SBIR-STTR Award

Mold growth in grain can produce deadly mycotoxins. SDCÆs nanocrystalline, solid state tin dioxide gas sensor will allow detection a signature gas given off during mold growth. This high sensitivity, high selectivity sensor system will allow real-time monitoring of grain in storage bins and during transportation. The initial target is detecting the growth of Aspergillus flavus in corn which produces aflatoxin. The FDA has a 20 ppb action limit on aflatoxin to insure food/feed safety. This project will optimize SDCÆs nanocrystalline gas phase sensors system. Sol gel coating of tin dioxide on an alumina substrate will produce a large number of active sites. Combinatorial chemistry will be used to add other compounds to the sol gel to improve selectivity to the signature gas given off by Aspergillus flavus. The operating conditions of the chip will be varied to further enhance sensitivity and selectivity. Commercial data analysis software will be adapted to further refine the detection system. Initially chips will be tested in a lab scale systems using know concentrations of signature gas and typical interferent gases. A lower detection limit of 1 ppb for the signature gas will be sought. Once optimized in the lab, a prototype will be evaluated in a real world environment using corn and Aspergillus flavus. Success will lead to beta site testing and the application to other mycotoxins, such as DON and other corps such as wheat and peanuts. OBJECTIVES: Sensor Development Corp intends to demonstrate the sensitivity and selectivity of its proprietary nanocrystalline tin oxide solid state gas phase sensor for detecting ppb levels of certain signature gases given off during mold growth in grain. The initial target mold is Aspergillus flavus which produces aflatoxin. The target grain is corn. The FDA has an action limit for aflatoxin of 20 ppb. SDC believes a signature gas for Aspergillus flavus mold growth has been identified. With the sensitivity and selectivity of our systems, it could provide real-time management of grain storage and reduce the losses. Since our sensor system measures the signature gas from mold, it does not rely on grain sampling with its inherent potential for inaccuracy and the cost/delay of the sampling process. Temperature and carbon dioxide measurements could indicate mold growth. In a later phase, we would attempt correlation of these measurements with our signature gas measurements. Selectivity for the signature gas in the presence of other gases typical in grain storage environments is necessary and SDC will test a variety of active coatings, chip operating conditions and chip signal processing software to give the optimum performance for Aspergillus flavus. SDC believes there will be a unique set of parameters for each distinct signature gas. SDC will use combinatorial chemistry to fabricate a number of active coatings using sol-gel techniques. This nanocrystalline active coating will be screened in a laboratory test system that was developed and qualified earlier for assessment of indoor air quality. This system will use simulant gases of known composition for our measurements. Initial operating conditions will be based on earlier work, while the data analysis software to be evaluated will be commercially available platforms adapted to our application. The hardware and test operating software are already in place and should require minimal modification to complete the Phase I objective of developing a sensor chip with a high sensitivity and high selectivity for the signature gas for aflatoxin production in corn, performing under optimized chip operating conditions, and successfully using commercial software adapted for this application. The second objective for the Phase I program will be to test this sensor system in a real world environment by collaborating with Purdue University. Purdue possesses expertise in mold growth in corn both at the lab scale and 500 bushel bin scale. They will grow the mold on corn in the lab scale, and measure the gases produced using the optimized sensor from our lab scale work. Following completion of this task, we will evaluate the overall sensor performance in preparation for Phase II which would involve field-testing first in pilot bins at Purdue and then at beta test sites at major grain handling firms. Successful completion of this Phase I will open up other mold based applications in grain storage, such as DON produced by Fusarium, and other food supplies such as peanuts and wheat. In fuel ethanol production, aflatoxin in the DDG would also be a serious problem. APPROACH: Sensor Development Corp will use its platform technology, a nanocrystalline tin dioxide solid state gas phase sensor system, to detect a signature gas for the presence of aflatoxin in corn storage. The Phase I effort will start with the fabrication of a number of sensor chips. These chips will be made by spin coating an alumina substrate with tin dioxide sol gels and then calcining under controlled temperature and time conditions. This will produce a nanocrystalline surface with numerous reaction sites, hence the high sensitivity. The composition of the sol gel will be modified by adding additional active metals to enhance selectivity in the presence of interferent gases. In addition to adding these metals to the sol gel, the composition of the sol gel can be varied. A combinatorial chemistry approach will be used to optimize performance of the sensors. The chip will have a platinum heater element printed, on its backside for chip heating, and a temperature sensor on the front side to monitor chip operating temperature. Chip fabrication will be done at Case Western Reserve University, using state-of-the-art equipment and experienced personnel. For sensors optimization, the fabricated chip will be evaluated in a lab test system which will expose the chip to controlled gas concentrations and compositions. Measurements of the specific net conductance (SNC), the change of the sensor in response to exposure to a test gas, will later be used to determine the concentration of the target gases. The system uses LabVIEW (National Instruments) to control the test operation and monitor the data output. During testing, the gas concentrations and compositions are varied along with the operating conditions. The optimum performance will be established from the variables of active coating composition, operating conditions, and data analysis. Software from commercial data analysis platforms will be used to analyze the data and correct for drift and noise if present. This optimized chip will then be installed in a test device, already prototyped by FloCell and delivered to Purdue University where it will be incorporated in a laboratory scale system for measuring the signature gas from actual mold growth in corn. The sensor chip, protected from dust, will be used to measure the concentration of the gas present. Knowing the bin volume and gas exchange rate, the high sensitivity of the system will allow comparison of the diluted signature gas with that determined by conventional analysis, the latter requiring representative sampling of the corn, then sample preparation and finally, reacting the resultant analyte with mycotoxin specific antibodies. The color change indicates the mycotoxin concentration. This method, Enzyme Link Immunosorbent Assay (ELISA), is approved by (GIPSA). Depending on the results from single chip testing in Phase I, multiple chips in an array format can be used to enhance the selectivity in Phase II. Each chip will have a different active coating or operating condition. Software manipulation of the data will further enhance the selectivity

Aflatoxin was discovered in groundnut meal which killed over 10,000 turkeys in England. Poultry are extremely sensitive to aflatoxin B1 with turkeys being more sensitive than chickens. Later research discovered that poultry are the most susceptible food animal species to the toxic effects of aflatoxin. Based on epidemiological data and knowledge of liver biochemistry it is suggested that humans fall somewhere in the middle of this lethality range. Lethality in humans has also been documented. In 1974, nearly 10 per cent of 1,000 patients died from suspected acute aflatoxin poisoning. Aflatoxicosis outbreaks (2004) in eastern Kenya resulted in 317 cases and 125 deaths. Specific medical implications of aflatoxin are related to their carcinogenic properties. Aflatoxin is listed as a Group I carcinogen by the International Agency for Research on Cancer. It is has been demonstrated in animal species to be the most potent liver carcinogen known, and is implicated as a cause of human primary heptatocellular carcinoma. This form of cancer is one of the most common forms of cancer in China, Saharan Africa, and Southeast Asia and causes at least 250,000 deaths annually worldwide, especially in developing countries. The human body also metabolizes aflatoxin to several compounds including aflatoxin M1 which is secreted in both mother?s milk and urine. Studies in Africa confirm infant exposure to aflatoxin via mother?s milk as well as the ability of aflatoxin in blood to cross the human placenta. Approximately 4.5 billion people around the world are exposed to virtually unregulated amounts of the toxin on a daily basis. The substance is so toxic that it is one of 19 contaminants along with mercury and DDT for which the US Food and Drug Administration imposes strict tolerance levels with only trace amounts allowed. In addition to the health impact of detecting the mycotoxins early, there is a significant economic impact. The Midwest drought in 2005 triggered an outbreak of poisonous aflatoxin at thousands of corn farms, spurring regulators and food companies to greatly expand testing of grain and milk. During the last US drought, many dairies were forced to dump aflatoxin-contaminated milk from cows fed corn contaminated with this toxin. The concern for aflatoxin-contaminated corn led company officials at the Quaker Oats breakfast cereal plant in Cedar Rapids, Iowa, to assay every truckload of corn entering the plant for aflatoxin. The value of this solution is evident by the fact that two major grain storage companies are committed to serve as beta test sites for the product. OBJECTIVES: The proposed research has several goals. The long term objective is to provide a tool for the protection of United States food and feed grains from contamination by aflatoxins, potent hepatocarcinogens produced by Aspergillus flavus. Though this fungus is generally an ubiquitous soil saprophyte, it can infect globally important grains, particularly oilseeds such as corn under certain conditions. These toxins are frequently carcinogenic and represent a direct threat to human health. The work will broaden the toolset for study of these important materials and provide a basis for a "real-time" sensor detecting volatile organic compounds generated by the aflatoxin producing microorganism Aspergillus flavus. The specific aims of this work are: 1) Identify unique volatile compound(s) associated with aflatoxin production by Aspergillus flavus growing on a defined liquid growth medium such as agar and then on corn. 2) Establish and optimize norms for stability, reproducibility, and ruggedness of the sensor. 3) Improve sensor response and device reliability, and develop device software, and 4) operate the sensor device to obtain selective analysis of volatiles over toxigenic-inoculated corn. A number of A. flavus strains (toxigenic and atoxigenic) will be employed in this study. Microbiological growth assays on defined medium will determine the volatiles produced by each of the test fungi on corn meal and agar. During a three-week incubation period seeds will be removed to determine weight and aflatoxin levels. A series of seed controls will be used (no fungal inoculum) to determine the volatiles present, if any. Aflatoxin-Volatile profile determinations will be made using separate experiments utilizing nonsterile and sterilized corn and cottonseed as growth medium for the test fungi. Assays will be performed separately on non-sterile and sterile seeds with inoculations of an equal mixture of atoxigenic and toxigenic strains of A. flavus. This will determine the effect of the growth of the atoxigenic strain, which is competing for the same nutrients as the toxigenic strain, on aflatoxin production and volatile profiles of the toxin producing strain. The most prevalent volatiles with the highest concentrations produced will be used as test markers in the Sensor Development Corporation (SDC) test system. Using these gases the SDC sensor device will be designed to analyze selected test gases. Significant upgrades will be added to the sensor device used in the Phase I project. These will include state-of-the-art application of the tin oxide coating on the wafer and screen-printing the circuitry on each side of the sensor wafer. In addition the sensor device will have enhanced software management of the conductance data from the individual wafers. At the completion of this project the specific marker volatiles from A. flavus will be known, as well as the common interferant gases that exist in a corn bin. The project will be a success with the demonstrated repeated performance of a robust sensor device which can selectively detect with high sensitivity the identified marker gases for toxigenic A.flavus. APPROACH: Several techniques have been used to identify volatile compounds in different samples including tenax cartridge trapping methods and Solid \Phase Micro-Extraction (SPME). Two commercially available SPME fibers suitable for volatile analysis available from Supelco ( Bellefonte, PA) will be examined for this study. These are poly(dimethylsiloxane) (PDMS; 100 ?m), and divinylbenzene/carboxen/poly(dimethylsiloxane) (DCP, 50/30 ?m). The SPME fiber is inserted into the headspace above the fungal sample manually. Adsorption is timed for 1 h. SPME fibers are desorbed at 230C for 2 min in the injection port of an HP5890/5989A GC-MS (Hewlett-Packard, Palo Alto, CA) with a HP-5 (cross-linked 5% phenyl methyl silicone, Hewlett Packard, Palo Alto, CA) column (50 m, 0.2 mm i.d., 0.5 ?m film thickness). Positive identification of a component will be performed in two corn varieties inoculated with two molds by comparison of its retention time or Retention Index (RI) and mass spectrum with that of an authentic compound (when available). The RI for each identified compound is calculated using a series of straight-chain alkanes (C5-C20). Tentatively identified compounds will be uniquely identified on the basis of the mass spectra from the Wiley (v.7 NIST98) library of mass spectral database (Palisade Corp., Newfield, NY). A. flavus, whose toxigenic strains produce aflatoxin, exhibits a marker target gas associated with those toxigenic strains, the sesquiterpene MVOC, C15H24, Humulene. To confirm the conclusion regarding selectivity, will use sensor response data to conduct a simulated 2-gas demonstration by sequentially testing two different sensor wafers in the same chamber using the same pair of gases at identical concentrations in each instance. Various combinations of four gases and two sensor wafers (one un-catalyzed and the other catalyzed), at three operating temperatures. Though these sensors will be tested simultaneously in one test chamber, the sequential procedure essentially represented that condition. These data will be used to calculate the "measured" concentration of each individual gas. A mixture of inteferant gases and susquiterpenes will also be tested using two catalyzed sensors at different temperatures. Combinatorial Chemistry techniques may be used to select 2-catalyst combinations