Chem. Anal. (Warsaw), 51, 949 (2006)
Enhancing the Selectivity of Amperometric Nitric Oxide
Sensor over Ammonia and Nitrite by Modifying
Gas-Permeable Membrane with Teflon AF®
by Wansik Cha and Mark E. Meyerhoff*
Department of Chemistry, University of Michigan,
930 N. University Avenue, Ann Arbor, Michigan 48109-1055
Keywords:
Nitric oxide; Amperometric gas sensor; Selectivity; Gas permeable
membrane
A planar amperometric nitric oxide (NO(g)) sensor based on a platinized platinum (pPt)
working electrode (as anode) is one of the most sensitive NO detection methods reported to
date with sub-nmol L–1 detection limits. The use of an outer gas permeable membrane
(porous polytetrafluoroethylene (PTFE) membrane) in this sensor design has been shown to
impart superior NO selectivity over common interfering species present in biological samples,
such as nitrite and ascorbate. Recently, however, it has been recognized that ammonia (NH3(g))
present in biological samples, e.g., cell culture medium or blood, can interfere with NO
detection using this sensor configuration owing to the concomitant oxidation capability of
ammonia at the surface of the inner platinized platinum electrode. Herein, the selectivity of
such an amperometric NO sensor is investigated in detail over both ammonia and nitrite and
these results are compared to experimental data obtained with other types of amperometric
NO sensors (including commercial WPI, Inc. device). Further, it is demonstrated that the
NO selectivity of the planar-type NO sensor can be enhanced significantly by treating the
porous PTFE gas permeable outer membrane with a Teflon AF® solution. By filling the
pores of the outer membrane with Teflon AF®, the flux of ammonia and nitrite to the internal
working electrode is greatly reduced, while maintaining good permeability toward NO (g).
Oznaczanie tlenku azotu (NO(g)) przy u¿yciu planarnego amperometrycznego sensora wykorzystuj¹cego elektrodê pracuj¹c¹ z platynowanej platyny jest jedn¹ z najczulszych metod
oznaczania tego analitu, z granic¹ detekcji poni¿ej nanomola, opracowanych do tej pory.
Zastosowanie membrany przepuszczalnej dla gazów (membrana z porowatego poli(tetrafluoroetylenu) (PTFE)) do konstrukcji tego sensora, pozwoli³o uzyskaæ wysok¹ selektywnoœæ
na tlenek azotu, jednoczeœnie dyskryminuj¹c czêsto obecne w próbkach biologicznych
interferenty, takie jak: azotany(III) i askorbiniany. Jednak¿e ostatnio dowiedziono, ¿e amoniak
* Corresponding author. E-mail: mmeyerho@umich.edu; Fax: +1-734-647-4865
950
W. Cha and E. Meyerhoff
(NH3(g)) obecny w próbkach biologicznych, np. roztworze do hodowli komórek czy krwi,
mo¿e zak³ócaæ oznaczanie NO przy tej konfiguracji sensora, w zwi¹zku z równoleg³ym
utlenianiem amoniaku na powierzchni wewnêtrznej elektrody z platynowanej platyny.
W pracy przedstawiono badania nad selektywnoœci¹ amperometrycznego sensora NO
w obecnoœci zarówno amoniaku, jaki i azotanów(III). Wyniki te zosta³y porównane z wynikami otrzymanymi dla innych typów amperometrycznych sensorów NO (miêdzy innymi
handlowo dostêpnego urz¹dzenia WPI, Inc.). Ponadto pokazano, ¿e selektywnoœæ planarnego
sensora NO mo¿e byæ znacznie poprawiona poprzez poddanie dzia³aniu roztworem teflonu
AF® zewnêtrznej membrany wykonanej z porowatego PTFE. Po wype³nieniu porów
membrany Teflonem AF®, przep³yw amoniaku i azotanów (III) do elektrody pracuj¹cej
zosta³ znacznie zredukowany, podczas gdy przepuszczalnoœæ dla NO pozosta³a na dobrym
poziomie.
Nitric oxide (NO) is an endogenous radical species that has been implicated in
a variety of physiological processes, including vascular smooth muscle relaxation,
inhibition of platelet activation/adhesion, and neurotransmission [1–3]. Since NO
was identified as the endothelial-derived relaxing factor (EDRF) [4], a great deal of
effort over the past 20 years has been devoted to understanding its role in biological
systems. As a consequence, various NO detection techniques and procedures have
been developed; bioassays, electron spin resonance spectroscopy, spectrophotometric assays using Griess reagent (to detect NO’s oxidative product, nitrite) or hemoglobin, and a gas phase chemiluminescence reaction of NO with ozone [5, 6]. However, the measurement of NO in biological samples is difficult owing to its high
reactivity and its resulting short half-life [7]. Indeed, NO disappears rapidly in vivo or
in vitro by reacting with hemoglobin, thiols, superoxide as well as oxygen in biological samples to produce methemoglobin, S-nitrosothiols, peroxynitrite and nitrite,
nitrate or other nitrogen oxides, respectively [8]. Thus, simple and direct techniques
that can measure very low levels of NO in the presence of a complex biological
matrix are preferred.
Toward this goal, electrochemical NO measurements, particularly using amperometric NO sensors, have emerged as very useful tools for the direct and real-time
analysis of NO in vivo or in vitro without the need for any sample pretreatment
[9–13]. In these devices, NO diffuses through some type of outer coating or membrane and is oxidized at the surface of an inner working electrode. Miniaturized- or
micro-electrochemical versions of such probes can be positioned in proximity to the
NO source and provide a means to estimate the local surface levels of NO [12, 13].
Depending on the probe shape, amperometric NO sensors can even be inserted directly into tissues or blood vessels (via needle-type probes) [10, 11] or further modified
to detect NO-relevant molecules (e.g., S-nitrosothiols, using planar-type probes)
using immobilized catalytic species [14, 15]. To date, such NO sensors have been
developed to possess extremely high sensitivity, with the capability to quantitate
Enhancing the selectivity of amperometric nitric oxide sensor over ammonia and nitrite
951
sub-nmol L–1 levels of NO. Further, selectivity over other electroactive species is
typically achieved by adding outer polymer layers (e.g., gas permeable membrane
(GPM) or Nafion®) to retard the flux of nitrite and ascorbate, two key potential interferents capable of oxidizing at the same electrode potential used for detecting NO, to
the surface of the inner electrode (carbon fiber or Pt).
Recently, we have found that ammonia (NH3(g)) is one additional possible interferent species, particularly for the „planar”-type NO sensors fabricated with an inner
platinized Pt (pPt) working electrode and an outer porous PTFE-GPM. At neutral
pH, about 1% of the total ammonia (NH3/NH4+) present in a given sample exists in the
form of the dissolved non-ionic solute, i.e., NH3(aq) (pKa 9.25). Indeed, the oxidation
of ammonia by metal catalysts, including pPt, has been explored elsewhere and the
reaction products identified include N2, N2O and NO, depending on the potential
applied [16, 17].
Figure 1. Schematic of p-pPt electrode based amperometric NO sensor examined in detail in this work.
The outer PTFE gas permeable membrane can be further impregnated with varying amounts of
Teflon AF® to enhance NO selectivity
952
W. Cha and E. Meyerhoff
Herein, we investigate and compare the selectivity of various amperometric NO sensors (including planar NO sensors equipped with PTFE-GPM, commercial NO probes,
and carbon fiber-based electrodes reported in the literature) with respect to ammonia
as well as nitrite. Despite the previously reported high selectivity of NO sensors, the
presence of potential interfering molecules at µM-levels in biological samples can
still influence the amperometric current measured for detecting very low nmol L–1-levels of NO. Therefore, a quantitative estimate of the selectivity of given NO sensors for potential interfering agents is necessary for selection of the best NO sensor
for a given application. Further, for the planar-type NO sensor, it is demonstrated that
enhanced NO selectivity can be achieved simply by modifying the porous PTFE–
GPM with Teflon AF®, a copolymer of tetrafluoroethylene and 2,2-bis(trifluoroethylene)-4,5-difluoro-1,3-dioxole (Fig. 1).
EXPERIMENTAL
Materials
Ammonium chloride, ammonium sulfate, sodium nitrite and sodium L-ascorbate were purchased from
Sigma–Aldrich (St. Louis, MO) and used as received. Nickel(II)-tetramethoxyhydroxyphenylporphyrin
(Ni-TMHPP) was synthesized as described in literature [18]. Microporous poly(tetrafluoro-ethylene) (PTFE)
membranes (Tetratex®, pore size 0.07 µm, thickness ~18 µm) were obtained from Donalson Company, Inc.
(Minneapolis, MN). Nitric oxide, nitrogen and argon gases were purchased from Cryogenic Gases (Detroit,
MI). Various phosphate buffers including phosphate-buffered saline (PBS) were prepared as needed in the
laboratory. A nitric oxide stock solution (~ 2 mmol L–1) was prepared by bubbling pure NO gas through
oxygen-free PBS solution obtained with prior Ar(g) purging. All buffer chemicals were of analytical grade or
better and used as received from various suppliers. All solutions were prepared with 18 MW cm–1 deionized
distilled water by using Milli–Q filter (Millipore Corp., Billerica, Mass.).
Fabrication of various NO sensors and amperometric detection
The „planar” amperometric NO sensors assembled with a platinized Pt (pPt) working electrode and
a PTFE–GPM were fabricated by the method reported previously [15, 19], and denoted by the ‘p-pPt based
sensor’. Briefly, a planar pPt (p-pPt) disk (250-µm O.D.) sealed in a glass wall tubing and a Ag/AgCl wire
as the reference/counter electrode were employed to create the electrochemical cell. These two electrodes
were incorporated behind a PTFE–GPM as illustrated in Figure 1. To enhance NO selectivity primarily
against ammonia, the PTFE–GPM (0.12 cm2) was coated with 0.5 µL of Teflon AF® solution (1%, used as
received, Dupont Fluoroproducts, Wilmington, DE) and then dried before sensor fabrication. To examine the
influence of the amount of the coated Teflon AF® on selectivity, the coating procedure was repeated up to
4 times (e.g., 2-µL Teflon AF® coated) as required. All sensor polarization, calibration and subsequent
amperometric measurements were carried out at +0.75 V (vs Ag/AgCl) constant applied potential as
described in our previous work [19].
Another „planar” NO sensor that employed a glassy carbon (GC) working electrode (p-GC sensor)
instead of the inner pPt electrode was also prepared and used for NO detection in the same manner as
Enhancing the selectivity of amperometric nitric oxide sensor over ammonia and nitrite
953
described above for the p-pPt based sensor. The GC working electrode (1-mm diameter) sealed in PEEK was
purchased from ESA Inc. (EE040, Cypress Systems, Lawrence, KS), and used without any catalytic layer
after the following electrode surface regeneration procedure; fine polishing with 0.1-µm alumina powder,
cleaning with ultra-sonication and washing with DI water.
Two different commercial NO sensors (ISO sensor) were purchased from World Precision Instruments
(WPI) Inc. (ISO–NOPF200, Sarasota, FL) with about a year interval; the old- and new-ISO sensor were
obtained in 2005 and 2006, respectively and separately tested over 3-month periods as received. All sensors
were used after overnight polarization at the indicated applied potentials (+0.75 V or +0.86 V vs Ag/AgCl).
Due to the presence of the integrated reference electrode (Ag/AgCl), the amperometric output for these
commercial sensors was also collected in a two-electrode configuration only.
A bare carbon fiber (CF) was also used to construct an NO electrode (b-CF sensor), and this device
served as the control sensor for a widely used nickel(II) porphyrin-modified carbon fiber based NO sensor
(NiP-CF sensor), originally described by Malinski et al. [20]. The sensors were prepared with an exposed
electrode tip length of ~2 mm. Pyrolytic graphite carbon fibers (7-µm in diameter, WPI Inc., Sarasota, FL)
were used for the CF electrode fabrication as reported elsewhere [21, 22]. The fibers were electrochemically
activated before use by cycling an applied potential between –1.2 and +1.8 V (vs Ag/AgCl) at 100 mV s–1
scanning rate. The NiP-CF sensors were prepared as described elsewhere, and employed an outer Nafion®
coating to enhance selectivity over nitrite and other anions [20]. Briefly, a porphyrinic catalytic layer
was created on the activated CF electrode via electro-polymerization of Ni-TMHPP (50 mmol L–1)
in a N2-purged 0.1 mol L–1 NaOH solution by cycling the applied potential between –0.2 and +1.2 V
(vs Ag/AgCl) at a 100 mV s–1 scan rate. An outer Nafion® layer was created by dip coating the electrode five
times for 5 s per layer in 1.25% (wt%) Nafion® solution and the sensors were then dried for 5 min. All
amperometric currents for these CF based sensors were measured using a two-electrode configuration with
a Ag/AgCl (3 mol L–1 NaCl) reference electrode (MF–2052, BAS Inc., West Lafayette, IN).
Stock solutions of each analyte tested (NO, NH4+/NH3, nitrite and ascorbate) were prepared fresh daily.
Amperometric current was monitored at room temperature as a function of time using a highly sensitive
ammeter module (Chemical Microsensor I, Diamond General Development Co., Ann Arbor, MI). Sensor
response to NO and interferent species was typically determined using 50 mL of PBS (pH 7.3) solution that
was well stirred (magnetically) under ambient condition, which was repeatedly spiked with small volumes
of concentrated stock solutions containing the test species to change their bulk concentration in the buffer
solution.
Amperometric selectivity coefficient calculation
To quantitatively express the amperometric selectivity of the various NO sensors examined, a previously reported methodology was adopted [23]. For any amperometric device, the total current (It) can be
described by the linear combination of two terms proportional to the concentration (C) of the target analyte
(i, i.e., NO in this study) and the interfering species (j, ammonia or nitrite) as shown in equation 1. Then, the
constant B denotes the true amperometric sensitivity of the given sensor toward the analyte (NO).
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Further, the amperometric selectivity coefficient (kNO,j) can be experimentally obtained via use of the
separate solution method; current levels were recorded separately for test solutions containing the analyte
and the interfering species. Based on the ratio of amperometric sensitivity (DI/C) obtained for each species,
the selectivity coefficient was calculated by equation 2.
954
W. Cha and E. Meyerhoff
ki,jamp = (DIj/Cj)/(DIi/Ci)
(2)
where DIj = Ij – Ib and DIi = Ii – Ib; Ii – the current recorded for the analyte; Ij – the current recorded for the
interfering species; Ib – the basal current recorded for the blank solution.
Typically, two or more identical sensors of each type were tested. For an individual NO sensor, the
average values of amperometric sensitivity (DI/C) and selectivity coefficients were obtained from the multiple measurements (n > 3) for each species (NO, NH3 and nitrite). All selectivity coefficients obtained for
the same type of probes were finally averaged and reported (see Tab. 1 below). For convenience, the
amperometric selectivity coefficient was subsequently transformed into logarithmic form, i.e., log(kNO,j).
The ammonia (NH3) levels in test solutions were approximated to be 1% of the given levels of total NH4Cl
added owing to the two orders of magnitude difference in buffer solution pH (7.3) and pKa of ammonia
(9.25).
Table 1.
Selectivity coefficients over ammonia and nitrite, sensitivity, limit of detection, and response
time for various types of amperometric NO sensors examined in this work
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Calculated by assuming that the amount of NH3(aq) is 1% of total NH4+/NH3 at pH 7.3.
e
Porous PTFE–GPM (0.12 cm2) coated with 2.0 mL of 1% Teflon AF® solution.
f
ISO NO sensors purchased in different year.
g
Possibly multiple layers of selective membranes including Nafion coating [21, 31].
h
Reported LOD by manufacturer.
a
b
Two other parameters that were used to compare each sensor’s performance were the response time and
detection limit. The response time (RT) was calculated as the time required to reach 95% of the final steady-state response current when the NO concentration change from 10 to 120 nmol L–1 was tested; the limit of
detection (LOD) of each sensor was estimated to the lowest NO concentration where the observed
amperometric signal/noise > 3.
Enhancing the selectivity of amperometric nitric oxide sensor over ammonia and nitrite
955
RESULTS AND DISCUSSION
Enhanced NO selectivity of platinized-Pt-electrode based planar gas sensor
with Teflon AF® treatment on PTFE–GPM
As shown in Figure 2, the addition of ammonium chloride to the test buffer solution (PBS, pH 7.3) leads to an elevation in the amperometric current levels of a planar
NO sensor (platinized Pt (pPt) working electrode and reference electrode behind
microporous PTFE–GPM (Fig. 1). The calculated logarithm of the amperometric
selectivity coefficient of this conventional planar-pPt (p-pPt) based gas sensor is –3.1
for ammonium chloride or –1.1 for ammonia (log(kNO,j), j = NH4Cl(aq) or NH3(aq), respectively (Tab. 1). These values are calculated based on the amperometric responses
shown in Figure 2a and the nearly two orders of magnitude difference in the pKa
value of ammonia (9.25) and the test buffer pH value (7.3). At a higher pH of the test
solution, a larger amperometric current change is observed since the equilibrium shifts
to increase the amount of dissolved free ammonia gas, NH3(aq) (data not shown).
–1
80
80 nmol
nmol LL–1
40 nmol L–1
40 nmol L–1
20mmol
mmol L
120
–1
–1
–1
nmolLL–1
NO added to 1010nmol
1.1 mmol L–1
80mmol
mmol LL–1–11.1 mmol L
80
–1
Figure 2. Amperometric response of p-pPt electrode based NO sensor (Fig. 1) toward increasing NO and
NH4Cl concentrations (a) before and (b) after the modification of the microporous PTFE–GPM
with Teflon AF®
It is thought that NH3(aq) present in equilibrium with ammonium ion can effectively
diffuse through porous GPM as NH3(g) and oxidize on the pPt electrode. In fact, it has
been shown previously that the oxidation of ammonia on such catalytic surfaces can
956
W. Cha and E. Meyerhoff
initiate at voltages as low as +0.52 V (vs NHE) with the reaction products including
nitrogen at lower potentials and nitrogen oxides (N2O or NO) at higher oxidative
potentials [16, 17]. Since, the potential applied to the inner pPt working electrode of
the gas sensor configuration is +0.75 V vs Ag/AgCl, it is likely that some nitrogen
oxide species are produced at the pPt surface during the amperometric response to
ammonia. Therefore, when using the pPt electrode as in inner working electrode for
the construction of an NO sensor, interference from ammonia can surely occur.
Indeed, the p-pPt based NO sensor displays high background current levels when
inserted within cell culture medium or blood, to directly detect NO (data not shown).
The presence of ammonia is strongly suspected from the observation that by first
purging N2 through a culture medium, and collecting this gas phase in DI water, the
pH of the DI water increases substantially. In fact, the spontaneous degradation of
labile L-glutamine, an essential nutrient in cell culture, often accounts for the build-up
of ammonia in culture media [24, 25]. Further, the level of ammonia in normal blood
samples is known to be within the 10~30 µmol L–1 range (total of NH3/NH4+) [26].
Hence, to minimize the ammonia interference for NO measurements in such biological matrixes, various methods were explored in preliminary studies to reduce this
interference, including varying the pH of the internal solution of the NO sensor (from
30 mmol L–1 NaCl/0.3 mmol L–1 HCl solution that was originally reported [19]) and
applying a less positive potential to the platinized Pt electrode. Neither of these approaches was successful (data not shown). Indeed, lowering the applied potential to the
inner working electrode was found to cause a significant deterioration in the NO
selectivity owing to a more reduced sensitivity toward NO relative to ammonia.
An approach to enhance selectivity of the NO sensor that was found to be quite
effective is to modify the nature of the porous PTFE–GPM using a solution of Teflon AF® (an amorphous fluoro-copolymer with large free volume and high gas permeability [27]). As shown in Figure 2b, when the same sensor configuration is prepared
using the PTFE–GPM that has been impregnated with the Teflon AF® (Fig. 1), NO
selectivity is dramatically changed. After the membrane modification, the logarithm
of the selectivity coefficient of the p-pPt based sensor improves to –6.2 for ammonium ion at pH 7.3 (–4.2 for NH3(aq)), which is about a thousand-fold improvement
compared to the conventional p-pPt based sensor (data shown in Fig. 2a). Further, the
influence of varying amounts of Teflon AF® coated into the PTFE–GPM was further
investigated for two interfering agents, ammonia and nitrite. As shown in Figure 3,
although the thicker coating appears better for the selectivity enhancement, the calculated selectivity coefficient exhibits the greatest change after the first layer of coating
(0.5 µL of 1% Teflon AF® solution over 0.12 cm2 of PTFE–GPM) for both species.
It should be noted that even without the GPM modification, the NO selectivity over
nitrite is excellent, manifesting the effectiveness of microporous PTFE–GPM as
a gas selective membrane. However, the existence of nitrite interference at high concen-
Enhancing the selectivity of amperometric nitric oxide sensor over ammonia and nitrite
957
tration (~ mmol L–1-levels, data not shown) is likely due to the detection of trace non-ionic products (N2O3, NO2 or even NO) created in solution equilibrium, rather than
nitrite ion itself. Indeed, HNO2, the conjugate acid of nitrite (pKa 3.3) is known to be
in equilibrium with N2O3, which can further disproportionate into NO and NO2 [8].
In comparison, the measured logarithm of the selectivity coefficient against ascorbate, another ionic species, is –6.5 for the unmodified p-pPt based sensor and even
less (< –7.0) for the Teflon AF®-modified NO sensor.
Figure 3. Change of NO selectivity coefficient of p-pPt electrode based NO sensor for ammonia (as total
of NH3 and NH4+) and nitrite as function of varying amounts of Teflon AF® coated on microporous
PTFE–GPM
It should be noted that after the gas permeable membrane is modified with Teflon AF® the resulting sensors exhibit relatively little change in their sensitivity for
detecting NO (Tab. 1). Although it is thought that the porous structure of PTFE–GPM
is partially or fully filled with the Teflon AF® matrix, depending on the amount used
during modification, NO retains significant permeability through the membrane presumably due to its favorable partition coefficient into the hydrophobic fluoropolymer
matrix. Indeed, the hydrophobic nature of NO is well known; NO is approximately
nine-times more soluble in organic media than water [28]. Thus, the difference in
partitioning capability of gaseous species, e.g., NO vs ammonia, within the hydrophobic polymer phase that fills the pores of the PTFE membrane, likely results in
changing the gas flux of ammonia much more so than NO, and this yields the observed improvement in NO selectivity over ammonia.
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W. Cha and E. Meyerhoff
Comparison of selectivity and other performance parameters of various
amperometric NO sensors
To examine and compare the extent of ammonia/nitrite interference with other
types of amperometric NO sensors (in addition to the p-pPt electrode based device
developed in this laboratory [19]), various types of NO sensors were fabricated
and/or purchased, and then tested. These studies focused primarily on two other
designs widely used in previous literature reports to measure NO in biological
matrixes; the commercial ISO NO sensor by WPI, Inc. [29], and a carbon fiber electrode based sensor that employs an electropolymerized nickel porphyrin catalytic
layer along with an outer Nafion® coating (as originally reported by Malinski et al.
[20]). The selectivity coefficients for all sensors tested are summarized in Table 1.
For p-pPt electrode based sensor, the enhanced selectivity with the membrane modification yields a somewhat elongated response time, about two-fold longer, than the
unmodified sensor, but an insignificant sensitivity change as describe above.
Interestingly, a planar GC (p-GC) electrode based sensor exhibits the lowest NO
selectivity against ammonia among all the sensor configurations examined, but comparable selectivity vs nitrite. The overall results of the p-GC electrode based sensor
(no catalytic layer) are in contrast with those of p-pPt electrode based sensor; higher
limit of NO detection and low sensitivity, which supports the catalytic effect of the
platinized Pt electrode. In general, selectivity coefficients for the gas sensor prepared
with the p-GC electrode as the inner working electrode appear similar or poorer than
those of p-pPt sensors (Tab. 1). However, the carbon fiber (CF) electrode based NO
sensors (ISO and b-CF sensors) tend to exhibit less ammonia interference than the
unmodified p-pPt or p-GC electrode based sensors. This may reflect the intrinsic
slower ammonia oxidation kinetics on carbon fiber (pyrolytic graphite) than on the
other working electrodes. Even though the CF electrode based NO probes are claimed
to exhibit high selectivity by using polymeric coatings that help discriminate against
electroactive ionic species such as nitrite or ascorbate [20, 21], as shown in Figure 4,
they still respond to such interfering ions at elevated concentrations. It is unlikely that
interfering species exist in vivo at such high levels, e.g., nitrite plasma level,
0.1~0.5 µmol L–1 [30]; however, the local build-up of such species, particularly for
in vitro measurements, is still possible and may interfere with the NO sensor depending on its selectivity. Hence, it should be noted that when low-nmol L–1 levels of NO
are directly monitored within biological samples, the concentration fluctuation of
interfering species in sample medium should be minimized or taken into account via
a predictive test, based on the known selectivity and sensitivity of the given NO sensors.
As illustrated in Table 1 for the commercial NO (ISO) sensors, the selectivity
over ammonia and nitrite has been found to improve in the newer model, now com-
Enhancing the selectivity of amperometric nitric oxide sensor over ammonia and nitrite
959
parable to that obtained with the Teflon AF®-modified p-pPt electrode based sensor.
Such improvement is likely due to the application of multiple selective layers over
the surface of this sensor, including a Nafion® coating and a hydrophobic layer as
described elsewhere [21, 31]. Of course, as a commercial product, the exact membrane composition used is not reported by the manufacturer.
NaNO2 100 mmol L–1
NaNO2 260 mmol L–1
Figure 4. Amperometric responses of (a) commercial old-ISO NO sensor purchased in 2005 and
(b) NiP-CF electrode based sensor with increasing nitrite concentration in PBS (pH 7.3).
The applied potentials for both sensors are same at +0.75 V (vs Ag/AgCl)
Further, the variation in response times of the NO sensors listed in Table 1 can be
explained by the differences in the dimensions of the selective membranes. Primarily, thinner membrane structures obtained from coating processes with polymer solution (for CF electrode, and presumably for ISO sensors [21]) are likely to result in
a much faster sensor response time than the PTFE-GPM (~18-µm thickness) physically held on the working electrodes. Moreover, NO transport for p-pPt sensors and
ISO sensors is probably retarded due to the multiple layered structures including an
additional hydrophobic selective membrane (i.e., Teflon AF® coating and an unknown
hydrophobic membrane, respectively; also see above). Hence, further optimization
of response time can be envisioned by minimizing the number of layers and thickness
of the layers; for example, via direct coating of Teflon AF® layer on a working electrode. However, using such an approach to improve sensor performance will require
that the NO sensors be designed to maintain electrical contacts between the working
and reference/counter electrodes after the application of such a hydrophobic selective
coating, since in general such coatings are electrically non-conducting (high resistance).
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W. Cha and E. Meyerhoff
CONCLUSION
In summary, the selectivity of various amperometric NO sensors over ammonia
and nitrite was examined quantitatively by determining the selectivity coefficients
and correlating the selectivity observed with other sensor performance parameters
such as response time and limit of detection. A Teflon AF® coating on a microporous
PTFE–GPM significantly reduces the ammonia interference observed for a new p-pPt
electrode based NO sensor without loss of sensor sensitivity. Also this study has
shown that finite ammonia/nitrite interference exists for other types of amperometric
NO sensors and needs to be known, particularly when the direct and real-time NO
detection at nmol L–1 levels is attempted within biological samples where ammonia
and nitrite levels may fluctuate in µmol L–1-range and above.
Acknowledgements
This research was supported by the National Institutes of Health (EB000783 and EB004527).
REFERENCES
1. Keaney J.F.J., Simon D.I., Stamler J.S., Jaraki O., Scharfstein J., Vita J.A., and Loscalzo J., J. Clin.
Invest., 91, 1582 (1993).
2. Do K.Q., Benz B., Grima G., Gutteckamsler U., Kluge I. and Salt T.E., Neurochem. Int., 29, 213
(1996).
3. Mathews W. and Kerr S., J. Pharmacol. Exp. Ther., 267 (3), 1529-1537 (1993)
4. Ignarro L.J., Buga G.M., Wood K.S., Byrns R.E. and Chaudhuri G., Proc. Natl. Acad. Sci. U.S.A.,
84, 9265 (1987).
5. Archer S., FASEB J., 7 (2), 349-360 (1993).
6. Feelisch M. and Stamler J.S., Measurement of NO-related activities – Which assay for which
purpose?, in: Methods in nitric oxide research, [Feelisch M. and Stamler J.S., Eds], John Wiley &
Sons Ltd., Chichester, England 1996, p. 303–307.
7. Thomas D.D., Liu X., Kantrow S.P. and Lancaster Jr. J.R., Proc. Natl. Acad. Sci. U.S.A., 98, 355
(2001).
8. Miranda K.M., Espey M.G., Jourd’heuil D., Grisham M.B., Fukuto J.M., Feelisch M. and
Wink D.A., Chapter 3, The chemical biology of nitric oxide, in: Nitric oxide, biology and pathology,
[Ignarro LJ, Ed.], Academic Press, San Diego, CA, 2000, p. 41.
9. Bedioui F. and Villeneuve N., Electroanalysis, 15, 5 (2003).
10. Mas M., Escrig A. and Gonzalez-Mora J.L., J. Neurosci. Methods, 119, 143 (2002).
11. Vallance P., Bhagat K., MacAllister R., Patton S., Malinski T., Radomski M., and Moncada S.,
The Lancet, 346, 153 (1995).
12. Brovkovych V., Stolarczyk E., Oman J., Tomboulian P. and Malinski T., J. Pharm. Biomed. Anal.,
19, 135 (1999).
13. Lee Y., Yang J., Rudich S.M., Schreiner R.J. and Meyerhoff M.E., Anal. Chem., 76, 545 (2004).
14. Cha W., Lee Y., Oh B.K. and Meyerhoff M.E., Anal. Chem., 77, 3516 (2005).
Enhancing the selectivity of amperometric nitric oxide sensor over ammonia and nitrite
961
15. Cha W. and Meyerhoff M.E., Langmuir, in Press (2006)
16. de Mishima B.A.L., Lescano D., Holgado T.M. and Mishima H.T., Electrochim. Acta, 43, 395
(1998).
17. Wasmus S., Vasini E.J., Krausa M., Mishima H.T. and Vielstich W., Electrochim. Acta, 39, 23
(1994).
18. Malinski T., Ciszewski A., Bennett J. and Fish J.R., J. Electrochem. Soc., 138, 2008 (1991).
19. Lee Y., Oh B.K. and Meyerhoff M.E., Anal. Chem., 76, 536 (2004).
20. Malinski T., Taha Z., Grunfeld S., Burewicz A., Tomboulian P. and Kiechle F., Anal. Chim. Acta,
279, 135 (1993).
21. Zhang X., Cardosa L., Broderick M., Fein H. and Lin J., Electroanalysis, 12, 1113 (2000).
22. Friedemann M.N., Robinson S.W. and Gerhardt G.A., Anal. Chem., 68, 2621 (1996).
23. Wang J., Talanta, 41, 857 (1994).
24. Greenstein J.P. and Winitz M., Chapter 25. Glutamic acid and glutamine, in: Chemistry of
the amino acids, [Greenstein J.P. and Winitz M., Eds], Robert E. Krieger Publishing Co., Inc.,
Malabar, FL 1984, p. 1929.
25. Hassell T., Gleave S. and Butler M., Appl. Biochem. Biotech., 30, 30 (1991).
26. Barsotti R.J., J. Pediatr., 138 (1, Part 2), S11-S20 (2001).
27. Merkel T.C., Bondar V., Nagai K., Freeman B.D. and Yampolskii Y.P., Macromolecules, 32, 8427
(1999).
28. Shaw A.W. and Vosper A.J., J. Chem. Soc. Faraday Trans. I, 73, 1239 (1977)
29. NO Microsensors Instruction Manual: World Precision Instruments, Inc.; 2005.
30. Kelm M., Biochim. Biophys. Acta – Bioenergetics, 1411, 273 (1999).
31. Zhang X., Lin J., Cardoso L., Broderick M. and Darley-Usmar V., Electroanalysis, 14, 697 (2002).
Received May 2005
Accepted September 2006