Using Molecularly Imprinted Polymers for the Detection of Mycotoxins in Animal Feed

Adam Weinstein Dhilan BekahKai SlaughterSahaj Dhamija

Mycotoxins Have A Crippling Effect On Livestock Feed

25% of crops are affected annually with mycotoxins worldwide

~ 30 Million tons of animal feed

Ref [1-3]2

The most potent naturally occurring carcinogen in the world

Up to 30% of liver cancer in the world

Ref [34]3

Current Testing Methods Are Not Sufficient

ELISA Kits, Thin-layer chromatography, Black light

Quick tests must be followed up by confirmatory tests

Aflatoxin FDA limit is ~ 20 ppb

Ref [4-5]4

Molecularly Imprinted Polymers (MIPs): ‘Plastic Antibodies’

• Low Cost

• High Stability

• High Sensitivity

Ref [6-9]5

Our Solution: MIP-Cellulose Composite Film

Implemented in 5 steps:

1. Formation of porous cellulose film2. Polymerization of MIP around template

molecule3. Removal of template molecule4. Addition of animal feed to film5. Observation of bound aflatoxin under black

lightRef [10-11]

6

Formation of Cellulose-MIP Composite Film

Soxhlet Extraction

Ref [12-13]

7

Detection of aflatoxin in animal feed

8

Key Advantages

• Superior to current antibody detection designs- Cost- Chemical Stability

• On-site vs. In-lab

• Possibly adaptable to other relevant testing

Ref [8]9

Costs For Comparison

Material CostCNC $25/Kg

Methacrylic Acid $2/Kg

3-methacryloxypropyltrimethoxysilane $1/Kg

Ethyleneglycol dimethacrylate $1/Kg

5,7-Dimethoxycoumarin (DMC - Aflatoxin template)

$130/g

MIP Cost (Outsourced): $0.10-0.50/mg Antibody Cost: $100-1000/mg

Ref [14-19]

10

Market Analysis

Modern agriculture requires vast amounts of animal feed:• 119 Million tons produced• ~ $25 Billion spending annually in just the US

Worldwide business• Millions of tons of animal feed distributed worldwide

Ref [3, 20]

11

Future Directions

Other mycotoxins (i.e. Ochratoxins)

Other food industries affected by mycotoxins

Bacteria (i.e. Salmonella, E.Coli, Lysteria)

Ref [21]

12

Potential Partners

Questions?

References[1] “Mold and Mycotoxin Issues in Dairy Cattle: Effects, Prevention and Treatment - eXtension,” Mold and Mycotoxin Issues in Dairy Cattle: Effects, Prevention and Treatment - eXtension, 23-Sep-2016. [Online]. Available: http://articles.extension.org/pages/11768/mold-and-mycotoxin-issues-in-dairy-cattle:-effects-prevention-and-treatment. [Accessed: 02-Nov-2016].

[2] “Mycotoxins: Invisible Toxins with Visible Impacts”, 15-May-2015. [Online]. Available: https://agrilinks.org/blog/mycotoxins-invisible-toxins-visible-impacts. [Accessed: 02-Nov-2016].

[3] J. A. Crump, P. M. Griffin, and F. J. Angulo, “Bacterial Contamination of Animal Feed and Its Relationship to Human Foodborne Illness,” Clinical Infectious Diseases, vol. 35, no. 7, pp. 859–865, 2002.

[4] R. S. Adams et al, “Mold and Mycotoxin Problems in Livestock Feeding (Dairy Cattle Nutrition),” Dairy Cattle Nutrition (Penn State Extension), 2016. [Online]. Available: http://extension.psu.edu/animals/dairy/nutrition/forages/mycotoxins-nitrates-and-other-toxicity-problems/mold-and-mycotoxin-problems-in-livestock-feeding-1. [Accessed: 02-Nov-2016].

[5] “Mycotoxin FAQs - CPN-2002 - Crop Protection Network,” Crop Protection Network. [Online]. Available: http://cropprotectionnetwork.org/corn/mycotoxin-faqs/. [Accessed: 02-Nov-2016].

[6] “Molecularly imprinted polymer,” Wikipedia. 26-Jun-2016.

References (Cont’d)

[7] Regal, P., Díaz-Bao, M., Barreiro, R., Miranda, J. M., & Cepeda, A. (2015). Development of a novel molecularly imprinted stir-bar for isolation of aflatoxins. Proceedings of The 19th International Electronic Conference on Synthetic Organic Chemistry. doi:10.3390/ecsoc-19-d001hhhhhhhh

[8] M. J. Whitcombe et al., “The rational development of molecularly imprinted polymer-based sensors for protein detection,” Chem. Soc. Rev., vol. 40, no. 3, pp. 1547–1571, Mar. 2011.

[9] L. Uzun and A. P. F. Turner, “Molecularly-imprinted polymer sensors: realising their potential,” Biosens. Bioelectron., vol. 76, pp. 131–144, Feb. 2016.

[10] R. Devonshire, “A Step by Step Guide to Using a Franking Machine,” LinkedIn Pulse, 31-Jul-2015. [Online]. Available: https://www.linkedin.com/pulse/step-guide-using-franking-machine-ryan-devonshire. [Accessed: 03-Nov-2016].

[11] Huang, Jennifer, and Diab Elmashni. "Analysis of Aflatoxins Using Fluorescence Detection." (2011): n. pag. Thermo Fisher Scientific. Web.

[12] C. Bodhibukkana et al., “Composite membrane of bacterially-derived cellulose and molecularly imprinted polymer for use as a transdermal enantioselective controlled-release system of racemic propranolol,” J. Controlled Release, vol. 113, no. 1, pp. 43–56, Jun. 2006.

[13] “Soxhlet extractor,” Wikipedia. 30-Oct-2016.

References (Cont’d)

[14] https://www.alibaba.com/product-detail/Manufacturing-methacrylic-acid_549935670.html

[15] https://www.alibaba.com/product-detail/ShinEtsu-KBM-503-CAS-2530-85_60421020692.html?s=p

[16] https://www.alibaba.com/product-detail/Ethylene-glycol-dimethacrylate-EGDMA-factory-in_60450864212.html

[17] Sigma-Aldrich – DMC [Online]. http://www.sigmaaldrich.com/catalog/product/aldrich/116238?lang=en&region=CA

[18] M. J. Whitcombe, “The Rational Development of Molecularly Imprinted Polymer-Based Sensors for Protein Detection,” Chemical Society Reviews, vol. 40, pp. 1547–1571, 2011.

[19] Lohr, Rhiannon et al. "Improved Aneurysm Treatment With Nanocrystalline Cellulose Reinforced Polymers". 2015. Presentation.

[20] Mycotoxins: Risks in Plant, Animal. and Human Systems. CAST - Council for Agricultural Science and Technology, 2003.

[21] http://www.foodpoisonjournal.com/uploads/image/recall(3).jpg

References (Cont’d)

[22]    T. Piacham, C. Isarankura-Na-Ayudhya, and V. Prachayasittikul, “A simple method for creating molecularly imprinted polymer-coated bacterial cellulose nanofibers,” Chem. Pap., vol. 68, no. 6, pp. 838–841, Nov. 2013.

[23]    M. Jiang et al., “Aflatoxin B1 Detection Using a Highly-Sensitive Molecularly-Imprinted Electrochemical Sensor Based on an Electropolymerized Metal Organic Framework,” Toxins, vol. 7, no. 9, pp. 3540–3553, Sep. 2015.

[24]    A. Rachkov, S. McNiven, A. El’skaya, K. Yano, and I. Karube, “Fluorescence detection of β-estradiol using a molecularly imprinted polymer,” Anal. Chim. Acta, vol. 405, no. 1–2, pp. 23–29, Jan. 2000.

[25]    M. A. Klich, “Aspergillus flavus: the major producer of aflatoxin,” Mol. Plant Pathol., vol. 8, no. 6, pp. 713–722, Nov. 2007.

[26] J. C. C. Yu and E. P. C. Lai, “Molecularly imprinted polymers for ochratoxin a extraction and analysis,” Toxins, vol. 2, no. 6, pp. 1536–1553, Jun. 2010.

[27] F. Navarro-Villoslada, J. L. Urraca, M. C. Moreno-Bondi, and G. Orellana, “Zearalenone sensing with molecularly imprinted polymers and tailored fluorescent probes,” Sens. Actuators B Chem., vol. 121, no. 1, pp. 67–73, Jan. 2007.

[28] B. Schyrr, S. Pasche, G. Voirin, C. Weder, Y. C. Simon, and E. J. Foster, “Biosensors Based on Porous Cellulose Nanocrystal–Poly(vinyl Alcohol) Scaffolds,” ACS Appl. Mater. Interfaces ACS Applied Materials & Interfaces, vol. 6, no. 15, pp. 12674–12683, 2014

References (Cont’d)

[29] D. Flynn, “USDA: U.S. Foodborne Illnesses Cost More Than $15.6 Billion Annually | Food Safety News,” USDA: U.S. Foodborne Illnesses Cost More Than $15.6 Billion Annually, Aug-2014. [Online]. Available: http://www.foodsafetynews.com/2014/10/foodborne-illnesses-cost-usa-15-6-billion-annually/. [Accessed: 02-Nov-2016].

[30] “Burden of Foodborne Illness: Findings,” Estimates of Foodborne Illness in the United States, 2016. [Online]. Available: http://www.cdc.gov/foodborneburden/2011-foodborne-estimates.html. [Accessed: 02-Nov-2016].

[31] Counting chickens. (2011, July/August). Retrieved October 20, 2016, from http://www.economist.com/blogs/dailychart/2011/07/global-livestock-counts

[32] “What is the aflatoxin problem?”. [Online]. Available: http://www.aflatoxinpartnership.org/?q=aflatoxins. [Accessed: 02-Nov-2016]. 

[33] “Aflatoxins in Kenya’s food chain: Overview of what researchers are doing to combat the threat to public health”. [Online]. Available: https://news.ilri.org/2014/05/06/aflatoxins-in-kens-food-chain/

[34] T. Lore, "New ILRI study finds high levels of aflatoxin in milk and dairy feeds in Greater Addis Ababa milk shed", AgHealth, 2016. .

[35] “FDA Mycotoxin Regulatory Guidance: A Guide for Grain Elevators, Feed Manufacturers, Grain Processors and Exporters,” Aug-2011. [Online]. Available: https://www.ngfa.org/wp-content/uploads/NGFAComplianceGuide-FDARegulatoryGuidanceforMycotoxins8-2011.pdf. [Accessed: 26-Nov-2016].

Cellulose Porous Film Preparation

Foodborne Illness

Foodborne illness is extremely prevalent CDC estimates that every year, 1 in 6 Americans (48

million people) contracts a foodborne illness, 128,000 are hospitalized, and 3000 die

It is also extremely expensive The USDA estimates that foodborne illnesses annually

cost the US economy more than $15.6 billion In reality these costs are much higher

Aflatoxin DMC

CNC-PVA film formation • Glass and quartz slides cleaned by sonication in 2-propanol and rinsed with deionized water• Substrates immersed in bath of concentrated sulfuric acid for 2 min to hydrophilize surface by

revealing silanol groups on surface• Substrates rinsed for 5 min with DI water and dried in N2

• PVA dissolved in deionized water heating at 95°C for 10 h with stirring • PVA solution diluted to 1 mg/mL in DI water• Freeze-dried CNCs added to conc. of 4 mg/mL• Mixture sonicated for 1 h at room temperature• Thin films deposited on substrates by dip coating• Films dried by freeze-drying process

CNC–composite film formation• Carried out in the presence of template molecule• React with 3-MPS (10% w/w in toluene) at 80°C for 5 h• Wash film in methanol and dry• Solution with 12 mmol MAA, 0.05 mol EDMA, 2mmol of template, 0.7 mmol AIBN in DMF (2 mL)• Purge with nitrogen, close and polymerize at 60°C for 18 h• Soxhlet extraction to remove the template molecule (10% w/w acetic acid in methanol for 72 h)