The first objective is to develop new technologies for characterizing fundamental nanoscale processes. The second objective is to construct and characterize self-assembled nanostructures. The third objective is to develop devices and systems incorporating microfabrication and nanotechnology. The fourth objective is to develop a framework for economic, environmental and health risk assessment for nanotechnologies applied to food, agriculture and biological systems. The last objective is to produce education and outreach materials on nanofabrication, sensing, systems integration and application risk assessment.
We will develop an inexpensive electrochemical immunoarray biosensor to simultaneously detect aflatoxin and fumonisin, which are major mycotoxin co-contaminants of corn and other grains6-10. Our sensor platform can be readily adapted to allow simultaneous detection of different and/or additional toxins, limited only by the number of channels available in the sensor instrumentation. Our system can also be engineered to simultaneously detect several toxins in multiple samples.
We will design and fabricate a electrochemical immunoarray capable of simultaneous testing of two mycotoxins in one sample. The target toxin-specific antibodies will be immobilized on the immunoarray and tested with known levels of standard toxins to obtain calibration curves. The detection of toxins will be based on competitive immunoreactions between target toxins and their respective horseradish peroxidase (HRP)-labeled counterparts (HPR-toxins) (to be purchased from commercial sources) for limited binding sites available on the immobilized Abs. When the target toxins are present in the sample, they will compete with their corresponding HRP-toxins for limited Ab epitopes. Due to their relatively small size, the target toxins are more competitive than the much larger HRP-toxins, with fewer HRP-toxins binding to Abs34. The extent of target toxin-Ab immunocomplexes formed on the sensor surface will be proportional to the concentration of the target toxins present in the sample, while HRP-toxin-Ab formed is inversely proportional to target toxin concentrations. This idea is used in ELISA and other immunoassays, but not for electrochemical sensors. We will be the first to implement this sensing scheme for multiplexed mycotoxin detection. The HRP on the Ab-captured HRP-toxins will catalyze the oxidation of enzyme substrate o-phenylenediamine (o-PD) by hydrogen peroxide (H2O2) to produce electrochemically-active 2,2'-diaminoazobenzene in the enzymatic reaction, which can generate detectable current signals (other enzyme substrates could be used besides o-PD, such as tetra-methylbenzidine (TMB), 2,2'-Azino di-ethylbenzothiazoline-sulfonic acid (ABTS), 5-aminosalicylic acid (5AS) and di-aminobenzidine (DAB)). The more target toxins are present in the sample, the less HRP-toxins are bound to Abs, generating less electrochemically active enzymatic products and thus the resulting peak current signals will be lower. This decrease in peak current in the presence of target toxins will be sensitively measured and used to quantify the toxin concentrations.
2012/01 TO 2012/12<br/>
OUTPUTS: Among the most important issues facing grain and livestock producers in Wisconsin and elsewhere are preventing mycotoxin contamination of food and feed and reducing the deleterious effects of mycotoxins on livestock. Our focus in this project is to detect aflatoxins and fumonisons, the two most common co-contaminants of the number one grain crop in Wisconsin: corn. Early, rapid, and simultaneous detection of these co-contaminants would help avert potentially calamitous food safety and security problems. The presence of unacceptable levels of mycotoxins in food and feed grains also has global trade implications, which could result in a tremendous economic loss. We have started assembling units for synthesizing nanoparticles to modify our electrodes to be used in the electrochemical sensing system.
<br/>PARTICIPANTS: Not relevant to this project.
<br/>TARGET AUDIENCES: Academic and industry research and development personnel.
<br/>PROJECT MODIFICATIONS: Not relevant to this project.
IMPACT: The electrochemical detection sensor platform we develop will be broad-based such that mycotoxins other than aflatoxins and fumonisins and even entities other than mycotoxins can be detected rapidly, selectively, and sensitively.