Renewable-Reagent Enzyme Inhibition Sensor for RemoteMonitoring of Cyanide

Joseph Wang,* Baomin Tian, Jianmin Lu, Doug MacDonald, Jianyan Wang,andDengbai Luo

Department of Chemistry and Biochemistry, New Mexico State University, Las Cruces, NM 88003, USA

Received: June 19, 1998Final version: July 23, 1998

AbstractA renewable-reagent flow probe has been developed for the remote electrochemical biosensing of enzyme inhibitors. The new submersible deviceaddresses the challenges of continuously replacing the inhibited enzyme and consumed substrate. The internal delivery of microliter enzyme andsubstrate solutions is coupled to an in situ microdialysis sampling of the toxin, and an amperometric detection of the enzymatically generatedproduct. The new concept is demonstrated for the detection of micromolar concentrations of free cyanide in the presence of the enzyme tyrosinaseand its catechol substrate. The optimization of various physical and chemical parameters has resulted in a low detection limit of 2×10¹6 M cyanideand good precision (RSD¼ 5%). The new device holds great promise for in situ environmental and industrial monitoring of toxins.

Keywords: Cyanide, Remote monitoring, Renewable-reagent enzyme inhibition sensor, Tyrosinase, Catechol

1. Introduction

Cyanide is widely used and produced in many industrialprocesses, and can be found in effluents of metal plating shops,steel mills and mining operations [1]. In view of the extremetoxicity of cyanide, there are urgent needs for innovative devicesfor its detection. Such new devices should replace the traditionaltime-consuming methods commonly being used for measurementsof cyanide [2, 3]. Field measurements are preferred, as they affordthe option of a rapid warning capability, while avoiding errors anddelays of laboratory-based analyses.

Biosensors based on enzyme inhibition have been used formonitoring toxic substances, including cyanide [4–8], and arewell suited for on-site applications. The modulated biocatalyticactivity accrued from the enzyme-inhibitor interaction is commonlydetected from the decreased electrochemical response of thecorresponding substrate. Disposable (thick-film) inhibitionbiosensors have already been developed for discrete fielddetection of various toxins [9, 10]. In situ measurements, inwhich the target toxin is determined in its own environment, arepreferred since they provide an immediate detection of a suddentoxin contamination and hence the option of a proper correctiveaction.

In this article we describe the development and optimizationof a submersible electrochemical biosensor for in situ moni-toring of free cyanide. The adaptation of inhibition-basedbiosensors for a submersible/remote operation is very challenging,as it requires a continuous delivery of the substrate solution aswell as an in situ replenishment of the inhibited enzyme. Toaccomplish this goal we have relied on a new flow probe, involvingthe delivery of fresh tyrosinase-enzyme and catechol-substratesolutions, an on-line microdialysis sampling of cyanide, andamperometric detection of the modulated biocatalytic reaction(Fig. 1). The concept of renewable reagent electrochemicalsensing was introduced recently in connection to remoteadsorptive stripping measurements of trace metals [11]. In thefollowing sections we report on the systematic optimization andattractive analytical performance of the remote inhibition sensor.While the concept of renewable enzyme-inhibition probes ispresented within the context of cyanide detection, it could beextended to the remote monitoring of other toxins (e.g., organo-phosphate pesticides).

2. Experimental

2.1. Probe Design

A schematic diagram of the renewable-reagent electro-chemical probe is illustrated in Figure 1. The probe utilizes amicrodialysis sampling tubing (F) of molecular weight cut-off(MWCO) 18 000 and inner diameter of 200mm. The dialysistubing, which encompasses a band of 5 regenerated cellulose (RC)

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Electroanalysis1998, 10, No. 15 q WILEY-VCH Verlag GmbH, D-69469 Weinheim, 1998 1040-0397/98/1511-1034 $ 17.50þ.50/0

Fig. 1. Schematic diagram of the renewable-reagent enzyme biosensor forremote monitoring of toxins: A) working electrode; B) reference electrode;C) counter electrode; D and E) microsyringe pumps; F) microdialysissampling tubing; G) Y connector; H) Plexiglas sensor body; I) reagent ouletJ) waste collector; K) shielded cable; L) potentiostat.

hollow fibers, is 5 cm in length. The reagent inlet tubes from thesyringes are connected to the dialysis sampling tubing through aY-shape connector (G). The cylindrical Plexiglas sensor body (H,37-mm diameter, and 56-mm length) houses the glassy carbonworking electrode, Ag/AgCl reference electrode, platinum wirecounter electrode as well as the reagent delivery and drainagecapillaries. The working electrode [(A) 3.2-mm diameter, ModelMF 2012, BAS Inc.] was mechanically modified and insertedthrough an O-ring into a 5.7-mm diameter threaded hole in thetop of the sensor body. It terminates in a thin-layer cavity nearthe base of the cylinder. The associated ‘dead volume’, includingthe flow path from the Y-shape connector to the dialysis tubingand the working electrode cavity, is 16mL. A homemade Ag/AgClreference electrode (B), containing a 3 M NaCl inner solution, wasinstalled in a similar fashion. The platinum wire counter electrode(C; 41 mm long, 0.6 mm diameter) extends to the base of the probecylinder, then bends at a 908 angle and extends 20-mm towardthe working electrode cavity. Teflon tubing connects the ‘enzyme’and ‘substrate’ microsyringe pumps [(D and E) 2.5-mL, ModelsMD-0250 and MD-1000, BAS Inc.] to the Y-connector,and constitutes the reagent outlet (I), which leads to the wastereservoir (J).

2.2. Apparatus

The experiments were performed with an AUTOLAB Electro-chemical Instrument (Eco Chemie B.V., The Netherlands)equipped with a Compaq Deskpro 2000 personal computer(pentium processor). Amperometric measurements were carriedout with the General Purpose Electrochemical System (GPES)software for Windows.

2.3. Reagents

All chemicals were reagent grade. Potassium cyanide wasreceived from J.T. Baker Co., while all others were obtained fromSigma. The stock solution of tyrosinase (3000 activity units/mL)was prepared with a phosphate buffer (pH 7.4) and stored at 48Cin a refrigerator. The cyanide and catechol stock solutions (50 mM)were prepared daily with water.Caution: in view of the high toxicityof cyanide, all solution preparations should be carried out in thefume hood, and the experiments should be performed in a well-aerated room.Double deionized water (>18 MQ) was used in allexperiments. The Rio-Grande river water samples were collectedin Las Cruces (NM).

2.4. Procedure

The glassy carbon electrode was polished with alumina slurryand rinsed with water before it was placed into the probe cavity. Asshown in Figure 1, the three-electrode probe was then dipped in a200 mL beaker containing the stirred sample solution. Two syringepumps were employed to deliver the enzyme and substratesolutions through a Y-shape connector and dialysis tubing to theprobe cavity. The flow rate of both solutions was 20mL/min. Themeasurement protocol was similar to a flow injection operation.The substrate solution flowed towards the electrode compartmentof the probe. The tyrosinase solution was then injected byturning on the enzyme flow for 25 s while the substrate flow wasturned off. The injected volume of enzyme solution was 8.3mL.After the injection, the substrate flow was turned on, thus carryingthe enzyme plug through the Y-shape connector and towardsthe dialysis tubing (where the cyanide was sampled). The

amperometric response was recorded while holding the potentialat –200 mV (vs. Ag/AgCl).

Multiple peak-shape amperograms were obtained by repeatingthe enzyme injections at 3.5 minute intervals. All experiments wereperformed at room temperature.

3. Results and Discussion

The concept of renewable-reagent inhibition biosensors isillustrated in the following section using the inhibition of tyrosinaseby cyanide [7] in the presence of its catechol substrate. The newsubmersible sensor thus relies on the delivery of fresh enzymeand substrate solutions, the inhibition of their biocatalytic reactionby the cyanide ion ‘collected’ by the microdialysis sampling tube,and a downstream amperometric detection of the enzymaticallygenerated quinone product (Figure 1). The microdialysis tubingreadily ‘collects’ the very small cyanide ion from the surroundingsample, while hindering the transport of potential interferences(i.e., large organic inhibitors or particulates). Such operationsuccessfully addresses the challenge of in situ replacement of theinhibited enzyme and substrate reagent from the sensor body.

Initial experiment involving the continuous delivery and mergingof the tyrosinase and catechol solutions, resulted in the expected(decreased-substrate) response in the presence of cyanide, but werecoupled to a relatively large noise and a slowly drifting background.More attractive response characteristics were observed by per-forming in situ a flow injection type operation, involving aninjection of the enzyme solution into the flowing substrate solution(prior to the cyanide collection). This enzyme-injection modeoffered a stable baseline, peak-shaped readout, low noise level,good precision, and minimization of the enzyme consumption.

Figure 2 displays the amperometric response of the remote sensorin the absence (a) and presence (b) of 7:5 × 10¹5 M cyanide, withoutthe catechol in the ‘substrate’ solution (A), without tyrosinase inthe ‘enzyme’ solution (B), and when both tyrosinase and catecholare present (C). As expected, no amperometric signals are detectedin the absence of either the substrate or the enzyme (A,B). Incontrast, a large cathodic peak (associated with the reduction ofthe quinone product) is observed upon injecting the enzyme intothe substrate solution (C,a). This response decreases upon adding

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Fig. 2. Amperometric response of the submersible inhibition biosensor. A)presence of tyrosinase in the enzyme line but without catechol in thesubstrate line; B) without tyrosinase in enzyme line but with catechol insubstrate line; C) with both enzyme and catechol present in correspondinglines. Without (a) and with (b) 75mM cyanide in the external sample solution.Applied potential:¹200 mV; flow rate: 20mL=min; carrier line: 0.05 Mphosphate buffer containing 0 (A) and 0.1 mM (B,C) catechol; enzyme line: 0(B) and 27 (A,C) units/mL tyrosinase in 0.05 M phosphate buffer; injectiontime: 20 s.

7:5 × 10¹5 M cyanide to the surrounding sample (C,b), reflectingthe inhibited biocatalytic activity. The decrease in the quinonereduction peak serves as a measure of the cyanide concentrationin the external sample solution. Such readout is typical to flowinjection operations, with a sharp rise of the current upon the arrivalof the dispersed enzyme plug to the working-electrode compart-ment, and a slower current decay upon its departure. The tailingreflects the parabolic velocity profile that is characteristic of laminarflow in the reaction tube.

Optimal conditions for the operation of the new in situ cyanideprobe were investigated. As the probe relies on the coupling ofmicrodialysis sampling with an in situ flow injection operation,one should consider variables affecting the efficiency of themicrodialysis recovery, the extent of the dispersion and biocatalyticreactions in the flow injection manifold, and the subsequentamperometric detection. The influence of various physical para-meters is examined in Figure 3. For example, Figure 3A showsthe effect of the flow rate of the reagent(substrate)/receivingsolution upon the inhibition signal. The response decreases nearlylinearly upon increasing the flow rate between 10 and 40mL/minand then more slowly. Such a profile reflects the fact that the flowrate affects (in a different fashion) the various processes mentionedabove. Both the sampling efficiency and the extent of enzyme/inhibition reaction are expected to decrease at higher flow rates,while the amperometric response is expected to increase uponraising the rate of forced convection. Apparently, the former stepsdominate the resultant flow rate profile.

Figure 3B displays the effect of the injection time (of the enzymesolution) upon the inhibition signal. The range of injection timesexamined (2 to 30 s) correspond to volumes of 0.7 to 10mL. Theresponse increases rapidly upon raising the injection time between2 and 10 s, and nearly levels off above 20 s. Such dependenceis expected based on the extended reaction time and increasedbiocatalytic activity associated with the expanded enzyme zone.Most subsequent analytical work employed a flow rate andinjection time of 20mL/min and 25 s, respectively. Chemicalvariables affecting the response are examined in Figure 4. Asexpected, the composition of the reagent solutions strongly affectsthe sensor performance. The inhibition response rises sharplyto a steady-state value upon raising the substrate concentrationto 7× 10¹4 M (A) and the enzyme activity to 25 units/mL (B).Based on the optimization experiments described above, mostsubsequent analytical work employed a flow rate of 20mL/min, aninjection time of 25 s, a catechol concentration of 5× 104M, andenzyme activity of 36 units/mL. Such conditions offer a measure-ment frequency of 15 per hour and minimal consumption ofthe internal reagents (e.g., 4.5 enzyme units and ca. 1.0 mL of thesubstrate solution per hour of continuous operation).

The analytical performance of the optimized sensing protocolis illustrated in Figure 5. The sensor offers convenient andreproducible quantitation of low (micromolar) levels of cyanide.Figure 5A displays response peaks for increasing concentrationsof cyanide in 12:5 × 10¹6 M increments (b–j). As expected forperturbed biocatalytic reactions, the inhibition signal increasesrapidly with the toxin concentration at first, and then more slowly.The resulting calibration plot (also shown) displays linearity upto 5× 10¹5 M cyanide, and a curvature thereafter. The sensitivity(of the linear portion) corresponds to 7.2 nA/mM, and the detection

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Fig. 3. Effects of carrier/substrate solution flow rate (A) and injection time ofthe enzyme zone (B) upon the amperometric response for 75mM cyanide.Applied potential,¹200 mV; A) carrier: 0.05 M phosphate buffer containing0.5 mM catechol; enzyme line: 36 units/mL tyrosinase, injection flow rate:20mL=min; B) carrier: 0.05 M phosphate buffer containing 0.1 mM catechol,enzyme line: 27 units/mL tyrosinase; enzyme flow rate, 20mL=min; injectiontime (A) 25 s.

Fig. 4. Effects of catechol concentration (A) and enzyme activity (B) uponthe amperometric response for 75mM cyanide. A) enzyme line: 30 units/mLtyrosinase; B) carrier: 0.05 M phosphate buffer containing 0.5 mM catechol.Flow rate: 20mL=min; injection time: 20 s. Other conditions, as in Figure 2C.

Fig. 5. Analytical performance: A) calibration data; B) sample analysis;C) reproducibility. A) Amperometric response to successive 12:5mMincrements in the cyanide concentration (b–j), along with correspondingbackground signal (a). B) Response to a river water sample containingincreasing levels of cyanide in 6:25mM steps (a–d), along with the blankcatechol response of the sample (s) and a deionized water (w) solutions.C) Sensor response to the blank (a) and 25mM cyanide (b) solutions. Appliedpotential: ¹200 mV; carrier: 0.05 M phosphate buffer containing 0.5 mMcatechol; flow rate: 20mL=min; enzyme line: 36 units/mL tyrosinase;injection time: 25 s.

limit (based onS=N ¼ 3) is 2× 10¹6 M. Changes in the permeabilityof the sampling tubing (i.e., analyte dilution) may be useful forextending the linear range.

The attractive performance characteristics of the renewable-reagent inhibition biosensor are retained upon using real-worldsample matrices. For example, Figure 5B displays the sensorresponse to an untreated river (Rio Grande) water sample contain-ing increasing levels of cyanide (6.25 to 25:0 × 10¹6 M, a–d), aswell as the catechol signals for the unspiked sample (s) or artificial(deionized water) blank solution (w). The sensor respondsfavorably to these micromolar changes in the inhibitor concentra-tion, with sensitivity similar to that observed (in Figure 5A) forsynthetic samples. Notice also the similar response to the substrateusing the river and deionized water samples (s vs. w). Suchsimilarity in the substrate signals, along with the similar inhibitionresponse, indicate negligible matrix effects, and offers greatpromise for ex situ calibration steps. These similarities areattributed in part to the microdialysis sampling step, that ‘isolates’the target cyanide ion from the natural water sample, whileexcluding potential interferences (e.g., surfactants, particulates,algae). (One should, however, consider potential fouling of themembrane over prolonged exposures to such matrices.) Anotherunique feature of the flow-probe configuration (associated with itsinternal electrolyte ‘supply’) is that the electrochemical measure-ment is not affected by the sample ionic strength. Samplescontaining little or no naturally occurring electrolyte can be readilyanalyzed. The response is also very reproducible. A prolongedseries of 21 repetitive measurements of 2:5 × 10¹5 M cyanide overan 84 min period yielded a mean inhibition signal of 180 nA, and arelative standard deviation of 5 % (Figure 5C).

Figure 6 demonstrates the selectivity of the submersible cyanidesensor in the presence of sodium azide, a poison known for itsinhibitory action on tyrosinase [12]. While the sensor is notresponding to the 1× 10¹4 M azide additions (b,d), it readily detects1 × 10¹5 M changes in the cyanide level (c,e). Only a larger (mM)excess of azide had a small effect upon the response to micromolar

cyanide concentrations (not shown). In view of the small size ofthe azide species, the selectivity (reported in Figure 6) appears toreflect differences in the inhibitory action and not in the dialysissampling. Yet, the membrane permeability may be exploited forenhancing the selectivity in the presence of large inhibitors oftyrosinase.

4. Conclusions

We have demonstrated the optimization and characterizationof a submersible enzyme-inhibition biosensor, capable of remotelydetecting low levels of free cyanide. Challenges associated withthe in situ replenishment of the substrate and enzyme have beenaddressed through a novel probe design, coupling an in situmicrodialysis sampling of the toxin with an internal delivery ofthese reagents and a flow injection manifold. The sensor offers arapid return of the chemical information in a timely manner.Extension to remote monitoring of additional inhibitors isanticipated on the basis of the judicious choice of the enzyme,substrate and permeability of the sampling tubing. Multichannel(multienzymes) devices, connected to a suitable chemometricapproach [13], should lead to a multi-inhibitor detection capability.Remote biosensors of various substrates, such as phenols, shouldalso benefit from the renewable reagent capability (e.g., through anextended lifetime in comparison to devices based on immobilizedenzymes). More sophisticated probes, integrating all the necessarycomponents (including the electronic control, data processor andalarm capability) in a small sensor body are currently beingdesigned. Such realization of in situ, near real-time detection oftoxins, should have a major impact on environmental and industrialmonitoring.

5. Acknowledgements

JW acknowledges financial support from the US DOE,DOE WERC Program and the NM WRRI. Discussions withDr. K. Rogers are also acknowledged.

6. References

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2822.[7] J. Besombes, S. Cosiner, P. Labbe, G. Reverdy,Anal. Chim. Acta1995, 311,

255.[8] T.M. Park, E.I. Iwuoha and M.R. Smyth,Electroanalysis1997, 9, 1120.[9] J. Wang, V. Nascimento, S. Kane, K. Rogers, M. Smyth, L. Angnes,Talanta

1996, 43, 1903.[10] I. Hartley, J.P. Hart,Anal. Comm.1994, 31, 333.[11] J. Wang, J. Lu, D. Luo, J. Wang, M. Jiang, B. Tian,Anal. Chem.1997, 69,

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Fig. 6. Effect of azide upon the response to cyanide: a) blank (deionizedwater) solution; b) same as (a) but after adding 1× 10¹4 M sodium azide;c) same as (b) but after adding 1× 10¹5 M cyanide; d) same as (c) but afteradding 1× 10¹4 M sodium azide; e) same as (d) but after adding 1× 10¹5 Mcyanide. Applied potential:¹200 mV; carrier: 0.05 M phosphate buffer and0.5 mM catechol, flow rate: 20mL=min; enzyme line: 36 units/mL tyrosinase,injection flow rate: 20mL=min, injection time: 25 s.