Jaunal club 2 :Electrochemical Detection Of Nitric Oxide In Biological Fluids

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. 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. Nitric Oxide in Blood
epentdent Vasodilator (Acetylcholine, Bradyk
r stress
matory
xia

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. 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. Nitric Oxide in Blood
(oxyhaemoglobin) (methaemoglobin) (nitrate)
J. P. Wallis (2005)

7. (Adrian J. Hobbs ,2002)
Nitric Oxide in Blood

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. 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. 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. Electrodes
•Platinum wires, 100 µM in
diameter
•Multiwalled carbon
nanotubes
•Coated with ruthenium
•Coated with Nafion

12. Electrode

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. Blood Samples
•Rabbit aortic blood 3 mL
•Containing 7 units of
lyophilized heparin
•kept on ice for up to 30
min before use.

15. Chemical Reagents
•Prepared 100 µM solutions in
deionized water
–ascorbate
–L-cystine
–2,3-diphospho-D-glyceric
acid (DPG)
–sodium nitrite

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. 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. Results
• Selectivity of the Sensor for Nitric Oxide
100 µM solutions in deionized water

19. Responses to Changing Gas
Mixtures
Change Gas mixed
350 s
NO oxidation
Control

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. Discussion
• The blood-drop preparation described
here may represent a useful approach for
further investigation of the response of NO
levels

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. 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. 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