapid-screening diagnostic to prevent wasted time and labor on the more complex instrumentation. This sensor can be expanded to other EPA contaminants of interest and will operate equally as well for airborne analyses. Finally, this particular sensor has great potential as a monitor for chemical and biological warfare agents in the water supply.

Supplemental Keywords:

neurotoxin, cytotoxin, water supply, cyanobacterial bloom, toxic cyanobacteria, surface-enhanced Raman spectroscopy, nontoxic bacteria, cyanotoxin sensor, cylindrospermopsin, microcystin, anatoxin-a, saxitoxin, domoic acid, warfare agents, EPA, small business, SBIR,, RFA, Scientific Discipline, Water, POLLUTANTS/TOXICS, Environmental Chemistry, Microorganisms, Drinking Water, Environmental Engineering, Environmental Monitoring, cytotoxic effects, sensors, monitoring, neurotoxicity, cyanobacteria, drinking water system, drinking water contaminants, drinking water treatment, bacteria, drinking water distribution system, monitoring sensor

SBIR Phase I:

Neurotoxic/Cytotoxin Detection in Water Supplies During Sample Collection  | Final Report