Grantee Research Project Results
Final Report: A TEF-Based Dioxin Kit Utilizing Fluorescent Aptamers
EPA Contract Number: EPD06031Title: A TEF-Based Dioxin Kit Utilizing Fluorescent Aptamers
Investigators: Chandramouli, Bharat
Small Business: Eno River Labs, LLC
EPA Contact: Richards, April
Phase: I
Project Period: March 1, 2006 through August 31, 2006
Project Amount: $69,737
RFA: Small Business Innovation Research (SBIR) - Phase I (2006) RFA Text | Recipients Lists
Research Category: Small Business Innovation Research (SBIR) , SBIR - Waste , Hazardous Waste/Remediation
Description:
Eno River Labs, LLC proposed to develop an on-site testing kit for dioxins that used fluorescently tagged ssDNA aptamers for selective binding and subsequent screening/quantitative analysis of dioxin in soil samples. The resulting kit would be based on the use of aptamers, specific binding nucleic acids that can be engineered to bind to almost any compound with specificity and reliability. Aptamer mixes also can be designed to have different affinities to the analytes of interest. Phase I of the project was a proof-of-concept effort to generate a set of specific fluorescent DNA aptamers to bind 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the dioxin with the highest toxic equivalency factor (TEF). The dioxin-specific fluorescent aptamers were to be developed by the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) technique. During development, the dioxin-binding aptamers would be tested for their ability to bind specifically to 2,3,7,8-TCDD in the presence of other potential soil contaminants, such as polychlorinated biphenyls (PCBs).
Summary/Accomplishments (Outputs/Outcomes):
The feasibility of making an aptamer specific to 2,3,7,8-TCDD was not achieved because of the inability of the amino-TCDD analyte to bind to the Sepharose column at sufficient efficiency. The binding efficiency achieved was insufficient to ensure that the selected aptamer was binding to the 2,3,7,8-TCDD and not to the column. The proposed binding analyte, 4-aminoethyl-2,3,7,8-TCDD could not be used for the binding experiments, as it could not be synthesized in the required time/budget frame. The alternative molecule used, 4-amino-2,3,7,8-TCDD, proved significantly less reactive to the Sepharose resin. Although this was anticipated because of the lowered reactivity of the aromatic amine versus the aliphatic amine functionality, the contingency plan of greater reaction time, more mixing, and buffer compound recirculation was insufficient to ensure the 80–90 percent binding efficiency required for further progress. The difficulty in the synthesis of this analyte by two different organic synthesis companies, leading to a 3-month delay in the receipt of this analyte, meant that there was insufficient time to perform troubleshooting and devise alternate plans. Because of the lack of binding of the analyte to the Sepharose column, Eno River Labs could not carry out the steps specific to Aim 1 of the project—the production of the TCDD affinity matrix in preparation of the SELEX process of aptamer selection.
Conclusions:
Although the applicability of a dioxin field kit is readily apparent, the development of such a kit is problematic. It increasingly is clear that the complexity of these compounds and their hydrophilic nature make any easy extraction a very difficult prospect. The concept of developing an aptamer is still untested after this study; the single largest obstacle, in fact the only obstacle found to date, is the manufacture of a reliable standard with which to conduct the experiments. The cost savings of a dioxin field kit are not considerable enough to differentiate it from other existing dioxin screens available in the laboratory. In light of the decline in dioxin testing in the last 2 years, it is unlikely that there exists a market significant enough to spur the investment dollars that would be required to bring such a kit to market. Aptamers remain a promising tool, however, because once the aptamer is synthesized and characterized, it can be manufactured inexpensively and consistently. Through our initial feasibility study, Eno River Labs hase identified that the aptamer selection process is not optimized for nonpolar, hydrophobic, and nonreactive molecules. Aptamer production is easier for moderately polar-to-polar molecules with active functionalities that have some ability to be dissolved in an aqueous medium. Many pesticides, endocrine disruptors, natural/synthetic hormones, antibiotics, and drug residues fit this bill and will make excellent candidates for aptamer design. Eno River Labs will continue to pursue research in this area.
Supplemental Keywords:
small business, SBIR,dioxin, onsite environmental testing, ssDNA aptamers, soil testing, dioxin contamination, toxicity equivalence factor, TEF, aptamers, EPA, monitoring, analytical, PCB, assessment methods, contaminated sediment, field monitoring, soil sampling,, Scientific Discipline, Waste, Water, Contaminated Sediments, Environmental Chemistry, Environmental Monitoring, Environmental Engineering, dioxin, toxicity equivalency factor test kit, contaminated sediment, field monitoring, PCB, assessment methodsThe 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.