Joe Fazio BE REU @ SLU Dr. Shelley D. Minteer Kyle Sjöholm SLU Department of Chemistry Enzymatic...

Joe Fazio BE REU @ SLU

Dr. Shelley D. Minteer Kyle Sjöholm

SLU Department of Chemistry

Enzymatic Glucose Biofuel Cell:Concentration Studiesand Biocompatibility

Background

Enzymatic Biofuel cell: Enzymes Power biomedical

devices High power and current

density Incomplete oxidation

www.nano-biokit.com

Biofuel Cell Process

Reaction at anode produces protons

Electrons create current Protons diffuse to cathode Protons at cathode react

with oxygen

Mediated Electron Transfer (MET) Commonly used to

reduce overpotential

Facilitates ion transfer to electrode

NAD+

NADH

Gluconolactone

Glucose Dehydrogenase Entrapped in

Polymer

Glucose

060 Toray Paper

Electrode

Electrocatalyst

2e-

Modified Polymers Immobilize enzymes Extend functional lifetime

Microencapsulation: Support enzyme structure

Neutral pH Micellar environment Geometry Ion exchange

properties

Polymer encapsulation

Project Goals

Power Densities Hypoglycemic (3mM) Normal (5mM) Hyperglycemic (8mM)

Biocompatibility Bulk electrolysis Live/dead assay

• Biofilm formation

Basic Components

Anode: 060 Toray Paper electrodes Fuel: Glucose Enzyme: Glucose Dehydrogenase Cofactor: NAD+

Electrocatalyst: Poly(methylene green) (PMG) Modified polymer:

Nafion® Chitosan

Polymer modification Nafion®: Tetrabutylammonium bromide (TBAB) Chitosan

Hydrophobic Deacylation

Co-cast polymer and enzyme onto electrode

Soak electrodes in solution of glucose overnight

Electrode Preparation

Chitosanhttp://www.global-b2b-network.com/

Experimental Set-up

3, 5, 8mM glucose fuel NAD+, pH 7.4 phosphate buffer

Open circuit potential (~1000secs)

Linear sweep voltammetry (<1mV/sec)

Power density equation P=I*V

Diagram of Icell

+

-

V

Glass tube

Glass tube

Bioanode

Nafion PEM

4.5cm2 20% Pt GDE Cathode

Fuel Solution

Air

O-ring

O-ring

Potentiostat+

-

V

Glass tube

Glass tube

Bioanode

Nafion PEM

4.5cm2 20% Pt GDE Cathode

Fuel Solution

Air

O-ring

O-ring

Potentiostat

8mM Averages

Current Density, Amps/cm2

0 1e-5 2e-5 3e-5 4e-5 5e-5

Po

we

r D

en

sity

, Wa

tts/c

m2

0

2e-6

4e-6

6e-6

8e-6

3mM Averages

Current Density, Amps/cm2

0 1e-5 2e-5 3e-5 4e-5

Pow

er D

ensi

ty, W

atts

/cm

2

0

1e-6

2e-6

3e-6

4e-6

5e-6

6e-6

7e-6

Power Density Test Results

Average Maximum Power Density* µW/cm2

  3mM 5mM 8mM

Chitosan 2.87(±0.21) 2.82(±0.52) 3.32(±0.46)

Deacylated chitosan 6.04(±3.23) 6.15(±3.51) 7.52(±4.31)

Nafion® 0.28(±0.02) 0.29(±0.02) 0.33(±0.04)

5mM Averages

Current Density, Amps/cm2

0 1e-5 2e-5 3e-5 4e-5 5e-5

Pow

er D

ensi

ty, W

atts

/cm

2

0

1e-6

2e-6

3e-6

4e-6

5e-6

6e-6

7e-6

*errors are equal to one standard deviation

Deacylated ChitosanChitosanNafion

Biocompatibility, Bulk ElectrolysisTesting Bacteria culture

injected Hold fuel cell at 0.3V

and monitor current (3 days)

Time, seconds

0.0 5.0e+4 1.0e+5 1.5e+5 2.0e+5 2.5e+5

Cu

rre

nt,

Am

ps

0

1e-6

2e-6

3e-6

4e-6

5e-6

6e-6

Decreasing current Possible biofilm

formation

Biocompatibility, Live/dead AssayLive/Dead assay Cast polymer with bacteria

Gluconobacter SP33 Origami C4-AW genetically modified E. Coli

Fluorescent nucleic acid stains FITC filter- live bacteria TRITC filter- dead bacteria

Live/Dead Assay

Nafion® GluconobacterNafion® E. coli

Chitosan E. coli Deacylated chitosan Gluconobacter

Assay showed biocompatibility for all polymers.FITC filter

Olympus IX71 fluorescence microscope

TRITC filter image

Conclusions

Chitosan and Nafion® can immobilize GDH Chitosan provides higher power and current

densities Chitosan and Nafion® provide biocompatible

surface material

Future work

Temperature and pH studies Biocompatible modifications

Impact on current densities

Acknowledgements

National Science Foundation

Saint Louis University

Dr. Minteer

Minteer group Kyle Sjöholm Dr. Waheed

Rob Arechederra

References1) Akers, Moore, Minteer. “Development of Alcohol/O2 Biofuel Cells Using Salt-Extracted Tetrabutylammonium Bromide/Nafion Membranes to Immobilize

Dehydrogenase Enzymes.” Electrochimica Acta 50 (2005): 2521-2525.2) Arechederra, Robert, Shelley D. Minteer. “Organelle-based Biofuel Cells: Immobilized Mitochondria on Carbon Paper Electrodes.” Electrochimica Acta 53 (2008):

6698-6703.3) Atanassov, Plamen, et al. “Enzymatic Biofuel Cells. The Electrochemical Society Interface (2007).4) Beilke, Michael C., et al. “Enzymatic Biofuel Cells.” Micro Fuel Cells Principles and applications. T.S. Zhao. Publisher location: Elsevier, 2009. 179-242. print.5) Blackwell, Anne E, et al. “Comparison of Electropolymerized Thiazine Dyes as an Electrocatalyst in Enzymatic Biofuel Cells and Self Powered Sensors.”

Nanoscience and Nanotechnology 9.3 (2009): 1714-21.6) Bond, Alan M, et al. “A Role for Electrospray Mass Spectrometry in Electrochemical Studies.” Analytical Chemistry 67(1995):1691-1695.7) Cooney, M.J., et al. “Enzyme Catalysed Biofuel Cells.” Energy & Environmental Science 1 (2008): 320-337.8) Cox, James A., Thomas J. Gray, “Controlled-Potential Electrolysis of Bulk Solutions at a Modified Electrode: Application to Oxidations of Cysteine, Cystine,

Methionine, and Thiocyanate.” Analytical Chemistry 62(1990): 2742-2744.9) Crittenden, Scott R., Christian J. Sund, James J. Sumner. “Mediating Electron Transfer from Bacteria to a Gold Electrode via a Self-Assembled Monolayer.”

Langmuir 22(2006):9473-9476.10) Galassetti, Pietro R., et al. “Breath Ethanol and Acetone as Indicators of Serum Glucose Levels: An Initial Report.” Diabetes Technology & Therapeutics

7(2005):115-123.11) Hu, Qiang, A. Scott Hinman. “A Bulk Electrolysis Raman Spectroelectrochemical Cell Using a Rotating Electrode.” Analytical Chemistry 72(2000): 3233-3235.12) Ikeda, Tokuji. “A Novel Electrochemical Approach to the Characterization of Exidoreductase Reactions.” The Chemical Record 4(2004):192-203.13) Klotzbach, Tamara L., Michelle Watt, Yasmin Ansari, Shelley D. Minteer. “Improving the microenvironment for enzyme immobilization at electrodes by

hydrophobically modifying chitosan and Nafion® polymers.” Journal of Membrane Science 311(2008):81-88.14) “Live/Dead Baclight Bacteria Viability Kitis.” Molecular Probes Inc. 2004.15) Mano, Nicolas, “A 280µW cm-2 biofuel cell operating at low glucose concentration.” The Royal Society of Chemistry (2008):2221-2223.16) Martin, Georgianna L., Shelley D. Minteer, Michael J. Cooney. “Spatial Distribution of Malate Dehydrogenase in Chitosan Scaffolds.” Applied Materials & Interfaces

1 (2009):367-372.17) Minteer, Shelley D., Bor Yann Liaw, Michael J. Cooney. “Enzyme-based biofuel cells.” Current Opinion in Biotechnology 18 (2007):228-234.18) Moore, Christine M, et al. “Improving the Environment for Immobilized Dehydrogenase Enzymes by Modifying Nafion with Tetraalkylammonium Bromides.”

Biomacromolecules 5 (2004): 1241-1247.19) Subramanyam, Elango, Sidharthan Mohandoss, Hyun-Woung Shin. “Synthesis, Characterization, and Evaluation of Antifouling Polymers of 4-

Acryloyloxybenzaldehyde with Methyl Methacrylate.” Journal of Applied Polymer Science 112(2009):2741-2749.20) Tamaki, T., T. Ito, T. Yamaguchi. “Modelling of Reaction and Diffusion Processes in a High-surface-area Biofuel Cell Electrode Made of Redox Poluymer-grated

Carbon.” Fuel Cells 09 1(2009):37-43.21) Wang, J., et al. “The effects of amorphous carbon films deposited on polyethylene terephthalate on bacterial adhesion.” Biomaterials 25(2004):3163-3170.22) Heikkila, O., N Lundbom, M Timonen, P-H Groop, S Heikkinen, S Makimattila. “Hyperglycaemia is associated with changes in the regional concentrations of glucose

and myo-inositol within the brain.” Diabetologia 52(2009):534-540.23) Gupta, Sandeep, Eugene Chough, Jennifer Daley, Peter Oates, Keith Tornheim, Neil B. Ruderman, and John F. Keaney Jr. “Hyperglycemia increases endothelial

superoxide that impairs smooth muscle cell Na+-K+-ATPase activity.” Am J Physiol Cell Physiol 282(2002): C560-C566.24) Mason RM,Thomas G, Davies M. “Proteoglycan synthesis by human mesangial cells is depressed by hyperglycemic glucose concentrations.” Biochemical society

transactions 2(1992): 9625) Xiaoli, Ma, Yao Zihua, Shi Dagang. “Preparation and characterization of porous chitosan membranes and the localization of the activity of urease immobilized on it

by SEM and X-ray microanalysis.” Chemical Journal on Internet 7(2005): 45.