Endocrine Disruptors Detection Test

Figure 4: Endocrine disruptors (EDs) measurement

Detection test developement

Device optimization

• Develop an EDs detection test and kit.

Mainly found in industrialized areas, they lead to soil and groundwater pollution. They are suspected to alter endocrine and reproductive system functions of the living organisms (2).

From a physiological perspective, an endocrine disruptor (ED) is a natural or synthetic chemical substance (1).

3. Assays on new dyes to increase the test sensitivity.

2. Concentration of EDs from samples to reach the limits of dye absorbance detection.

1. Culture of Pleurotus ostreatus fungi in different conditions in order to increase enzymatic activity.

Optimization of a preliminary device

REFS.

COLLAB.

ABSTRACT

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BUDGET & TIMING

PRELIMINARY RESULTS & EXPECTED OUTCOMES

EXPERIMENTAL APPROACH

• Selection of new dyes

• Further expectations

• Optimize the test sensitivity already implemented by the previous Fils rouge team.

CONTEXT

AIMS

Guez Salomé Rodrigues Cécile Rose Solène

Endocrine Disruptors Detection Test

Guez Salomé, Rodrigues Cécile, Rose Solène

Wild pollution of EDs and their harmful effects

CONTEXT

Guez Salomé, Rodrigues Cécile, Rose Solène

Detections approaches

• Time consuming
• Need qualified personnel

Our solution

• Low-cost
• Fast
• Easy-to-use kit

Remediation approaches

• Can’t remove EDs completely (inefficacies)
• Expensive
• Limited specificity
• Can create toxic compounds

CONTEXT

Guez Salomé, Rodrigues Cécile, Rose Solène

How is our solution working ?

A first proof of concept:

• Pleurotus ostreatus produces ligninolytic enzymes.
• These enzymes degrade aromatic structures (lignin, EDs and Dyes).

CONTEXT

Detection test development

Assays on new dyes

EDs concentration

Culture of Pleurotus ostreatus in different conditions

Device optimization

Guez Salomé, Rodrigues Cécile, Rose Solène

Our main objectives:

AIMS

(3) Sánchez, C. (2010). Cultivation of Pleurotus ostreatus and other edible mushrooms. Applied Microbiology and Biotechnology, 85(5), 1321–1337. https://doi.org/10.1007/s00253-009-2343-7 (4) Palmieri, G., Giardina, P., Bianco, C., Fontanella, B., & Sannia, G. (2000). Copper induction of laccase isoenzymes in the ligninolytic fungus Pleurotus ostreatus. Applied and Environmental Microbiology, 66(3), 920–924. https://doi.org/10.1128/AEM.66.3.920-924.2000

Guez Salomé, Rodrigues Cécile, Rose Solène

Device optimization

• Measurement of ligninolytic enzymes production and activity by a spectroscopic analysis in order to determine the optimum culture conditions.

• Culture of Pleurotus ostreatus on two types of ligninolytic substrates (3).
• Use of Copper ions evaluation of its effect on ligninolytic enzymes production and activity (4).

1. Culture of Pleurotus ostreatus fungi in different conditions in order to increase enzymatic activity and production.

EXPERIMENTAL APPROACH

Device optimization

• Concentrations varying from 10µM to 200 µM.

• Spectroscopic analysis on seven dyes: Methyl orange, Tartrazine, Brillant blue, Bromophenol blue, Azorubine, Crystal violet and Brillant green.

3. Assays on new dyes to increase the test sensitivity

Low EDs concentration in the environment

Guez Salomé, Rodrigues Cécile, Rose Solène

• Concentration of EDs through binding on nylon membranes, and elution with acetonitrile (hydrophobic solution).

2. Concentration of EDs from samples to reach the limits of dye absorbance detection

EXPERIMENTAL APPROACH

Figure 4: Endocrine disruptors (EDs) measurement

• Test principle: measurement of dyes discoloration inhibition.

• Conduction of the test with dyes previously selected (during the optimization step).

• Analysis of 3 EDs : Bisphenol A, Oestradiol and Phthalate.

Detection test developement

Guez Salomé, Rodrigues Cécile, Rose Solène

EXPERIMENTAL APPROACH

Guez Salomé, Rodrigues Cécile, Rose Solène

GANTT CHART

BUDGET & TIMING

Guez Salomé, Rodrigues Cécile, Rose Solène

The budget has been estimated based on consumables, reagents but also, we have included the salaries of the three junior engineers working on the project.

BUDGET

BUDGET & TIMING

ABSORBANCE

CONCENTRATION (µM)

Test on sensitivity of several dyes

Guez Salomé, Rodrigues Cécile, Rose Solène

Brillant Green and Bromophenol Blue are the most sensitive dyes Brillant Green absorbance of 0,375 at 10 µM Bromophenol Blue absorbance of 0,192 at 5 µM

Preliminary results

PRELIMINARY RESULTS AND EXPECTED OUTCOMES

x ?

PRELIMINARY RESULTS AND EXPECTED OUTCOMES

Sensitivity increase by at least 500

x 15

x 30

Guez Salomé, Rodrigues Cécile, Rose Solène

• Increase of ligninolytic enzymes activities obtained by different culture conditions of Pleurotus

• Reach the limits of dye absorbance detection by concentration of EDs

• Increase of dye sensitivity

Further expectations

(1) Diamanti-Kandarakis, E., Bourguignon, J. P., Giudice, L. C., Hauser, R., Prins, G. S., Soto, A. M., Zoeller, R. T., & Gore, A. C. (2009). Endocrine-disrupting chemicals: An Endocrine Society scientific statement. Endocrine Reviews, 30(4), 293–342. https://doi.org/10.1210/er.2009-0002 (2) Puig. (2012). Departamento de Enxeñería Química Removal of Endocrine Disrupting Chemicals by the Ligninolytic Enzyme Versatile Peroxidase. (3) Sánchez, C. (2010). Cultivation of Pleurotus ostreatus and other edible mushrooms. Applied Microbiology and Biotechnology, 85(5), 1321–1337. https://doi.org/10.1007/s00253-009-2343-7 (4) Palmieri, G., Giardina, P., Bianco, C., Fontanella, B., & Sannia, G. (2000). Copper induction of laccase isoenzymes in the ligninolytic fungus Pleurotus ostreatus. Applied and Environmental Microbiology, 66(3), 920–924. https://doi.org/10.1128/AEM.66.3.920-924.2000

Guez Salomé, Rodrigues Cécile, Rose Solène

BIBLIOGRAPHY

Guez Salomé, Rodrigues Cécile, Rose Solène

It is nowadays well known that endocrine disruptors (EDs) are major pollutants of our environment. These natural or synthetic substances are suspected to be able to alter the functions of the endocrine system which can induce health issues like growth inhibition, sex differentiation, reproductive health disorders, cancers in both human and wildlife. The sources of EDs are wide and diverse (Pharmaceutical, care products, plastics, pesticides, herbicides, …). A lot of investment and efforts have been made to remove these pollutants, but the approaches developed do not generally lead to a complete removal of EDs from wastewater. If effective remediation constitutes a problem, detection is also another issue associated with EDs pollution. Indeed, existing tests are expensive, time-consuming and require highly skilled person. The project presented here has for aim to propose a low-cost, fast and easy-to-use kit, which is based on the competitive character between EDs and dyes to react with ligninolytic enzymes produced by Pleurotus ostreatus. Indeed, this fungus is well-known for its ability to degrade lignin through ligninolytic enzymes (laccase, peroxidase). Pleurotus ostreatus use these enzymes to degrade lignin (wood component), a complex structure of diverse phenylpropane randomly linked. Like lignin, EDs and dyes contain aromatic groups in their structures, that is why ligninolytic enzymes are capable of degrading EDs as well as dyes.

ABSTRACT

The strain of Pleurotus Ostreatus has been kindly provided by the food engineering laboratory of Sup'Biotech (LRPIA ) We are grateful for the help of : Mr Yates supervisor of Fil Rouge project and Mrs Saint-Pol our tutor and LRPIA manager

Guez Salomé, Rodrigues Cécile, Rose Solène

COLLABORATIONS & ACKNOWLEDGEMENTS