Electrochemical Detection of NitElectrochemical Detection of Nitric Oxide in Biological Fluidsric Oxide in Biological Fluids

METHODS IN ENZYMOLOGY, VOL. 396 ,2005

BARRY W. ALLEN, JIE LIU, and CLAUDE A. PIANTADOSI

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 NOS (eNOS or NOS3 or Constitutive / cNOS)

Generates NO in blood vessels and is involved with regulating vascular function

Three isoforms of NO synthase (NOS)

Furchgott R, Zawadzki J (1980).

Nitric Oxide in Blood•NO-depentdent Vasodilator (Acetylcholine, Bradykinin)•Sheer stress•Inflamatory•hypoxia

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 half life of aqueous NO in red cell-free

plasma in vitro is around 1 min (Rassaf et al., 2002).

2. Nitrosothiols or RSNOs

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• Hypertension• Obesity• Dyslipidemias (particularly hypercholesterolemia and hyp

ertriglyceridemia)• Diabetes (both type I and II)• Heart failure• Atherosclerosis • Cigarette smoking• Septicemia • Etc.

Introduction

• Electrochemistry– fluids in real time and in situ

• NO electrodes can be made small eno ugh to be used in vivo

• NO in biological fluids that are maint ained in contact with a gaseous envir

onment,• R elease of NO from blood cells as the

y move between regions of high and l ow PO

2 levels

Materials and Methods

Helix Diameter 1.

85mm

100 µM MM MMMM MMMM , 3 .mm long

S uspended a20 µL

drop of rabbit aortic blood

Electrodes

•P 100latinum wires, µM MM MMMM MMMM

•M ultiwalled carbon nanotubes•CMMMMM MMMM MMMMMMMMM•C oated with Nafion

Electrode

Gas flow

•Air–CO2

M( 20% O2

, 5% CO2

, 75% N2

)

•CO2

–nitrogen mixture (5%CO

2 , 95% N

2)

MMM MMMM MMM MMMMMMMMMM MM MMMMMMMM MMMe

Blood Samples

• 3Rabbit aortic blood mL•C M MMMMM MM MMMM7

MMMMMMM MMMMMMM • 30keptonicef or up t o mi n bef or e use.

Chemical Reagents

•P 100repared µM MMMMMMMMM MM M eionized water

–ascorbate–M-MMMMMMM– -MMMMMMMMM-M -MMMMMMMM MMMM 2 ,3(DPG)

– sodi umni tri te– sodium nitrate

Electrochemical Methods

•Amperometry•BAS 100 B/W potentiostat equipped

•+ 675 mV (vs. Ag/AgCl,)•T he electrodes were activated e

lectrochemically by applying altern ating potentials of 200 and 800

- mV for 250 ms each at 500 ms in tervals for a total of 120 s

The data were not used

•the composite resistance of the electrochemical cell was m easured three times, final avera

ge was more than 1 0 % greater t hanthei ni t i al average,foul edor tha tthe bl ooddrophaddri ed,

•T he bl oodwas not fl ui d• thedropdi dnot fi l l the hel i x,

Results

• Selectivity of the Sensor for Nitric Oxide

1 0 0 µ M solutions in deionized water

Responses to Changing GMM M MMMMMMM

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 to 400 s aft er the flowing gas was change

d– spike was followed by a continuou

s signal of 1–2 nA

Discussion

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

Discussion

• limiting the potential or by applying coatings to the electrode that exclude species that have certain characteristics of charge or size

• always useful to confirm anyexperimental result by using other nonelectrochemical methods

Conclusions•M MMMMMM MMMMMMMMM MMMMMMMMMMMMM

MM MMMMMM MMM MM MMMMMMMM MM MMMMM t nMconcentrati ons of NOacti vi t

y•F 200 400rom to s after a suspen

deddropof rabbi t arteri al bl ood was exposed to a decrease in ambie

MM MM2

•a nexperimentalcondition—hypoxia—inwhi chhypoxemi a coul dbe i nMMMMM MM MMM MMMMMMMM MMMMM MMMMM

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 release to a pa rticular source in the blood, for exam

ple, the red cells or the plasma