Enzyme Entrapment in Polyaniline Biosensors Observed via Fluorescence Anisotropy and AntiquenchingLouis R. NemzerNova Southeastern UniversityAPS March MeetingQ45.3 – 3/5/14

Elevation = 1 mElevation = 1600 m

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.POLYANI

LINE

Emeraldine Base (EB)

Leucomeraldine Base (LEB)

Glucose Oxidase

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

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

Hydrophobic Collapse

Coiled conformation in a

poor solvent (PBS)

Extended conformation in a

good solvent (NMP)

--- - -

+

+ +

+

GOx nucleated

EB Exciton

R P McCall et al. PRB 1990X Tang 1991

G D Scholes and G Rumbles. Nature Materials 2006

L T Sein et al. Synthetic Metals 2009

Mechanical “Confinement”

Collapsed Conformation

Globally: Fluorophores more isolated

Locally: Forced Planarization

Fluorescence Antiquenching

Emeraldine base suspended in NMP exhibiting fluorescence antiquenching with increasing amounts of added PBS

(λexcite = 295 nm)

Increasing PBS

Fluorescence Quenching

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)

Increasing PBS

Fluorescence (Anti)quenching

Thin Films

Fluorescence Anisotropy

450nm -> 510 nm

Polarized Photon

GOx

Polarized(?) Photon

The polarization anisotropy of the emitted photons depends on the ratio of the

fluorescence lifetime (τ) and the rotational correlation time (φ)

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)

Fluorescence Anisotropy

𝑟=𝑟 0

1+ 𝜏𝜑

=𝐼𝑉𝑉 −𝐺∗ 𝐼𝑉𝐻𝐼𝑉𝑉+2∗𝐺∗ 𝐼𝑉𝐻

Fluorescence Anisotropy

SAMPLEAnisotr

opyτ/φ

GOx in PBS 0.09010.

2

EB+GOx in NMP

Solution0.102 8.8

EB+GOx Film 0.9940.0

1

LITERATURE

GOx in Solution 8.4

GOx in Glass 5.8

A. M. Hartnett, C. M. Ingersoll, G. A. Baker, and F. V. Bright. Kinetics and Thermodynamics of Free Flavins and the Flavin-Based Redox Active Site within Glucose Oxidase Dissolved in Solution or Sequestered

within a Sol-Gel-Derived Glass. Anal Chem 71 1215-1224 (1999)

3 Order of Magnitude Decrease!

ConclusionsFluorescence 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.

THANK YOU!