1. Experimental studies for electrochemical and enzymatic detection of biological active sulfur compounds Authors: Niculina Dragomir, Ligia Chelmea Coordinators: Conf. Dr. Mihaela Badea, Conf. Dr. Marius Moga Faculty of Medicine, University Transilvania from Brasov

2. Motivation and purpose sulfur in organic compounds  different properties, depending on the function in which it is located. The paper aims to optimize biochemical methods for detection of biologically active compounds with sulfur  spectral analysis enzymatic catalysis (in the presence of peroxidase)  electrochemical methods analysis of the different behaviour of sulfur compounds: cysteine thiourea

3. Cysteine In scleroproteins of animal tissues (skin, hair, nails, hooves) Food sources: pork, poultry, eggs, dairy products, peppers, garlic, onions, wheat Industrial sources : hydrolysis feathers Structure of proteins - bind metal ions: Zn, Ni, Fe, Pb, Hg, Cd Applications precursor in the food industry (food additive E920), pharmaceuticals, cosmetics cysteine ​​ derivatives, multiple protective actions against hyperlipidemia, hyperglycemia and oxidative stress - drugs

4. Oxidized form - cystine Biological functions - thiol Cysteine ​​ -cystine system - reversible redox system Cysteine

5. Thiourea composed harmful, dangerous for the environment specific biological properties of thyroid tissue - transport inhibitor organic iodine Applications Intermediate in organic synthesis and medicinal - hyperthyroidism For the production of synthetic resins Vulcanization accelerator Corrosion inhibitor

6. Spectral analysis of enzyme kinetics Apparatus spectrophotometer (λ= 492 nm) Reaction conditions temperature: 25 ◦ C pH = 7.4 (phosphate buffer)

7. Principle H 2 O 2 + 2H + + 2e - 2H 2 O peroxidase Colourless substrat Coloured product

8. Substrate/chromophore (reduced form) Product (oxidized) 5-aminosalicylic acidBrown o-phenylenediamineOrange 4-chloro-1-naphtholBlue o-toluidine Blue o-dianisidineYellow-orange 3,3 '-diaminobenzidine Brown Principle

9. Principle Variation of extinction in studies of inhibition / activation of enzyme E xtinction Time E xtinction Time C0C0 C1 C2 C 2 >C 1 Activator C1C1 C2C2 C0C0 C 2 >C 1 Inhibitory

10. Results The equations of the regression lines Dependencies of %EA (reaction rate) vs concentrations of analytes

11. double behaviour of the organic sulfur compounds in presence of enzymatic media: 3,3 '-diaminobenzidine - peroxidase - hydrogen peroxide cysteine  ​​ activator thiourea  inhibitor A laborious method, consuming enzyme (expensive, non-reusable) Partial conclusions

12. Electrochemical analysis Apparatus Screen-printed electrodes with cobalt phthalocyanine (as mediator) Differentiated pulse voltammetry (DPV) AUTOLAB – soft GPES 4.0 Magnetic stirrer University of Perpignan via Domitia, France

13. Principle redox processes  electrochemical methods

14. Procedure 1. Preparation of solutions: buffer system watery cysteine ​​ / thiourea 2. Testing sensors stabilitation measuring method: differential pulse voltammetry (DPV) optimum potential (mV) for detection the optimum pH for detection 3. Plotting the calibration curve

15. Cysteine pH = 7

16. Thiourea acetate buffer - pH = 5.6phosphate buffer – pH = 7 Intensity (A) Potential (mV)

17. Thiourea Thiourea pH = 5,6

18. Partial conclusions sulfur compounds / DPV signals cysteine ​​ - pH = 7 - phosphate buffer system thiourea - pH = 5.6 - acetate buffer system Different peaks (specific intervals)  possibility of simultaneous detection of real samples

19. Conclusion  studies of aqueous standard samples  subsequent analysis of real samples.  mechanisms of action in peroxidase systems: activator – inhibitor different pH  electrochemical detection methods Using screen-printedelectrodes with cobalt phthalocyanine (as mediator)  cheap and fast detection of biological active compounds with sulfur in aqueous samples.

20. Acknowledgements The authors thank Prof. Dr. Jean-Louis Marty (Perpignan Via Domitia University of France) for providing electrochemical transducers used in experimental studies.

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