A fiber tip label free biological sensing platform for in-vivo applications

1. A fiber tip label free biological sensing platform for in-vivo applications Alexandre François Kristopher J. RowlandTanya M. Monro SPIE Photonic West – BIOS 2013

2. Our motivation Develop an in-vivo sensing platform for: Biomedical diagnostic applications Studying fundamental biological processes in-vivo(probing a cell or its local environment) Himmelhaus et al., Biosensors & Bioelectronics (2009)

3. Whispering Gallery Modes • When light is trapped in a microsphere by Total Internal Reflection (TIR), it might circulate within the sphere along its circumference. These modes are so-called Whispering Gallery Modes (WGM).

4. Whispering Gallery modes for sensing Any modification of the microspheres optical properties (adsorption of molecules at its surface) results in modification of the WGM resonance frequency. WGM frequencies are influenced by both the resonator’s shape (radius, sphericity) and optical properties (index of refraction, absorption coefficient and scattering). Courtesy of J.U. Nockel (http://www.uoregon.edu/~noeckel/microlasers.php)

5. Why is WGM interesting for biosensing ? • label-free • highly sensitive (single molecule detection demonstrated) and can give access to other information such as the orientation of the attached molecule • non-destructive to the sample • highly selective (through surface functionalisation of the micro sphere surface) • applicable to various targets (sensitivity depends on the target’s molecular weight) Vollmer et al., Nature Methods (2008), Noto et al., J. Bio. Phys (2007), Armani et al., Science (2007)

6. Microresonators High Q WGM sensor Low Q WGM sensor • External excitation via optical coupling with an eroded optical fibre or prism (Q>107) • Require a tedious tuning of the gap between the microsphere and an optical fibre taper (nm scale). • Microsphere/microtoroid diameter ranging from 40 to 300 mm – low free spectral range. • Internal excitation with fluorescent dye/quantum dots – can be operated above the lasing threshold of the gain medium • Allow a reduction in size to increase the WGM wavelength shift (down to 3mm Ø) which in turns results in higher sensitivity (Dl/Dn). • Many resonance peaks available, large free spectral range Vollmer et al., Nature Methods (2008), Armani et al., Science (2007), Francois et al, APL (2008), Astratov et al., APL (2004), Beier et al., J Opt Soc Am B (2010)

7. Polymer microspheres can be easily doped or coated with a gain medium Microspheres preparation • Fluorescent dye (Nile Red) in xylene on top of an aqueous polystyrene beads solution. • Left under agitation with magnetic stirrer until the xylene evaporates WGM fluorescence modulated emission spectra Francois et al., APL (2009) & (2008) Kuwata-Gonokami et al., Opt. Mat.,(1998)

8. WGM resonators combined with MOF • The idea is to combine the unique light guiding properties of MOF for the excitation and collection of WGM in dye doped resonators. • Simple (auto alignment of the sphere with the fiber’s core) & robust sensor architecture • Dip & in-vivo sensing application possible (doesn’t require a microfluidic flow cell) Gregor et al., APL (2010), Francois et al., APL (2011)

9. Experimental setup • Allows excitation/collection of the fluorescence emission through the suspended core Microstructured Optical Fiber (MOF)

10. How does the fiber influence the modes? Almost perfect match between the resonator and the fiber hole: Øsphere=16mm Øhole=15mm Larger resonator diameter compared to the fiber hole: Øsphere=20mm Øhole=15mm Complete mismatch between the fiber hole and resonator diameter: Øsphere=25mm Øhole=15mm Enhancement of specific modes depending on their polarization are observed when resonator is sitting into a hole of a suspended core MOF, depending on the mismatch between the resonator and the MOF’s hole diameter.

11. Doped microresonators can be turned into microscopic laser sources once the pump power overcomes the losses. Produces resonance features with higher signal to noise ratio = Higher Q factor = Higher resolution WGM laser

12. Dip refractive index sensing • single microsphere (Ø~10.54mm) attached to a glass slide within a microfluidic flow cell • (b) WGM spectra of a single microsphere (Ø~9.91mm) attached to a MOF tip • In both cases we reached similar RI sensitivities, 56.93nm/RIU and 45.49nm/RIU • However the Q factor (Q ~ l/Dl) seems lower when the resonator is attached (Q ~ 500) • Q factor (Q ~ l/Dl) of the microsphere (20 mm Ø) deposited onto the MOF dip is about 11000 when lasing compared to Q ~ 1600 below the lasing threshold • Sensitivity Dl/Dn= 30nm/RIU

13. Sensing performance of microspheres: a comparison We assumed that 1/50 of the FWHM can be resolved using high resolution spectrometer and autocorrelation functions.

14. Deposition of positively and negatively charged polyelectrolyte in solution layer by layer by simply dipping the sensor into the different solutions. No flow cell – no agitation. Monitoring deposition of polymers onto the sensor in real time nL and ns are the refractive index of the deposited layer (1.45) and the resonator (1.69) respectively First bi-layer (PAH/PSS) is about 3 nm, the second about 3.8 nm which is good agreement with reported thicknesses of PAH/PSS bi layer deposited under the same conditions. Lösche et al., Macromolecules (1998) Decher, Science (1997)

15. In an in-vivo sensing situation, it is likely that no or very little flow will be available around the sensor. Therefore all kinetic process will be eventually limited by diffusion. Biotin-Neutravidin specific interaction From the kinetic results, we found that the surface density achieved at the steady state with the 400 nM concentration is about 236 ng/cm2 which is about half of the density of a full neutravidin monolayer (415 ng/cm2). No neutravidin is detectable below 4nM concentration (220ng/mL) Sciacca et al., Nanomedecine (2012)

16. Conclusion • We demonstrated the ability of a single dye doped polymer microsphere to be turned into a microlaser at the tip of a suspended core optical fibre. • We showed that lasing microresonators enable enhanced sensor resolution in individual measurements and can also to be used to detect a specific analyte, neutravidin in this case down to a concentration of 25 nM in an experimental setting mimicking condition for in-vivo sensing. • Beyond these results, we demonstrate that this simple and robust sensing platform can be used as a dip sensor, and we envision that it can be used to perform an immunoassay in area that are present difficult to access with existing sensors. Future work & perspective • Demonstration of the detection of a clinically relevant biomolecule in-vivo • Possibility of multiplexed sensing using multiple microresonators

17. Acknowledgments • This work was supported by the Australian Research Council (ARC), Defense Science and Technology Organisation (DSTO), the South Australian State Government and The University of Adelaide • Tanya Monro acknowledges the support of an ARC Federation Fellowship

18. MOF losses