Enzymes Encapsulation in Peptide-based Nanodroplets for the Degradation of Organophosphate

The use of pesticides in agriculture has widely spread in order to increase the yield while maintaining food quality and safety.

The use of pesticides allows for effective and rapid elimination of a wide variety of pests, which severely harm agricultural crops.

However, the widespread use of pesticides is of increasing concern bearing various ecological impacts, as well as leading to various health impacts due to low-level chronic exposure.

One particularly toxic type of chemical pesticides is the organophosphate (OP)-based pesticides, which have contributed to improvements in agricultural productivity across the world by controlling insects, nematodes, and plant pathogens.

Notably, OP-based pesticides affect the nervous system by interfering with the activity of, acetylcholinesterase, the enzyme that regulates the neurotransmitter acetylcholine.

Inhibition of acetylcholinesterase by OP-based pesticides may cause a variety of health problems, including hormonal changes, neurological, respiratory, reproductive, and endocrine effects, as well as cancer.

Uncontrolled discharge of OPs into the environment has led to their accumulation in sediments and soil, making them the secondary source of pollution affecting both human health and ecosystems.

Today, the conventional approaches for OPs degradation include chemical decontamination, such as, hydrolysis, oxidation, or reduction, and photochemical methods that are based on light radiation to degrade OPs. However, the current methods are costly, often aggressive and not entirely safe, in term of chemical residues.

Therefore, there is a need for on-site, cost-effective, eco-friendly technologies for the degradation of OPs into harmless compounds.

This need has motivated our research aimed to develop technologies based on OP degrading enzymes, such as phosphotriesterase.

Here we propose to design and develop microfluidic approach to encapsulate OP degrading enzyme in peptide-based nanodroplets to efficiently act as supportive and protective nanocarriers to enhance the durability and activity of the encapsulated enzyme. We will use Boc-Phe-Phe-OH (BocFF) and FmocFF that assembles into nanospheres and hydrogel nanodroplets respectively.

These peptide nanostructures were shown to have notable durability in a wide range of pH values and temperatures and will serve as enzyme nanocarriers. In addition, they are environmentally friendly, easily synthesized, inexpensive, and biocompatible materials.

This complex and interdisciplinary approach requires the combination of techniques from the fields of biochemistry biophysics and material science, which will be incorporated into one unique platform – microfluidics.

Microfluidic device can produce discrete monodispersed droplets at a rate of over 100 s−1, with volumes as small as several picoliters. Under these conditions, each droplet can serve as an individual nano-reactor.

We will analyze the encapsulated system and its efficiency using both microscopy and spectroscopy tools, such as high-resolution scanning electron microscopy, dynamic light scattering and zeta potential.

The stability and activity of the enzyme will be further studied using enzymatic kinetic tests under various extreme environmental condition, including a wide range of temperatures (20-80°C), pH (3-10) and in the presence of proteases such as trypsin. This pioneering approach can pave the way for more essential studies in various fields.

Furthermore, by scaling this microfluidic platform, high throughput screening can be readily done, thus reducing costs and time.