Benito-Lopez, Fernando

Curto, Vincenzo F.

Diamond, Dermot

MacFarlane, Douglas R.

Ranganathan, Vijayaraghavan

Scheuermann, Stefan

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UNIVERSITY	  COLLEGE	  DUBLIN	  	  	  	  	  	  DUBLIN	  CITY	  UNIVERSITY	  	  	  	  	  	  TYNDALL	  NATIONAL	  INSTITUTE	  	   Vincenzo F. Curto1, S. Scheuermann1, V. Ranganathan2, D. MacFarlarne2,  D. Diamond1, F. Benito-Lopez1 1CLARITY: Centre for Sensor Web Technologies, National Centre for Sensor Research,, School of Chemical Sciences, Dublin City University, Dublin 9, Ireland 2School of Chemistry, Monash University, Clayton, Victoria 3800, Australia vincenzo.curto2@mail.dcu.ie  Ionic Liquids in biosensors: influence of  hydrated choline based ILs on the bio-functionality of Lactate Oxidase REFERENCES [1] M Armand, F Endres, DR MacFarlane, H Ohno, B Scrosati, Ionic-liquid materials for the electrochemical challenges of the future, Nature Materials, 8(2009) 621-9. [2] Moniruzzaman M., et al., Activation and stabilization of enzymes in ionic liquids.  Organic & Biomolecular Chemistry, 2010, 8, 2887–2899 [3] Fujita K., et al., Solubility and Stability of Cytochrome in Hydrated Ionic Liquids: Effect of Oxo Acid Residues  and Kosmotropicity, Biomacromolecules 2007, 8, 2080-2086 [4] Khodagholy D., et al., Organic electrochemical transistor incorporating an ionogel as a solid state electrolyte for lactate sensing, J. Mater. Chem., 2012. 22, 4440-4443.     .    ACKNOWLEDGMENT  Temperature Scan of LOx in PBS and Choline dhp Secondary structure Thermal denaturation and aggregation of LOx in PBS and Choline dhp at 222 nm Science Foundation Ireland (SFI) under the CLARITY CSET award (Grant 07/CE/I1147) and Research Career Start Programme 2010 fellowship form Dublin City University. Marie Curie Actions International Reintegration Grant (IRG) (PIRG07-GA-2010-268365) and Irish Research Council for Science, Engineering and Technology. Marie Curie International Reintegration Grant (Grant PIRG06-GA-256367 CELLTOX).  RESULTS – Residual Activity RESULTS – Circular Dichroism Colorimetric enzyme activity assay    ILs/LOx stock solutions were stored at 5  °C and 37 °C INTRODUCTION CONCLUSION and FUTURE WORK The use of conventional aqueous electrolyte on the realisation of biosensor can give some life-time issues related to the volatility of water, limited temperature range and the very narrow pH range for protein stability.[1]   Ionic Liquids (ILs) are low melting point salts (<100°C) that represent a new class of non-aqueous solvents composed entirely of ions, which have been extensively used in biocatalysis. [2]  It was shown that certain proteins are soluble, stable and remain active for up to 18 months when dissolved in hydrated ILs. [3]  In fact, the addition of small amounts of water to ILs strongly influences the protein solubility while the main properties of the selected Ionic liquid are retained. In the present study, the structural and the catalytic properties of LOx were investigated using Circular Dichroism and colorimetric  assay, respectively.  The final aim of this study is the realisation an electrochemical biosensor for the sensing of lactic acid in sweat. [4] In this work we studied the structural conformation and residual activity of LOx enzyme when dissolved in conventional PBS and hydrated choline ionic liquids. Circular Dichrosim showed that hydrated choline dhp is capable to retain the secondary structure and enhance the heat induced denaturation of the enzyme. Moreover, residual activity experiments revealed that the bio-functionality of the enzyme is strongly influenced by the nature of the anion of ILs.  Future work will focus on the study of the kinetic constants and the integration of choline dhp in to a biocompatible polymeric matrix, such as chitosan and Gelatin A. -10000 -8000 -6000 -4000 -2000 0 2000 4000 6000 8000 10000 12000 14000 16000 18000 190 200 210 220 230 240 250 mean residue ellipticity [deg cm2  dmol-1]  Wavelength [nm] 25°C before heating 95°C 25°C after heating -5000 -4000 -3000 -2000 -1000 0 1000 2000 3000 4000 5000 6000 7000 190 200 210 220 230 240 250 mean residue ellipticity [deg cm2  dmol-1]  Wavelength [nm] 25 °C before heating 95°C 25°C after heating (1) 25 °C (2) 95 °C (3) 25 °C α-helix 11 4 8 β-sheet 34 28 37 Turns 24 24 25 Unordered 30 32 30 NRMSD 0.087 0.067 0.116 (1) 25 °C (2) 95 °C (3) 25 °C α-helix 28 12 18 β-sheet 23 30 30 Turns 20 24 28 Unordered 30 32 23 NRMSD 0.011 0.042 0.039 Choline dhp PBS 435 440 445 450 455 460 465 -10000 -9000 -8000 -7000 -6000 -5000 25 35 45 55 65 75 85 95 HT [V] mean residue ellipticity [deg cm2  dmol-1] Temperature [°C] 25°C to 95°C 95°C to 25°C HT 25°C to 95°C HT 95° to 25°C 490 495 500 505 510 515 520 525 530 535 -5000 -4500 -4000 -3500 -3000 -2500 25 35 45 55 65 75 85 95 HT [V] mean residue ellipticity [deg cm2  dmol-1]  Temperature [°C] PBS Choline dhp 0 25 50 75 100 0 30 60 90 120 150 Normalised enzyme velocity (v/vo) % time/day 0 25 50 75 100 0 30 60 90 120 150 Normalised enzyme velocity (v/vo) %  time/day IL	  free	  Choline	  Cl	  Choline	  dhp	  Choline	  Nitrate	  Choline	  formate	  Choline	  tartrate	  Choline	  levulinate	  Choline	  dibutylphosphate	  

Ionic liquids in biosensors: influence of hydrated choline based ILs on the bio-functionality of lactate oxidase

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