J Biol Inorg Chem (2015) 20:385–393 DOI 10.1007/s00775-014-1171-0
ORIGINAL PAPER
A sensitive and stable amperometric nitrate biosensor employing Arabidopsis thaliana nitrate reductase Palraj Kalimuthu • Katrin Fischer-Schrader Gu¨nter Schwarz • Paul V. Bernhardt
•
Received: 3 April 2014 / Accepted: 5 June 2014 / Published online: 2 July 2014 Ó SBIC 2014
Abstract Nitrate reductase (NR) from the plant Arabidopsis thaliana has been employed in the development of an amperometric nitrate biosensor that functions at physiological pH. The anion anthraquinone-2-sulfonate (AQ) is used as an effective artificial electron transfer partner for NR at a glassy carbon (GC) electrode. Nitrate is enzymatically reduced to nitrite and the oxidized form of NR is electrochemically reduced by the hydroquinone form of the mediator (AQH2). The GC/NR electrode shows a pronounced cathodic wave for nitrate reduction and the catalytic current increases linearly in the nitrate concentration range of 10–400 lM with a correlation coefficient of 0.989. Using an amperometric method, a low detection limit of 0.76 nM (S/N = 3) was achieved. The practical application of the present electrochemical biosensor was demonstrated by the determination of nitrate concentration in natural water samples and the results agreed well with a standard spectroscopic method. Keywords Nitrate reductase Voltammetry Molybdenum
Responsible Editors: Jose´ Moura and Paul Bernhardt.
Electronic supplementary material The online version of this article (doi:10.1007/s00775-014-1171-0) contains supplementary material, which is available to authorized users. P. Kalimuthu P. V. Bernhardt (&) School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane 4072, Australia e-mail: [email protected] K. Fischer-Schrader G. Schwarz Department of Chemistry and Center for Molecular Medicine, Institute of Biochemistry, Cologne University, Zu¨licher Str. 47, 50674 Cologne, Germany
Abbreviations AQ Anthraquinone-2-sulfonate CV Cyclic voltammetry FAD Flavin adenine dinucleotide GC Glassy carbon MV Methyl viologen NADH Nicotinamide adenine dinucleotide NHE Normal hydrogen electrode NR Nitrate reductase
Introduction Nitrate has a widespread distribution within environmental, food, industrial and physiological systems. Nitrate in the environment originates from wastewater, nitrogen fertilizers and other natural degradation processes [1–3]. Excess nitrate leaching in environmental water systems may cause eutrophication of lakes resulting in excessive growth of weeds and algae and reduced dissolved oxygen levels which dramatically affect aquatic life [4, 5]. In humans the consumption of high levels of nitrate leads to hemoglobin disorders including methemoglobinemia and also the toxicological problems of nitrate associated with the formation of harmful N-nitroso compounds [6–8]. Therefore, monitoring nitrate levels is of importance and there is an increased demand for the development of accurate, rapid and sensitive analytical procedures. Electrochemical methods for nitrate determination have attracted much attention in analytical chemistry [9, 10] for several reasons including low-cost, minimal sample preparation, ease of operation and rapid measurement compared to wet chemical methods [11]. However, electrochemical methods that reduce nitrate directly at a (chemically modified) electrode suffer from poor stability, sensitivity
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Scheme 1 Catalytic nitrate reduction mechanism at the active site of nitrate reductase
NO3-
O S
A
OH-
Cys
O S
IV
IV
O HN H 2N
N
H N N H
S Mo OH S
N
N H
H2 N
O S
S Mo O S N O O O OPO32-
Cys
B
VI
O
H N
N
N H
HN H2 N
123
H N
OPO32-
C
and most importantly poor selectivity with interference from other equally common ions such as nitrite, chlorate, sulfate and phosphate being problematic [12–15]. Mo-dependent nitrate-reducing enzymes (nitrate reductases, NRs), that are selective for nitrate, offer an immediate solution to the development of nitrate-specific analytical biosensors. There are several types of Mo-dependent nitrate reductases; from both prokaryotes and eukaryotes [16, 17]. Eukaryotic nitrate reductases (from plants, algae and fungi) are structurally very different from their bacterial NR analogues although each still bears a Mo ion at its active site, but in this case it is coordinated to a single bidentate molybdopterin ligand (Scheme 1). Eukaryotic nitrate reductases are multi-redox center enzymes responsible for the biological conversion of nitrate to nitrite coupled to NADH oxidation during nitrogen assimilation. These enzymes possess three distinct cofactors (FAD, heme, and the Mo active site) bound to distinct and independently folded domains [18– 20]. Therefore, NR enzymes provide an ideal catalyst for the development of highly selective and sensitive nitrate biosensors [21–26]. The NR from the plant Arabidopsis thaliana is the subject of this paper. In the catalytic reaction, nitrate binds to the catalytically active Mo(IV) form displacing an hydroxido/aqua ligand and forming the Michaelis complex (Scheme 1A). Upon oxidation of the Mo(IV) center to Mo(VI), one N–O bond of nitrate is broken, leaving an oxido ligand on the Mo ion and nitrite is released (Scheme 1B). After completion of the oxidative half reaction the active Mo(IV) form is regenerated by two-electron reduction (from the native reduced nicotinamide partner, Scheme 1C) or, in an electrochemical system, by reduced artificial electron transfer mediators [27]. Direct electron transfer between an enzyme and an electrode [28] requires the redox cofactors to be in
O HN
O
2e-, H+
Cys
S Mo O S
NO2-
O OPO32-
proximity to the electrode and this is often not achievable due to their location being buried within the folded polypeptide. Small molecular weight redox-active mediators have been used as a suitable alternative as they can communicate with both the enzyme (homogeneous electron transfer) and the electrode (heterogeneous electron transfer). An ideal redox-active mediator should possess high stability in both its oxidized and reduced forms, and there should be no side reactions with the enzyme nor its substrate (in this case nitrate) and product (nitrite). In previous publications [21, 22, 24, 25, 29, 30] the cationic low redox potential mediator methyl viologen (MV, or 4,40 -dimethyl-4,40 -dipyridinium, E0 -430 mV vs. NHE) and analogues have been employed due to their large driving force, rapid homogeneous reduction rates and facile heterogeneous electron transfer. Generally, these compounds have been co-immobilized with NR (either bacterial or fungal) by different methods including adsorption, cross-linking, entrapment, electro-polymerization and covalent binding on different substrates such as gold, glassy carbon and edge plane pyrolytic graphite electrodes. However, some drawbacks have been reported. Enzyme activity was lost completely within a short period of time when NR was co-adsorbed with viologen compounds on an electrode surface [31, 32]. Further, no catalysis was observed while MV was confined within a polymer film due to restricted mobility of mediator [22]. Therefore, selection of mediator and the immobilization method are both crucial for the construction of effective biosensors. Moreover, it was found that NR biosensors employing commercially available nitrate reductases (e.g., from the fungus Aspergillus niger) have decreased activity due to the low purity of enzyme [33, 34]. In the present study, we report a nitrate biosensor with purified truncated plant NR (from Arabidopsis thaliana)
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387
O
OH
SO3-
SO3-
2e-, 2H+ -230 mV O
AQ
OH
e-
,H
+
e-
,H
+
AQH2
nitrate biosensor based on plant NR and AQ as a relatively high-potential mediator reduced at a glassy carbon working electrode. The GC/NR electrode was successfully used for the selective quantification of nitrate in natural water samples and results were validated with an established spectroscopic method.
OH SO3-
O
AQH
Scheme 2 Redox reaction of anthraquinone-2-sulfonate (AQ) at pH 6 (potential vs. NHE)
employing a glassy carbon (GC) electrode [35]. The truncated NR contains only two cofactors; the Mo active site and heme, but no FAD. Very recently, we demonstrated the pronounced electrocatalytic voltammetry of this enzyme with mediating cosubstrates such as methyl viologen (E0 430 mV), benzyl viologen (E0 -324 mV) and anthraquinone-2-sulfonate (AQ, E0 -230 mV vs. NHE) [36]. Among these three mediators, we have chosen the compound with the highest redox potential AQ (Scheme 2) as electron transfer partner for NR in the present nitrate biosensor. AQ is typically a two-electron, two-proton acceptor. Naturally its redox potential is pH dependent although it has been shown that both one-electron redox steps lie very close in potential leading to an apparent 2-electron response it its cyclic voltammogram [37]. Scheme 2 illustrates the overall reaction of the mediator that involves two-electron and twoproton reduction of AQ to dihydroanthraquinone-2-sulfonate (AQH2) and subsequent reoxidation of AQH2 to AQ [38]. However, we recently showed that electrochemically reduced AQH2 is a single-electron donor to truncated NR [36] due to the enzyme only being able to receive a single electron from the mediator at its heme cofactor, which then is relayed to the Mo active site. This process must occur twice to fully reactivate the enzyme. In any practical reductase-based biosensor that might operate in an aerobic environment, competitive reduction of oxygen, or other reducible species in solution, is a potential issue for low-potential mediators such as viologens [32] as well as their potential inhibiting properties [31]. Very importantly, viologen compounds may react with enzymeproduced nitrite in the reaction layer to generate an additional catalytic reduction current and a positive error. Also for practical applications where nitrite may coexist with nitrate in analytical samples, this can also lead to errors [39]. In this paper, an amperometric method is used to estimate the lowest detection limit of an electrochemical
Materials and methods Arabidopsis thaliana nitrate reductase was purified from a heterogeneous expression system in E. coli as previously described [35]. Potassium nitrate, potassium nitrite and anthraquinone-2-sulfonate (Na? salt) were purchased from Aldrich and were used as received. All other reagents used were of analytical grade purity and used without further purification. All solutions were prepared with ultrapure water (resistivity 18.2 MX cm) from a Millipore Milli-Q system. Bis-Tris-acetate (50 mM) in the presence of 50 mM KCl as inert electrolyte was used for experiments at pH 7. The mixture of buffers (20 mM MES buffer pH 5.5–6.7, 20 mM Bis-Tris buffer pH 5.8–7.2 and 20 mM Tris buffer pH 7.0–9.0) were used for pH-dependent experiments and the desired pH was obtained with dilute acetic acid or NaOH. The natural water samples, collected locally from a lake and river, were diluted with 50 mM Bis-Tris buffer (pH 7) before analysis. Electrochemical and spectral measurements and electrode cleaning Cyclic voltammetry (CV) experiments were performed at 25 °C using a BAS 100B/W electrochemical workstation. A three-electrode system was employed comprising a glassy carbon (GC) disk working electrode (0.057 cm2), a Pt wire counter electrode, and an Ag/AgCl reference electrode (?196 mV vs. NHE). Potentials are cited versus NHE. Experiments were carried on solutions that had been purged with argon for 30 min. The GC electrode was polished with 0.50 and 0.05 lm alumina slurry and then rinsed thoroughly with water. The electrode was then sonicated in water for 5 min to remove adsorbed alumina particles and dried in a stream of nitrogen. UV–Vis spectra were recorded at room temperature with a Perkin-Elmer Lamda 35 Spectrophotometer. The sample was mounted in a 1-cm path length quartz cuvette. The variation of catalytic cathodic peak current versus nitrate concentration was fit to Michaelis–Menten kinetics (where KM,app is the apparent Michaelis constant, ilim is the plateau (steady state) current and imax is the maximal current under substrate saturating conditions ([NO3-] » KM,app) [28].
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ilim ¼
imax ½NO 3 KM;app þ ½NO 3
ð1Þ
The pH dependence of the catalytic current was modeled by Eq. (2) which is applicable for an active form of the enzyme that is switched off by either a deprotonation (pKa1) or protonation (pKa2) [40] where iopt is the maximum catalytic current at the pH optimum ilim ðpHÞ ¼
iopt : 1 þ 10ðpHpKa1 Þ þ 10ðpKa2 pHÞ
ð2Þ
GC/NR electrode preparation A 3 lL droplet of NR (36 lM) in 50 mM assay buffer [50 mM Bis-Tris-acetate (pH 7.0), 50 mM KCl, 5 mM magnesium acetate, and 1 mM CaCl2] was pipetted onto the conducting surface of an inverted, freshly polished GC working electrode and this was allowed to dry to a film for about 20 min at 4 °C. To prevent protein loss through desorption, the electrode surface was carefully covered with a small piece of perm-selective dialysis membrane (*1 9 1 cm, molecular weight cut off 3,500 Da), presoaked in water. The dialysis membrane was pressed onto the electrode with a Teflon cap and fastened to the electrode with a rubber O-ring. The resulting modified electrode was stored at 4 °C in 50 mM Bis-Tris buffer (pH 7.0) when not in use. The enzyme was confined to a thin layer beneath the membrane while nitrate, nitrite and AQ were able to diffuse across the membrane. A cartoon representation of the biosensor electrode is shown in Scheme 3.
nitrite
AQH2
NRox e-
NRox: nitrite NRint
AQH AQH2
NRred: nitrate NRred enitrate
AQH
Membrane
GC electrode
Scheme 3 The electron transfer mechanism of NR in the presence of AQ and nitrate
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Chemical nitrate analysis The nitrate concentrations determined in natural water samples employed in the present electrochemical sensor (Figure S1) were cross-checked with a well-established spectrophotometric method in which acid catalyzed nitration of salicylic acid yields a colored product (kmax = 410 nm) at pH [12 [11]. Briefly, 0.2 mL of the water sample was pipetted into 50-ml Erlenmeyer flasks and mixed thoroughly with 0.8 ml of 5 % (w/v) salicylic acid in concentrated H2SO4. After incubation for 20 min at room temperature, 19 ml of 2 M NaOH was cautiously added to raise the pH above 12 (caution exothermic). On cooling to room temperature and dilution volumetrically, the absorbance at 410 nm was measured with a spectrophotometer. The salicylic acid–H2SO4 reagent was made afresh before the experiment. The method of standard addition was employed by adding known amounts of nitrate to each sample. A linear increase in the absorption at 410 nm was observed with increasing quantities of added nitrate which enabled the original nitrate concentration of the sample to be determined by back-extrapolation (Supporting information Figure S2). The main advantage of this method is that potential interferents such as ammonium, nitrite and chloride ions are not reactive so selective quantification of nitrate is possible.
Results and discussion Mechanism of NR electrocatalysis NR is confined to a thin layer on GC electrode surface entrapped beneath a membrane (MW cutoff 3,500 Da), whereas the small MW substrates/products (nitrate and nitrite) and AQ are under diffusion control and may cross the membrane. As mentioned above, generally AQH2 is a two-electron and two-proton reductant, but recently we found that AQH2 acts as single-electron donor to NR [36]. The semiquinone AQH is thermodynamically unstable with respect to disproportionation, but this is not significant as the products of disproportionation (2AQH AQ ? AQH2) are both components of the catalytic cycle either in homogeneous reaction with NR (AQH2) or reduction at the electrode low potential (AQ) [36, 37]. As shown in Scheme 3, during the reductive half reaction, AQH2 reduces fully oxidized NRox in consecutive, one-electron, outer-sphere electron transfer reactions via an intermediate form NRint.1 In the oxidative half reaction, NRred in its (MoIV form) converts the coordinated nitrato 1
In all cases we assume that the heme cofactor of NR relays electrons rapidly from AQH2 to the Mo active site.
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ligand to nitrite which then dissociates from the active site. The detailed kinetics of this system was discussed in our recent publication [36]. Catalytic voltammetry The mediated catalytic voltammetry of NR in the presence of nitrate and AQ is shown in Fig. 1. In the absence of AQ, the GC/NR electrode shows no direct voltammetric responses from the NR redox-active cofactors in 50 mM Bis-Tris buffer solution (Fig. 2, curve a) and none in the presence of nitrate. It is assumed that the redox cofactors (heme and Mo) are deeply buried inside the protein and
thus not accessible for electron transfer to the electrode surface. However, the GC/NR electrode shows a pair of asymmetric peaks centered at -230 mV (vs. NHE) in the presence of 25 lM AQ (without nitrate) in 50 mM Bis-Tris buffer solution (pH 7) with a peak to peak separation of 80 mV (Fig. 1a). This current is governed by the diffusion of both oxidized and reduced forms of AQ between the electrode and bulk solution. Upon addition of 200 lM nitrate to the same solution, a pronounced cathodic wave emerges and the anodic wave vanishes (Fig. 1b). This voltammetry is characteristic of a catalytic homogeneous reaction coupled to heterogeneous electron transfer (ECcat mechanism) where nitrate is reduced enzymatically yielding the oxidized form of NR, which is reduced again by electro-generated AQH2. AQ concentration dependence
Fig. 1 CVs obtained for AQ (25 lM) in the absence (a, red) and presence (b, green) of 200 lM nitrate at the GC/NR electrode in 50 mM Bis-Tris buffer (pH 7) at a sweep rate of 5 mV s-1
0
I/ μΑ
–1
a
The sigmoidal voltammograms are indicative of an electrochemical steady state, i.e., the intermediate AQH2 is consumed (by homogeneous reaction with NRox) at the same rate that it is generated at the electrode surface and the forward and reverse sweeps are virtually identical.
–2
f
–3
–500
–400
–300
Figure 2 illustrates the effect of different AQ concentrations in the presence of 1 mM nitrate at the GC/NR electrode. In the absence of AQ, the GC/NR electrode does not show any catalytic response towards nitrate in the potential window of -150 and -550 mV (curve a). On the other hand, a sigmoidal catalytic wave emerges upon increasing the concentration of AQ in the presence of 1 mM nitrate (curves b ? f). The catalytic current is dependent upon the diffusion of AQ to the electrode surface, reaction between nitrate and NR, and the reaction between AQH2 and NRox. The limiting current (ip, at low potential) increases linearly with AQ concentrations within the range of 0–15 lM. Theoretically, the plateau current at low potential (ip) current is directly proportional to [AQ] only at low AQ concentrations (Eq. 3) and high nitrate concentrations [28]; [NR*] and [AQ*] denote bulk concentrations, DAQ is the AQ diffusion coefficient and the other terms have their usual meaning. This equation breaks down when the mediator concentration becomes too high; in this case beyond *15 lM AQ. qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ip ¼ FA[AD 2k3 DAQ ½NR ð3Þ
–200
Nitrate concentration dependence
E/ mV vs. NHE
Fig. 2 CVs obtained for varying AQ concentrations a 0, b 5, c 10, d 15, e 20 and f 25 lM in the presence of 1 mM nitrate at GC/NR electrode in 50 mM Bis-Tris buffer, pH 7 at a sweep rate of 5 mV s-1
The cathodic current response of the GC/NR electrode as function of nitrate concentration and in the presence of 25 lM AQ was examined. As the electrode potential is
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1.0
2.0
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0.6
|Imax| / μA
|Ilim| / μA
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0.4
0.5
0.2
0.0
0.0
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800
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pH
Concentration (μM) Fig. 3 Plot of the baseline subtracted (absolute) limiting cathodic current (at low potential) for the GC/NR electrode in presence of 25 lM AQ as a function of nitrate concentrations in 50 mM Bis-Tris buffer, pH 7 at a scan rate of 5 mV s-1
5
Fig. 4 pH dependence of the maximum absolute catalytic current for 400 lM nitrate in the presence of 25 lM AQ at the GC/NR electrode in 60 mM mixed buffer (MES, Bis-Tris and Trizma) at a sweep rate of 5 mV s-1
pH dependence lowered to -500 mV, the catalytic reduction current eventually reaches a limiting value (ilim) and the magnitude of the limiting current value becomes larger with increasing nitrate concentration in accord with Michaelis–Menten kinetics. It is noted that transient (peak-shaped) catalytic voltammetry was observed for nitrate concentrations 10–400 lM (see Fig. 1b as an example) indicating that the electrocatalytic current is somewhat limited by nitrate diffusion at low concentrations. However, at higher nitrate concentrations ([800 lM) the transient voltammogram becomes sigmoidal (see Fig. 2) where the concentration of nitrate within the reaction layer is constant during the sweep and the forward and reverse sweeps are virtually identical. As shown in Fig. 3 the catalytic reduction current is approximately linear for nitrate concentration from 10 to 400 lM with a correlation coefficient of 0.9890 and after that approaches an asymptote as the enzyme becomes saturated with nitrate. The apparent Michaelis–Menten constant was found to be 212 lM which is similar to the value obtained by solution assay (197 lM) using NADH as electron donor [35]. We note that at the lower concentrations of nitrate, the voltammetry is not steady state so the values of ip only approximate the true steady-state current. Overall the behavior indicates that NR is functioning natively on GC electrode and that diffusion of nitrate across the membrane is not rate limiting. Further, we have estimated the sensitivity of the GC/NR electrode from its catalytic reduction current and found it to be 14 nA/lM. The present biosensor shows better sensitivity compared to biosensors that were loaded with commercially available NR enzymes entrapped within different polymer modified electrodes (5.5 nA/lM [33] and 7.3 nA/lM [22]).
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The optimal pH for the GC/NR electrode was determined over the pH range of 5.5–9.0. Figure 4 depicts the pH profile of the GC/NR electrode with 400 lM nitrate and 25 lM AQ in 60 mM mixed buffers. The maximum catalytic current was found to be at pH 7 so this pH was chosen for all subsequent measurements. Further, the bell-shaped pH profile was modeled with Eq. (2) where protonation and deprotonation events at the active site lead to a loss of activity. Approximate pKa values of 6.6 and 8.1 were obtained. Moreover, it is also noted that all changes in CV peak potential and currents as a function of pH were reversible. Amperometric determination of lower detection limit Figure 5a depicts the amperometric i–t curve for catalytic nitrate reduction at the GC/NR electrode in a homogeneously stirred 50 mM Bis-Tris buffer solution at an applied potential of -400 mV vs. NHE. An initial steadystate current response was observed in the presence of 10 lM nitrate and subsequent 10 lM nitrate additions at intervals of 100 s led to a step in the current with a steady state reached within ca. 30 s (Fig. 5a). An expansion of the i–t curve is shown in Figure S3. The steady-state current increased linearly with nitrate concentration from 10 to 70 lM and the detection limit was found to be 0.76 nM (S/ N = 3) (Fig. 5b) using a standard procedure [41]. The detection limit and linear dynamic range of GC/NR electrode towards nitrate reduction were compared with those recently reported with NR-based nitrate biosensor electrodes. The detection limit lies in the range of 0.4–6.7 lM and dynamic range between 3 and 300 lM which is comparable with other studies [22, 25, 29, 31–33].
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A
B
3 nA
Y = 0.257 X + 2.0 2 R = 0.9857
20
|I| / nA
15
|I|
Fig. 5 a Amperometric i– t curve (absolute current shown) for the determination of nitrate at GC/NR electrode in stirred 50 mM Bis-Tris buffer solution. Each addition increased the concentration by 10 lM nitrate at regular intervals of 100 s at an applied potential of -400 mV vs. NHE. b Linear response range for nitrate
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10 5 0
400
600
800
1000
1200
0
Time (sec)
20
40
60
80
Concentration (μM)
Interference studies
Oxygen
Oxidoanions
The GC/NR biosensor was tested in aerobic solution to see if the higher potential mediator AQ could selectively reduce NR but not O2. The data for an unmodified GC electrode (Supporting Information Figure S7) show that, in the absence of AQ but in aerated solution, a large cathodic current emerges in the region -300 to -500 mV vs. NHE at pH 7 from non-specific O2 reduction (to H2O2) at the electrode. As expected, in the absence of oxygen but in the presence of AQ and nitrate a catalytic sigmoidal wave is seen at the GC/NR electrode (Figure S8a). Upon introduction of oxygen (Figure S8b) a significant enhancement of current due to oxygen reduction (both direct and mediated by AQH2) is seen and cannot be avoided so it is still necessary to purge the solution of oxygen before using this biosensor in nitrate analysis to avoid co-reduction of oxygen and false positive readings.
Solis and coworkers [39] have reported an enzyme biosensor for nitrate with methyl viologen as electron donor on a GC electrode. They found that the methyl viologen radical cation (and its two-electron reduced neutral form) reacts with nitrite leading to an enhanced electrochemical signal and a false positive signal in nitrate analysis. This is relevant because samples for nitrate analysis often also contain nitrite. In another report, a Nafion-immobilized GC electrode containing NR and MV exhibits a large interfering response to chlorate and to a lesser extent nitrite ions [31]. Therefore, it was important to check the interference effects towards these common anions here. In fact no significant enhancement in current was observed upon addition of 2 mM of nitrite and phosphate (supporting information Figure S4). This also illustrated that non-specific redox reactions (at the electrode) of these ions do not occur at the GC/NR electrode and that AQ does not react with them either. Chlorate does lead to a slightly enhanced current (Figure S4, curve f) when a tenfold excess of chlorate:nitrate is present which shows that NR does exhibit some chlorate reductase activity. A catalytic current is seen when chlorate is the only substrate present (Figure S5) and this is indeed due to NRcatalyzed chlorate reduction as no effect on the AQ voltammetry by chlorate is observed in the absence of NR (Figure S6). The level of chlorate interference is quite small considering the large (tenfold) relative concentration of chlorate examined (Figure S4, curve f) and evidently nitrate reductase activity is dominant. Therefore, the selective determination of nitrate is possible with in the presence of common interferants at the GC/NR electrode and this may have practical applications.
Stability of the biosensor The stability of the GC/NR electrode was assessed by repeated use and also long-term storage. The CVs for 400 lM nitrate with 25 lM AQ in 50 mM Bis-Tris buffer solution were recorded at 3 min. intervals to evaluate the stability of the biosensor. It was found that the cathodic peak for nitrate reduction remained the same with a relative standard deviation of 1.6 % for 10 repeat measurements. To check the long-term storage stability of the biosensor, the enzyme-modified electrode was kept in 50 mM Bis-Tris buffer solution in a refrigerator (4 °C). No apparent decrease in the catalytic current response of nitrate was observed over the first 2 days and after 1 week the current had decreased by 18 % and after 2 weeks the current decreased by 32 %. This lowering in electrocatalytic activity could be due to degradation of NR or leakage from the inner membrane solution. It is
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Table 1 Determination of nitrate in natural water samples using the CG/NR biosensor and also compared with the standard spectroscopic method (all results are a mean of three determinations) Sample
GC/NR method [NO3-]/mM
RSD (%)
Spectroscopic method [NO3-]/mM
RSD (%)
River water
1.68
2.1
1.74
2.5
Lake water
2.45
2.4
2.37
1.9
worth comparing the stability of the present nitrate biosensor with others reported in the literatures. Scheller and coworkers [32] have reported an amperometric nitrate biosensor with a bacterial NR (Pseudomonas stutzeri) and found that the sensor lost the stability within 2 days due to inactivation of NR. Similarly, Glazier and coworkers [31] reported that their NR biosensor lost *50 % of activity within a day at room temperature. No attempt appeared to have been made to prolong the biosensor by storing in a refrigerator and degradation of the NR enzyme was assumed to be the cause for loss of activity [31]. Nevertheless, our results demonstrate that the present GC/NR nitrate biosensor shows good stability compared to others reported to date. Determination of nitrate in natural water samples The GC/NR electrode was applied to the determination of nitrate in natural water samples (river and lake water in the Brisbane area). The water samples collected were analyzed for nitrate using the present biosensor and validated by a reported spectroscopic method (Figure S2) [11]. The water samples were diluted with 50 mM Bis-Tris buffer and then directly used for analysis without any pretreatment. The method of standard additions was employed by injecting known amounts of nitrate to each water sample within the linear range and measuring the increase in catalytic current which enabled the original nitrate concentration to be determined by back-extrapolation to zero current (Supporting Information Figure S1). Further, we did not observe any interference signals in the water samples from nonspecific reduction reactions at the electrode. Table 1 shows the results of nitrate determination in river and lake water samples using the present electrochemical biosensor. The nitrate concentrations determined using the present electrochemical biosensor are in good agreement with the spectroscopic method (Figure S2). Relative errors between the results obtained with the present method and the spectroscopic methods were within acceptable limits. The obtained results demonstrate that the present electrochemical biosensor is suitable for the determination of nitrate in analytical samples.
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Conclusions We have developed a highly selective and sensitive nitrate biosensor based on truncated plant nitrate reductase from Arabidopsis thaliana using a GC electrode and an artificial electron transfer partner AQ. The NR enzyme retains its native activity and shows a pronounced electrocatalytic reduction response for nitrate. The present biosensor shows an excellent dynamic range (10–400 lM), and sensitivity (14 nA/lM) and low detection limit (0.76 nM). Further, the construction of this biosensor is very easy and rapid compared to most of other reported nitrate biosensors. Overall, the results presented here show that the present electrochemical biosensor is selective, sensitive and relatively stable allowing an accurate determination of nitrate in analytical samples. Acknowledgments We gratefully acknowledge support from the Australian Research Council (DP120101465).
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ORIGINAL PAPER
A sensitive and stable amperometric nitrate biosensor employing Arabidopsis thaliana nitrate reductase Palraj Kalimuthu • Katrin Fischer-Schrader Gu¨nter Schwarz • Paul V. Bernhardt
•
Received: 3 April 2014 / Accepted: 5 June 2014 / Published online: 2 July 2014 Ó SBIC 2014
Abstract Nitrate reductase (NR) from the plant Arabidopsis thaliana has been employed in the development of an amperometric nitrate biosensor that functions at physiological pH. The anion anthraquinone-2-sulfonate (AQ) is used as an effective artificial electron transfer partner for NR at a glassy carbon (GC) electrode. Nitrate is enzymatically reduced to nitrite and the oxidized form of NR is electrochemically reduced by the hydroquinone form of the mediator (AQH2). The GC/NR electrode shows a pronounced cathodic wave for nitrate reduction and the catalytic current increases linearly in the nitrate concentration range of 10–400 lM with a correlation coefficient of 0.989. Using an amperometric method, a low detection limit of 0.76 nM (S/N = 3) was achieved. The practical application of the present electrochemical biosensor was demonstrated by the determination of nitrate concentration in natural water samples and the results agreed well with a standard spectroscopic method. Keywords Nitrate reductase Voltammetry Molybdenum
Responsible Editors: Jose´ Moura and Paul Bernhardt.
Electronic supplementary material The online version of this article (doi:10.1007/s00775-014-1171-0) contains supplementary material, which is available to authorized users. P. Kalimuthu P. V. Bernhardt (&) School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane 4072, Australia e-mail: [email protected] K. Fischer-Schrader G. Schwarz Department of Chemistry and Center for Molecular Medicine, Institute of Biochemistry, Cologne University, Zu¨licher Str. 47, 50674 Cologne, Germany
Abbreviations AQ Anthraquinone-2-sulfonate CV Cyclic voltammetry FAD Flavin adenine dinucleotide GC Glassy carbon MV Methyl viologen NADH Nicotinamide adenine dinucleotide NHE Normal hydrogen electrode NR Nitrate reductase
Introduction Nitrate has a widespread distribution within environmental, food, industrial and physiological systems. Nitrate in the environment originates from wastewater, nitrogen fertilizers and other natural degradation processes [1–3]. Excess nitrate leaching in environmental water systems may cause eutrophication of lakes resulting in excessive growth of weeds and algae and reduced dissolved oxygen levels which dramatically affect aquatic life [4, 5]. In humans the consumption of high levels of nitrate leads to hemoglobin disorders including methemoglobinemia and also the toxicological problems of nitrate associated with the formation of harmful N-nitroso compounds [6–8]. Therefore, monitoring nitrate levels is of importance and there is an increased demand for the development of accurate, rapid and sensitive analytical procedures. Electrochemical methods for nitrate determination have attracted much attention in analytical chemistry [9, 10] for several reasons including low-cost, minimal sample preparation, ease of operation and rapid measurement compared to wet chemical methods [11]. However, electrochemical methods that reduce nitrate directly at a (chemically modified) electrode suffer from poor stability, sensitivity
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Scheme 1 Catalytic nitrate reduction mechanism at the active site of nitrate reductase
NO3-
O S
A
OH-
Cys
O S
IV
IV
O HN H 2N
N
H N N H
S Mo OH S
N
N H
H2 N
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S Mo O S N O O O OPO32-
Cys
B
VI
O
H N
N
N H
HN H2 N
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H N
OPO32-
C
and most importantly poor selectivity with interference from other equally common ions such as nitrite, chlorate, sulfate and phosphate being problematic [12–15]. Mo-dependent nitrate-reducing enzymes (nitrate reductases, NRs), that are selective for nitrate, offer an immediate solution to the development of nitrate-specific analytical biosensors. There are several types of Mo-dependent nitrate reductases; from both prokaryotes and eukaryotes [16, 17]. Eukaryotic nitrate reductases (from plants, algae and fungi) are structurally very different from their bacterial NR analogues although each still bears a Mo ion at its active site, but in this case it is coordinated to a single bidentate molybdopterin ligand (Scheme 1). Eukaryotic nitrate reductases are multi-redox center enzymes responsible for the biological conversion of nitrate to nitrite coupled to NADH oxidation during nitrogen assimilation. These enzymes possess three distinct cofactors (FAD, heme, and the Mo active site) bound to distinct and independently folded domains [18– 20]. Therefore, NR enzymes provide an ideal catalyst for the development of highly selective and sensitive nitrate biosensors [21–26]. The NR from the plant Arabidopsis thaliana is the subject of this paper. In the catalytic reaction, nitrate binds to the catalytically active Mo(IV) form displacing an hydroxido/aqua ligand and forming the Michaelis complex (Scheme 1A). Upon oxidation of the Mo(IV) center to Mo(VI), one N–O bond of nitrate is broken, leaving an oxido ligand on the Mo ion and nitrite is released (Scheme 1B). After completion of the oxidative half reaction the active Mo(IV) form is regenerated by two-electron reduction (from the native reduced nicotinamide partner, Scheme 1C) or, in an electrochemical system, by reduced artificial electron transfer mediators [27]. Direct electron transfer between an enzyme and an electrode [28] requires the redox cofactors to be in
O HN
O
2e-, H+
Cys
S Mo O S
NO2-
O OPO32-
proximity to the electrode and this is often not achievable due to their location being buried within the folded polypeptide. Small molecular weight redox-active mediators have been used as a suitable alternative as they can communicate with both the enzyme (homogeneous electron transfer) and the electrode (heterogeneous electron transfer). An ideal redox-active mediator should possess high stability in both its oxidized and reduced forms, and there should be no side reactions with the enzyme nor its substrate (in this case nitrate) and product (nitrite). In previous publications [21, 22, 24, 25, 29, 30] the cationic low redox potential mediator methyl viologen (MV, or 4,40 -dimethyl-4,40 -dipyridinium, E0 -430 mV vs. NHE) and analogues have been employed due to their large driving force, rapid homogeneous reduction rates and facile heterogeneous electron transfer. Generally, these compounds have been co-immobilized with NR (either bacterial or fungal) by different methods including adsorption, cross-linking, entrapment, electro-polymerization and covalent binding on different substrates such as gold, glassy carbon and edge plane pyrolytic graphite electrodes. However, some drawbacks have been reported. Enzyme activity was lost completely within a short period of time when NR was co-adsorbed with viologen compounds on an electrode surface [31, 32]. Further, no catalysis was observed while MV was confined within a polymer film due to restricted mobility of mediator [22]. Therefore, selection of mediator and the immobilization method are both crucial for the construction of effective biosensors. Moreover, it was found that NR biosensors employing commercially available nitrate reductases (e.g., from the fungus Aspergillus niger) have decreased activity due to the low purity of enzyme [33, 34]. In the present study, we report a nitrate biosensor with purified truncated plant NR (from Arabidopsis thaliana)
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O
OH
SO3-
SO3-
2e-, 2H+ -230 mV O
AQ
OH
e-
,H
+
e-
,H
+
AQH2
nitrate biosensor based on plant NR and AQ as a relatively high-potential mediator reduced at a glassy carbon working electrode. The GC/NR electrode was successfully used for the selective quantification of nitrate in natural water samples and results were validated with an established spectroscopic method.
OH SO3-
O
AQH
Scheme 2 Redox reaction of anthraquinone-2-sulfonate (AQ) at pH 6 (potential vs. NHE)
employing a glassy carbon (GC) electrode [35]. The truncated NR contains only two cofactors; the Mo active site and heme, but no FAD. Very recently, we demonstrated the pronounced electrocatalytic voltammetry of this enzyme with mediating cosubstrates such as methyl viologen (E0 430 mV), benzyl viologen (E0 -324 mV) and anthraquinone-2-sulfonate (AQ, E0 -230 mV vs. NHE) [36]. Among these three mediators, we have chosen the compound with the highest redox potential AQ (Scheme 2) as electron transfer partner for NR in the present nitrate biosensor. AQ is typically a two-electron, two-proton acceptor. Naturally its redox potential is pH dependent although it has been shown that both one-electron redox steps lie very close in potential leading to an apparent 2-electron response it its cyclic voltammogram [37]. Scheme 2 illustrates the overall reaction of the mediator that involves two-electron and twoproton reduction of AQ to dihydroanthraquinone-2-sulfonate (AQH2) and subsequent reoxidation of AQH2 to AQ [38]. However, we recently showed that electrochemically reduced AQH2 is a single-electron donor to truncated NR [36] due to the enzyme only being able to receive a single electron from the mediator at its heme cofactor, which then is relayed to the Mo active site. This process must occur twice to fully reactivate the enzyme. In any practical reductase-based biosensor that might operate in an aerobic environment, competitive reduction of oxygen, or other reducible species in solution, is a potential issue for low-potential mediators such as viologens [32] as well as their potential inhibiting properties [31]. Very importantly, viologen compounds may react with enzymeproduced nitrite in the reaction layer to generate an additional catalytic reduction current and a positive error. Also for practical applications where nitrite may coexist with nitrate in analytical samples, this can also lead to errors [39]. In this paper, an amperometric method is used to estimate the lowest detection limit of an electrochemical
Materials and methods Arabidopsis thaliana nitrate reductase was purified from a heterogeneous expression system in E. coli as previously described [35]. Potassium nitrate, potassium nitrite and anthraquinone-2-sulfonate (Na? salt) were purchased from Aldrich and were used as received. All other reagents used were of analytical grade purity and used without further purification. All solutions were prepared with ultrapure water (resistivity 18.2 MX cm) from a Millipore Milli-Q system. Bis-Tris-acetate (50 mM) in the presence of 50 mM KCl as inert electrolyte was used for experiments at pH 7. The mixture of buffers (20 mM MES buffer pH 5.5–6.7, 20 mM Bis-Tris buffer pH 5.8–7.2 and 20 mM Tris buffer pH 7.0–9.0) were used for pH-dependent experiments and the desired pH was obtained with dilute acetic acid or NaOH. The natural water samples, collected locally from a lake and river, were diluted with 50 mM Bis-Tris buffer (pH 7) before analysis. Electrochemical and spectral measurements and electrode cleaning Cyclic voltammetry (CV) experiments were performed at 25 °C using a BAS 100B/W electrochemical workstation. A three-electrode system was employed comprising a glassy carbon (GC) disk working electrode (0.057 cm2), a Pt wire counter electrode, and an Ag/AgCl reference electrode (?196 mV vs. NHE). Potentials are cited versus NHE. Experiments were carried on solutions that had been purged with argon for 30 min. The GC electrode was polished with 0.50 and 0.05 lm alumina slurry and then rinsed thoroughly with water. The electrode was then sonicated in water for 5 min to remove adsorbed alumina particles and dried in a stream of nitrogen. UV–Vis spectra were recorded at room temperature with a Perkin-Elmer Lamda 35 Spectrophotometer. The sample was mounted in a 1-cm path length quartz cuvette. The variation of catalytic cathodic peak current versus nitrate concentration was fit to Michaelis–Menten kinetics (where KM,app is the apparent Michaelis constant, ilim is the plateau (steady state) current and imax is the maximal current under substrate saturating conditions ([NO3-] » KM,app) [28].
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ilim ¼
imax ½NO 3 KM;app þ ½NO 3
ð1Þ
The pH dependence of the catalytic current was modeled by Eq. (2) which is applicable for an active form of the enzyme that is switched off by either a deprotonation (pKa1) or protonation (pKa2) [40] where iopt is the maximum catalytic current at the pH optimum ilim ðpHÞ ¼
iopt : 1 þ 10ðpHpKa1 Þ þ 10ðpKa2 pHÞ
ð2Þ
GC/NR electrode preparation A 3 lL droplet of NR (36 lM) in 50 mM assay buffer [50 mM Bis-Tris-acetate (pH 7.0), 50 mM KCl, 5 mM magnesium acetate, and 1 mM CaCl2] was pipetted onto the conducting surface of an inverted, freshly polished GC working electrode and this was allowed to dry to a film for about 20 min at 4 °C. To prevent protein loss through desorption, the electrode surface was carefully covered with a small piece of perm-selective dialysis membrane (*1 9 1 cm, molecular weight cut off 3,500 Da), presoaked in water. The dialysis membrane was pressed onto the electrode with a Teflon cap and fastened to the electrode with a rubber O-ring. The resulting modified electrode was stored at 4 °C in 50 mM Bis-Tris buffer (pH 7.0) when not in use. The enzyme was confined to a thin layer beneath the membrane while nitrate, nitrite and AQ were able to diffuse across the membrane. A cartoon representation of the biosensor electrode is shown in Scheme 3.
nitrite
AQH2
NRox e-
NRox: nitrite NRint
AQH AQH2
NRred: nitrate NRred enitrate
AQH
Membrane
GC electrode
Scheme 3 The electron transfer mechanism of NR in the presence of AQ and nitrate
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Chemical nitrate analysis The nitrate concentrations determined in natural water samples employed in the present electrochemical sensor (Figure S1) were cross-checked with a well-established spectrophotometric method in which acid catalyzed nitration of salicylic acid yields a colored product (kmax = 410 nm) at pH [12 [11]. Briefly, 0.2 mL of the water sample was pipetted into 50-ml Erlenmeyer flasks and mixed thoroughly with 0.8 ml of 5 % (w/v) salicylic acid in concentrated H2SO4. After incubation for 20 min at room temperature, 19 ml of 2 M NaOH was cautiously added to raise the pH above 12 (caution exothermic). On cooling to room temperature and dilution volumetrically, the absorbance at 410 nm was measured with a spectrophotometer. The salicylic acid–H2SO4 reagent was made afresh before the experiment. The method of standard addition was employed by adding known amounts of nitrate to each sample. A linear increase in the absorption at 410 nm was observed with increasing quantities of added nitrate which enabled the original nitrate concentration of the sample to be determined by back-extrapolation (Supporting information Figure S2). The main advantage of this method is that potential interferents such as ammonium, nitrite and chloride ions are not reactive so selective quantification of nitrate is possible.
Results and discussion Mechanism of NR electrocatalysis NR is confined to a thin layer on GC electrode surface entrapped beneath a membrane (MW cutoff 3,500 Da), whereas the small MW substrates/products (nitrate and nitrite) and AQ are under diffusion control and may cross the membrane. As mentioned above, generally AQH2 is a two-electron and two-proton reductant, but recently we found that AQH2 acts as single-electron donor to NR [36]. The semiquinone AQH is thermodynamically unstable with respect to disproportionation, but this is not significant as the products of disproportionation (2AQH AQ ? AQH2) are both components of the catalytic cycle either in homogeneous reaction with NR (AQH2) or reduction at the electrode low potential (AQ) [36, 37]. As shown in Scheme 3, during the reductive half reaction, AQH2 reduces fully oxidized NRox in consecutive, one-electron, outer-sphere electron transfer reactions via an intermediate form NRint.1 In the oxidative half reaction, NRred in its (MoIV form) converts the coordinated nitrato 1
In all cases we assume that the heme cofactor of NR relays electrons rapidly from AQH2 to the Mo active site.
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ligand to nitrite which then dissociates from the active site. The detailed kinetics of this system was discussed in our recent publication [36]. Catalytic voltammetry The mediated catalytic voltammetry of NR in the presence of nitrate and AQ is shown in Fig. 1. In the absence of AQ, the GC/NR electrode shows no direct voltammetric responses from the NR redox-active cofactors in 50 mM Bis-Tris buffer solution (Fig. 2, curve a) and none in the presence of nitrate. It is assumed that the redox cofactors (heme and Mo) are deeply buried inside the protein and
thus not accessible for electron transfer to the electrode surface. However, the GC/NR electrode shows a pair of asymmetric peaks centered at -230 mV (vs. NHE) in the presence of 25 lM AQ (without nitrate) in 50 mM Bis-Tris buffer solution (pH 7) with a peak to peak separation of 80 mV (Fig. 1a). This current is governed by the diffusion of both oxidized and reduced forms of AQ between the electrode and bulk solution. Upon addition of 200 lM nitrate to the same solution, a pronounced cathodic wave emerges and the anodic wave vanishes (Fig. 1b). This voltammetry is characteristic of a catalytic homogeneous reaction coupled to heterogeneous electron transfer (ECcat mechanism) where nitrate is reduced enzymatically yielding the oxidized form of NR, which is reduced again by electro-generated AQH2. AQ concentration dependence
Fig. 1 CVs obtained for AQ (25 lM) in the absence (a, red) and presence (b, green) of 200 lM nitrate at the GC/NR electrode in 50 mM Bis-Tris buffer (pH 7) at a sweep rate of 5 mV s-1
0
I/ μΑ
–1
a
The sigmoidal voltammograms are indicative of an electrochemical steady state, i.e., the intermediate AQH2 is consumed (by homogeneous reaction with NRox) at the same rate that it is generated at the electrode surface and the forward and reverse sweeps are virtually identical.
–2
f
–3
–500
–400
–300
Figure 2 illustrates the effect of different AQ concentrations in the presence of 1 mM nitrate at the GC/NR electrode. In the absence of AQ, the GC/NR electrode does not show any catalytic response towards nitrate in the potential window of -150 and -550 mV (curve a). On the other hand, a sigmoidal catalytic wave emerges upon increasing the concentration of AQ in the presence of 1 mM nitrate (curves b ? f). The catalytic current is dependent upon the diffusion of AQ to the electrode surface, reaction between nitrate and NR, and the reaction between AQH2 and NRox. The limiting current (ip, at low potential) increases linearly with AQ concentrations within the range of 0–15 lM. Theoretically, the plateau current at low potential (ip) current is directly proportional to [AQ] only at low AQ concentrations (Eq. 3) and high nitrate concentrations [28]; [NR*] and [AQ*] denote bulk concentrations, DAQ is the AQ diffusion coefficient and the other terms have their usual meaning. This equation breaks down when the mediator concentration becomes too high; in this case beyond *15 lM AQ. qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ip ¼ FA[AD 2k3 DAQ ½NR ð3Þ
–200
Nitrate concentration dependence
E/ mV vs. NHE
Fig. 2 CVs obtained for varying AQ concentrations a 0, b 5, c 10, d 15, e 20 and f 25 lM in the presence of 1 mM nitrate at GC/NR electrode in 50 mM Bis-Tris buffer, pH 7 at a sweep rate of 5 mV s-1
The cathodic current response of the GC/NR electrode as function of nitrate concentration and in the presence of 25 lM AQ was examined. As the electrode potential is
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Concentration (μM) Fig. 3 Plot of the baseline subtracted (absolute) limiting cathodic current (at low potential) for the GC/NR electrode in presence of 25 lM AQ as a function of nitrate concentrations in 50 mM Bis-Tris buffer, pH 7 at a scan rate of 5 mV s-1
5
Fig. 4 pH dependence of the maximum absolute catalytic current for 400 lM nitrate in the presence of 25 lM AQ at the GC/NR electrode in 60 mM mixed buffer (MES, Bis-Tris and Trizma) at a sweep rate of 5 mV s-1
pH dependence lowered to -500 mV, the catalytic reduction current eventually reaches a limiting value (ilim) and the magnitude of the limiting current value becomes larger with increasing nitrate concentration in accord with Michaelis–Menten kinetics. It is noted that transient (peak-shaped) catalytic voltammetry was observed for nitrate concentrations 10–400 lM (see Fig. 1b as an example) indicating that the electrocatalytic current is somewhat limited by nitrate diffusion at low concentrations. However, at higher nitrate concentrations ([800 lM) the transient voltammogram becomes sigmoidal (see Fig. 2) where the concentration of nitrate within the reaction layer is constant during the sweep and the forward and reverse sweeps are virtually identical. As shown in Fig. 3 the catalytic reduction current is approximately linear for nitrate concentration from 10 to 400 lM with a correlation coefficient of 0.9890 and after that approaches an asymptote as the enzyme becomes saturated with nitrate. The apparent Michaelis–Menten constant was found to be 212 lM which is similar to the value obtained by solution assay (197 lM) using NADH as electron donor [35]. We note that at the lower concentrations of nitrate, the voltammetry is not steady state so the values of ip only approximate the true steady-state current. Overall the behavior indicates that NR is functioning natively on GC electrode and that diffusion of nitrate across the membrane is not rate limiting. Further, we have estimated the sensitivity of the GC/NR electrode from its catalytic reduction current and found it to be 14 nA/lM. The present biosensor shows better sensitivity compared to biosensors that were loaded with commercially available NR enzymes entrapped within different polymer modified electrodes (5.5 nA/lM [33] and 7.3 nA/lM [22]).
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The optimal pH for the GC/NR electrode was determined over the pH range of 5.5–9.0. Figure 4 depicts the pH profile of the GC/NR electrode with 400 lM nitrate and 25 lM AQ in 60 mM mixed buffers. The maximum catalytic current was found to be at pH 7 so this pH was chosen for all subsequent measurements. Further, the bell-shaped pH profile was modeled with Eq. (2) where protonation and deprotonation events at the active site lead to a loss of activity. Approximate pKa values of 6.6 and 8.1 were obtained. Moreover, it is also noted that all changes in CV peak potential and currents as a function of pH were reversible. Amperometric determination of lower detection limit Figure 5a depicts the amperometric i–t curve for catalytic nitrate reduction at the GC/NR electrode in a homogeneously stirred 50 mM Bis-Tris buffer solution at an applied potential of -400 mV vs. NHE. An initial steadystate current response was observed in the presence of 10 lM nitrate and subsequent 10 lM nitrate additions at intervals of 100 s led to a step in the current with a steady state reached within ca. 30 s (Fig. 5a). An expansion of the i–t curve is shown in Figure S3. The steady-state current increased linearly with nitrate concentration from 10 to 70 lM and the detection limit was found to be 0.76 nM (S/ N = 3) (Fig. 5b) using a standard procedure [41]. The detection limit and linear dynamic range of GC/NR electrode towards nitrate reduction were compared with those recently reported with NR-based nitrate biosensor electrodes. The detection limit lies in the range of 0.4–6.7 lM and dynamic range between 3 and 300 lM which is comparable with other studies [22, 25, 29, 31–33].
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A
B
3 nA
Y = 0.257 X + 2.0 2 R = 0.9857
20
|I| / nA
15
|I|
Fig. 5 a Amperometric i– t curve (absolute current shown) for the determination of nitrate at GC/NR electrode in stirred 50 mM Bis-Tris buffer solution. Each addition increased the concentration by 10 lM nitrate at regular intervals of 100 s at an applied potential of -400 mV vs. NHE. b Linear response range for nitrate
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The GC/NR biosensor was tested in aerobic solution to see if the higher potential mediator AQ could selectively reduce NR but not O2. The data for an unmodified GC electrode (Supporting Information Figure S7) show that, in the absence of AQ but in aerated solution, a large cathodic current emerges in the region -300 to -500 mV vs. NHE at pH 7 from non-specific O2 reduction (to H2O2) at the electrode. As expected, in the absence of oxygen but in the presence of AQ and nitrate a catalytic sigmoidal wave is seen at the GC/NR electrode (Figure S8a). Upon introduction of oxygen (Figure S8b) a significant enhancement of current due to oxygen reduction (both direct and mediated by AQH2) is seen and cannot be avoided so it is still necessary to purge the solution of oxygen before using this biosensor in nitrate analysis to avoid co-reduction of oxygen and false positive readings.
Solis and coworkers [39] have reported an enzyme biosensor for nitrate with methyl viologen as electron donor on a GC electrode. They found that the methyl viologen radical cation (and its two-electron reduced neutral form) reacts with nitrite leading to an enhanced electrochemical signal and a false positive signal in nitrate analysis. This is relevant because samples for nitrate analysis often also contain nitrite. In another report, a Nafion-immobilized GC electrode containing NR and MV exhibits a large interfering response to chlorate and to a lesser extent nitrite ions [31]. Therefore, it was important to check the interference effects towards these common anions here. In fact no significant enhancement in current was observed upon addition of 2 mM of nitrite and phosphate (supporting information Figure S4). This also illustrated that non-specific redox reactions (at the electrode) of these ions do not occur at the GC/NR electrode and that AQ does not react with them either. Chlorate does lead to a slightly enhanced current (Figure S4, curve f) when a tenfold excess of chlorate:nitrate is present which shows that NR does exhibit some chlorate reductase activity. A catalytic current is seen when chlorate is the only substrate present (Figure S5) and this is indeed due to NRcatalyzed chlorate reduction as no effect on the AQ voltammetry by chlorate is observed in the absence of NR (Figure S6). The level of chlorate interference is quite small considering the large (tenfold) relative concentration of chlorate examined (Figure S4, curve f) and evidently nitrate reductase activity is dominant. Therefore, the selective determination of nitrate is possible with in the presence of common interferants at the GC/NR electrode and this may have practical applications.
Stability of the biosensor The stability of the GC/NR electrode was assessed by repeated use and also long-term storage. The CVs for 400 lM nitrate with 25 lM AQ in 50 mM Bis-Tris buffer solution were recorded at 3 min. intervals to evaluate the stability of the biosensor. It was found that the cathodic peak for nitrate reduction remained the same with a relative standard deviation of 1.6 % for 10 repeat measurements. To check the long-term storage stability of the biosensor, the enzyme-modified electrode was kept in 50 mM Bis-Tris buffer solution in a refrigerator (4 °C). No apparent decrease in the catalytic current response of nitrate was observed over the first 2 days and after 1 week the current had decreased by 18 % and after 2 weeks the current decreased by 32 %. This lowering in electrocatalytic activity could be due to degradation of NR or leakage from the inner membrane solution. It is
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Table 1 Determination of nitrate in natural water samples using the CG/NR biosensor and also compared with the standard spectroscopic method (all results are a mean of three determinations) Sample
GC/NR method [NO3-]/mM
RSD (%)
Spectroscopic method [NO3-]/mM
RSD (%)
River water
1.68
2.1
1.74
2.5
Lake water
2.45
2.4
2.37
1.9
worth comparing the stability of the present nitrate biosensor with others reported in the literatures. Scheller and coworkers [32] have reported an amperometric nitrate biosensor with a bacterial NR (Pseudomonas stutzeri) and found that the sensor lost the stability within 2 days due to inactivation of NR. Similarly, Glazier and coworkers [31] reported that their NR biosensor lost *50 % of activity within a day at room temperature. No attempt appeared to have been made to prolong the biosensor by storing in a refrigerator and degradation of the NR enzyme was assumed to be the cause for loss of activity [31]. Nevertheless, our results demonstrate that the present GC/NR nitrate biosensor shows good stability compared to others reported to date. Determination of nitrate in natural water samples The GC/NR electrode was applied to the determination of nitrate in natural water samples (river and lake water in the Brisbane area). The water samples collected were analyzed for nitrate using the present biosensor and validated by a reported spectroscopic method (Figure S2) [11]. The water samples were diluted with 50 mM Bis-Tris buffer and then directly used for analysis without any pretreatment. The method of standard additions was employed by injecting known amounts of nitrate to each water sample within the linear range and measuring the increase in catalytic current which enabled the original nitrate concentration to be determined by back-extrapolation to zero current (Supporting Information Figure S1). Further, we did not observe any interference signals in the water samples from nonspecific reduction reactions at the electrode. Table 1 shows the results of nitrate determination in river and lake water samples using the present electrochemical biosensor. The nitrate concentrations determined using the present electrochemical biosensor are in good agreement with the spectroscopic method (Figure S2). Relative errors between the results obtained with the present method and the spectroscopic methods were within acceptable limits. The obtained results demonstrate that the present electrochemical biosensor is suitable for the determination of nitrate in analytical samples.
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Conclusions We have developed a highly selective and sensitive nitrate biosensor based on truncated plant nitrate reductase from Arabidopsis thaliana using a GC electrode and an artificial electron transfer partner AQ. The NR enzyme retains its native activity and shows a pronounced electrocatalytic reduction response for nitrate. The present biosensor shows an excellent dynamic range (10–400 lM), and sensitivity (14 nA/lM) and low detection limit (0.76 nM). Further, the construction of this biosensor is very easy and rapid compared to most of other reported nitrate biosensors. Overall, the results presented here show that the present electrochemical biosensor is selective, sensitive and relatively stable allowing an accurate determination of nitrate in analytical samples. Acknowledgments We gratefully acknowledge support from the Australian Research Council (DP120101465).
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