Guan H. Tan, Ng Khan Loon, Khor Sook Mei and Lee See Mun

1. Electrochemical preparation and characterization of gold nanoparticles graphite electrode: Application to antioxidant analysis Guan H. Tan, Ng Khan Loon, Khor Sook Mei and Lee See Mun

2. Contents • Introduction • Literature reviews • Objectives • Experimental • Results and discussion • Conclusions • References

3. Introduction • Graphite • Good electrical conductance • Renewable surface • Chemical inertness • Abundantly available (such as used batteries) Graphite possess a honeycomb laminar structure Source from www.pixshark.com The beta carbon of the upper layer is positioned above the cavity, therefore it possesses a free valence electron, which can form Van der Waals interaction with metals (Appy et al., Prog. Surf. Sci., 2014 vol. 89, pp. 219)

4. Introduction Limitations with graphite - High activation overpotential (Wring et al., Analyst, 1992, 117, pp 1215), which limits its application in electroanalysis. • To overcome this, the surface of the graphite could be modified with nobel metal nanoparticles such as goldand platinum. Anti-oxidant analysis required high oxidation potential window, instead of reduction potential Gold Silver Source : F. Campbell, R. Compton., The use of nanoparticles in electroanalysis: an updated review. Analytical and bioanalytical chemistry, 2010, 396, Pg 241

5. Introduction - 1-step preparation: Auric acid solution and Cyclic voltammetry - High reproducibility with simple control parameter (deposition cycles, and Auric acid concentration) • Does not require any incubation time. • Surface can be easily cleaned using tape and re-used. • Less chemical usage

6. Introduction Anti-oxidant • Anti-oxidant compounds are substances that can inhibit oxidation caused by free radicals, peroxide, and oxygen. Electro-active compound that could be analyzed using electrochemical method Electron donating peroxide Anti-Oxidants Natural Synthetic Myricetin Quercetin Rutin Tocopherol Propyl gallate Butylated hydroxyanisole (BHA) Butylated hydroxytoluene (BHT) Tert-butylhydroquinone (TBHQ)

7. Introduction Myricetin • Abundantly available in fruits and vegetables such as grape, tomato, cabbage and carrot ( Huang et al., Toxicology in vitro. 2010, pp 21) • Possess anti-oxidant properties, which is significant to health: • a) anti-cancer (Shiomi et al., Food Chem., 2013, 139, pp 910) • b) therapeutic potential for diabetes mellitus( Li et al., Food science and human wellness, 2012, pp 19) • Analytical procedure used in the analysis of myricetin in food • Liquid chromatography (Flores et al., Food Composition and Analysis, 2015, 39 , pp 55) • Gas chromatography ( Kumar et al., Analytica Chimica Acta, 2009, 631, pp 177) Accurate and precise method but tedious sample preparation and not possible for field analysis Alternatively – electrochemical method could provide a rapid, and possible for on-field analysis (screen printed electrode)

8. Introduction BHA, BHT and TBHQ • Anti-oxidants used as the additives in food • International food safety standards, limit the usage at 200 ppm in edible oil products, biscuits, chewing gum, cream-based products,etc. Butylated hydroxytoluene (BHT) Tert - Butylhydroquinone (TBHQ) Butylated hydroxyanisole (BHA) Safety assessment study – high concentration level in food above 3000 ppm could promote cancer (G. M. Williams, M. J. Iatropoulos, and J. Whysner, Food Chem. Toxicol., 1999, 37, pp 1027)

9. Literature reviews AOx: ascorbic oxidase, GOx: glucose oxidase Significance of the modified graphite electrode studies - Renewable surface ( Ref. 2) - Disposable sensor (Ref.3, 5 and 6) - Improved performance – increase surface area, electrocatalytic, and overpotential (Ref. 1,4, and 5)

10. Literature reviews

11. Objectives The objectives of this research study : • Fabrication of a graphite electrode from a used battery and surface modification with gold nanoparticles. • Electrochemical and morphology study of the fabricated working electrode to assess the electrode performance. • Application of the surface modified working electrode in anti-oxidant analysis and determination in food samples.

12. Experimental design

13. Experimental Surface polish with emery paper and alumina silicate powder Used battery PTFE insulation Fabrication of Au-NPs/graphite Graphite rod, diameter 3mm Dry in oven at 130 °C 1.0 mM HAuCl4 Cyclic voltammetry (electrodeposition) 4, 8, 12, 16, 20 and 24 cycles Surface Activation (0.5 M H2SO4) Cleaning – Sonication in UPW and ethanol

14. Experimental – Electrode characterization • Gold nanoparticles size • Distribution • Element analysis by EDXrF • Cyclic voltammetry - scan rates study • Ferri/ferro cyanide redox • Nyquist Plot • Randless-circuit

15. Application of Au-NPs/graphite electrode Method development flow Method validation - LOD, LOQ, Linearity, Buffer and electrolyte optimization. Example Britton-Robinson buffer, Phosphate buffer, Experimental sample analysis pH optimization • Electrochemical technique • Linear sweep voltammetry • Square wave voltammetry

16. Results and discussion Results and discussions

17. Results and discussion Constant peak current – thermodynamic favorable nucleation growth of gold. Anodic scan - oxidation • Gold nanoparticles deposition Graphite 0.7109 V 8 cycles Reduction 0.5497 V 16 cycles Peak potential shifted toward more positive potential suggesting a favorable deposition of the Au on the metal rather than carbon substrate.

18. Results and discussion suppression in Au oxide formation after 20 CV scan 1st CV scan • Activation of Au-NPs/graphite in 0.5 M H2SO4 inactivated activated 20th CV scan Reduction of Au Peak bare The CV of the ferri/ferro redox of the activated Au-NPs/graphite showed an improvement in the overpotential (51mV). Au-NPs without activation impedes the performance of the Au-NPs/graphite Peak potential difference (Epa - Epc) / V, activated = 78 mV, Bare and inactivated = 183 mV

19. Results and discussion D Bare graphite A 24th Deposition cycles Morphology evaluation with FE-SEM 180 nm 8th Deposition cycles B 75 nm 16th Deposition cycles C 110 nm Au peak

20. Results and discussion • Electrochemical characterization Effective surface area (A) (Randless-Sevcik) Heterogeneous electron transfer (HET)rate (Laviron equation) Electron transfer resistance Randless Circuit fitting (c)

21. Results and discussion 0.1M Ferricyanide solution Activated Au-NPs/graphite • Electrochemical characterization (cont.) inactivated Au-NPs/graphite Bare graphite Anodic potential at 0.3210 V (solid line) shifted to 0.2698 V (dotted line) Nyquist Plot – at16th deposition cycle The overpotential of the activated Au-NPs/graphite was improved and the measured current was much higher than the bare graphite. The peak separation between the anodic and cathodic is much closer to the theoretical 59.16 mV (Nernst equation).

22. Results and discussion Highest peak current at pH 2 • Application to anti-oxidant analysis – myricetin Method: Square wave voltammetry (SWV) Electrolyte: Britton-Robinson Buffer 0.1M At 0.0591, n =1 Nernst slope

23. Results and discussion From Nernst equation, it suggests an equal ratio of electron to proton transfer, i.e. n=1. A B 3 oxidation potential Oxidation mechanism at peak , 0.4 V The first oxidation occurs at the 2nd hydroxyl group of pyrogallol group also reported by Goncalo et. al and Komorskyet al. C. Goncalo et. al. J. Mol. Chem., 2010, 16, 863 S. Komorsky-Lovrić et. al.,Electrochim. Acta, 2013, 98, 53.

24. Application to anti-oxidant analysis – myricetin Results and discussion Au-NPs /graphite Bare graphite Au-NPs /graphite Au-NPs/graphite - Improvement in the sensitivity of myricetin analysis. Bare graphite

25. Results and discussion SWV of myricetin with concentration increment corresponding to 0.2, 0.4, 0.6, 0.8 and 1.0 µg mL-1. The detection limit (LOD)of myricetin = 0.4 µg mL-1 The limit of quantitation (LOQ) was calculated based on 10 times the standard deviation of LOD (n=5). The LOQ of myricetin = 0.8 µg mL-1

26. Results and discussion • To test the method accuracy and precision in sample analysis, solutions of myricetin in ethanol were prepared at 0.8 and 1.0 µg mL-1 ( n=5) Analysis in green tea samples (n =2)

27. Other Anti-Oxidants in Food • TBHQ (Tertiary Butyl Hydroquinone) • BHA (Butylated Hydroxy Anisole) • BHT (Butylated Hydroxy Toluene)

28. Results and Discussion BHT BHA Au-NPs/Graphite TBHQ Bare graphite

29. Results and Discussion Linear correlation of peak current against concentration Linear sweep voltammetry (LSV) analysis of TBHQ, BHA and BHT standards BHA (b) BHA (a) BHT 64 µg mL-1 4 µg mL-1 TBHQ TBHQ Blank BHT

30. Results and Discussion Analysis of TBHQ, BHA and BHT in food samples using linear sweep voltammetry and Au-NPs/graphite working electrode. (n =5) All within the allowed limits of 200 mg/kg

31. Conclusions • In this study a gold nanoparticles graphite electrode was successfully fabricated from a used battery graphite. • The electrochemical and morphology characterization showed improvement in the effective surface area, overpotential and heterogeneous electron transfer rate of the Au-NPs/graphite electrode. • It can be inferred that at the 16th deposition cycle the Au-NPs/graphite electrode reached an optimum performance. • The Au-NPs/graphite was successfully applied in the myricetin analysis using square wave voltammetry. The electrode sensitivity was improved by 2.5 fold when compared to the bare graphite. The LOD and LOQ were determined at 1.26 x 10-6 mol L-1 and 2.51 x 10-6 mol L-1 • The 3 anti-oxidants can be simultaneously analyzed using the Au-NPs/graphite electrode. It was successfully applied in the determination of TBHQ, BHA, and BHT in some food samples.

32. Acknowledgment • This work was financially supported by : - The University of Malaya Research Grant (UMRG-ProgrammeRP012C-14SUS/PG177-2014B), - Fundamental Research Grant Scheme (FRGS) from the Ministry of Higher Education of Malaysia (MOHE) FP014-2013A and FP058-2014A. Publication • This work has been accepted by the journal of Analytical Sciences: • Khan Loon Ng, See Mun Lee, Sook Mei Khor, Guan Huat Tan. 2015. Electrochemical preparation and characterization of gold nanoparticles graphite electrode: Application to myricetin antioxidant analysis. Analytical Sciences. Accepted for publication. (ISI-Cited Publication)

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