Nitrate-selective optical sensor applying a lipophilic fluorescent potential-sensitive dye

Analytica Chimica Acta 449 (2001) 81–93

Nitrate-selective optical sensor applying a lipophilicfluorescent potential-sensitive dye

Christian Hubera, Ingo Klimantb,1, Christian Krausea,Tobias Wernera, Otto S. Wolfbeisa,∗

a Institute of Analytical Chemistry, Chemo- and Biosensors, University of Regensburg, D-93040 Regensburg, Germanyb Institute of Analytical Chemistry, University of Technology, A-8010 Graz, Austria

Received 6 June 2001; received in revised form 7 August 2001; accepted 21 August 2001

Abstract

An optical sensor has been developed for continuous determination of nitrate that is based on a polymer-stabilized emul-sion system consisting of a hydrogel with entrapped plasticizer droplets. The droplets contain a cationic potential-sensitivefluorescent dye (PSD) located near its surface. The cationic PSD also acts as an anion-exchange catalyst that extracts nitrateout of the aqueous solution to form a complex (PSD+/NO3

−) that moves into the plasticizer droplet due to its lipophilicity.This process causes a decrease of micro-polarity near the dye, resulting in an increase in the fluorescence intensity of the PSD.In a second detection scheme, the fluorescence intensity information of the PSD is converted into a fluorescence phase shiftby mixing into the membrane particles that contain an inert long-lifetime reference luminophore. The second sensing schemeis referred to as “dual lifetime referencing”. Both schemes can be used for reversible sensing of nitrate in the 0.1–50 mMrange, with response times in the order of 3 min. © 2001 Elsevier Science B.V. All rights reserved.

Keywords: Emulsion sensor; Nitrate sensor; Potential-sensitive dye; Dual lifetime referencing; Reference beads

1. Introduction

The determination of nitrate in drinking water isof continuing interest in the environmental sciences.Since nitrate can be converted to toxic nitrite by nitratereductases occurring in the digestive systems of humaninfants, nitrate-contaminated water can be a serioushazard. Nitrite is known to convert hemoglobin intomethemoglobin, a substance incapable of transportingoxygen and, hence, especially hazardous to infants as

∗ Corresponding author. Tel.:+49-941-9434065;fax: +49-941-9434064.E-mail address: [email protected](O.S. Wolfbeis).

1 Co-corresponding author.

it induces the “blue baby syndrome”. Another concernabout nitrate ingestion is the possibility that nitrites inthe stomach and intestines may cause the formation ofN-nitroso compounds, many of which are known to beanimal carcinogens. Biochemical studies in humanshave shown that nitrite in water combines with aminoacids to form these compounds [1,2].

Common sources of nitrate contamination includethe over-use of fertilizers, animal wastes, septic tanks,municipal sewage treatment systems, and decayingplant debris. Since nitrates are extremely water-solu-ble, they easily penetrate soil to reach the water sup-ply [3]. The presence of nitrate in drinking water mayalso indicate that ground water contains other com-pounds which may be health hazards, e.g. agriculturalchemicals such as pesticides. EU directives such as

0003-2670/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved.PII: S0003-2670(01)01363-0

82 C. Huber et al. / Analytica Chimica Acta 449 (2001) 81–93

80/778/EEC allow a maximum permissible nitratevalue of 50 mg l−1 in drinking water [4]. Therefore,detection methods for nitrate between 5 and 500mg l−1 are required.

Conventional methods for the determination ofnitrate involve commercially available, irreversiblecolor test strips based on the reduction of nitrate to ni-trite, followed by diazotation of aromatic compounds[5]. Other methods include the monitoring of nitratethrough its intrinsic UV absorption at 215 nm usingquartz fibers [6] or enzymatically by reduction withnitrate reductase and NADPH, and measuring theconsumption of NADPH spectrophotometrically [7].

There have been several reports on the developmentand application of nitrate ion-selective electrodes(ISEs) and ion-sensitive field-effect transistors (IS-FETs) [8–11]. A few optical sensor membranes werealso described [12–16]. Almost all of these sensors arebased on ion-exchangers such as TDMACl and saltsof tris(1,10-phenanthroline)nickel(II). These schemesare based on a pH-dependent co-extraction mecha-nism displaying Hofmeister-type selectivities [14,16].Recently, a new ion-exchanger based on a lipophiliciodocobalt(III)salen complex was used [17]. Elec-trodes based on these compounds are nitrate-sensitivewith stable potentials. However, selectivity againfollows the Hofmeister pattern, but compared to elec-trodes based on classical ion-exchangers, a slightlyimproved selectivity of nitrate versus chloride andnitrite was obtained [17].

An optical nitrate sensor has previously been pre-sented that is composed of plasticized poly(vinylchloride) (PVC), TDMACl and a PSD [18,19], butstill is not highly selective. However, most of theapplications described require reagents to reduceinterferences by certain anions, such as for precip-itation of chloride or hydrolysis of pyrophosphate[20,21]. Recently, a chloride-selective optical sensorwas presented that is based on a polymer-stabilizedemulsion system consisting of a hydrogel with en-trapped plasticizer droplets [22]. The droplets con-tain a neutral chloride-selective ionophore and apolarity-sensitive cationic fluorescent dye (PSD)located near the droplet surface. A nitrate-selectivemembrane was prepared that is based on a similarsensing mechanism. The membrane exists in the formof a “plasticizer-in-hydrogel” emulsion with a signif-icantly enlarged interfacial area compared to planar

PVC membranes. Unlike in previous nitrate-sensitiveoptical sensors, a PSD is used as both the ion transferagent and optical transducer.

The optical sensors described so far are based onmeasurement of light intensity. Recently, a new andgeneral logic to reference fluorescence intensity sig-nals by decay time measurement has been introduced[23,24]. This scheme, referred to as dual-lifetime ref-erencing (DLR), uses two luminophores of differentdecay times, but similar excitation spectra. In contrastto the alternative ratiometric methods, where lumines-cence excitation or emission is measured at two wave-lengths, this scheme uses a pair of luminophores (oneindicator, one reference dye) having different decaytimes, but overlapping excitation spectra. The phaseshift Φm of the overall luminescence obtained at asingle frequency depends on the ratio of intensities ofboth the reference luminophore (which is constant)and that of the indicator dye. Hence, the measured av-erage phase shift reflects the intensity of the indicatordye and, consequently, the analyte concentration.

In this contribution, we show how to adapt theDLR scheme to PSD-based anion sensing by com-bining an analyte-insensitive�s-lifetime luminophore(contained in nano-beads) with the nitrate-sensitivens-lifetime PSD. The reference dye used displaysdecay times in the�s time domain so to simplify theopto-electronic system.

2. Experimental

2.1. Chemicals

The PSD, 4-(4-(dihexadecylamino)styryl-N-meth-ylpyridinium iodide (DiA) was from MolecularProbes (Eugene, OR, USA). The reference standardruthenium(II)-tris-phenanthroline (Ru(phen)) and theion-exchanger TDMACl were obtained from Aldrich(Steinheim, Germany).

Hydrogel (Hypan, type HN80) was from HymedixInternational, Inc. (Dayton, NJ, USA). The plasti-cizer 2-cyanophenyl dodecyl ether (CPDDE) wassynthesized as described earlier [25]. All inorganicsalts, sodium dodecyl sulphate (SDS) and the solventdimethylsulfoxide (DMSO) were of analytical grade(Merck, Darmstadt, Germany). The pH was adjustedto the desired value using a sodium phosphate buffer

C. Huber et al. / Analytica Chimica Acta 449 (2001) 81–93 83

(cH2PO−4

+ cHPO2−4

= 13 mM). The buffers were ad-

justed to constant ionic strength (I = 230 mM) usingsodium fluoride as background electrolyte (unlessotherwise stated) [26]. Aqueous solutions wereprepared from air-saturated distilled water.

2.2. Membrane preparation

2.2.1. Preparation of Ru(phen) incorporatedin a sol–gel matrix

Tetramethoxysilane (TMS; 4.94 ml, 33.3 mM) wasadded to a solution of 7.12 mg of Ru(phen) in 4 mlof methanol and 2 ml of distilled water. The sol–gelprocess was started by adding 0.4 ml of 0.1 M HCl.After a 10 min condensation reaction, the solventswere removed within a few seconds at 30 Torr and100◦C. The dense sol–gel obtained was powdered andheated to 300◦C for 10 h. After cooling, the powderwas dispersed in 10 ml of methanol and a new sol–gelprocess was started by adding 2.47 ml of TMS, 1 mlof distilled water and 0.2 ml of 0.1 M HCl. Whilestirring, the solution was heated to 100◦C to dryness(ca. 10 min) and then kept at 300◦C for 2 h.

2.2.2. Preparation of emulsion membranesVarious cocktails were prepared by dissolving vary-

ing quantities of Hypan HN80, the non-quenchingplasticizer CPDDE and DiA in DMSO (see Table 1).The cocktail of sensor membrane M8 differs fromthe others in that phosphorescent reference particleswere added; 150�l of the respective cocktail wasdropped onto a dust-free, 125�m polyester supportof 25 mm diameter. After leaving the membranes

Table 1Composition of cocktails for making nitrate-selective membranes

% (w/w)a DMSO (�l) Sol–gel particles (mg)

HN80 (mg) CPDDE (mg) DiA (�g)

M1 10 (97.8) 0.21 (2.1) 10.3 (0.10) 270 –M2 10 (94.8) 0.54 (5.1) 10.8 (0.10) 280 –M3 10 (89.8) 1.12 (10.1) 11.4 (0.10) 290 –M4 10 (84.6) 1.81 (15.3) 12.0 (0.10) 300 –M5 10 (79.7) 2.54 (20.3) 13.11 (0.10) 320 –M6 10 (89.8) 1.13 (10.1) 5.7 (0.05) 280 –M7 10 (89.7) 1.13 (10.1) 17.1 (0.15) 300 –M8 10 (89.8) 1.12 (10.1) 11.4 (0.10) 300 5.2

a Per weight matrix.

in a water saturated atmosphere for 12 h to form a“plasticizer-in-hydrogel” emulsion, they were exten-sively washed with water to remove any DMSO. Themembranes were conditioned by cycling between50 mM nitrate and analyte-free phosphate buffer untila constant fluorescence signal was obtained.

2.2.3. Instrumentation and measurementsFluorescence excitation and emission spectra as

well as response curves of the sensing membraneswere acquired with an Aminco–Bowman Series 2luminescence spectrometer (SLM-Aminco, Rochester,NY, USA) equipped with a continuous wave 150 Wxenon lamp as a light source.

Phase angle measurements were performed witha system described previously [27]. A dual-phaselock-in amplifier (DSP 830, Stanford Research Inc.,Stanford) was used for sine wave modulation of thelight emitting diode (LED) at a frequency of 75 kHzand for detection. The optical system consists of ablue LED (λmax, 470 nm; NSPB 500, from Nichia,Nurnberg, Germany), equipped with a blue bandpassfilter (FITCA, Schott, Mainz, Germany), a bifurcatedglass fiber bundle (Ø, 2 mm) and a red-sensitive PMTmodule (H5701-02, Hamamatsu, Herrsching, Ger-many) equipped with a long-pass (550 nm cut-off)filter (Schott, Mainz, Germany). The tip of the fiberbundle was placed at the wall of the flow-through cellcontaining the sensing membrane.

Fluorescence studies on sensor membranes wereperformed with a home-made flow-through cellmounted in the fluorometer [22]. Solutions of knownpH and concentrations of nitrate were passed throughthe cell at a rate of 1 ml min−1. Sample solutions were


transported by a Minipuls-3 peristaltic pump (Gilson,Vilers-Bel, France) via silicone tubing of 1.0 mm i.d.

The pH values of the solutions were checked usinga digital pH meter (Knick, Berlin, Germany) cali-brated at 22± 2◦C with standard buffers of pH 7.0and 4.0 (Merck, Darmstadt, Germany). Calibrationgraphs were fitted using a Boltzmann function. Theconcentration causing a 50% decrease in fluorescenceintensity [c1/2(NO3

−)] was calculated by determiningthe point of inflection of the calibration graph.

3. Results

3.1. Choice of materials

The preparation of polymer-stabilized emulsionsystems has been described previously [22]. Astructured hydrogel was chosen as the hydrophilicphase forming an emulsion on adding a plasticizer(the lipophilic phase) in a water-saturated atmo-sphere. The hydrogel used — a partially hydrolyzedpolyacrylonitrile — was described as an ideal material

Fig. 1. Top: cross-section of sensor membrane; bottom: schematic representation of the micro-environment of DiA before (A) and after(B) extraction of nitrate from the aqueous phase into the plasticizer droplet.

to form emulsions with plasticizers [28] since it stabi-lizes the plasticizer droplets without the need foradding an emulsifier. The colorless, high-boiling,water-insoluble plasticizer CPDDE was chosen aslipophilic phase. In contrast to the frequently usedNPOE, it does not quench fluorescence. The struc-tured hydrogel surrounds the lipophilic plasticizerdroplets, thus, forming a micro-emulsion with a largehydrophilic/lipophilic interfacial area as is schemati-cally represented in Fig. 1. A large specific surface isachieved since the droplet diameters were estimatedto be<1�m.

The styryl dye 4-(4-(dihexadecylamino)styryl-N-methylpyridinium iodide (DiA) was used as the PSD.This dye has an amphiphilic structure due to the pres-ence of both a positively charged nitrogen atom and alipophilic backbone. The long alkyl chains prevent acomplete exchange of DiA between the lipophilic andhydrophilic phase, but still enable the displacementof the dye at the hydrophilic/lipophilic interface sincethe PSD is not covalently immobilized. The signalchange is assumed to be the result of a change ofthe micro-environment of the PSD at the hydrogel/


Fig. 2. Fluorescence excitation and emission spectra of M3 when exposed to 200 mM of NaF (pH 7.1) (A), and 50 mM NaNO3 (pH 7.1) (B).

plasticizer interface. The fluorescence quantum yieldof DiA is low in a hydrophilic environment and highin apolar solvents and plasticizers [22].

Unlike in other nitrate sensors, DiA is also used asthe ionophore. Due to its positively charged nitrogenit can act as an anion carrier similar to the well knownquaternary ammonium ions. The response mechanismis schematically illustrated in Fig. 1.

3.2. Response to nitrate

The fluorescence excitation and emission spectraof membrane M3 in the absence and presence ofnitrate are shown in Fig. 2. The fluorescence intensity

Fig. 3. Response time, relative signal change and reversibility of sensor membrane M3 at various nitrate concentrations.

of the excitation and emission bands increases and isshifted bathochromically by 5 nm in the presence ofnitrate.

Solutions of constant pH and ionic strength, butvarying nitrate concentrations were pumped throughthe flow-through cell. The relative signal change,reversibility and response times are shown in Fig. 3.On exposure to nitrate, the fluorescence intensityincreases and this effect is fully reversed on expo-sure to buffer. The forward response time (t90) is inthe range of 2–3 min, while the reverse response is7–8 min. Nitrate partitions more easily into the mem-brane and less readily diffuses out, which is attributedto the lipophilic character of nitrate.


Fig. 4. Calibration plots of sensor membranes M1 to M5. The plasticizer contents are 2% (�), 5% (�), 10% (�), 15% (�), and 20%(-) (w/w), respectively.

3.3. Effect of membrane compositionon the sensor response

An increase in the fraction of the plasticizer (mem-branes M1 to M5) leads to a distinct increase in signalchange, as can be seen in Fig. 4. The background fluo-rescence of the dye remaining in the more hydrophilicenvironment is reduced on increasing the fraction ofplasticizer, and this results in a greater signal change.However, the dynamic range and point of inflection(c1/2(NO3

−) did not alter significantly. No response

Fig. 5. Calibration plots of sensor membranes M3, M6 and M7. The dye contents are 0.05% (�), 0.10% (�), and 0.15% (�) (w/w),respectively.

to nitrate was given by membranes containing noplasticizer.

By increasing the dye concentration (M6, M3, M7),and hence, increasing the adsorption sites for nitrate,a shift of the dynamic range to lower nitrate concen-trations is observed along with a greater signal changeas can be seen from Fig. 5. However, a dye fractionof >0.15% (w/w) (M7) results in deep red membranesand a smaller signal change. This decrease at high dyeconcentrations is attributed to self-quenching of thedye in the lipophilic phase. The results, including the


Table 2Effect of the fraction of plasticizer (w/w) and the dye concentration on the signal change�S on going from 0 to 50 mM nitrate

Plasticizer fraction (%) (w/w) Dye concentration (%) (w/w)

2 (MNO3 1) 5 (MNO3 2) 10 (MNO3 3) 15 (MNO3 4) 20 (MNO3 5) 0.05(MNO3 6)

0.10(MNO3 3)

0.15(MNO3 7)

c1/2(NO3−) (mM) 3.5 4.1 4.1 3.7 4.1 9.9 4.1 3.1

�S (%) 31.3 65.6 88.6 114.2 115 53.3 88.6 96.7

points of inflection and the signal changes (�S), aresummarized in Table 2.

3.4. Cross-sensitivity towards interferinganions and pH

Interferences by anions occurring in drinking waterwere investigated. Concerning the sensing mechanism,we assume that the extraction of anions is governed bytheir lipophilicity which correlates with their hydrationenergy. Hydrophilic ions such as sulfate and hydrogencarbonate do not seriously affect the response of mostexisting nitrate optical sensors or electrodes. If, how-ever, such sensor membranes are exposed to chlorideor more lipophilic anions, a distinct response follow-ing the Hofmeister pattern can be observed [19,29].

The new kind of sensor membranes presented heredo not respond to chloride even at concentrationsof about 200 mM. It appears that the [DiA+Cl−]ion-pair is not lipophilic enough to move into theplasticizer. Consequently, there is no change in the

Fig. 6. Calibration plot of membrane M3 to nitrate and other anions (I− (�), NO3− (�), Br− (�), Cl− (�)).

Table 3Selectivity coefficients of membrane M3 to anions

Iodide Bromide Chloride Sulfate

logKoptNO3

−,A (MNO3 3)a +1.8 −2.3 −3.7 −4

a Selectivity coefficients logKoptNO3

−,A obtained by the separatesolution method (10 mM of the sodium salts, pH 7.1).

micro-environment of the dye. The sensors suffer,however, from strong interference by iodide (similarto commercial electrodes). Selectivity coefficients(logK

optNO3

−,A) relative to nitrate as determined by theseparate solution method are given in Table 3.

The calibration plots for iodide, bromide and chlo-ride were compared with the calibration plot for nitrateand are shown in Fig. 6. The data show that chlorideand bromide significantly interfere in the presence ofnitrate, thus, dramatically lowering the fluorescenceintensity, as can be seen in Fig. 7. The addition ofnegative charge density (chloride or bromide) causesa competition with nitrate for the positively charged


Fig. 7. Cross-sensitivity of membrane M3 to chloride and bromide in the presence of 5 mM nitrate. The accumulation rate of 5�M SDSin the membrane (line (A)) can be decreased adding a polyelectrolyte and covering the membrane with a dialysis membrane (line (B)).

DiA, resulting in a decrease in fluorescence intensity.As discussed before, we assume that the [DiA+Cl−]and the [DiA+Br−] ion-pairs are not lipophilic enoughto move in the lipophilic phase. We can speculate thatthis leads to a displacement of the dye further into thehydrogel resulting in a decrease in intensity.

The addition of the ion-exchanger TDMACl tothe membrane had no beneficial effect on sensorresponse. The signal change on exposure to nitratewas not increased, but the membrane showed a sig-nificant response to chloride even in the absence ofnitrate. Additionally, the fluorescence of the mem-branes was decreased dramatically, probably becausethe positively charged TDMACl shifted DiA furtherout of the plasticizer.

Drinking water can contain anionic surfactants in aconcentration of up to 0.2 mg l−1 and this value is evenhigher in surface water. Therefore, its effect on the sen-sor was investigated. We find that sodium dodecyl sul-fate (SDS), an anionic surfactant, is accumulated in theplasticizer, thereby changing the micro-environmentof DiA. This results in an increase in its fluorescence(Fig. 7, graph A). Consequently, sensor membrane M3is not suitable for detection of nitrate in the presence ofSDS. To overcome cross-sensitivity to SDS and in or-der to apply this sensor to SDS-contaminated samples,it was tried to keep SDS away from the membrane.

To accomplish this, a sensor spot with a diameter of5 mm was placed on the top of a fiber bundle covered

with a dialysis membrane. This fiber bundle wasimmersed into a sample containing a 0.5% (w/w) solu-tion of SuperflocTM C-589, a strong cationic polyelec-trolyte with a polyacrylamide structure and an averagemolecular weight of 200,000. The polyelectrolyte wasadded to the sample with the intention that SDS wouldform associates with the polyelectrolyte which thenwould not penetrate the dialysis membrane due to theirhigh molecular weight. Nitrate, on the other hand, wasassumed to be able to diffuse to the membrane. In-deed, fluorescence increases on addition of nitrate, butthe response time is largely increased and the dynamicrange is shifted to higher nitrate concentrations. Thiscan be seen in Fig. 7, where plot B displays an increasein fluorescence following addition of SDS. However,compared to plot A, where no polyelectrolyte wasadded, the rate is strongly diminished. Consequently,the penetration of SDS through the dialysis membraneis not prevented by adding a polyelectrolyte to thesample, even though the diffusion rate is diminished.

The response mechanism of PSD-based membranesis virtually independent of pH, even though somePSD systems described in literature show a distinctpH response [18]. The pH of surface water can varyfrom 5 to 9. Therefore, the response of the membranein the presence of 5 mM nitrate was investigated inthe pH range 4.5 to 9. In this range, the fluorescenceintensity decreases by around 3.5% which can beattributed to changes in the partitioning coefficient


of the dye in the hydrogel/plasticizer system and,possibly, changing hydration of the hydrogel.

4. Dual lifetime referenced (DLR)optical nitrate sensor

Fluorescence intensity is often used as an analytedependent-parameter since its measurement is appar-ently simple in terms of instrumentation. However,its accuracy is often compromised by adverse effectssuch as drifts of the opto-electronic system and varia-tions in the optical properties of the sample includingfluorophore concentration, turbidity, coloration andrefractive index. Recently, a new and general logic toreference fluorescence intensity signals by decay time

Fig. 8. Phase shift of the overall luminescence (Φm), the reference (Φref) and the indicator (Φ ind). Fluorescence of the indicator in (A)absence of analyte (high) and (B) presence of analyte (low).

measurement has been presented [23,24]. In contrastto the most common ratiometric method, where lu-minescence excitation or emission is measured at twowavelengths, this scheme uses a pair of luminophores(one indicator, one reference dye) having differentdecay times and similar excitation spectra. The phaseshift Φm of the overall luminescence obtained at asingle frequency depends on the ratio of intensities ofthe reference luminophore and the indicator dye andcan be represented as the superposition of the singlesine wave signals of the indicator and the referenceluminophore (see Fig. 8).

The reference luminophore gives a constant back-ground signal (ref) while the fluorescence signal ofthe indicator (ind) depends on the analyte concentra-tion. The average phase shiftΦm directly reflects the


intensity of the indicator dye and, consequently, theanalyte concentration.

Several sensors based on this technology have beendescribed recently [23,30]. In the present case, thenitrate-sensitive DiA acts as the indicator dye and ananalyte-insensitive�s-lifetime luminophore (a ruthe-nium dye in a sol–gel matrix) acts as the referencedye. By exciting both dyes with a sinusoidally lightof a blue LED (λ, 470 nm), the fluorescence intensityinformation can be converted [23,30,31] into a phaseshift as represented by Eq. (1):

cotΦm = Aref cosΦref + Aind

Aref sinΦref

= cotΦref + 1

sinΦref

Aind

Aref(1)

whereAref is the amplitude of the reference standard,Aind the amplitude of the PSD andΦ the phase angleof either the overall signal (Φm), the reference (Φref),or the PSD (Φ ind), respectively.

The reference luminophore added (in form of smallbeads) is expected to meet the following criteria: (a) adecay time in the�s or ms range; (b) spectral proper-ties including decay time, fluorescence quantum yieldand spectral shape that are not affected by the analyteor any other substances in the sample; and (c) spectraloverlap with the indicator.

Transition metal–ligand probes with ruthenium orosmium as central ion and polypyridyls as ligandsdisplay long decay times and can be used as the

Fig. 9. Fluorescence emission spectra of M3 when exposed to 200 mM NaF (pH 7.1) (A), 50 mM NaNO3 (pH 7.1) (B) and the emissionspectrum of Ru(phen) encapsulated in a sol–gel matrix (C). The transmission of long-pass filters OG550 is inserted in the graph as dottedline.

reference luminophore [32]. The ruthenium com-plexes are particularly well suited due to their goodquantum yields and their decay times in the 0.5–6.0�srange [33,34]. However, their luminescence is oftenquenched by redox-active compounds [35] and bymolecular oxygen [36]. To avoid this, the dyes have tobe encapsulated in a material that shields them fromquenchers so as to warrant a constant background sig-nal. In this application, the ruthenium phenanthrolinecomplex encapsulated in beads of a dense glass pre-pared by a modified sol–gel process [37] was used asthe reference luminophore. These particles — whichare not quenched by oxygen and also are inert toorganic solvents such as dimethyl sulfoxide (DMSO)— were added to the sensor cocktail to obtain sensormembrane M8.

Fig. 9 displays the spectral match of the PSD andthe reference particles. A typical time trace of emul-sion membrane M8, when exposed to different con-centrations of nitrate is shown in Fig. 10. On exposureto nitrate, the fluorescence intensity of M8 increases.This increase in intensity leads to a decrease in thephase angle because the short-lived fluorescence ofDiA can be detected among the constant referencesignal of the ruthenium complex (see also Fig. 8A).The signal changes are fully reversed when exposedto analyte-free buffer.

The sensing characteristics of membrane M8 aresummarized in Table 4 and compared with the in-tensity data of an identically prepared membrane


Fig. 10. Response time, relative signal change and reversibility sensor membrane M8 at various nitrate concentrations (pH 7.1; IS 230 mM).

without reference luminophore particles (membraneM3). Both the dynamic range and the point of inflec-tion of M8 are shifted to higher analyte concentrationsin the presence of reference particles. Furthermore,the change in the phase angle of M8 was smaller thanexpected when exposed to nitrate. The phase angleof membrane M8 decreases from 35.3 to 30.7◦ whenexposed to 50 mM nitrate. The fluorescence intensityof a membrane without reference beads increases,however, by about 114% in the presence of 50 mM ni-trate. Therefore, according to Eq. (1) and determiningthe phase angle (Φref) of the reference luminophorein absence of the PSD to be 50◦, a phase shift of

Table 4Figures of merit of membrane M8 and a comparison of the data with M3 (containing no reference particles)

Nitrate Iodide Salicylate Bromide Chloride Sulfate

M8c1/2(A−) (mM) 8.6 1.4 –a –a –a –a

�Φ (◦)b −4.5 −8.6 −1.6 −0.4 +0.03 0logK

optNO−

3 ,A– +1.28 −0.95 −1.3 Less than−2 Less than−3

M3c1/2(A−) (mM) 4.1 0.45 – 9.5 –a –a

�S (%)c 114 208 – 26 2 1logK

optNO−

3 ,A– +1.8 – −0.7 Less than−2 Less than−3

a Not determinable.b �Φ = Φ50 mM NO3

− − Φ0 mM NO3− .

c �S: signal change between 0 and 50 mM nitrate.

9.5◦ was expected. Obviously, the addition of the ref-erence beads changes the response characteristics ofmembrane M8. It can be assumed that the plasticizerdroplets are larger if reference particles are added,and this would result in a smaller interfacial area and,consequently, a smaller signal change.

The calibration plots of sensor membrane M8for chloride, bromide, salicylate, nitrate and iodideare shown in Fig. 11. The selectivity coefficientslogK

optNO−

3 ,A, determined via the separate solution

method at a nitrate concentration of 10 mM, are listedin Table 4. Again, M8 suffers from chloride interfer-ence in the presence of nitrate.


Fig. 11. Calibration plots of membrane M8 for anions (I− (�), NO3− (�), salicylate (Sal) (�), Br− (�), Cl− (—).

5. Conclusions

Sensors for the determination of nitrate basedon a PSD a presented. Unlike conventional opticalnitrate-selective membranes, the PSD is used as bothan optical transducer and an ionophore. The interfacialarea is increased compared to conventional PVC mem-branes by preparing polymer-stabilized emulsion sys-tems with small plasticizer droplets. This has a favor-able effect in that the sensors display fast response andhigh signal changes. The signal changes of the sensormembranes are fully reversible and display an appro-priate dynamic range as well as low cross-sensitivityto pH. However, chloride and lipophilic anions —especially anionic surfactants such as SDS — inter-fere seriously. The cross-sensitivity to SDS can bereduced to some extent by the addition of a cationicpolyelectrolyte.

In the second part, we show that the DLR sensorscheme is an attractive alternative to intensity-basedsensing. By adding an inert reference luminophorewith a decay time in the�s range the fluorescenceintensity information can be converted into a fluores-cence phase shift. However, the addition of referenceparticles disturbs the formation of small plasticizerdroplets since smaller signal changes and shiftsin the working range are observed. Therefore, thedynamic range of the nitrate sensor no longer meetsthe requirements for determining nitrate in groundwater.

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