Alexandre François Sabrina Heng Roman Kosteki Tanya M . Monro

1. Enzyme detection by surface plasmon resonance using specially engineered spacers and plasmonic labelling Alexandre François Sabrina Heng Roman Kosteki Tanya M. Monro

2. Outline New SPR fiber sensor architecture Application for enzyme sensing

3. Traditional SPR systems – Kretschmann configuration Kretschmann prism configuration (angular interrogation) Reflectivity measured as function of incidence angle Sequence measurements require precise temperature control High quality sensor chip (gold coated quartz slides, high precision required for the gold coating) • Label free Sensing • Refractive index sensor - High sensitivity (10-5 RIU) • Fast detection (within the order of seconds) • Real time kinetics measurements (constant of dissociation, affinity…) http://ultrabio.ed.kyushu-u.ac.jp/A9912/katudo/smelldog-e/SPR3-e.jpg

4. Alternative Optical fibers SPR systems Coupling between the incoming light and the plasmonic wave following the same mechanism as described by Krestchmann. Spectral interrogation rather than angular (broadband source for excitation) SPR characterisation performed by transmission/optical losses measurements Same restriction concerning the metallic coating Jorgenson, R. C., and Yee, S. S., “A fiber-optic chemical sensor based on surface plasmon resonance”, Sensors and Actuators B, 12, 213-220 (1993)

5. Novel optical fiber SPR systems Similar coupling mechanism between incoming light and surface plasmon wave as standard SPR optical fibers Point detection of the re-emitted light from the plasmonic wave by surface scattering at the sensing region Enables multiple sensing regions for both dynamic self referencing and multiplexed sensing

6. Novel optical fiber SPR systems from concept to fabrication

7. How it works Inlet Outlet PDMS flow cell Spectrometer Transmitted light White light source Intensity Spectrometer Scattered light Intensity 532nm cw laser Wavelength Wavelength Allows to perform both SPR & fluorescence sensing simultaneously

8. SPR sensor fabrication Surface roughness = Key element for SPR scattering Silver deposited using modified Tollens reaction Silver nitrate solution (Transparent solution) Silver oxide (Brown precipitate) Reduction of silver ammonia to silver metal (1) 2 AgNO3 + 2 KOH → Ag2O (s) + 2 KNO3 + H2O Ag2O (s) + 4 NH3 + 2 KNO3 +H2O (l) → 2 Ag(NH3)2NO3 + 2 KOH CH2OH(CHOH)4CHO + 2Ag(NH3)2+ + 3OH–→ 2Ag(s) + CH2OH(CHOH)4CO2- + 4NH3 + H2O Silver ammonia complex (Transparent solution) (2) (3) (1) (2) (3) Kretschmann, E., “The angular dependence and the polarisation of light emitted by surface plasmons resonance on metals due to roughness”, Optics Communications, 5, 331-336 (1972) Raether, H., “Surface plasmons on smooth and rough surfaces and on gratings”, Berlin, New York, Springer-Verlag (1988)Boehm, J., François, A., Ebendorff-Heidepriem, H., and Monro, T. M., “Chemical Deposition of Silver for the Fabrication of Surface Plasmon Microstructured Optical Fibre Sensor”, Plasmonics, 6, 133-136 (2011)

9. SPR sensor fabrication Tollens reaction Sputtering reaction Similar SPR response obtained from sputtered glass slide and Tollens coated glass slides Surface roughness ~ 5nm for a 60nm thick Ag film with Tollens reaction, ~ 2nm with sputtering Boehm, J., François, A., Ebendorff-Heidepriem, H., and Monro, T. M., “Chemical Deposition of Silver for the Fabrication of Surface Plasmon Microstructured Optical Fibre Sensor”, Plasmonics, 6, 133-136 (2011)

10. Evanescent field capture mode vs transmission • Same information obtained from transmission & evanescent field measurements. • Higher Signal to Noise ratio in the evanescent field measurements = Higher detection limit. François, A., Boehm, J., Oh, S. Y., Kok T. and Monro T. M., “Collection mode surface plasmon fibre sensors: A new biosensing platform”, Biosensors and Bioelectronics, 26, 3154-3159 (2011) White, I., M. and Fan, X., “On the performance quantification of resonant refractive index sensors”, Optics Express, 16, 1020-1028 (2008)

11. SPR signal as function of the silver coating thickness 22nm • No dependency of the SPR signal measured using the evanescent field mode as function of the thickness of the metallic coating • Ease the sensor fabrication 40nm Increasing silver coating thickness 55nm 65nm 132nm François, A., Boehm, J., Oh, S. Y., Kok T. and Monro T. M., “Collection mode surface plasmon fibre sensors: A new biosensing platform”, Biosensors and Bioelectronics, 26, 3154-3159 (2011)

12. Refractive index sensitivity Dl/Dn = 7.9×10-4 RIU Comparable to previous published SPR fiber sensor (2.5×10-4 -7.5×10-5 RIU) Jorgenson, R. C., and Yee, S. S., “A fiber-optic chemical sensor based on surface plasmon resonance”, Sensors and Actuators B, 12, 213-220 (1993)

13. Small biomolecule sensing • Small biomolecules (below 50kDa) sensing remains a challenge with SPR due to their low molecular weight. • Proteins such as enzymes are known to have a major role in many biological processes. • Enzymes are involved in numerous diseases spanning from gastro intestinal disorder to cancer and can consequently be used as specific biomarker for diagnostic purposes. DiMagno, E. P., Go, V. L. W. and Summerskill, W. J., “Relations between pancreatic enzyme outputs and malabsorption in severe pancreatic insufficiency”, The New England J. of Med., 288, 813-815 (1973) Watson, M. A., Stewart, R. K., Smith, G. B., Massey, T. E. and Bell, D. A., “Human glutathione S-transferase P1 polymorphisms: relationship to lung tissue enzyme activity and population frequency distribution”, Carcinogenesis, 19, 275-280 (1998) Sidransky, D., “Emerging molecular markers of cancer”, Nat. Rev. Cancer, 2, 210-219 (.2002)

14. Enzyme sensing concept Label released from the surface upon cleaving of the spacer Spacer + label binding Intensity Sensor surface Wavelength

15. Spacer synthesis & characterization Trypsin cleavage group Trypsin cleaves peptide chains mainly at the carboxyl side of the amino acid arginine Carboxylic group Amino group ~ 7 nm MW = 1509.8 g/mol (calc) Histidine provides higher water solubility Alamine neutral amino acid

16. Spacer synthesis & characterization Fragment 1: Molecular weight (M): 554 Fragment 2: Molecular weight (M): 1016

17. SPR fiber sensor preparation PAH PSS PAH PSS PAH Silver 1st step Polyelectrolyte coating 2nd step Spacer immobilisation 3rd step Qdot immobilisation 4th step Enzyme cleaving A 100mg/mL trypsin solution in PBS buffer (Phosphate buffered saline (PBS) yielding a pH of 7.4.) was injected into the flow cell after the Qdots immobilisation, at 5mL/min (150mL) François, A., Boehm, J., Oh, S. Y., Kok T. and Monro T. M., “Collection mode surface plasmon fibre sensors: A new biosensing platform”, Biosensors and Bioelectronics, 26, 3154-3159 (2011)

18. Combined SPR/fluorescence sensing DlSPR~15nm upon spacer immobilization cross -linking of the spacer. DlSPR~40nm upon Qdots immobilisation and subsequent enzyme cleaving Qdots surface density calculated from fluorescence measurements ~38 fmol/mm2

19. Conclusion future work • New SPR fiber sensor achitecture based on surface scattering of plasmonic wave estabished • Proof of concept of enzyme sensing using SPR and specially engineered spacers has been established • Non specific binding blocking not required since a release from the surface is measure • Combined SPR / fluorescence sensing as a secondary confirmation and analytic evaluation Future work • Novel spacer design to prevent cross-linking onto the sensor surface • Assessment of the detection limit • Sensor surface re-generation & application to antibodies-antigen based immunoassays

20. Acknowledgments • This work was supported by the ARC, DSTO, the SA State Government and The University of Adelaide Assessment of the detection limit • Tanya Monro acknowledges the support of an ARC Federation Fellowship