enhancing the selectivity of amperometric nitric oxide sensor...

Chem. Anal. (Warsaw), 51, 949 (2006)

Keywords: Nitric oxide; Amperometric gas sensor; Selectivity; Gas permeablemembrane

Enhancing the Selectivity of Amperometric Nitric OxideSensor 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

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 todate 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 toimpart 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 NOdetection using this sensor configuration owing to the concomitant oxidation capability ofammonia at the surface of the inner platinized platinum electrode. Herein, the selectivity ofsuch an amperometric NO sensor is investigated in detail over both ammonia and nitrite andthese results are compared to experimental data obtained with other types of amperometricNO sensors (including commercial WPI, Inc. device). Further, it is demonstrated that theNO selectivity of the planar-type NO sensor can be enhanced significantly by treating theporous PTFE gas permeable outer membrane with a Teflon AF® solution. By filling thepores of the outer membrane with Teflon AF®, the flux of ammonia and nitrite to the internalworking electrode is greatly reduced, while maintaining good permeability toward NO(g).

* Corresponding author. E-mail: [email protected]; Fax: +1-734-647-4865

Oznaczanie tlenku azotu (NO(g)) przy u¿yciu planarnego amperometrycznego sensora wyko-rzystuj¹cego elektrodê pracuj¹c¹ z platynowanej platyny jest jedn¹ z najczulszych metodoznaczania tego analitu, z granic¹ detekcji poni¿ej nanomola, opracowanych do tej pory.Zastosowanie membrany przepuszczalnej dla gazów (membrana z porowatego poli(tetrafluo-roetylenu) (PTFE)) do konstrukcji tego sensora, pozwoli³o uzyskaæ wysok¹ selektywno�æna tlenek azotu, jednocze�nie dyskryminuj¹c czêsto obecne w próbkach biologicznychinterferenty, takie jak: azotany(III) i askorbiniany. Jednak¿e ostatnio dowiedziono, ¿e amoniak

950 W. Cha and E. Meyerhoff

Nitric oxide (NO) is an endogenous radical species that has been implicated ina variety of physiological processes, including vascular smooth muscle relaxation,inhibition of platelet activation/adhesion, and neurotransmission [1�3]. Since NOwas identified as the endothelial-derived relaxing factor (EDRF) [4], a great deal ofeffort over the past 20 years has been devoted to understanding its role in biologicalsystems. As a consequence, various NO detection techniques and procedures havebeen developed; bioassays, electron spin resonance spectroscopy, spectrophotomet-ric assays using Griess reagent (to detect NO�s oxidative product, nitrite) or hemo-globin, and a gas phase chemiluminescence reaction of NO with ozone [5, 6]. How-ever, the measurement of NO in biological samples is difficult owing to its highreactivity and its resulting short half-life [7]. Indeed, NO disappears rapidly in vivo orin vitro by reacting with hemoglobin, thiols, superoxide as well as oxygen in biologi-cal samples to produce methemoglobin, S-nitrosothiols, peroxynitrite and nitrite,nitrate or other nitrogen oxides, respectively [8]. Thus, simple and direct techniquesthat can measure very low levels of NO in the presence of a complex biologicalmatrix are preferred.

Toward this goal, electrochemical NO measurements, particularly using ampero-metric NO sensors, have emerged as very useful tools for the direct and real-timeanalysis 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 mem-brane and is oxidized at the surface of an inner working electrode. Miniaturized- ormicro-electrochemical versions of such probes can be positioned in proximity to theNO 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 direc-tly into tissues or blood vessels (via needle-type probes) [10, 11] or further modifiedto detect NO-relevant molecules (e.g., S-nitrosothiols, using planar-type probes)using immobilized catalytic species [14, 15]. To date, such NO sensors have beendeveloped to possess extremely high sensitivity, with the capability to quantitate

(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 równoleg³ymutlenianiem amoniaku na powierzchni wewnêtrznej elektrody z platynowanej platyny.W pracy przedstawiono badania nad selektywno�ci¹ amperometrycznego sensora NOw obecno�ci zarówno amoniaku, jaki i azotanów(III). Wyniki te zosta³y porównane z wyni-kami otrzymanymi dla innych typów amperometrycznych sensorów NO (miêdzy innymihandlowo dostêpnego urz¹dzenia WPI, Inc.). Ponadto pokazano, ¿e selektywno�æ planarnegosensora NO mo¿e byæ znacznie poprawiona poprzez poddanie dzia³aniu roztworem teflonuAF® zewnêtrznej membrany wykonanej z porowatego PTFE. Po wype³nieniu porówmembrany Teflonem AF®, przep³yw amoniaku i azotanów (III) do elektrody pracuj¹cejzosta³ znacznie zredukowany, podczas gdy przepuszczalno�æ dla NO pozosta³a na dobrympoziomie.

951Enhancing the selectivity of amperometric nitric oxide sensor over ammonia and nitrite

sub-nmol L�1 levels of NO. Further, selectivity over other electroactive species istypically 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 inter-ferents capable of oxidizing at the same electrode potential used for detecting NO, tothe surface of the inner electrode (carbon fiber or Pt).

Recently, we have found that ammonia (NH3(g)

) is one additional possible inter-ferent species, particularly for the �planar�-type NO sensors fabricated with an innerplatinized Pt (pPt) working electrode and an outer porous PTFE-GPM. At neutralpH, about 1% of the total ammonia (NH3/NH4

+) present in a given sample exists in theform of the dissolved non-ionic solute, i.e., NH

3(aq) (pK

a 9.25). Indeed, the oxidation

of ammonia by metal catalysts, including pPt, has been explored elsewhere and thereaction products identified include N

2, N

2O 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 ofTeflon AF® to enhance NO selectivity


Herein, we investigate and compare the selectivity of various amperometric NO sen-sors (including planar NO sensors equipped with PTFE-GPM, commercial NO probes,and carbon fiber-based electrodes reported in the literature) with respect to ammoniaas well as nitrite. Despite the previously reported high selectivity of NO sensors, thepresence of potential interfering molecules at µM-levels in biological samples canstill 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 sen-sors for potential interfering agents is necessary for selection of the best NO sensorfor a given application. Further, for the planar-type NO sensor, it is demonstrated thatenhanced NO selectivity can be achieved simply by modifying the porous PTFE�GPM with Teflon AF®, a copolymer of tetrafluoroethylene and 2,2-bis(trifluoroethy-lene)-4,5-difluoro-1,3-dioxole (Fig. 1).

EXPERIMENTAL

Materials

Ammonium chloride, ammonium sulfate, sodium nitrite and sodium L-ascorbate were purchased fromSigma�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 thelaboratory. A nitric oxide stock solution (~ 2 mmol L�1) was prepared by bubbling pure NO gas throughoxygen-free PBS solution obtained with prior Ar(g) purging. All buffer chemicals were of analytical grade orbetter and used as received from various suppliers. All solutions were prepared with 18 MW cm�1 deionizeddistilled 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 anda PTFE�GPM were fabricated by the method reported previously [15, 19], and denoted by the �p-pPt basedsensor�. Briefly, a planar pPt (p-pPt) disk (250-µm O.D.) sealed in a glass wall tubing and a Ag/AgCl wireas the reference/counter electrode were employed to create the electrochemical cell. These two electrodeswere incorporated behind a PTFE�GPM as illustrated in Figure 1. To enhance NO selectivity primarilyagainst ammonia, the PTFE�GPM (0.12 cm2) was coated with 0.5 µL of Teflon AF® solution (1%, used asreceived, Dupont Fluoroproducts, Wilmington, DE) and then dried before sensor fabrication. To examine theinfluence of the amount of the coated Teflon AF® on selectivity, the coating procedure was repeated up to4 times (e.g., 2-µL Teflon AF® coated) as required. All sensor polarization, calibration and subsequentamperometric measurements were carried out at +0.75 V (vs Ag/AgCl) constant applied potential asdescribed 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


described above for the p-pPt based sensor. The GC working electrode (1-mm diameter) sealed in PEEK waspurchased from ESA Inc. (EE040, Cypress Systems, Lawrence, KS), and used without any catalytic layerafter 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 wereobtained in 2005 and 2006, respectively and separately tested over 3-month periods as received. All sensorswere 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 thesecommercial 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 deviceserved 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 exposedelectrode 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 electrochemicallyactivated 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 layerwas 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 fivetimes for 5 s per layer in 1.25% (wt%) Nafion® solution and the sensors were then dried for 5 min. Allamperometric currents for these CF based sensors were measured using a two-electrode configuration witha 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 sensitiveammeter module (Chemical Microsensor I, Diamond General Development Co., Ann Arbor, MI). Sensorresponse to NO and interferent species was typically determined using 50 mL of PBS (pH 7.3) solution thatwas well stirred (magnetically) under ambient condition, which was repeatedly spiked with small volumesof concentrated stock solutions containing the test species to change their bulk concentration in the buffersolution.

Amperometric selectivity coefficient calculation

To quantitatively express the amperometric selectivity of the various NO sensors examined, a previo-usly reported methodology was adopted [23]. For any amperometric device, the total current (It) can bedescribed 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, theconstant B denotes the true amperometric sensitivity of the given sensor toward the analyte (NO).

(1)�&N%�&,

Q

MM

DPS

ML�LW

¦�

Further, the amperometric selectivity coefficient (kNO,j) can be experimentally obtained via use of theseparate solution method; current levels were recorded separately for test solutions containing the analyteand the interfering species. Based on the ratio of amperometric sensitivity (DI/C) obtained for each species,the selectivity coefficient was calculated by equation 2.


ki,jamp = (DIj/Cj)/(DIi/Ci)

where DIj = Ij � Ib and DIi = Ii � Ib; Ii � the current recorded for the analyte; Ij � the current recorded for theinterfering 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, theaverage values of amperometric sensitivity (DI/C) and selectivity coefficients were obtained from the mul-tiple measurements (n > 3) for each species (NO, NH3 and nitrite). All selectivity coefficients obtained forthe same type of probes were finally averaged and reported (see Tab. 1 below). For convenience, theamperometric 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 NH4Cladded owing to the two orders of magnitude difference in buffer solution pH (7.3) and pKa of ammonia(9.25).

(2)

Table 1. Selectivity coefficients over ammonia and nitrite, sensitivity, limit of detection, and responsetime for various types of amperometric NO sensors examined in this work

a Applied potential during measurement.b Limit of detection.c Response time for NO.d 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.

Two other parameters that were used to compare each sensor�s performance were the response time anddetection 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 ofdetection (LOD) of each sensor was estimated to the lowest NO concentration where the observedamperometric signal/noise > 3.

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RESULTS AND DISCUSSION

Enhanced NO selectivity of platinized-Pt-electrode based planar gas sensorwith Teflon AF® treatment on PTFE�GPM

As shown in Figure 2, the addition of ammonium chloride to the test buffer solu-tion (PBS, pH 7.3) leads to an elevation in the amperometric current levels of a planarNO sensor (platinized Pt (pPt) working electrode and reference electrode behindmicroporous PTFE�GPM (Fig. 1). The calculated logarithm of the amperometricselectivity coefficient of this conventional planar-pPt (p-pPt) based gas sensor is �3.1for ammonium chloride or �1.1 for ammonia (log(kNO,j), j = NH4Cl(aq) or NH3(aq), respec-tively (Tab. 1). These values are calculated based on the amperometric responsesshown 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 testsolution, a larger amperometric current change is observed since the equilibrium shiftsto increase the amount of dissolved free ammonia gas, NH3(aq) (data not shown).

80 nmol L�1

20 mmol L�1

40 nmol L�1

10 nmol L�1

1.1 mmol L�180 mmol L�1

Figure 2. Amperometric response of p-pPt electrode based NO sensor (Fig. 1) toward increasing NO andNH4Cl concentrations (a) before and (b) after the modification of the microporous PTFE�GPMwith Teflon AF®

It is thought that NH3(aq) present in equilibrium with ammonium ion can effectivelydiffuse through porous GPM as NH

3(g) and oxidize on the pPt electrode. In fact, it has

been shown previously that the oxidation of ammonia on such catalytic surfaces can

80 nmol L�1

120 mmol L�1

40 nmol L�1

NO added to 10 nmol L�1

1.1 mmol L�1

80 mmol L�1


initiate at voltages as low as +0.52 V (vs NHE) with the reaction products includingnitrogen at lower potentials and nitrogen oxides (N

2O or NO) at higher oxidative

potentials [16, 17]. Since, the potential applied to the inner pPt working electrode ofthe gas sensor configuration is +0.75 V vs Ag/AgCl, it is likely that some nitrogenoxide species are produced at the pPt surface during the amperometric response toammonia. Therefore, when using the pPt electrode as in inner working electrode forthe construction of an NO sensor, interference from ammonia can surely occur.

Indeed, the p-pPt based NO sensor displays high background current levels wheninserted 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 firstpurging N2 through a culture medium, and collecting this gas phase in DI water, thepH of the DI water increases substantially. In fact, the spontaneous degradation oflabile L-glutamine, an essential nutrient in cell culture, often accounts for the build-upof ammonia in culture media [24, 25]. Further, the level of ammonia in normal bloodsamples 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 biologi-cal matrixes, various methods were explored in preliminary studies to reduce thisinterference, including varying the pH of the internal solution of the NO sensor (from30 mmol L�1 NaCl/0.3 mmol L�1 HCl solution that was originally reported [19]) andapplying a less positive potential to the platinized Pt electrode. Neither of these approa-ches was successful (data not shown). Indeed, lowering the applied potential to theinner working electrode was found to cause a significant deterioration in the NOselectivity 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 quiteeffective is to modify the nature of the porous PTFE�GPM using a solution of Tef-lon AF® (an amorphous fluoro-copolymer with large free volume and high gas permea-bility [27]). As shown in Figure 2b, when the same sensor configuration is preparedusing the PTFE�GPM that has been impregnated with the Teflon AF® (Fig. 1), NOselectivity is dramatically changed. After the membrane modification, the logarithmof the selectivity coefficient of the p-pPt based sensor improves to �6.2 for ammo-nium ion at pH 7.3 (�4.2 for NH3(aq)), which is about a thousand-fold improvementcompared to the conventional p-pPt based sensor (data shown in Fig. 2a). Further, theinfluence of varying amounts of Teflon AF® coated into the PTFE�GPM was furtherinvestigated for two interfering agents, ammonia and nitrite. As shown in Figure 3,although the thicker coating appears better for the selectivity enhancement, the calcu-lated 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 overnitrite is excellent, manifesting the effectiveness of microporous PTFE�GPM asa gas selective membrane. However, the existence of nitrite interference at high concen-


tration (~ mmol L�1-levels, data not shown) is likely due to the detection of trace non--ionic products (N

2O

3, NO

2 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 bein equilibrium with N

2O

3, which can further disproportionate into NO and NO

2 [8].

In comparison, the measured logarithm of the selectivity coefficient against ascor-bate, another ionic species, is �6.5 for the unmodified p-pPt based sensor and evenless (< �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 totalof NH3 and NH4

+) and nitrite as function of varying amounts of Teflon AF® coated on microporousPTFE�GPM

It should be noted that after the gas permeable membrane is modified with Tef-lon AF® the resulting sensors exhibit relatively little change in their sensitivity fordetecting NO (Tab. 1). Although it is thought that the porous structure of PTFE�GPMis partially or fully filled with the Teflon AF® matrix, depending on the amount usedduring modification, NO retains significant permeability through the membrane presu-mably due to its favorable partition coefficient into the hydrophobic fluoropolymermatrix. Indeed, the hydrophobic nature of NO is well known; NO is approximatelynine-times more soluble in organic media than water [28]. Thus, the difference inpartitioning capability of gaseous species, e.g., NO vs ammonia, within the hydro-phobic polymer phase that fills the pores of the PTFE membrane, likely results inchanging the gas flux of ammonia much more so than NO, and this yields the obser-ved improvement in NO selectivity over ammonia.


Comparison of selectivity and other performance parameters of variousamperometric NO sensors

To examine and compare the extent of ammonia/nitrite interference with othertypes of amperometric NO sensors (in addition to the p-pPt electrode based devicedeveloped in this laboratory [19]), various types of NO sensors were fabricatedand/or purchased, and then tested. These studies focused primarily on two otherdesigns widely used in previous literature reports to measure NO in biologicalmatrixes; the commercial ISO NO sensor by WPI, Inc. [29], and a carbon fiber elec-trode based sensor that employs an electropolymerized nickel porphyrin catalyticlayer 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 modi-fication yields a somewhat elongated response time, about two-fold longer, than theunmodified sensor, but an insignificant sensitivity change as describe above.

Interestingly, a planar GC (p-GC) electrode based sensor exhibits the lowest NOselectivity against ammonia among all the sensor configurations examined, but com-parable 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; higherlimit of NO detection and low sensitivity, which supports the catalytic effect of theplatinized Pt electrode. In general, selectivity coefficients for the gas sensor preparedwith the p-GC electrode as the inner working electrode appear similar or poorer thanthose of p-pPt sensors (Tab. 1). However, the carbon fiber (CF) electrode based NOsensors (ISO and b-CF sensors) tend to exhibit less ammonia interference than theunmodified p-pPt or p-GC electrode based sensors. This may reflect the intrinsicslower ammonia oxidation kinetics on carbon fiber (pyrolytic graphite) than on theother working electrodes. Even though the CF electrode based NO probes are claimedto exhibit high selectivity by using polymeric coatings that help discriminate againstelectroactive 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 thatinterfering 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 forin vitro measurements, is still possible and may interfere with the NO sensor depend-ing on its selectivity. Hence, it should be noted that when low-nmol L�1 levels of NOare directly monitored within biological samples, the concentration fluctuation ofinterfering species in sample medium should be minimized or taken into account viaa predictive test, based on the known selectivity and sensitivity of the given NO sen-sors.

As illustrated in Table 1 for the commercial NO (ISO) sensors, the selectivityover ammonia and nitrite has been found to improve in the newer model, now com-


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 overthe surface of this sensor, including a Nafion® coating and a hydrophobic layer asdescribed elsewhere [21, 31]. Of course, as a commercial product, the exact mem-brane 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 beexplained by the differences in the dimensions of the selective membranes. Prima-rily, thinner membrane structures obtained from coating processes with polymer solu-tion (for CF electrode, and presumably for ISO sensors [21]) are likely to result ina much faster sensor response time than the PTFE-GPM (~18-µm thickness) physi-cally held on the working electrodes. Moreover, NO transport for p-pPt sensors andISO sensors is probably retarded due to the multiple layered structures including anadditional hydrophobic selective membrane (i.e., Teflon AF® coating and an unknownhydrophobic membrane, respectively; also see above). Hence, further optimizationof response time can be envisioned by minimizing the number of layers and thicknessof the layers; for example, via direct coating of Teflon AF® layer on a working elec-trode. However, using such an approach to improve sensor performance will requirethat the NO sensors be designed to maintain electrical contacts between the workingand reference/counter electrodes after the application of such a hydrophobic selectivecoating, since in general such coatings are electrically non-conducting (high resis-tance).


CONCLUSION

In summary, the selectivity of various amperometric NO sensors over ammoniaand nitrite was examined quantitatively by determining the selectivity coefficientsand correlating the selectivity observed with other sensor performance parameterssuch as response time and limit of detection. A Teflon AF® coating on a microporousPTFE�GPM significantly reduces the ammonia interference observed for a new p-pPtelectrode based NO sensor without loss of sensor sensitivity. Also this study hasshown that finite ammonia/nitrite interference exists for other types of amperometricNO sensors and needs to be known, particularly when the direct and real-time NOdetection at nmol L�1 levels is attempted within biological samples where ammoniaand 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).

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Received May 2005Accepted September 2006

enhancing the selectivity of amperometric nitric oxide sensor...

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