Bioethical Considerations

a) Is the technology easy to use without extensive scientific knowledge or resources?

Yes, we intentionally designed safeTEA with accessibility in mind so it could be applied in resource-constrained communities.

b) Could people easily access or recreate our system?

While recreating the plasmid and aptamer complex itself would require a background in bioengineering, the creation of the toxin-capturing aptamers has been designed to be regeneratable with very little scientific knowledge or laboratory resources.

a) How could someone use this technology to cause harm?

Our technology allows the user to bind and separate Aflatoxin B1 from an aqueous solution, which has the potential to be isolated. Such isolation does have the potential to be used to create a concentrated AFB1 solution that can be used maliciously. For example, the Aflatoxin could be taken off the filter post-extraction and potentially be added to food, water, or an individual’s skin, opening up varying avenues for additional exposure. These types of malicious actions can be identified through detectable exposure techniques for acute cases, and those individuals would be able to receive treatment, but it would not be traceable to the perpetrator.

b) Is the health or ecological harm that our system could cause preventable or reversible?

Ecological harm from Aflatoxins depends on how it is disposed of. Our system is compostable, which we chose due to the fact that the half-life of AFB1 is about 5 days [1]. However, we recognize that this half-life is dependent on soil temperature and environmental factors that can vary across communities. The system, if disposed of improperly, also has the potential to impact natural water wells, be eaten by wildlife, or cause harm to plants.

a) Could our system be used in a large-scale attack on a group of people?

Since AFB1 and other mycotoxins are naturally present in many food products, someone could theoretically introduce the captured toxins into a community's diet or water source as a way to effectively poison a large group of people, that could appear as a natural occurrence. This, however, would be difficult to do without noticing, as accumulating enough mycotoxin to poison a community would require the collection of a large and likely noticeable amount of used filters. Also, the process of releasing the bound toxin from the filter could prove to be difficult without specialized equipment. It would require intense technical knowledge to release and concentrate AFB1, and we expect it to be an involved and long process to achieve. Therefore, we determined that it is unlikely someone would have the time and resources to maliciously obtain AFB1 concentrates from our system.

b) Would such an attack be readily noticeable?

There are likely not many ways to notice an attack before one occurs. Any health testing performed on the victim(s) would indicate the toxin, but it could be explained by the natural presence of the toxin in any consumed food products. Therefore, it would be unlikely that an actor could be identified. Despite this likelihood, acute aflatosis is a serious health event that impacts entire global communities, so divergence from this pattern for acute aflatosis in a single targeted individual is likely to raise concerns regarding malicious intent.

a) What defensive or preventative measures can be taken to recognize misuse?

We would like to implement a design into our system that indicates when the filter is saturated. Therefore, any malicious actor could be identified through the filter they used to isolate AFB1. We are also exploring the binding affinity between the aptamer and the bound toxin to give us a better understanding about the risk, and possible methods of releasing the bound toxin. We would like to further investigate what infrastructures should be in charge of the distribution of this technology, and create tracking capabilities in where the product is being distributed. There is the capability for our system to come with a comprehensive training to professionals and communities to whom we would be providing this technology, including safety protocols for handling and disposal.

Our deliverable requires the usage of a cellulose filtration system to remove the aptamer-toxin system from a solution. The potential for filter-to-skin contact provides a safety concern for users, and creates the possibility for it to be used as a weapon to harm someone. Our system currently is for the capture and removal of Aflatoxin B1, but our design is intended to be interchangeable with other aptamers with different binding targets. Risk assessment will be dependent on the target molecules used with our system, which changes the associated risks of adverse health effects if the filter comes into contact with skin. However, we expect that isolating such a toxin from the aptamer system would require extensive molecular knowledge, laboratory use, and time in order to create a concentrated toxin stock.

In the scope of our current application to target Aflatoxin B1 (AFB1), the risks associated with dermal exposure of this toxin has been determined to cause an increase in hepatic tumor development in rat studies [2]. However, such tumorigenesis requires consistent, long-term exposure to the toxin, an exposure that would become noticeable. Additionally, the adverse effects of skin exposure to AFB1 have been found to be decreased with natural remedies, such as treatment from the O. Sanctum leaf [3], commonly used as a medicinal agent primarily used in India. Given the ability of skin exposure risk to be mitigated with local medicinal agents, and the long-term consistent exposure needed to produce increased cancer risk, we determined that this aspect of our deliverable is low-risk. Because there are many simpler ways to cause immediate and lasting harm to someone, we expect low rates of malicious use in terms of dermal exposure of AFB1.

Since aptamers are able to have highly specific binding to target molecules, they have the potential to be used as a way to create concentrated stocks of harmful substances that aren’t normally able to be isolated outside of a laboratory. There is potential for certain wash steps to be applied to release the aptamer-toxin complex, indicating that more testing needs to be done to quantify the risk. However, we identified that such an endeavor would be difficult for a general consumer to perform, due to the process requiring specialized knowledge, time, and access to molecular tools in order to create a concentrated toxin solution.

Another possible exposure could occur from the cellulose filter posthumously releasing the aptamers and toxin, allowing the flow-through to contain parts of our plasmid, aptamers or unbound toxin.

Although we can’t necessarily quantify the effects of aptamer ingestion without further experimentation on mammalian species, literature indicates that DNA will denature in the stomach due to the low pH of stomach acid [4]. There is a possibility that if the aptamer is bound to AFB1, once denatured in the stomach it AFB1 could be released and cause genotoxic effects, as if it were still in the liquid. Although this is a possibility, our team determined that the efficacy of our aptamer and filtration system can still reduce overall exposure, even if some flow through occurs. Therefore, using our system can provide lower concentrations of AFB1 in the consumed liquid in total than unfiltered liquid, which indicates a low level of concern.

• Binding affinity testing would consist of quantifying the binding strength of the aptamer and AFB1 molecule in relevant food-bearing liquids. This would consist of using a cellulose filter with the aptamer-toxin compound on it. This would then be placed through different wash steps with different liquids to test their capacity for releasing the toxin via analysis of the flow through.
• We also propose a lifecycle assessment to quantify the environmental effects of our system once composted. It is known that AFB1 is able to degrade in soil [1], but its degradation rate is unknown while attached to our aptamer system. We would place a filter with our toxin-aptamer compound in a closed system soil environment, and quantify how much AFB1 is left on the filter paper at varying time intervals up to a few weeks. We would be able to then quantify how much AFB1 has been released by our aptamers.

• Detailed matrices for risk assessment and biosafety would include accurately instructing users of the product to dispose of the filter properly to prevent affecting their own dermis and the environment. This is a key intervention point to prevent misuse of our product due to accidental misuse, which would in turn reduce exposure.