Direct detection of S-nitrosothiols using planar amperometric nitric oxide sensor modified with polymeric films containing catalytic copper species

Article abstract of DOI:10.1021/ac048192u

The direct amperometric detection of S-nitrosothiol species (RSNOs) is realized by modifying a previously reported amperometric nitric oxide gas sensor with thin hydrophilic polyurethane films containing catalytic Cu(II)/(I) sites. Catalytic Cu(II)/(I)-mediated decomposition of S-nitrosothiols generates NO_(g) in the thin polymeric film at the distal tip of the NO sensor. Three different species are examined to create the catalytic layer: (1) a lipophilic Cu(II)-ligand complex; (2) Cu(II)-phosphate salt; and (3) small (3-μm) metallic Cu⁰ particles. All three catalytic layers yield reversible amperometric response in proportion to the concentration of S-nitrosothiols (e.g., nitrosocysteine, nitrosoglutathione, S-nitroso-N- acetylcysteine, S-nitrosoalbumin) present in the aqueous test solution. Sensitivity toward the different RSNO species is dependent on the respective catalytic rates of decomposition of the RSNO species by reactive Cu(I), accessibility of the species into the polyurethane layer containing the catalyst, the level of reducing agents (ascorbate) used in solution to help generate reactive Cu(I) species, and the concentration of metal ion complexing agents present in the test solution (e.g., EDTA). Under optimized conditions, all RSNO species can be detected at ≤ μM levels, with sensor lifetimes of at least 10 days for the sensors based on Cu(II)-phosphate and Cu⁰ particles. It is further shown that the new RSNO sensors can be used to assess the "NO-generating" ability of fresh blood samples by effectively detecting the total level of reactive RSNO species present in such samples.

Full text of DOI:10.1021/ac048192u

Anal. Chem. 2005, 77, 3516-3524
Direct Detection of S-Nitrosothiols Using Planar
Amperometric Nitric Oxide Sensor Modified with
Polymeric Films Containing Catalytic Copper
Species
Wansik Cha, Youngmi Lee,† Bong Kyun Oh, and Mark E. Meyerhoff*
Department of Chemistry, University of Michigan, 930 North University Avenue, Ann Arbor, Michigan 48109-1055
and several species are known to enhance NO production by the
endothelial cells (e.g., bradykinin, ATP, etc.).3,4 Further, nitric
oxide’s transient nature has led to the recognition that NO is
possibly stored and transferred throughout the body in the form
of RSNOs, species that exhibit physiological lifetimes comparable
to that of EDRF.5,6 Indeed, nitrosylation of sulfhydryl groups in
amino acids, peptides, and proteins has been shown to provide
NO-like biological activities including vascular smooth muscle
relaxation, neurotransmission, and inhibition of platelet aggre-
gation.7-9 Such species, including low molecular weight RSNOs
(e.g., S-nitrosocysteine (CysNO) and S-nitrosoglutathione (GSNO))
as well as S-nitrosoproteins, have all been found in mammalian
systems, especially in blood plasma.9-12 In addition, it has been
suggested that S-nitrosoalbumin (AlbSNO) is the predominant
reservoir for NO in mammalian blood.10 RSNOs are also reportedly
involved in the trans-S-nitrosylation of proteins by transferring the
NO+ moiety,13,14 a process that may regulate the activity of
enzymes and receptors by a reversible post-translational modifica-
tion.5,15,16
The direct amperometric detection of S-nitrosothiol spe-
cies (RSNOs) is realized by modifying a previously re-
ported amperometric nitric oxide gas sensor with thin
hydrophilic polyurethane films containing catalytic Cu(II)/
(I) sites. Catalytic Cu(II)/(I)-mediated decomposition of
S-nitrosothiols generates NO(g) in the thin polymeric film
at the distal tip of the NO sensor. Three different species
are examined to create the catalytic layer: (1) a lipophilic
Cu(II)-ligand complex; (2) Cu(II)-phosphate salt; and
(3) small (3-µm) metallic Cu0 particles. All three catalytic
layers yield reversible amperometric response in propor-
tion to the concentration of S-nitrosothiols (e.g., nitroso-
cysteine, nitrosoglutathione, S-nitroso-N-acetylcysteine,
S-nitrosoalbumin) present in the aqueous test solution.
Sensitivity toward the different RSNO species is depend-
ent on the respective catalytic rates of decomposition of
the RSNO species by reactive Cu(I), accessibility of the
species into the polyurethane layer containing the catalyst,
the level of reducing agents (ascorbate) used in solution
to help generate reactive Cu(I) species, and the concen-
tration of metal ion complexing agents present in the test
solution (e.g., EDTA). Under optimized conditions, all
RSNO species can be detected at e1 µM levels, with
sensor lifetimes of at least 10 days for the sensors based
on Cu(II)-phosphate and Cu0 particles. It is further
shown that the new RSNO sensors can be used to assess
the “NO-generating” ability of fresh blood samples by
effectively detecting the total level of reactive RSNO
species present in such samples.
Most of the biological effects of RSNO are mediated by the
homolytic cleavage of the S-N bond leading to the release of
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S-Nitrosothiols (RSNOs) are a group of bioactive molecules
generated in vivo through the nitrosylation of thiols by oxidative
intermediates of endogenous nitric oxide (NO) (e.g., N2O3 and
NO+) that form under physiological conditions.1 Nitric oxide has
been identified as the endothelium-derived relaxing factor (EDRF),2
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* To whom correspondence should be addressed. E-mail: mmeyerho@
umich.edu.
† Current address: Department of Chemistry, University of Tennessee,
Knoxville, TN 37996-1600.
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3516 Analytical Chemistry, Vol. 77, No. 11, June 1, 2005
10.1021/ac048192u CCC: $30.25 © 2005 American Chemical Society
Published on Web 04/19/2005

NO. The decomposition of RSNOs is dependent on various factors,
such as intensity of light, solution pH, metal ion concentration,
and the presence of various reductants (e.g., ascorbate and
thiols).17-22 In aqueous solution, exposure to visible light or certain
metal ions leads to the rapid decomposition of RSNOs. Williams
et al. reported that RSNOs can undergo a Cu(II)-catalyzed reaction
involving reduction of Cu(II) to Cu(I) by thiolate impurities or
added reducing agents and subsequent reduction of the RSNO
to RS- and NO by the Cu(I) species.17,23-25 Thiolate formed from
this reaction can reduce the Cu(II) species also formed, regen-
erating Cu(I), yielding a catalytic cycle. Even a trace level of Cu(II)
ions can greatly facilitate this reaction, including copper ions
bound to peptides or proteins.26 The reduction of Cu(II) to Cu(I)
is a crucial first step for such decomposition, and the subsequent
formation of a Cu(I) intermediate complex (five- or six-membered
ring structures) with the RSNO species is also postulated to be
involved in the decomposition mechanism.23 The reaction of
RSNOs via this pathway can be completely suppressed by adding
a multidentate Cu(II) chelator such as EDTA to the solution.17,23
To detect RSNOs in biological systems, a variety of methods
have been suggested. Most techniques depend on redox or
substitution reactions of RSNOs to produce detectable products
including other forms of nitrogen oxides (NO, NO2-, NO3-). These
species can be monitored via either spectrophotometric or
fluorometric assays, as well as chemiluminescence or electro-
chemical methods.19,27,28 The rate and extent of these reactions
are dependent on reaction conditions (e.g., pH), as well as the
specific nature of the R group of the RSNO species.19,25,29 In
addition, the presence of interfering compounds often found in
biological fluids can influence the accuracy in assaying the given
RSNO species.19,28,30 Therefore, separation steps, such as electro-
phoresis and HPLC, are frequently combined to avoid possible
influence/interference from nitrite and other species (disulfides,
thiols, or antioxidants) or proteins during analytical detection.27
While current analytical methods provide good sensitivity, the
intrinsic lability of RSNOs makes sample pretreatment steps
difficult to implement.27,31,32 Hence, the development of simple and
reliable RSNO detection methods is still of great interest.
Figure 1. Amperometric detection scheme based on catalytic
copper species within the outer polymeric layer of proposed RSNO
sensor.
Recent research in this laboratory has demonstrated the
possibility of preparing more thromboresistive coatings by incor-
porating Cu(II) sites within polymeric films.33,34 When in contact
with fresh blood, endogenous RSNO species in the blood can be
catalytically converted to NO via the Cu(II)/Cu(I) redox chemistry
described above, creating a locally elevated level of NO at the
polymer/blood interface. The improved biocompatibility of the
films is due to the potent inhibitory effect of NO on platelet
activation and adhesion.35 However, to use such polymers to
enhance the blood compatibility of biomedical devices in clinical
practice, it will be necessary to prove whether blood levels of
endogenous reactive RSNO species do not vary greatly from
subject to subject.
Herein we propose the concept of a direct and real-time RSNO
sensor by combining the catalytic NO-generating capability of
polymer films containing copper species with a highly sensitive
amperometric NO sensor described previously.36,37 While elec-
trochemical RSNO detection using commercially available am-
perometric NO probes has been suggested,19 RSNO measure-
ments in this earlier work were accomplished by triggering
solution-phase RSNO decomposition by adding Cu(II) ions and
thiols as reagents to the bulk sample solution.19 In the present
work, various catalytic polymeric films containing copper species
are used to modify the surface of the amperometric NO detector,
thereby creating true sensors that respond reversibly to the levels
of RSNO species in test samples. As illustrated schematically in
Figure 1, the Cu(II)-mediated decomposition of RSNOs is achieved
within a thin hydrophilic polymer layer (polyurethane (PU)) at
the distal tip of the improved NO sensor, leading to the production
of NO in this confined region. The NO generated can diffuse
through the gas-permeable membrane of the NO sensor to a
planar platinized platinum anode, where oxidation of NO takes
place. Three different catalytic layers are examined to prepare
RSNO sensors including a lipophilic Cu(II)-ligand complex, a
relatively insoluble copper-phosphate salt, and small (3-µm)
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M., Stamler, J. S., Eds.; John Wiley: Chichester, U.K., 1996; pp 521-539.
(28) Cook, J. A.; Kim, S. Y.; Teague, D.; Krishna, M. C.; Pacelli, R.; Mitchell, J.
B.; Vodovotz, Y.; Nims, R. W.; Christodoulou, D.; Miles, A. M. Anal. Biochem.
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Chem. 2004, 76, 545-551.
Analytical Chemistry, Vol. 77, No. 11, June 1, 2005 3517

metallic Cu0 particles, each incorporated into a thin PU layer. It
will be shown that sensors prepared with all three types of films
respond to various RSNO species at submicromolar levels and
that those prepared with copper phosphate and metallic copper
particles maintain amperometric response for at least 10 days.
Further, it will be demonstrated that the resulting devices can be
used to detect the presence and relative levels of NO-generating
RSNO species within fresh blood samples.
BSA concentration, sulfhydryl level, and S-nitroso-BSA concentra-
tion were determined by the BCP dye-binding method,41 Ellman
method,42 and chemiluminescence measurements of NO liberated
by Cu(II)-catalyzed decomposition, respectively. In this study, only
the actual AlbSNO concentrations are reported based on the above
analysis. It was found that more than 80% of the initially reduced
BSA species contained free thiol groups, and more than 60% of
the total BSA was converted to the desired S-nitroso-BSA.
Fabrication of Amperometric NO/RSNO Sensors. The
amperometric NO gas sensors used in this work were composed
of a platinized Pt working electrode (Pt disk (with 250-µm o.d.))
sealed in glass wall tubing (with 2-mm o.d.) and a Ag/AgCl wire
(127-µm o.d.) as reference/counter electrode. These two elec-
trodes were incorporated behind a PTFE gas-permeable mem-
brane (18-µm thickness, 0.07-µm pore size, Tetratex). A detailed
preparation procedure for such sensors was reported previously.36
To create control NO sensors for blood measurements that
respond to NO similarly to the RSNO sensors, a thin HPU (SP90A-
100) film (∼4-µm thickness) was attached over the PTFE
membrane using an O-ring to hold the dual layer of membranes
in place.
To fabricate the amperometric RSNO sensors, the additional
catalytic polymer layer was prepared separately (see below) and
physically held over the PTFE membrane of the NO sensor (see
Figure 1a in ref 36 for basic sensor configuration) with an O-ring.
Depending on the specific type of Cu catalyst used, i.e., Cu(II)-
DTTCT, Cu(II)-phosphate, and metallic Cu0 particles, the sensors
will be referred to as type I, type II and type III, respectively. All
sensors were polarized at +0.75 V versus Ag/AgCl for at least 12
h before use, and all subsequent amperometric measurements
were carried out using the same applied potential. Calibration
curves and current recordings were obtained as described in our
previous work.36
Immobilization of Copper Catalysts in PU Polymer Matrix.
A catalytic polymer layer containing the Cu(II)-DTTCT complex
was obtained by wetting a PTFE membrane filter (pore size, 1.2
µm) with THF solution containing Cu(II)-DTTCT and HPU
(SP60D-20). The Cu(II)-DTTCT (4 mg, 5.7 µmol) was dissolved
in 2 mL of warm THF and then mixed with 40 mg of HPU. The
clear cocktail solution (0.5 mL) was poured and evenly distributed
over the entire surface of a PTFE membrane filter on a glass slide
and allowed to dry slowly. A piece of this membrane was cut and
mounted on the NO sensor to create the type I RSNO sensor.
To immobilize inorganic copper ion sources, 20 mg of Cu(II)-
phosphate (for type II sensors) or Cu0 particles (type III sensors)
was mixed with 2 mL of THF solution containing HPU (SP90A-
100) (80 mg). The solution was then sonicated to obtain a fine
dispersion. An aliquot of this cocktail slurry (0.5 mL) was spread
in a glass casting plate (area, ∼25 cm2) and dried using a
mechanical shaker to prevent particle aggregation. A small section
of this film was then attached to the NO sensor using an
appropriate O-ring. A second pure HPU film (∼4-µm thickness)
was also affixed to the sensor as an outermost layer to block direct
contact of copper sources with the bulk sample solution.
Detection of RSNOs with Polymer Film Modified Am-
perometric NO Sensors. All preliminary RSNO measurements
EXPERIMENTAL SECTION
Materials. Microporous poly(tetrafluoroethylene) (PTFE)
membranes (Tetratex, pore size 0.07 µm, thickness ∼18 µm) and
PTFE membrane filters (SM11803, pore size 1.2 µm, 16.6 cm2
area) were obtained from Donalson Co., Inc. (Minneapolis, MN)
and Sartorius (Goettingen, Germany), respectively. Silicone rubber
(SR) (RTV3140) was from Dow Corning Corp. (Midland, MI),
hydrophilic polyurethanes (HPU) (Tecophilic SP60D-20 or SP90A-
100; 20 and 100 wt % water-uptake capability, respectively) were
from Thermedics Polymer Products (Wilmington, MA), and
o-nitrophenyloctyl ether and poly(vinyl chloride) (PVC) were from
Fluka (Buchs, Switzerland). All chemicals were of analytical grade
or better and used as received from the suppliers. Various
phosphate buffers including phosphate-buffered saline (PBS) were
prepared as needed in the laboratory. Cu(II)-dibenzo[e,k]-2,3,8,9-
tetraphenyl-1,4,7,10-tetraazacyclododeca-1,3,7,9-tetraene (Cu(II)-
DTTCT) was synthesized as described in the literature.38 Cu(II)-
phosphate and Cu0 particles (dendritic, 3-µm average particle size)
were purchased from Riedel-de Hae¨n (Seelze, Germany) and
Sigma-Aldrich (St. Louis, MO), respectively. All solutions were
prepared with 18 MΩ cm-1 deionized distilled water by using a
Milli-Q filter system (Millipore Corp., Billerica, MA).
Preparation of S-Nitrosothiols. Crystalline S-nitroso-N-
acetylpenicillamine (SNAP) was synthesized according to a
reported procedure39 using a 5 mM solution of N-acetylpenicil-
lamine containing 10 µM EDTA. Solutions (5 mM each) of other
S-nitrosothiols (CysNO, GSNO, S-nitroso-N-acetylcysteine (SNAC))
were also prepared as previously described.40 Briefly, equal
volumes of fresh 10 mM monothiol in 120 mM H2SO4 and 10 mM
NaNO2 (with 20 µM EDTA) were mixed at room temperature.
Unless noted otherwise, these solutions were directly injected into
PBS (pH 7.4) to obtain the desired concentration of RSNOs. The
concentrations and stabilities of the synthesized RSNOs were
determined by using a chemiluminescence NO analyzer (NOA;
Seivers 280, Boulder, CO).
S-Nitrosylation of the Cys-34 in bovine serum albumin (BSA)
was achieved by modifying the method of Stamler.5 BSA was first
reduced using dithiothreitol before S-nitrosylation. After incubation
with equimolar dithiothreitol at 37 °C for 1 h, the BSA solution in
0.1 M Tris-HCl buffer (pH 8.0) was filtered three times with 10
µM EDTA solution to remove remaining dithiothreitol using a
centrifugal filter device (molecular weight cutoff 30 000, Amicon
Ultra, Millipore Corp.). The reduced BSA was then reacted with
the acidified NaNO2 solution. After standing for 30 min, the
solution was neutralized with 1 M NaOH and filtered three times
with PBS (pH 7.4) solution containing 10 µM EDTA. The total
(38) Chandra, S.; Sharma, H. K. Ind. J. Chem. 1998, 37A, 1074-1078.
(39) Field, L.; Dilts, R. V.; Ravichandran, R.; Lenhert, P. G.; Carnahan, G. E. J.
Chem. Soc., Chem. Commun. 1978, 249-250.
(41) Pinnell, A.; Northam, B. Clin. Chem. 1978, 24, 80-86.
(42) Riddles, P. W.; Blakeley, R. L.; Zerner, B. Methods Enzymol. 1983, 91, 49-
(40) Stamler, J. S.; Loscalzo, J. Anal. Chem. 1992, 64, 779-785.
60.
3518 Analytical Chemistry, Vol. 77, No. 11, June 1, 2005

were performed in PBS buffer (pH 7.4) containing 10 µM EDTA
and 10 µM ascorbate (added as reducing agent) and in a 100-mL
amber reaction vessel at room temperature. Each RSNO solution
was prepared fresh and used within 2 h. Current signals were
monitored using a microchemical sensor analyzer (Diamond
General, Ann Arbor, MI) in stirred solutions with polarization of
the platinized platinum electrode of the sensor at +0.75V (vs Ag/
AgCl). In some experiments, background NO levels in the bulk
test solutions were monitored simultaneously using a plain
unmodified NO sensor placed into the same sample solution as
with the modified sensor. The measured current levels of RSNO
sensors at specific times were converted to equivalent NO
concentrations based on prior NO calibration data of the RSNO
sensor.
For stability studies, the sensors’ amperometric responses
toward both NO (calibration) and SNAP were recorded periodi-
cally and compared to differentiate whether decreased response
occurs due to deterioration of NO response or from loss of
catalytic activity in the HPU polymeric layer.
Figure 2. Influence of polymer film matrix containing Cu(II)-DTTCT
in type I sensors on direct amperometric response toward SNAP and
NO (inset). Sample solutions contain 10 µM EDTA and 10 µM
ascorbate in pH 7.4 PBS solution.
Estimation of Copper Ion Leaching from Catalytic Poly-
meric Films. To correlate the change in sensor response with
the dissolution of copper-containing catalytic sites, the copper
content within the polymeric films and soaking solutions were
measured after given time periods by using standard atomic
absorption spectroscopy or ICPMS methods. The polymeric
matrixes were dissolved with concentrated sulfuric acid and
filtered before analysis.
previously to function catalytically in generating NO from RSNO
species when incorporated into plasticized PVC films.33 Earlier
studies suggested that the ligated copper ions in this particular
complex are able to carry out the same catalytic redox chemistry
as free Cu(II) ions.33 At the same time, any copper species that
can provide a reservoir of low concentrations of free Cu(II) or
Cu(I) ions within the polymeric film at the surface of the NO
sensor could also be employed to convert RSNO species to NO.
Detection of RSNOs in Blood. For direct detection of
endogenous RSNO species in blood, fresh sheep blood was
obtained from Extracorporeal Membrane Oxygenation (ECMO)
laboratory at the University of Michigan Medical School. Heparin
was administered in the venous line of adult sheep to elevate the
activated clotting time (measured in seconds) from an average
range of 140-160 s (pre-heparin) to over 250 s (post-heparin), to
decrease the propensity of blood clotting after obtaining the
samples. Blood (∼100 mL) was always drawn from an arterial line
placed in the left carotid artery and then immediately isolated from
ambient light by wrapping the syringe with aluminum foil. The
amperometric measurements were made as quickly thereafter as
possible (always < 2 h). The temperature of the sampled blood
was maintained at 37 °C both before and during RSNO detection.
Two electrodes, a “control” NO sensor (no copper catalytic
layer but with a thin HPU film over the PTFE membrane of the
NO sensor) and a type II RSNO sensor were used in most of these
blood experiments. Each sensor was first calibrated with respect
to their inherent response to NO in PBS buffer. Then, the
amperometric signals of both sensors were stabilized at 37 °C
with the sensors placed into the same N2-saturated 75-mL PBS
(pH 7.4) solution. Finally, 25 mL of the fresh whole blood sample
was added to the PBS solution to yield a 25% (v/v) dilution under
a N2 atmosphere, and the amperometric responses of each sensor
to the sample were monitored.
Toward this end, a relatively insoluble Cu(II) salt, i.e., cupric
43
phosphate (Ksp ) ∼10-37
)
(type II sensor) and metallic Cu0
particles (type III sensor) were also examined as candidate
materials within the outer polymer film of the RSNO sensor. In
the case of Cu0 particles, continuous slow corrosion of the particles
in the presence of a phosphate buffered saline (typical sample
test phase) could generate the necessary copper ions required to
yield amperometric RSNO response.44 In general, these different
species were tested by adding them to an appropriate polymer
coating at 10-20 wt % levels.
The selection of the appropriate polymer matrix that contains
the various copper species was found to be critical to achieve
optimal RSNO sensitivity. Indeed, the permeability of this polymer
matrix to the RSNO species as well as any reducing agents used
to maintain high levels of Cu(I) species in the layer is a key
element that dictates the degree of sensor response to RSNO
species. Any NO generated in this layer due to the catalytic
reaction must be able to diffuse quickly up to the underlying gas-
permeable PTFE membrane of the NO sensor. In preliminary
experiments, several possible polymer films were examined to
confine the copper catalysts. Figure 2 illustrates how the specific
nature of the film layer can dramatically affect the observed
amperometric RSNO response. In this example, type I sensors
were prepared using either PVC, silicone rubber, or HPU as the
polymer matrix containing 10 wt % Cu(II)-DTTCT and all
employed a thin PTFE membrane treated with each catalyst-doped
polymer matrix as the outer catalytic film. As shown, a thin silicone
RESULTS AND DISCUSSION
Catalytic Polymer Matrix. The proposed RSNO sensor
design requires the use of immobilized copper species within a
thin polymeric film in direct contact with the surface of the PTFE
membrane of the NO sensor. Initial efforts focused on the use of
the Cu(II)-DTTCT complex (type I sensor), which was shown
(43) Sillen, L. G.; Martell, A. E. Stability Constants of Metal-ion Complexes; The
Chemical Society: London, 1964.
(44) Drogowska, M.; Brossard, L.; Menard, H. J. Electrochem. Soc. 1992, 139,
2787-2793.
Analytical Chemistry, Vol. 77, No. 11, June 1, 2005 3519

rubber outer coating actually yields an RSNO sensor with the most
sensitive response to solution-phase NO (see inset), but very little
amperometric response toward SNAP, a relatively stable low
molecular weight RSNO test species. A plasticized PVC coating
containing the Cu(II)-DTTCT species yields a sensor with the
lowest sensitivity to NO and also very poor response to SNAP. In
contrast, a sensor made with an outer film of HPU provides
modest direct amperometric NO response but a substantially
enhanced sensitivity toward SNAP compared to either of the other
two matrixes. Hydrophilic polyurethanes with poly(ethylene oxide)
soft segments are well known to swell in water and possess good
permeability toward small solute molecules.45,46 Indeed, the
polymer (HPU, Tecophilic SP60D-20) employed in this experiment
has a high water uptake of 20 wt %, compared to ∼0.1 and <0.6%
for SR and plasticized PVC, respectively.47,48 It is believed that
the partially swollen HPU allows the effective partitioning of the
SNAP and ascorbate into the layer, thereby generating more NO,
and closer to the PTFE membrane of the amperometric gas
sensor.
Figure 3. Typical real-time current recordings for type III sensor in
response to low levels of three endogenous RSNO species. Sample
solutions contain 10 µM EDTA and 10 µM ascorbate as a reducing
agent, along with varying RSNO concentrations at room temperature.
levels of Cu(II) ions in the presence of the analyte RSNO species.
It is well known that such bulk phase decomposition reactions
can be completely suppressed by adding EDTA to the sample
phase to chelate any free copper ions.17,23 It was found that, in
the presence of 10 µM EDTA, the amperometric NO sensors used
in this work did not detect any NO in the solution phase while a
corresponding RSNO sensor placed in the same solution re-
sponded to the added RSNO species (see Figure 1s in Supporting
Information). However, at much higher levels of EDTA in the
sample solution, a reduced response to the RSNO species is
observed owing to greater chelation of copper ions within the
catalytic layer (see below).
Amperometric Responses to RSNO Species. To investigate
the relationship between the recorded current and RSNO sample
concentration, all sensors’ responses at +0.75 V (vs Ag/AgCl)
were recorded as increasing concentrations of the various RSNO
species were added to PBS solution containing 10 µM ascorbate
and 10 µM EDTA. Figure 3 illustrates the typical amperometric
responses of a type III sensor at the low concentration range of
CysNO, GSNO, and AlbSNO species, while panels a-c of Figure
4 illustrate representative complete calibration curve data for type
I, type II, and type III sensors toward these same three species,
as well as for SNAC, SNAP, and nitrite. As shown in Figure 3,
typical response times required to achieve 95% of the steady-state
current following changes in RSNO levels are less than 5 min. As
expected, response and recovery (see below) times are dependent
on the physical dimensions (especially thicknesses) of the various
membranes/films and electrolyte layers that comprise the final
sensor configuration.50 All sensors exhibited fully reversible and
reproducible amperometric responses (see Figure 5 for example
of type II and III sensors’ reversible response toward SNAP) when
changing from low to high and then back down to lower
concentrations of a given RSNO species.
Measurement Conditions. The following rate equation ap-
plies for most copper ion mediated RSNO decomposition reac-
tions:17
rate ) k[Cu2+][RSNO]
(1)
However, at low RSNO concentrations, zero-order kinetics is often
observed due to the absence of thiolate (RS-) reducing species
within the given RSNO preparation (as impurity). Since reducing
equivalents are required initially to generate Cu(I) species, the
reaction rate is limited by the lack of reducing equivalents.23,25
Moreover, the oxidation of Cu(I) to Cu(II) by ambient oxygen
can compete with Cu(I)-mediated RSNO decomposition.25 Thus,
for the proposed RSNO sensor, a low level of exogenous reducing
agent should be added to the sample phase to maximize the
formation of reactant Cu(I) species within the catalytic polymer
layer. While various thiol compounds can serve in this capacity,
they can also activate NO release from RSNO via transnitrosylation
reactions.23 To avoid this complication, ascorbate was chosen as
the primary reducing agent for the studies described herein. The
level of ascorbate added to the test solution must be relatively
low, since higher concentrations of ascorbate can promote NO
release from RSNO species,24,49 presumably by a direct redox
reaction. Therefore, to maintain enhanced levels of Cu(I) species
within the catalytic HPU layer of sensors, most experiments
employed fresh 10 µM ascorbate (final concentration) in the
sample phase. It was shown in separate experiments using
chemiluminescence NO detection that this level of ascorbate does
not generate NO from RSNO species.
The proposed RSNO sensor can also detect any solution-phase
NO present. Therefore, the sample phase must not contain trace
As illustrated in Figure 4a-c, type III sensors were found to
be much more sensitive than type I sensors in terms of the current
response versus RSNO concentration change, even though type
I sensors are more sensitive ((∼12 pA/nM) than type III (∼6 pA/
nM) in their direct response toward NO (see two different y-axis
(45) Lamba, N. M. K.; Woodhouse, K. A.; Cooper, S. L.; Lelah, M. D. Polyurethanes
in biomedical applications; CRC Press: Boca Raton, 1998.
(46) Lyman, D. J.; Loo, B. H. J. Biomed. Mater. Res. 1967, 1, 17-26.
(47) DeLassus, P. T.; Whiteman, N. F. In Polymer Handbook, 4 ed.; Brandrup, J.,
Immergut, E. H., Grulke, E. A., Eds.; Wiley-Interscience: Hoboken, NJ, 1999;
Vol. 1.
(48) Chan, A. D. C.; Li, X.; Harrison, D. J. Anal. Chem. 1992, 64, 2512-2517.
(49) Holmes, A. J.; Williams, D. L. H. J. Chem. Soc., Perkin Trans. 2 2000, 1639-
1644.
(50) Hitchman, M. L. In Measurement of Dissolved Oxygen; Wiley: New York,
1978; pp 103-111.
3520 Analytical Chemistry, Vol. 77, No. 11, June 1, 2005

Figure 5. Dynamic recordings illustrating real-time reversibility of
type II and type III RSNO sensors in amperometric response to
varying concentrations of SNAP.
may be more sequestered in the hydrophobic hard segment of
the HPU matrix than free copper ions, and this may limit its
catalytic function in the type I sensor configuration. On the other
hand, in the presence of oxygen and in pH 7.4 PBS, Cu0 metal
corrodes slowly in a complex manner,51 to yield passive layer of
Cu2O and Cu(II) compounds (CuO, Cu(OH)2, or Cu3(PO4)2),44
which can generate low levels of Cu(I)/Cu(II) due to their
solubility or further dissolution.52 Thus, during the measurements,
a small but continuous flux of copper ions from immobilized
inorganic catalyst (either Cu3(PO4)2 or Cu°) within the polymer
film is expected to form a dynamic catalytic layer within/or at
the surface of the HPU film/solution interface of the type II and
III sensors. Indeed, the corrosion of metallic Cu0 to Cu(II) is
known to occur via Cu(I) formation, so both Cu(I) and Cu(II)
can coexist within the polymer film of type III sensor.44,52 By
separate ICP-mass spectrometry analysis, the saturation level of
the 10 mM phosphate buffer solution in contact with the inorganic
copper phosphate sites used in the type II sensor was estimated
to be below 2 µM at 37 °C (below 1 µM at room temperature).
Hence, copper ions from both copper phosphate (type II sensor)
and metallic copper (type III sensor) are likely to exist at certain
levels in the HPU matrix and decompose the water-soluble RSNOs
more effectively than the lipophilic Cu(II)-DTTCT species used
in the type I sensor.
Figure 4. Typical calibration curves for three types of RSNO
sensors toward various RSNO species and nitrite. (a) Cu(II)-DTTCT
in HPU catalytic layer (type I); (b) Cu(II)-phosphate in HPU catalytic
layer (type II); and (c) metallic Cu0 particles in HPU catalytic layer
(type III). PBS (pH 7.4) solutions containing 10 µM EDTA and 10 µM
ascorbate were spiked with increasing levels of RSNOs to obtain the
calibration plots. Insets in each panel show expanded view of low-
concentration calibration data for all three sensors.
As shown in Figure 4a-c, all RSNO sensors yield concentra-
tion-dependent amperometric responses toward the RSNO species
tested, but the relative response toward the various RSNO test
species is quite different. The order of amperometric sensitivity
is CysNO > SNAP > GSNO g SNAC ∼ AlbSNO. This response
pattern is essentially the same regardless of the nature of the
copper catalytic layer employed. There are two possible factors
that contribute to this response pattern. First, the diffusivity of
the RSNOs within the HPU film should be dependent on the
molecular weight of a given RSNO,46 and this can influence the
overall sensor sensitivity, especially the relatively small ampero-
metric response observed for AlbSNO, a macromolecule. Second,
the inherent reactivities arising from the structural characteristics
of the RSNOs also appear to be critical. N-Acetylation in SNAP,
SNAC, and GSNO has been considered as a structural feature
scales in figure). This observation suggests that the difference in
sensor-to-sensor sensitivity toward a given RSNO species origi-
nates from the different NO-generating capability of the catalytic
layer. Initially it was assumed that the actual species responsible
for RSNO decomposition was the Cu(II)-DTTCT for type I and
low levels of free copper ions within the HPU matrix for type II
or III sensors. The lipophilic Cu(II)-DTTCT in the type I sensors
was shown previously to exhibit catalytic function within a
hydrophobic polymer matrix.33,34 However, the Cu(II)-DTTCT
(51) Taylor, A. H. J. Electrochem. Soc. 1971, 118, 854-859.
(52) Ives, D. J. G.; Rawson, A. E. J. Electrochem. Soc. 1962, 109, 458-462.
Analytical Chemistry, Vol. 77, No. 11, June 1, 2005 3521

that hampers the RSNO decomposition by blocking the formation
of a cyclic intermediate complex (five- or six-membered ring
structures) between the given RSNO and Cu(I)/Cu(II).17,23 Inter-
estingly, the observed amperometric sensitivity sequence for the
RSNO sensors is quite comparable to the reported trend for the
measured second-order RSNO decomposition rate constants (see
eq 1) (without additional thiols) reported previously.17 Such
measured rate constants exhibit a large disparity between CysNO
(∼24 500 ( 500 M-1
s
-1) and SNAC/GSNO (∼0 M-1
s
-1).17
However, in this study, since exogenous reducing agents are
employed, the rate for decomposition of SNAP, SNAC, and GSNO
is likely to be enhanced (compared to their reported rate constants
with Cu(II) in the absence of exogenous reducing equivalents),
owing to faster recycling of Cu(II) to Cu(I) species in the catalytic
layer by ascorbate added to the bulk solution.
For all RSNO sensor designs tested, there is essentially no
response toward inorganic nitrite in the range of 0.1-100 µM.
Although a previous report34 suggests that nitrite can be catalyti-
cally reduced to NO by Cu(II)-DTTCT or free copper ions, such
a reaction only occurs at a much higher concentration range (e.g.,
>1 mM) of nitrite and reducing agents, i.e., ascorbate.
Factors Influencing RSNO Sensor Sensitivity. Several
experimental variables can influence the sensor sensitivity due
to their effect on the Cu(II)-mediated RSNO decomposition
chemistry. The most important factors appear to be the concentra-
tion of reducing agent (ascorbate) and metal chelator (EDTA) in
the sample phase, as well as the specific nature of the buffer salts
in the sample, oxygen levels, and catalyst leaching from the HPU
polymeric layer. The latter determines the lifetimes of the RSNO
sensors (see below).
Figure 6. Influence of sample phase EDTA concentration (a) and
ascorbate level (b) on the amperometric signal of type II and III
sensors in response to constant SNAP at 2.0 µM. For (a), ascorbate
was present at a constant level of 10 µM in PBS; for (b), EDTA was
present at a constant level of 10 µM in PBS.
To verify the influence of ascorbate or EDTA levels, the con-
centration of both species in the sample phase were varied. As
shown in Figure 6, the level of both species can have a significant
effect on the amperometric response of the RSNO sensors. In-
creasing EDTA levels in the sample phase decreases the ampero-
metric response toward a given concentration (2 µM) of SNAP,
as shown for types II and III sensors (Figure 6a). Since free copper
ions within the catalytic layer are likely responsible for the NO
generation process for these two types of sensors, this effect is
anticipated. Type I sensors also exhibit a similar dependence on
EDTA levels (data not shown); however, at this point it is not yet
clear whether this effect is due to scavenging of low levels of free
copper ions or direct ligation of EDTA to the Cu(II)-DTTCT
complex within the polymeric layer, and concomitant inhibition
of the catalytic reaction that can be carried out by the complex.
In contrast, increasing levels of ascorbate in the sample
solution enhance the reactive Cu(I) species in the catalytic layer,
thereby yielding faster NO generation rates and greater ampero-
metric sensitivity toward RSNOs. This is illustrated in Figure 6b
for type II and type III sensor responses to SNAP. A much greater
effect is seen for the type II sensors based on cupric phosphate
as the source of catalytic ions. Indeed, as mentioned above,
corrosion of Cu0 in type III sensors already yields a significant
quantity of Cu(I) species, and hence even in the absence of
reducing equivalents, there is a very large amperometric response
toward 2 µM SNAP.
particular, the presence of chloride ions in the PBS buffer is very
beneficial with respect to the responses observed with type III
sensors (see Figure 2s in Supporting Information). The addition
of chloride in PBS dramatically enhances the amperometric
response toward SNAP compared to phosphate buffers that do
not contain chloride ions. This can be explained by the fact that
the dissolution kinetics of Cu0 change substantially in saline media
since preformed surface Cu2O dissolves more readily under such
conditions.44,53 Even though phosphate ions are known to inhibit
corrosion of Cu0 by forming a passive layer containing Cu3(PO4)2,44
the concentration of chloride ions present in the PBS used in this
study (100 mM) is in a region where chloride can sufficiently
induce the breakdown of this passive layer and promote the
continuous dissolution of Cu0 by forming CuCl2- as the predomi-
nant cuprous species.51,54,55 Such species can be further oxidized
to cupric complexes under aerated conditions.56 In addition,
chloride ions are known to form a more stable complex with
cuprous ions (log â°2 ) 5.68 for CuCl2-) than cupric ions in
aqueous media.43,57
The sensitivity of the type III sensor to SNAP in the presence
of oxygen is somewhat less than in the absence of oxygen (see
(53) Ives, D. J. G.; Rawson, A. E. J. Electrochem. Soc. 1962, 109, 462-466.
(54) Puigdomenech, I.; Taxen, C. Thermodynamic Data for Copper: Implication
for the Corrosion of Copper under Repository Conditions; Swedish Nuclear
Fuel and Waste Management Co.: Stockholm, 2000.
(55) Lee, H. P.; Nobe, K. J. Electrochem. Soc. 1986, 133, 2035-2043.
(56) Hine, F.; Yamakawa, K. Electrochim. Acta 1970, 15, 769-781.
Given ions and oxygen levels present in the test solution also
influence the amperometric response of the RSNO sensors. In
3522 Analytical Chemistry, Vol. 77, No. 11, June 1, 2005

Figure 2s in Supporting Information). Two factors are likely
responsible for this effect. First, the steady-state level of Cu(I)
species in the catalytic layer will be oxygen dependent, since
oxygen reacts quickly with free Cu(I) to generate Cu(II) species.
Second, oxygen also reacts directly with NO, and hence, higher
levels of RSNO generate greater surface concentrations of NO
that can be partially scavenged by reaction with oxygen. All three
types of sensors display a similar influence of oxygen on the
measured amperometric RSNO response. Fortunately, for in vitro
measurements, the level of oxygen can be readily controlled to
essentially constant levels.
RSNO Sensor Lifetimes. To ascertain the practical lifetime
of the RSNO sensors, their sensitivity (pA/nM) changes in
response to SNAP were monitored with time. A relatively fast loss
(∼30% reduction after 1 d) in sensitivity toward SNAP is observed
for type I sensors, but little change in response is found for type
II and type III sensors (∼20 and ∼0% reduction after 10 d,
respectively) (see Figure 3s in Supporting Information). The
change in response of the type I sensors correlates with loss of
copper species from the polymer film (as measured by atomic
absorption; see Figure 4s). This loss of sensitivity and loss of
copper species is accelerated by soaking the sensor in solutions
containing EDTA and or thiols (see again Figure 4s). The
influence of free thiols suggests that the presence of reducing
agents can affect the binding constant for copper ions, with Cu(I)
being much larger in size, and therefore bound less strongly to
the DTTCT ligand. Hence, loss of copper ions from the ligand
complex may well be responsible for the limited lifetime of type
I sensors.
In contrast, type II and III sensors give relatively stable
amperometric responses to RSNO species over an extended period
of time, even when constantly soaked in PBS buffer containing
10 µM ascorbate and 10 µM EDTA (see Figure 3s b,c in
Supporting Information). This is presumably due to the presence
of a larger reservoir of total copper (in excess or continuously
replenished with the newly generated ions from the dissolution
of copper phosphate or the corrosion of metallic copper). However,
even in these cases, the continuous leaching of copper ions is
expected; for example, type II and III catalytic layers were found
to saturate the 10 mM phosphate buffers with ∼2 µM copper level
at 37 °C. Therefore, the rate of loss of copper ions will be enhanced
when the sensor is in frequent contact with a large volume of
fresh solution. For the type III sensor, the copper content within
the catalytic layer is most abundant among three types of sensors
and likely supplies enough Cu(I) and Cu(II) through corrosion
on the high surface area of metallic particles. This explains why
type III sensors exhibit the highest sensitivity toward RSNO over
a long period of time (at least 15 days; data not shown).
Figure 7. Direct detection of endogenous RSNOs in fresh sheep
blood. (a) Two sensors (NO sensor and RSNO sensor utilizing copper
phosphate as a catalyst) were initially stabilized at 37 °C in 75 mL of
PBS under a N2 environment. Upon injection of 25 mL of fresh sheep
blood, only RSNO sensor displays significant amperometric current
change. In (b), SNAP was added to a separate diluted blood solution
at increasing concentrations to demonstrate the amperometric re-
sponse to this nonendogenous RSNO in the presence of blood. EDTA
and ascorbate were not added to the buffer.
reported NO concentrations in plasma from human blood.10 The
RSNO sensor, however, yields response equivalent to a change
of ∼25 nM in effective NO levels at the surface of the sensor
(note: the RSNO sensor is precalibrated for its direct response
to NO). No reducing equivalents were added to the blood samples
in these experiments since fresh blood is known to possess a pool
of potential reducing agents, such as protein-SH (452 µM), GSH
(2.8 µM), cysteine (9.7 µM), and ascorbate (1-10 µM).58,59 Similar
amperometric responses were observed for multiple experiments
with fresh sheep blood and when type III sensors were employed
in such measurements (see Figure 5s in Supporting Information).
Figure 7b shows the amperometric response to added SNAP
for a type II RSNO sensor and a control NO sensor placed in PBS
buffer with a sample of diluted sheep blood added as well. Clearly,
even in the absence of added reducing equivalents, very large
response to the added SNAP is observed, above and beyond the
initial response to the endogenous RSNOs already present in the
sample.
Direct Detection of RSNOs in Blood. To demonstrate the
practical capability of the proposed RSNO sensors to detect
endogenous RSNOs, both a control NO sensor and a type II RSNO
sensor were employed to measure NO and RSNO levels, respec-
tively, in diluted fresh sheep blood samples. As illustrated in
Figure 7a, only the RSNO sensor exhibits a significant elevation
in amperometric NO response upon the injection of sheep blood
into PBS (pH 7.4) at 37 °C. The NO sensor within the same sample
detects only a low-nanomolar level of NO, which matches the
CONCLUSIONS
It has been demonstrated here that direct, real-time measure-
ments of RSNO species can be achieved by modifying a planar
(58) Jones, D. P.; Carlson, J. L.; Mody, V. C.; Cai, J.; Lynn, M. J.; Sternberg, P.
Free Radical Biol. Med. 2000, 28, 625-635.
(57) Wang, M.; Zhang, Y.; Muhammed, M. Hydrometallurgy 1997, 45, 53-72.
(59) May, J. M. Free Radical Biol. Med. 2000, 28, 1421-1429.
Analytical Chemistry, Vol. 77, No. 11, June 1, 2005 3523

amperometric NO gas sensor with an outer polymeric coating that
contains catalytic copper species. Nitrosothiols that diffuse into
the polymeric film can undergo catalytic decomposition to gener-
ate NO via complexed (e.g., DTTCT ligated) copper ions or local
levels of Cu(II) or Cu(I) ions produced by cupric phosphate or
metallic copper particles dispersed within the coating. All three
types of modified sensors yield amperometric currents propor-
tional to the concentrations of various RSNOs, including low
molecular weight RSNOs as well as AlbSNO, and exhibit fully
reversible electrochemical response. Sensitivity varies for the
different S-nitrosothiol species, with the largest response observed
for CysNO. The NO concentration changes observed for the
RSNO sensor when in contact with fresh blood are a measure of
the total RSNO species present that are reactive with the copper
catalyst at the surface of the sensor. Indeed, all endogenous RSNO
species in the blood sample can contribute to this amperometric
response, including S-nitrosoproteins (primarily AlbSNO) and low
molecular weight RSNO species (GSNO and CysNO), but with
the different sensitivities as described above.
Several applications are envisioned for the RSNO sensor
concept described here. As demonstrated already above, such
sensors can be used to assess the relative levels of total reactive
RSNO species in physiological fluids, and this measurement
technique could be employed to predict the effectiveness of new
copper-based polymer coatings as a means to reduce thrombosis
on the surface of biomedical implants for patients that possess
adequate levels of circulating RSNO species in their blood.
Further, if the response and recovery times can be enhanced via
use of thinner polymeric films, it should be possible to use the
RSNO sensors as sensitive flow-through detectors in HPLC or
other separation methods that will be able to quantitate the
individual RSNO species in physiological samples. Finally, the
sensor configuration described here can also provide a simple tool
to assess the stability and purity of given nitrosothiol preparations,
given their propensity to decompose in the presence of light as
well as trace levels of copper ions in solution phase.
ACKNOWLEDGMENT
This research was supported by the National Institutes of
Health under Grant EB-00783.
SUPPORTING INFORMATION AVAILABLE
(Figure 1s) Simultaneous detection of both SNAP and NO with
type II sensor and NO sensor in the same sample solution; (Figure
2s) The effects of oxygen level and buffer composition on
amperometric response of type III RSNO sensor toward SNAP;
(Figure 3s) Long-term monitoring of RSNO sensors’ amperometric
sensitivity to SNAP; (Figure 4s) Changes in the copper content
in HPU films with time as a result of leaching from the film of
type I sensor; (Figure 5s) Response of type III RSNO sensor when
in contact with diluted fresh blood. This material is available free
of charge via the Internet at http://pubs.acs.org.
Received for review December 7, 2004. Accepted March
24, 2005.
AC048192U
3524 Analytical Chemistry, Vol. 77, No. 11, June 1, 2005