Joint research project

Development of new disposable nano-biosensor platforms for the detection of neurotoxic chemicals

Project leaders
Ferdinando Febbraio, Rabeay Younes Abdelfatah Hassan
Agreement
EGITTO - ASRT - Academy of Scientific Research and Technology
Call
CNR/ASRT biennio 2018-2019 2018-2019
Department
Biomedical sciences
Thematic area
Biomedical sciences
Status of the project
New

Research proposal

Addressing the problems related to the widespread presence of pesticides in the environment and the persistence of several of them (five of the nine new persistent organic pollutants (POPs) (Kannan, Yun et al. 2010) are pesticides), resulting in their bio-accumulation in the bodies of animals and humans, represents one of the most important challenges of this century (Ploteau, Antignac et al. 2016). Differently from other harmful chemicals, introduced into the environment by natural processes, or produced as by-products of industrial processes and other human activities, pesticides are designed to be toxic and are used as poisons to kill organisms. They are extensively applied over large areas in agriculture and in urban settings, becoming the most diffuse form of chemical pollution in the world. There is now an increasing awareness in the scientific community that the abundance of such chemical contaminants in the environment is the cause of many disorders that affect the environment and human health.
The detection and monitoring of pesticides has improved significantly in the last few years (Bras, Santos et al. 2000; Jeannot, Sabik et al. 2000; Ahmadi, Shahsavari et al. 2008). Technological advances, such as Surface-Activated Chemical Ionization (SACI) (Cristoni et al. 2007), which increases the sensitivity and accuracy of GC- LC-MS measurements, in association with methodologies, such as Quick Easy Cheap Effective Rugged and Safe (QuEChERS) (Payá et al. 2007), allow the identification of hundreds of these substances in a single shot. However, their detection in the environment still remains difficult, because are so widely distributed in the world and can be found in locations as diverse as pristine forests and the blood of arctic animals, wind, rain and other weather phenomena contributing to their diffusion. Consequently, in the last years, the scientific community has directed considerable research towards the development of new detection systems, such as biosensors, to replace conventional GC- and LC-MS technologies for the measurement of the contamination levels of these chemicals in the environment, in food and the human body fluids and tissue. Basically, biosensors involve bio-recognition element(s) such as enzymes, receptors, nucleic acids, or antibodies in the contact with an appropriate transducer (Morales-Narvaez, Baptista-Pires et al. 2017). Depending on the method of signal transduction, biosensors may be divided into four basic techniques: optical, mass, thermal and electrochemical biosensors (Gopel W 1995). The importance of electrochemical biosensors results from their high specificity, ease of fabrication and use with low cost and high sensitivity, which allow the on-line detection of a broad spectrum of analytes in complex matrices (e.g. blood, serum, urine or food) (U. Bilitewski 1993; Kariate Sudhakara Prasad 2009). On the other hand, the interesting properties of nanomaterials to provide suitable nano-structured platforms for biomolecules immobilization, maintaining their bioactivity, facilitating the electron transfer between their redox center and electrode surfaces, have led to the construction of nanostructured electrochemical biosensors (Hassan and Wollenberger 2016).
To achieve the monitoring of the organophosphate pesticides (OP) in the environment (water and liquid food) using a safe and easy to use electrochemical biosensor based on very stable electroactive enzymes, nanostrucured of disposable screen printed electrodes have been suggested for this study. The advantages to use stable enzyme is related to the possibility of their use in operative conditions. Moreover, OPs represent a large family of compounds that include also nerve agents, extending the use of such type of biosensors also to security fields.
The Italian research group at the IBP-CNR, have high expertise in enzymology, biotechnology and biochemistry. In the last years they studied a carboxylesterase enzyme (EST2) isolated from thermophilic source, belonging to the hormone sensitive lipase (HSL) family, that includes several acetylcholinesterases (AChE), in order to use as bioreceptor in biosensors for organophosphate pesticides detection. EST2 is obtainable in large amount by expression in mesophilic host, and easy purified by using thermo-precipitation steps. This enzyme shows very long time stability and an appropriate resistance and activity at different pH values and temperatures, as well as a good stability in the presence of low concentrations of organic solvents and detergents. Furthermore, the EST2 3D structure has been solved giving the possibility of having a model structure for in silico studies such as molecular docking predictions. Our studies demonstrated that is sensitive to the irreversible inhibition by OPs, showing a better selectivity and affinity with respect acethylcholinesterases, the main target of OP (Febbraio et al., 2008; Febbraio et al., 2011; Carullo et al., 2015).
On the other hand, the Egyptian group has the high experience to develop nanostrucured electrochemical biosensors that use different mutants of EST2 (multi-enzymatic biosensor), showing different specificity toward OPs, characterized by the Italian group, to be used for the detection of OPs in water and liquid food.

Research goals

An international and complementary team work, which is consisting of experts in biosensors, microbiology and bioelectrochemistry, is responsible for achieving the following tasks:
Isolation and purification of selective enzymes that will be used as bio-recognition elements for detection the target toxic compounds.
Selection of new enzymes with different specificity toward Ops
Development of electrochemical strategies for the assay of EST2 mutants in presence of OP for their detection
Synthesis and characterizations of nano-composites (metal nano-wires decorated with metal oxides nano particles) the disposable biosensors platforms using screen printed electrodes.
Identification the enzymatic inhibition mechanism with Ops.
Biosensors validations and application in different food and environmental samples.

This project will open an important scientific and technological collaboration that will continue also in the future submission of common projects. Moreover, the proposed research is expected to be appealing for industrial interactions, since it addresses innovative researches for important environmental and health problems.

Last update: 29/03/2024