Measurement of Nitric Oxide in Single Cells and Tissue Using a Porphyrinic Microsensor

互联网2013-12-31

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Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

This unit describes the preparation and applications of porphyrinic sensors for quantitative measurement of nitric oxide (NO) in single cells and in tissues. The determination of NO is based on the electrochemical oxidation of NO on a carbon fiber electrode covered with a thin layer of a conducting polymeric metalloporphyrin catalyst, overlaid with another thin film of Nafion, a cation exchange material. The electric current generated during NO oxidation on the surface of the polymeric porphyrin is linearly proportional to the concentration of NO, so this current is used as an analytical signal which can be measured in either the amperometric or the voltammetric mode. Both methods provide a quantitative signal. This unit describes the electrochemical setup for measurement of NO in single cells and tissue. Support protocols describe porphyrin synthesis, sensor preparation, and sensor calibration.

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Basic Protocol 1: Measurement of Nitric Oxide in Single Cells and Tissue with a Porphyrinic Microsensor
Support Protocol 1: Preparation and Calibration of Porphyrinic Sensors
Support Protocol 2: Preparation of Bare Carbon‐Fiber Electrodes
Support Protocol 3: Preparation of TMHPPNi Electrochemical Coating Solution
Support Protocol 4: Preparation of Saturated NO Solution
Reagents and Solutions
Commenatry
Literature Cited
Figures

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Materials

Basic Protocol 1: Measurement of Nitric Oxide in Single Cells and Tissue with a Porphyrinic Microsensor

Materials

Modified HBSS (see recipe )
Biological matrix: cells (grown on 25‐mm tissue culture dish) or tissue of interest
Chemical agonist for constitutive nitric oxide synthase (cNOS; optional)
Selective inhibitor for inducible nitric oxide synthase (iNOS; optional)
0.1 mM L‐arginine in recipemodified HBSS
Saturated nitric oxide (NO) solution (see protocol 5 )

Temperature‐controlled 3‐ml microscope stage (Olympus)
Porphyrinic sensor (see protocol 2 )
Stereotactic micromanipulators, resolution 1 µm or better
Platinum wire (platinum auxiliary or counter electrode; 0.5‐ to 1‐mm diameter, 3‐cm length)
Silver/silver chloride electrode (SSCE; 1‐ to 2‐mm‐diameter silver wire covered with thin layer of silver chloride; Princeton Applied Research) or saturated calomel electrode (SCE; 2‐mm diameter; Princeton Applied Research)
Fast‐response (≤1 µsec) voltammetric analyzer (Princeton Applied Research, PAR 273)
Low‐diameter (e.g., 1‐mm) copper wires shielded with copper mesh
Faraday cage (optional; Princeton Applied Research)
Chart recorder, x ‐y recorder, or computer with appropriate software (Electrochemistry Software, Princeton Applied Research)
Nanopipet, picopipet, or femtopipet (World Precision Instruments; optional)
Microscopes: stereoscopic (for measurements in tissue) and inverted (for measurements in single cells)

Support Protocol 1: Preparation and Calibration of Porphyrinic Sensors

Materials

0.1 M NaOH
TMHPPNi electrochemical coating solution (see protocol 4 )
5% (w/v) Nafion solution in alcohols (Aldrich)
Absolute ethanol
Saturated NO solution (1.74 mM at 0°C; see protocol 5 )
Physiological buffer solution, pH 7.4 (optional)

Electrolytic cells: 19 × 48–mm, 2‐dram (∼10‐ml) clear glass vials (Kimble) with a Teflon cap with four holes
Multifiber or single‐fiber carbon electrode (see protocol 3 )
Platinum wire (platinum auxiliary or counter electrode; 0.5‐ to 1‐mm diameter, 3‐cm length)
Silver/silver chloride electrode (SSCE; 1‐ to 2‐mm‐diameter silver wire covered with thin layer of silver chloride; Princeton Applied Research) or saturated calomel electrode (SCE; 2‐mm diameter; Princeton Applied Research)
Fast‐response (≤1 µsec) voltammetric analyzer (Princeton Applied Research, PAR 273)
Nitrogen or argon gas tank
1‐ to 2‐mm‐i.d. glass pipet
Flatbed x ‐y recorder or computer with appropriate software
Vacuum oven at 40°C

Support Protocol 2: Preparation of Bare Carbon‐Fiber Electrodes

Materials

Conductive silver epoxy (P/N EG 8020; AIT), parts A and B
Beeswax
Nonconductive epoxy (2‐TON, Devcon Plexus; for single‐fiber electrodes only)
Violin rosin
0.5‐ to 1‐mm‐i.d. open‐ended glass capillaries
Carbon fibers (6‐ to 7‐µm diameter; specific resistivity 12 ohm⋅cm; Amoco Performance Products)
Bare copper or silver wire (1‐mm diameter)
Emery paper
Vacuum dryer at 40°C
Propane microburner

Support Protocol 3: Preparation of TMHPPNi Electrochemical Coating Solution

Materials

Propionic acid (Aldrich)
Silicon grease (Fisher)
Reagent‐grade vanillin (Aldrich)
Pyrrole (Aldrich), distilled immediately before use
Methylene chloride (Aldrich)
Methanol (Aldrich), ice cold
1:1 (v/v) methylene chloride/methanol
Gradient of 200:1 to 40:1 (v/v) methylene chloride/methanol
Dimethylhydrofuran (Aldrich), room temperature and ice cold
Nickel(II) acetate tetrahydrate (Aldrich)
0.1 M NaOH

Boiling stones
250‐ and 500‐ml single‐neck round‐bottom flasks with female 24/40 joint (Pyrex)
Heating mantles for 250‐ and 500‐ml round‐bottom flasks
Claisen adapter with three 24/40 joints, one male leading to two female (Pyrex)
Tap water–cooled reflux condenser with 24/40 male joint (Pyrex)
24/40 male ground‐glass stopper (Pyrex)
Rubber hoses
Small glass funnels
60‐ml medium‐coarseness frits (Pyrex) and rubber adapter to fit a 1‐liter Erlenmeyer flask
1‐liter side‐arm Erlenmeyer flasks (Pyrex)
Florisil chromatography column
Silica‐gel chromatography column
Rotary evaporator
Ground‐glass hose adapter with 24/40 male joint (Pyrex)
Tygon tubing
Nitrogen gas tank
Electrolytic cell: 19 × 48–mm, 2‐dram (∼10‐ml) clear glass vial (Kimble) with a Teflon cap with four holes
0.5‐ to 1.0‐mm‐i.d. glass pipet

Support Protocol 4: Preparation of Saturated NO Solution

Materials

4 M NaOH
Laboratory‐generated or commercial NO (Fisher)
Methyl red
0.1 M sodium phosphate buffer, pH 7.40 ( appendix 2A )
1 M KMnO₄ (optional)

Two 500‐ml purge cylinders (Fisher)
5‐ml conical Pyrex reaction vial with 14/10 threaded, screw‐cap rubber septum
Teflon‐coated magnetic spin vane to fit conical vial
22‐G syringe needles
Hot plate or heating mantle equipped with a stirrer
Nitrogen or argon tank (99% v/v purity) equipped with hose and 22‐G input syringe needle

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Figures

Figure 7.14.1 Electrochemical deposition of a polymeric TMHPPNi film on the surface of a carbon electrode. Multiple‐scan cyclic voltammograms were recorded with a scan rate of 100 mV/sec. Peak I_a indicates oxidation of Ni(II) to Ni(III) and peak I_c indicates reduction of Ni(III) to Ni(II). Rising current (II_a ) indicates catalytic oxidation of water. Peaks A and B are due to oxidation of substituent(s) of the porphyrinic ring.

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Figure 7.14.2 Typical voltammetric and amperometric curves showing response of the sensor to NO in homogenous solution. (A ) Differential pulse voltammogram obtained from 0.2 µM NO. E _p is the peak potential. (B ) Amperograms obtained at different concentrations of NO.

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Figure 7.14.3 Nitric oxide concentration measured with porphyrinic sensor placed at different distances from the membrane surface of a single bovine aorta endothelial cell. The porphyrinic sensor was placed at the indicated distance from the membrane surface of the cell, and NO release was stimulated with 1 µM calcium ionophore A23187.

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Figure 7.14.4 Typical amperograms showing NO release from single bovine aorta endothelial cells either (A ) isolated from culture or (B ) maintained in culture. The porphyrinic sensor was placed 2 µm from the membrane surface of the cell, and NO release was stimulated with 1 µM calcium ionophore A23187 (arrow).

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Figure 7.14.5 Nitric oxide release measured near endothelial cells of rabbit mesenteric artery (A ) and aorta (B ). The porphyrin sensor was placed 2 ± 0.2 µm from the membrane surface of the endothelium, and NO release was stimulated by the application of 1 µM calcium ionophore A23187 (arrow).

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Videos

Literature Cited

Literature Cited
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