Louis R. Nemzer Nova Southeastern University APS March Meeting Q45.3 – 3/5/14

1. Enzyme Entrapment in Polyaniline Biosensors Observedvia Fluorescence Anisotropy and Antiquenching Louis R. NemzerNova Southeastern University APS March MeetingQ45.3 – 3/5/14 Elevation = 1600 m Elevation = 1 m

2. Introduction • The purpose of this study was to verify the physical entrapment of glucose oxidase (GOx) enzyme within a polyaniline thin film biosensor, and confirm that charge donation can occur between them. POLYANILINE Emeraldine Base (EB) Glucose Oxidase Leucomeraldine Base (LEB)

3. Glucose Monitoring System US Patent #8,326,389: “A System for in vivo Biosensing Based on the Optical Response of Electronic Polymers.” A J Epstein and L R Nemzer

4. β-D-glucose + EB → D-glucono-1,5-lactone+ LEB D-glucono-1,5-lactone + OH-→ Gluconic Acid

5. Hydrophobic Collapse + + - - - - - + + GOx nucleated Extended conformation in a good solvent (NMP) Coiled conformation in a poor solvent (PBS)

6. EB Exciton R P McCall et al. PRB 1990 X Tang 1991

7. Collapsed Conformation Mechanical “Confinement” Locally: Forced Planarization Globally: Fluorophores more isolated L T Sein et al. Synthetic Metals 2009 G D Scholes and G Rumbles. Nature Materials 2006

8. Fluorescence Antiquenching Increasing PBS Emeraldine base suspended in NMP exhibiting fluorescence antiquenching with increasing amounts of added PBS (λexcite = 295 nm)

9. Fluorescence Quenching Increasing PBS Quenching of EB Exciton. The slight shift of the peak emission wavelength toward longer wavelengths at higher PBS concentrations is a solvent effect. (λexcite = 450 nm)

10. Fluorescence (Anti)quenching

11. Thin Films

12. Fluorescence Anisotropy Polarized(?) Photon Polarized Photon GOx The polarization anisotropy of the emitted photons depends on the ratio of the fluorescence lifetime (τ) and the rotational correlation time (φ) 450nm -> 510 nm

13. Successful Entrapment Entrapment increases the rotational correlation time (φ) and decreases the fluorescence lifetime (τ). Both effects lead to a decrease in τ/φ, which corresponds to an increase in polarization anisotropy. Noriega et al. Nature Materials 12, 1038–1044 (2013)

14. Fluorescence Anisotropy

15. Fluorescence Anisotropy 3 Order of Magnitude Decrease! • A. M. Hartnett, C. M. Ingersoll, G. A. Baker, and F. V. Bright. Kinetics and Thermodynamics of Free Flavinsand the Flavin-Based Redox Active Site within Glucose Oxidase Dissolved in Solution or Sequestered within a Sol-Gel-Derived Glass. Anal Chem71 1215-1224 (1999)

16. Conclusions • Fluorescence anisotropy and antiquenching were used to confirm the physical entrapment and charge donation capability of enzymes immobilized in polyaniline thin films. • The increase in the fluorescence intensity indicates that the polymer chains undergoes an extended-to-coiled conformation transition. • The 3-order-of-magnitude decrease in τ/φ indicates that the enzyme is entrapped and donating charge to the polymer matrix. • These are both prerequisites for a successful biosensor.

17. THANK YOU!