Diabetes: chronic disease when the pancreas does not produce enough insulin
or ... IDF Diabetes Atlas 4th edition, © International Diabetes Federation, 2009. 3
...
Ionogel-biosensor: Novel Ionogels Based on Ionic Liquids as platforms to measure Enzyme Activity of Glucose Oxidase in Real Time Caroline Barry
Adaptive Sensors Group, National Centre for Sensor Research (NCSR) Dublin City University, Dublin 9, Ireland UNIVERSITY COLLEGE DUBLIN DUBLIN CITY UNIVERSITY TYNDALL NATIONAL INSTITUTE
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Outline •
Diabetes
•
Ionic liquids
•
Ionogels
•
Colorimetric and electrochemical analysis
•
Conclusion
•
Future outlook
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Background of Project • Diabetes: chronic disease when the pancreas does not produce enough insulin or the body cannot effectively use the insulin it produces. •
Insulin is a hormone that regulates blood sugar levels.
•
Types of diabetes: • • •
•
Type 1 diabetes Type 2 diabetes Gestational diabetes
11.6% of total health care budget in 2010.
IDF Diabetes Atlas 4th edition, © International Diabetes Federation, 2009
.
http://www.healthline.com/images/staywell/213.jpg
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Prevalence (%) estimates of diabetes (20-79), 2010
IDF Diabetes Atlas 4th edition, © International Diabetes Federation, 2009
.
UNIVERSITY COLLEGE DUBLIN DUBLIN CITY UNIVERSITY TYNDALL NATIONAL INSTITUTE
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Prevalence (%) estimates of diabetes (20-79), 2030
IDF Diabetes Atlas 4th edition, © International Diabetes Federation, 2009
.
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Current Technology
Walmart relion blood glucose monitor
OneTouch ultra blood glucose biosensor
GlucoSure blood glucose monitor
Accu-Chek active meter
Glucowatch®
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Ionic liquids •
Ionic Liquids (ILs) are low melting point salts (c reac>ons Electrolytes Separa>on science Polymer chemistry Solvent replacement
• Bio‐applica(ons • • • • • •
Enzyme catalyst Whole cell biotransforma>on An>microbial ac>vity Gene therapy DNA Extrac>on Immunosensors
• Glucose Oxidase • •
Enzyma>c catalysis (Wu et al., 2009) Electrochemical characteriza>on (Shan et al., 2009)
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Preparation of Ionogel
Ionogels: Ionic liquid polymer gels
Photopolymerized UV light
+ Polymer poly(N-isopropylacrlamide) N,N-methylene-bis(acrylamide)
-
+
+
-
+ +
-
Photoinitiator [P6, 6, 6, 14]DCA
[P66614][dca] ionogel 35000
PNIPAm
30000 Photopolymerized UV light
25000
[P66614][dca]
Photoinitiator
20000 15000 10000
Intensity (a.u)
40000
5000 0 2200
2000
1800
1600
Wavenumber cm-1
1400
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1200
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Colorimetric Assay for Glucose Oxidase (GOx)
Rxn cocktail: 1.7% (w/v)Glucose. Rxn cocktail: 1.7% (w/v)Glucose. 0.17mM o-dianisidine, 10ul peroxidase 0.17mM o-dianisidine, 10ul peroxidase enzyme enzyme, 10ul GOx http://www.sigmaaldrich.com/life-science/metabolomics/enzymatic-kits.html
Reaction stopped with H2SO4
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Enzyme Assay of Glucose Oxidase (GOx) (Ionogel)
0.17mM o-dianisidine in buffer,10µl peroxidase enzyme GOx ionogel
0.17mM o-dianisidine in buffer,10µl peroxidase enzyme, ionogel (-GOx)
0.17mM o-dianisidine in buffer,10ul peroxidase enzyme, GOx ionogel 40µl of glucose (10% w/v)
0.17mM o-dianisidine in buffer,10ul peroxidase enzyme, ionogel (-GOx) 40µl of glucose (10% w/v)
Reaction stopped with H2SO4
Reaction stopped with H2SO4
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Glucose Oxidase Assay (GOx on ionogel) Enzyme Unit/ml 15µmol/min/ml 9.8µmol/min/ml
Leaching of Enzyme from Ionogel 1.2
Enzyme Unit/ml 7.6µmol/min/ml 3.6µmol/min/ml Absorbance (a.u.)
1 0.8 0.6
Day 1
0.4
Day 4
0.2 0 ‐0.2
0
1
2
3
4
5
6
Time (min)
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Principle of Electrochemical Biosensors
Harper, A.C. 2005. Modified Electrodes for Amperometric Determination of Glucose and Glutamate Using Mediated Electron Transport. Thesis. PhD. Virginia Polytechnic Institute and State University UNIVERSITY COLLEGE DUBLIN DUBLIN CITY UNIVERSITY TYNDALL NATIONAL INSTITUTE
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Characterization of Ionogel for Electrochemical Determination [P6,6,6,14][dca] ionogel
70000 [P6,6,6,14][dca] ionogel + Fc
60000
Fc GOx
40000
[P6,6,6,14][dca] ionogel + GOx
30000
Intensity (a.u.)
50000
20000 10000 0 2200
1700
1200
Wavelength (cm‐1)
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Electrochemical results of Ionogel
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Conclusion
•
Two ionogels: colorimetric and electrochemical.
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Colorimetric assay shows that the enzyme is active in ionogel.
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Electrochemical: Incorporated ferrocence and GOx into ionogel, results show a correlation between the enzyme in buffer and enzyme in ionogel.
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Future Outlook
•
Determination of the ‘life time’ of the enzyme.
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Optimisation of the electrochemical and colorimetric procedure.
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Integrate the assay onto a microfluidic chip.
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Acknowledgments •
Professor Dermot Diamond
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Dr. Fernando Benito-López and Dr. Robert Byrne
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Adaptive Sensors group (DCU)
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Cytec Industries
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Science Foundation Ireland (grant 07/CE/I1147)
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