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Electrochemical Detection ofElectrochemical Detection of
Nitric Oxide in Biological FluidsNitric Oxide in Biological Fluid...
Nitric Oxide in Blood
Name Description
Neuronal
NOS
(nNOS or NOS1)
Produces NO in
neuronal tissue
in both the central and
...
Nitric Oxide in Blood
epentdent Vasodilator (Acetylcholine, Bradyk
r stress
matory
xia
Nitric Oxide in Blood
• NO has a half-life of about 4 s in biological
fluids and is oxidized to nitrite and nitrate
anions
Nitric Oxide in Blood
NO may be present in the blood in at least 2 active
Forms
1. Aqueous form as a dissolved gas
The hal...
Nitric Oxide in Blood
(oxyhaemoglobin) (methaemoglobin) (nitrate)
J. P. Wallis (2005)
(Adrian J. Hobbs ,2002)
Nitric Oxide in Blood
Why Detection of NO in Blood
Diseases or Conditions Associated with
Abnormal NO Production and Bioavailability
• Hypertens...
Introduction
•Electrochemistry
–fluids in real time and in situ
•NO electrodes can be made
small enough to be used in vivo...
Materials and Methods
Helix Diameter
1.85 mm
100 µM in diameter , 3 mm long.
Suspended a 20
µL
drop of rabbit
aortic blood
Electrodes
•Platinum wires, 100 µM in
diameter
•Multiwalled carbon
nanotubes
•Coated with ruthenium
•Coated with Nafion
Electrode
Gas flow
•Air–CO2 mixture (20% O2, 5%
CO2, 75% N2)
•CO2–nitrogen mixture (5%CO2,
95% N2)
Gas flow was maintained at
consta...
Blood Samples
•Rabbit aortic blood 3 mL
•Containing 7 units of
lyophilized heparin
•kept on ice for up to 30
min before us...
Chemical Reagents
•Prepared 100 µM solutions in
deionized water
–ascorbate
–L-cystine
–2,3-diphospho-D-glyceric
acid (DPG)...
Electrochemical Methods
•Amperometry
•BAS 100 B/W potentiostat
equipped
•+675 mV (vs. Ag/AgCl,)
•The electrodes were
activ...
The data were not used
•the composite resistance of
the electrochemical cell was
measured three times, final
average was m...
Results
• Selectivity of the Sensor for Nitric Oxide
100 µM solutions in deionized water
Responses to Changing Gas
Mixtures
Change Gas mixed
350 s
NO oxidation
Control
Responses to Changing
Gas Mixtures
•Responses to Changing Gas
Mixtures
–NO oxidation signals were first
detected from 200 ...
Discussion
• The blood-drop preparation described
here may represent a useful approach for
further investigation of the re...
Discussion
• limiting the potential or by applying
coatings to the electrode that exclude spe
cies that have certain chara...
Conclusions
•A highly sensitive
electrochemical system can b
e designed to detect nM
concentrations of NO activity
•From 2...
Conclusions
•we did not measure the
change in either ambient PO2
or blood-drop PO2 in this
deliberately kept low in order
...
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Electrochemical Detection Of Nitric Oxide In Biological Fluids

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Electrochemical Detection Of Nitric Oxide In Biological Fluids

  1. 1. Electrochemical Detection ofElectrochemical Detection of Nitric Oxide in Biological FluidsNitric Oxide in Biological Fluids METHODS IN ENZYMOLOGY, VOL. BARRY W. ALLEN, JIE LIU, and CLAUDE A. PIANTADOSI
  2. 2. Nitric Oxide in Blood Name Description Neuronal NOS (nNOS or NOS1) Produces NO in neuronal tissue in both the central and peripheral nervous system. Inducible NOS (iNOS or NOS2) Can be found in the immune system used by macrophages in immune defence against pathogens. Endothelial Three isoforms of NO synthase (NOS) Furchgott R, Zawadzki J (1980).
  3. 3. Nitric Oxide in Blood epentdent Vasodilator (Acetylcholine, Bradyk r stress matory xia
  4. 4. Nitric Oxide in Blood • NO has a half-life of about 4 s in biological fluids and is oxidized to nitrite and nitrate anions
  5. 5. Nitric Oxide in Blood NO may be present in the blood in at least 2 active Forms 1. Aqueous form as a dissolved gas The half life of aqueous NO in red cell-free plasma in vitro is around 1 min (Rassaf et al., 2002). 2. Nitrosothiols or RSNOs
  6. 6. Nitric Oxide in Blood (oxyhaemoglobin) (methaemoglobin) (nitrate) J. P. Wallis (2005)
  7. 7. (Adrian J. Hobbs ,2002) Nitric Oxide in Blood
  8. 8. Why Detection of NO in Blood Diseases or Conditions Associated with Abnormal NO Production and Bioavailability • Hypertension • Obesity • Dyslipidemias (particularly hypercholesterolemia and hypertriglyceridemia) • Diabetes (both type I and II) • Heart failure • Atherosclerosis • Cigarette smoking • Septicemia • Etc.
  9. 9. Introduction •Electrochemistry –fluids in real time and in situ •NO electrodes can be made small enough to be used in vivo •NO in biological fluids that are maintained in contact with a gaseous environment, •Release of NO from blood cells as they move between regions
  10. 10. Materials and Methods Helix Diameter 1.85 mm 100 µM in diameter , 3 mm long. Suspended a 20 µL drop of rabbit aortic blood
  11. 11. Electrodes •Platinum wires, 100 µM in diameter •Multiwalled carbon nanotubes •Coated with ruthenium •Coated with Nafion
  12. 12. Electrode
  13. 13. Gas flow •Air–CO2 mixture (20% O2, 5% CO2, 75% N2) •CO2–nitrogen mixture (5%CO2, 95% N2) Gas flow was maintained at constant rate
  14. 14. Blood Samples •Rabbit aortic blood 3 mL •Containing 7 units of lyophilized heparin •kept on ice for up to 30 min before use.
  15. 15. Chemical Reagents •Prepared 100 µM solutions in deionized water –ascorbate –L-cystine –2,3-diphospho-D-glyceric acid (DPG) –sodium nitrite
  16. 16. Electrochemical Methods •Amperometry •BAS 100 B/W potentiostat equipped •+675 mV (vs. Ag/AgCl,) •The electrodes were activated electrochemically b y applying alternating potent ials of 200 and 800 mV for 2
  17. 17. The data were not used •the composite resistance of the electrochemical cell was measured three times, final average was more than 10% greater than the initial avera ge, fouled or that the blood d rop had dried, •The bloodwas not fluid
  18. 18. Results • Selectivity of the Sensor for Nitric Oxide 100 µM solutions in deionized water
  19. 19. Responses to Changing Gas Mixtures Change Gas mixed 350 s NO oxidation Control
  20. 20. Responses to Changing Gas Mixtures •Responses to Changing Gas Mixtures –NO oxidation signals were first detected from 200 to 400 s after the flowing gas was chang ed –spike was followed by a continuous signal of 1–2 nA
  21. 21. Discussion • The blood-drop preparation described here may represent a useful approach for further investigation of the response of NO levels
  22. 22. Discussion • limiting the potential or by applying coatings to the electrode that exclude spe cies that have certain characteristics of ch arge or size • always useful to confirm anyexperimental result by using other nonelectrochemical methods
  23. 23. Conclusions •A highly sensitive electrochemical system can b e designed to detect nM concentrations of NO activity •From 200 to 400 s after a suspended drop of rabbit arte rial blood was exposed to a d ecrease in ambient PO2 •an experimental condition—
  24. 24. Conclusions •we did not measure the change in either ambient PO2 or blood-drop PO2 in this deliberately kept low in order to prevent drying of the blood drop, PO2 will change slowly. • we cannot assign this

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