Physics and Chemistry of the Earth 50–52 (2012) 252–261

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Physics and Chemistry of the Earth

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Determination of selected persistent organic pollutants in wastewaterfrom landfill leachates, using an amperometric biosensor

Philiswa N. Nomngongo a, J. Catherine Ngila a,⇑, Titus A.M. Msagati a, Bhekumuzi P. Gumbi b,Emmanuel I. Iwuoha c

a Department of Applied Chemistry, University of Johannesburg, PO Box 17011, Doornfontein 2028, Johannesburg, South Africab School of Chemistry, University of KwaZulu Natal, Westville Campus, P. Bag X54001, Durban 4000, South Africac Sensor Lab, Chemistry Department, University of the Western Cape, Bellville 7535, South Africa

a r t i c l e i n f o a b s t r a c t

Article history:Available online 19 August 2012

Keywords:Amperometric biosensorHorseradish peroxidaseEnzyme inhibitionLeachatePersistent organic pollutants

1474-7065/$ - see front matter � 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.pce.2012.08.001

⇑ Corresponding author. Tel.: +27 11 5596196; fax:E-mail address: jcngila@uj.ac.za (J. Catherine Ngila

Landfill leachates that contain persistent organic pollutants (POPs) are a big threat to groundwatersystems and are projected to have hazardous effects in the long term if proper management strategiesof the landfills are not put in place by those responsible. Monitoring the levels of POPs in landfill leachatesis very crucial. This work presents an amperometric biosensor for determination of selected POPs in land-fill leachates. The biosensor is based on kinetic inhibition of horseradish peroxidase (HRP). The enzymewas immobilised by electrostatic attachment on a polyaniline-modified Pt electrode surface. SelectedPOPs inhibited HRP enzyme activity and the decrease in the enzyme activity was used to determine theseenvironmental pollutants. Selected polybrominated diphenyl ethers (PBDEs), polybrominated biphenyls(PBBs) and polychlorinated biphenyls (PCBs) were the analytes of choice because they are commonlyfound in South Africa water systems. Limits of detection for the amperometric biosensor were establishedas 0.014, 0.018, 0.022, 0.016 and 0.019 lg l�1 for BDE-100, PBB-1, PCB-1, PCB-28 and PCB-101,respectively. The HRP biosensor system gave different linear ranges for; BDE-100 (0.424–25.8 lg l�1),PBB-1 (0.862–13.4 lg l�1), PCB-1 (0.930–18.1 lg l�1), PCB-28 (0.730–15.7 lg l�1) and PCB-101 (0.930–27.1 lg l�1). Inhibition studies on HRP biosensor response toward the reduction of H2O2 in the absenceand presence of the selected POPs were carried out to investigate the inhibition kinetics and its mecha-nism. The results obtained indicated that the inhibition mechanism was competitive for PBDEs andnon-competitive for biphenyls (PCBs and PBBs). The application of the biosensor was tested on wastewa-ter samples obtained from landfill leachate for determination of selected POPs. The leachate sampleswere found to contain PCB-28 (0.28 ± 0.03 lg l�1) and PCB-101 (0.31 ± 0.02 lg l�1). The samples werealso analysed by GC–MS as a cross-check method and the two sets of results were in close agreement.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Current management practice for landfill leachate in South Africaprioritises the disposal rather than the actual treatment of landfillleachate. In this regard, current disposal methods for raw leachateinclude evaporation, irrigation to available land, recirculation backto the landfill, or disposal to an available sewer line. The develop-ment of a National Waste Management Strategy for South Africa in1999 (DEAT, 1999) promulgated a paradigm shift of the landfill-mind-set to consider end-of-pipe treatment (Joubert et al., 1999),as opposed to mere dilution principles. Tighter national legislation,more stringent disposal by-laws, an environmental demand fortreatment-at-source, and the unavailability of the sewer-disposaloption, now demand that on-site treatment of landfill leachate be

ll rights reserved.

+27 11 5596425.).

carried out. Additionally, it is by now widely known that the levelsof persistent organic pollutants (POPs) in landfill leachates can behundred times higher than the levels found in domestic sewage.

Persistent organic pollutants (POPs) in water pose a consider-able risk to the environment and human health even at extremelylow concentrations. These pollutants are characterised by markedpersistence against chemical or biological degradation, high envi-ronmental mobility and strong tendency for bioaccumulation inthe food chain (Katsoyiannis and Samara, 2004). Persistent organicpollutants consist of a wide range of compounds which areproduced by industrial activities. These include polybrominateddiphenyl ethers (PBDEs), polybrominated biphenyls (PBBs) andpolychlorinated biphenyls (PCBs), among others.

In view of major concerns regarding the toxicity of PBDEs andPCBs, there is a growing need for innovative ways to monitor theseenvironmental pollutants. It should noted that leachates are a bigthreat to groundwater systems and are projected to have hazardous

Table 1Gas oven temperature program.

Initial temperature(�C)

Ramp(�C min�1)

End temperature(�C)

Hold time(min)

80 – 80 280 50 140 1.5

140 20 220 1220 2 280 0300 30 300 10

P.N. Nomngongo et al. / Physics and Chemistry of the Earth 50–52 (2012) 252–261 253

effects in the long term if proper management strategies are not putin place by those responsible. Many methods have been proposed forthe determination of POPs. The existing methods for detection ofPOPs utilise gas chromatography, GC (Vonderheide, 2009) and highperformance liquid chromatography, HPLC (Vonderheide, 2009),both coupled to different types of detectors such as electron capturedetector, ECD (Wang et al., 2006) or mass spectrometry, MS(Tadeo et al., 2009) for GC; and DAD/UV–Vis (Vilaplana et al.,2009) and MS (Bacaloni et al., 2009) for the HPLC method. Althoughthe conventional chromatography-based methods have high accu-racy and low detection limits, they are, however, sophisticated andrequire skilled operators. They also require a sample preparationstep before detection, which is time consuming and tiresome. Forthis reason, rapid and simpler methods are required for analysis ofthese environmental pollutants. Electrochemical biosensors offeran alternative method. This is because electrochemical techniquesare easy to use, require minimal sample preparation and are easilyminiaturised.

During the past decade biosensors have been employed asuseful monitoring devices in the environmental programmes(Rodriguez-Mozaz et al., 2006; Suwansa-Ard et al., 2005). This isdue to the advantages they possess, such as minimising the samplepretreatment, reducing cost and time of analysis as well as display-ing sufficient sensitivity and selectivity. Recently, attention hasturned to the enzyme inhibition-based biosensors (Yang et al.,2008). The latter is used to determine the concentration of inhibi-tors in the sample by measuring the degree of inhibition with low-er limits of detection. Very few studies have explored the biosensorinhibition approach which offers an indirect method for detectionof organic pollutants at trace levels to improve detection levels.

This work therefore aims to explore the application of thePt/PANi/HRP biosensor according to the following objectives:

� Enzyme kinetic determination of PBDEs, PBBs and PCBs.� The inhibitory effect of POPs on the electrochemical reduction

of hydrogen peroxide (H2O2, substrate) by HRP biosensor.� Determination of POPs in landfill leachate samples.� Cross-check results obtained from the biosensor with those

from a gas chromatography–mass spectrometry (GC–MS)technique.

This study forms part of wastewater management throughmonitoring of the levels of organic substances in leachates dis-charged by landfills.

2. Materials and methods

2.1. Reagents

All chemicals used in this work were of analytical grade unlessotherwise stated. Dichloromethane (DCM), hexane methanol,2,20,4,40,6-pentabromodiphenyl ether (BDE-100), octabromodiphe-nyl ether (octaBDE) mixture of isomers, 2,4,40-trichlorobiphenyl(PCB-28), 2,20,4,5,50-pentachlorobiphenyl (PCB-101), 2-chlorobi-phenyl (PCB-1), 2-bromobiphenyl (PBB-1) disodium hydrogenphosphate (dehydrated) (99%) and sodium dihydrogen phosphate(hydrated) (99%) were obtained from Sigma–Aldrich (South Africa)Ltd. Hydrogen peroxide (H2O2) (30% v/v) was obtained from MerckChemical (Pty) Ltd. The H2O2 (30%) stock solution was stored in arefrigerator at 4 �C. The H2O2 working solutions were freshlyprepared from the stock solution. Phosphate buffer solution(PBS, 0.1 M, pH 7.0) was prepared by mixing appropriate amountsof 0.1 M NaH2PO4 and 0.1 M Na2HPO4. The pH was adjusted with0.1 M NaH2PO4 or 0.1 M Na2HPO4. The PBS was used as supportingelectrolyte for electrochemical measurements.

2.2. Instrumentation

All electrochemical experiments were performed using eitherBAS100W Electrochemical Analyser (Bioanalytical Systems, WestLafayette, IN) or BASi epsilon Electrochemical Analyser equippedwith BASi cell stand (Bioanalytical Systems, West Lafayette, IN).A 15 ml electrochemical cell with a conventional three electrodesystem consisting of platinum electrode as the working electrode(A = 0.0177 cm2), platinum wire as the auxiliary electrode, andAg/AgCl (saturated 3 M NaCl) electrode as the reference electrode.Supporting electrolyte solution was degassed with argon gas forelectrochemical experiments. All measurements were performedat room temperature (20–25 �C). All Fourier transform infrared(FT-IR) and ultraviolet–visible (UV–Vis) spectra were recorded onPerkinElmer Spectrum 100 FT-IR spectrometer equipped withUniversal Attenuated Total Reflectance (ATR) attachment with adiamond crystal and Perkin Elmer Lambda 35 UV–Vis spectrome-ter, respectively.

Gas chromatography–mass spectrometry analysis was per-formed on Thermo Finnigan Trace GC equipped with Thermo Finn-igan ion trap mass spectrometer detector Polaris-Q (ThermoElectron Corporation, Waltham, MA) equipped with autosampler.The separation of the analytes was achieved using fused silicaSGE forte GC capillary column coated with BPX5 (stationary phase5% phenyl polysilphenylene-siloxane, 0.25 mm i.d., 0.25 lm filmthickness, 30 m length). The temperatures for the GC–MS interfaceand ion source were 280 �C and 250 �C, respectively. The massspectrometer was operated in the electron ionisation (EI) modeat 70 eV and helium (flow rate 1 ml min�1) was used as carriergas. Full-scan data acquisition was performed over the mass rangeof m/z 150–596. The GC oven temperature programme is presentedin Table 1.

2.3. Immobilisation of horseradish peroxidase

Horseradish peroxidase immobilisation by electrostatic attach-ment followed the steps described by Nomngongo et al. (2011)and Songa et al. (2009). The biosensor was made by depositing50 ll of 2.0 mg ml�1 of horseradish peroxidase (HRP) on a PANI-modified Pt electrode surface. Fig. 1 shows the schematic diagramfor the preparation of the biosensor.

2.4. Spectrometric characterisation of PANi/HRP film

After enzyme immobilisation, the film was scraped gently fromthe platinum electrode surface. The ATR required not more than2 mm3 of PANi/HRP film (to cover the ATR diamond window).The film, free HRP and PANi film were analysed by FT-IR-ATR spec-trometer. The film was then placed on the surface of the ATR crys-tal and the spectrum was recorded within the wave number rangeof 400–4000 cm�1.

The PANi/HRP film, free HRP and PANi film were dissolved inDMF–PBS mixture, PBS and DMF, respectively, and the solutionswere analysed with UV–Vis spectrophotometry. The resulting

Pt Pt/PANi Pt/PANi/HRP

Aniline

HRP

Polymerization Reduction ofPANi at -0.5 V

Oxidation of PANi at +0.65 V

Pt/PANi

Fig. 1. Schematic representation of the preparation of Pt/PANi/HRP biosensor.

254 P.N. Nomngongo et al. / Physics and Chemistry of the Earth 50–52 (2012) 252–261

solutions were placed in a quartz cuvette of 1 cm path length. TheUV–Vis spectrum was recorded from 200 to 900 nm.

2.5. Electrochemical response to hydrogen peroxide (H2O2) by the Pt/PANi/HRP biosensor

Amperometric measurements were performed in stirred sys-tems by applying a potential of �200 mV to the Pt/PANi/HRP elec-trode (working electrode). Current–time data were recorded aftersteady-state current was reached. The difference between the stea-dy-state current (Iss) upon addition of H2O2, and the backgroundcurrent (I0), was reported as current I. After each experiment, theelectrode was rinsed with double-distilled water and kept in theworking buffer solution at 4 �C.

2.6. Determination of persistent organic pollutants

2.6.1. Procedure for POPs determinationAn aliquot (200 ll) of BDE-100 stock solution (50 mg l�1 in iso-

octane) was first dissolved in 200 ll methanol, then 9.8 ml PBS wasadded with the resulting concentration as 1.0 mg l�1. A concentra-tion of 0.1 mg l�1 was prepared by further diluting an aliquot of10 ml containing 1.0 mg l�1, in a volumetric flask. Amperometricmeasurements for enzyme inhibition by BDE-100 were carriedout in an electrochemical cell containing 2.0 ml of 0.1 M PBS (pH7.02) and constant concentration H2O2 (0.5 mM) with continuousstirring. The experiments were carried out at �0.20 V againstAg/AgCl (3 M NaCl) while allowing the steady-state current to beattained. The desired volume of the inhibitor stock solution (10 lof 1.0 mg l�1 BDE-100) was then added using a micropipette withcontinuous stirring until a steady-state current was obtained. Aftereach experiment the enzyme electrode activity was regenerated byrinsing the electrode with double distilled water. Stock solutions(100 mg l�1) of PCB-1, PCB-28 and PCB-101 were prepared by dis-solving 1.0 mg of each compound in 100 ll methanol then dilutingit to 10 ml in a volumetric flask using PBS (0.1 M, pH 7). The stocksolution (1000 mg l�1) of PBB-1 was prepared by dissolving 75 llin 100 ll of methanol then diluting it to 100 ml in a volumetricflask using PBS (0.1 M, pH 7). Working solutions of PCB-1, PCB-28, PCB-101 and PBB-1 were prepared by serial dilution of thestock solutions in PBS.

2.6.2. Inhibition studiesThe inhibition mechanism was studied by investigating the

relationship between the Pt/PANi/HRP response current to H2O2

(substrate) concentration in the absence and presence of the inhib-itor. The biosensor’s response to various H2O2 concentrations in thepresence of an inhibitor was done as follows: the HRP electrodewas first incubated in PBS containing a known concentration

(IC50) of each inhibitor for 20 min followed by the successive addi-tion of H2O2 concentrations.

Selectivity of the biosensor is very important for the construc-tion and application of the biosensor in actual landfill leachatesamples. Amperometric measurements of Cu2+, Fe2+, Cd2+, Pb2+

and phenol as interferents were measured under the same exper-imental conditions. The effect of the interferents on the signaldetection was investigated by adding 0.5 mg l�1 BDE-100 or PCB-101 in 0.5 mM H2O2 in PBS, and different concentration levels ofinterferents.

2.6.3. Analysis of real samplesLandfill leachate samples were collected in polyethylene con-

tainers from the Mariannhill Landfill outside Durban (EthekwiniMunicipality’s Durban Solid Waste, DSW), and stored in the fridgeat 4 �C. Determination of POPs in the leachate samples was per-formed by two methods, namely amperometric biosensor andGC–MS. The latter was used as a cross-check for the results ob-tained from the fabricated amperometric biosensor. For ampero-metric analysis, determination of POPs in leachate water sampleswas done using standard addition method. The samples were fil-tered to remove solid particles and then spiked with 0.1 mg l�1

of each standard solution using standard addition method, fol-lowed by amperometric measurements.

For GC–MS analysis, the landfill leachate sample was filtered toremove the solid particles. The organic compounds were extractedusing C-18 solid-phase extraction (SPE) cartridges. Prior to use, thecartridges were activated with 5 ml methanol followed by 5 mlwater. After sample loading the SPE cartridges were rinsed with5 ml water. The organics were eluted with 5 ml hexane and theextract was concentrated to about 2 ml under nitrogen followedby GC–MS analysis.

3. Results and discussion

3.1. Spectrometric characterisation of PANi/HRP Film

The stability of HRP after immobilisation was investigated byFTIR-ATR and UV–Vis spectroscopy. The FTIR-ATR spectra of (A) freeHRP, (B) PANI/HRP film and (C) PANI film are presented in Fig. 2:

� In curve A the absorption bands at 1 642 cm�1 and 1 530 cm�1

were assigned to the stretching of amide groups (I and II) ofHRP, respectively (Songa et al., 2009; Sun et al., 2004, 2007;Ma et al., 2007). The amide I band (1700–1600 cm�1) is due toC@O stretching vibrations of the peptide linkages. The amideII band (1620–1500 cm�1) on the other hand results from acombination of NAH in plane bending and CAN stretchingvibrations of the peptide groups (Sun et al., 2007; Ma et al.,2007).

Fig. 2. FT-IR-ATR spectra of (A) free HRP; (B) PANi/HRP film; and (C) PANI film.

P.N. Nomngongo et al. / Physics and Chemistry of the Earth 50–52 (2012) 252–261 255

� It can be observed from curve C that PANI had no absorptionpeaks at the absorption region of amide I and amide II bandsas in the case of HRP and PANi/HRP. Hence, the IR peaks at 1643 cm�1and 1 533 cm�1 for PANi/HRP film (curve B) are thusattributed to those of amide I and II bands of HRP, occurringat similar positions as in the free HRP.� The similarities of the two spectra of free HRP and PANi/HRP

(curve A and B) showed that HRP retained the essential featuresof its secondary structure on the surface of Pt/PANI modifiedelectrode. Sun et al. (2004) reported that when HRP is dena-tured, it shows completely different spectral characteristics inthe amide I and II regions.

UV–Vis spectroscopy is an effective method to investigate thecharacteristic structure of proteins (Yin et al., 2009). For this rea-son, the possible change in the intense peak (Soret peak) of HRPwas monitored by UV–Vis spectroscopy. Fig. 3 shows UV–Vis spec-tra of free HRP (PBS), PANI (in DMF) and PANi/HRP (in PBS–DMFmixture). The UV–Vis spectrum of free HRP in curve A shows aSoret absorption band at 400 nm (Yin et al., 2009). The same wasobserved in PANi/HRP spectrum (curve B) at 404 nm. The slightshift in Soret band for PANi/HRP may be due to the interactionbetween PANI film and HRP during immobilisation (Songa et al.,

Fig. 3. UV–Vis spectra of (A) free HRP in PBS; (B) PANi/HRP in PBS–DMF mixture;and (C) PANI in DMF.

2009). The UV–Vis spectrum (curve B) confirms that the interac-tions of PANI film and HRP do not destroy the structure of theenzyme (Songa et al., 2009). Therefore it can be concluded thatthe HRP was attached and retained its biological activity afterimmobilisation on the Pt/PANI modified electrode (Songa et al.,2009; Yin et al., 2009). The UV–Vis spectrum of PANI in curve Cdoes not have an absorption band at 400 nm; this shows that theabsorption band (404 nm) observed in curve B is entirely due tothe presence of HRP.

3.2. Amperometric detection of H2O2

The principle of an amperometric biosensor was based on themeasurement of the current produced when H2O2 is reduced byHRP at a constant applied potential. The possible mechanism ofPANI-mediated HRP reduction of H2O2 is presented in Fig. 4. Inthe first step of the mechanism, H2O2 in solution diffuses to thesurface of the film where it is reduced by the immobilised HRPand the latter becomes oxidised to form HRP-I (also known asCompound-I). The latter in turn oxidises PANI to give HRP-II (Com-pound II). The HRP resting state, Fe-III is then regenerated via thisintermediate of HRP-II, Compound-II. The partially oxidised PANI+

(emeraldine) is then electrochemically reduced at the electrode toleucoemeraldine (fully reduced form) yielding an enhanced reduc-tion current (Liu and Ju, 2002; Dhand et al., 2011; Iwuoha et al.,1997). The magnitude of the reduction current produced by theelectrode reaction depends on the bulk concentration of the sub-strate, H2O2.

Amperometric responses of the Pt/PANi/HRP biosensor wereinvestigated by consecutively increasing the concentration ofH2O2 at a working potential of �200 mV. Fig. 5 presents a typicalsteady-state current–time plot obtained with the fabricated bio-sensor upon successive additions of 10 ll of 0.01 M H2O2 into2.0 ml PBS resulting in a calibration plot, given as an inset. It wasobserved that, upon the addition of H2O2 into the PBS, the reduc-tion current rises sharply to reach a steady-state value. In addition,the biosensor attained 95% steady-state current within 5 s aftereach addition of 0.010 M H2O2 aliquot. This observation implieda fast response of the fabricated biosensor.

The biosensor’s responses (current signals) were linear toincreasing concentration of H2O2 in the range 0.05 mM to3.17 mM with a correlation coefficient of 0.9991 (n = 18); sensitiv-ity of 1.75 lA mM�1; and a detection limit of 36.8 nM (0.0368 lM)(estimated as signal-to-noise ratio of 3).

Fig. 4. The possible mechanism of PANI-mediated HRP reduction of H2O2, where Fe3+ is the ferric HRP in resting state; Fe�þ

4þ@O stands for oxyferryl HRP-I (Compound I);Fe��

4þAOH is the hydroxyferryl HRP-II (Compound II); and PANI0/+ stands for leucoemeraldine/emeraldine cation radical redox couple (Iwuoha et al., 1997; Dhand et al., 2011).

Fig. 5. Amperometric responses of Pt/PANi/HRP to successive additions of 10 ll0.05 mM of hydrogen peroxide (inset shows the calibration curve). Potential:�0.2 V; supporting electrolyte: 0.1 M PBS (pH 7.02).

256 P.N. Nomngongo et al. / Physics and Chemistry of the Earth 50–52 (2012) 252–261

3.3. Detection of selected brominated flame retardants andpolychlorinated biphenyls in model solution

Fig. 6 shows the typical amperometric Pt/PANi/HRP biosensorresponse to successive additions of 10 ll of 0.01 M H2O2 (r) fol-lowed by successive additions of aliquots (ll) of BDE-100 standardsolutions (0.1 mg l�1) (i) in PBS. The terms ‘r’ and ‘i’ represents theincreasing response signal recorded after the addition of H2O2, andinhibitory effect (decrease in signal) upon the addition of BDE-100,respectively. It can be seen that after each addition of BDE-100, theresponse current decreased. The decrease in response current afterthe addition of BDE-100 indicated that the latter inhibits the activ-ity of HRP. All the four POPs (PBB-1, PCB-1, PCB-28 and PCB-101)were measured in the range 0.1–44.2 lg l�1. The linear ranges, lim-its of detection (LOD ¼ 3�SD

m1), limits of quantification (LOQ ¼ 10�SD

m )as well as regression coefficients for each compound are presentedin Table 2.

The fact that the biosensor could be used to perform severalmeasurements for each analyte without showing a decline in cur-rent intensity suggests that the type of inhibition is reversible asthe performance is not affected. This is because HRP recovers itsactivity once BDE-100 (inhibitor) is absent in the supportingelectrolyte (that is, after the biosensor is reconditioned by placingit in the supporting electrolyte in between the inhibitor measure-ments). Thus, the background current of the Pt/PANi/HRP biosen-sor was restored to the original value of PBS in the absence ofBDE-100 (Songa et al., 2009; Vidal et al., 2008). The response timeof the fabricated biosensor was observed to reach 95% of itsmaximum response after about 5–7 s. As shown in Table 2, the

1 SD refers to the standard deviation of the blank signal (n = 8) obtained in PBS(SD = 4.8 � 10�5 lA) m is the slope of the calibration curve.

fabricated Pt/PANi/HRP biosensor exhibits a long linear range,relatively high sensitivity and low LODs for all the tested POPs.

Table 2 shows that the biosensor had the highest sensitivity to-wards the determination of BDE-100 in aqueous media. However,due to the high hydrophobicity of the octaBDE, no observableresults were recorded. OctaBDE dissolves only in selected hydro-phobic organic solvents (DCM). There is therefore a need to devel-op an organic-phase biosensor for detection of highly hydrophobicPBDEs. Organic-phase biosensors are known to offer a favourable

Fig. 6. Typical amperometric responses of Pt/PANi/HRP biosensor to successiveadditions of 0.01 mM H2O2 (r) and BDE-100 (i); applied potential of �0.20 V;supporting electrolyte of 0.1 M PBS (pH 7.02); where ‘r’ and ‘i’ represents theincreasing response due to the addition of H2O2 and inhibitory effect of BDE-100,respectively.

Fig. 7. Calibration curve showing the inhibition of HRP activity by BDE-100.

P.N. Nomngongo et al. / Physics and Chemistry of the Earth 50–52 (2012) 252–261 257

environment for the detection of water-insoluble analytes and lessinterference from water-soluble analytes (Wu et al., 2004, 2007).Since it was not possible to determine octaBDE in aqueous media,methanol was used as a supporting electrolyte (instead of PBS) forthe electrochemical signal measurements. Electrocatalytic reduc-tion of H2O2 was found to be very slow and the biosensor wasnot as stable as in aqueous media. The degree of stability wasfound to affect both reproducibility and repeatability. Since thebiosensor showed the highest sensitivity to BDE-100 and verylow response to non-aqueous POps, it was concluded that in aque-ous media only hydrophilic PBDEs could be analysed using the fab-ricated biosensor. The determination of the octaBDE mixture ofisomers was not successful.

3.3.1. Inhibition studiesThe values of the steady-state current before (I0) and after (Ii)

the additions of an inhibitor were determined from the recordedamperograms similar to that presented in Fig. 6. The inhibition per-centage (I%) was calculated as per Eq. (1). In order to obtain theinhibitor’s concentration that causes 50% inhibition (IC50),1.0 mg l�1 was used instead of 0.1 mg l�1.

I% ¼ I0 � Ii

I0� 100% ð1Þ

where I% is the inhibition percentage; I0 is the steady state currentbefore addition of an inhibitor; Ii is the steady state current afteraddition of an inhibitor.

The I% values obtained were used to compare the inhibitoryeffect of POPs on immobilised HRP enzyme’s activity. A calibrationcurve constructed by plotting the inhibition percentage of HRPactivity against the concentration of BDE-100 is shown in Fig. 7.

Table 2A summary of analytical characteristics and regression parameters for calibration curves f

POPs Linear range (ppb) Sensitivity (lA ppb�1)

BDE-100 0.424–25.8 9.38 � 10�3 ± 0.005OctaBDE Not detected –PBB-1 0.862–13.4 7.56 � 10�3 ± 0.002PCB-1 0.930–18.9 6.24 � 10�3 ± 0.004PCB-28 0.730–15.7 8.29 � 10�3 ± 0.006PCB-101 0.930–27.1 6.95 � 10�3 ± 0.005

It was observed that the degree of inhibition increased with anincrease in concentration of an inhibitor. From Table 3, it can beseen that the sequence of inhibition to HRP activity is as follows:BDE-100 > PCB-101 > PCB-28 > PCB-1 > PBB-1. The inhibition per-centages as well as the inhibitor concentration leading to 50% inhi-bition (IC50) are presented in Table 3.

3.3.2. Investigation of inhibition kinetics and inhibition mechanismThe biosensor’s response to various H2O2 concentrations re-

corded (Fig. 8) in the absence and presence of BDE-100 (curve A)were interpreted using the Lineweaver–Burk plots (curve B). Inthe absence of the 0.407 mg l�1 (IC50) BDE-100, a fast response tothe additions of different concentrations of H2O2 was observedwhereas in the presence of 0.407 mg l�1 BDE-100, it was slow.The experiment was repeated for other POPs studied in this workand similar observations were made. Sariri et al. (2006a, 2006b)and Zaton and De Aspuru, 1995 suggested that the inhibition ofHRP activity by phenyl-containing compounds could be due tothe incorporation of the phenyl group into enzyme molecules atthe haem periphery thus leading to a decrease in HRP activity.The slope and the y-intercept values of the linear plots were usedto calculate the kinetic parameters (apparent Michaelis–Mentenconstants Kapp

M

� �and maximum current (Imax). The Kapp

M and Imax

values in the absence and presence of selected POPs are presentedin Table 4.

Enzyme kinetic parameters (KappM and Imax) in Table 4 were used

to estimate the inhibition mechanism of POPs to HRP activity. Itcan be observed from Table 4 that the presence of BDE-100 had apositive effect on the Kapp

M value but the Imax value remainedunchanged (1.2 lA) irrespective of the absence or presence of theinhibitor. This indicates that BDE-100 bound to the same HRP ac-tive sites (causing decrease in the amount of free HRP) is availablefor H2O2 binding, thus increasing the Kapp

M (Amine et al., 2006).Based on these results it was concluded that the inhibition

mechanism in this case is competitive inhibition. The latter occurs

or determination of POPs.

LOD (ppb) LOQ (ppb) R2

0.014 0.048 0.9983– – –0.018 0.059 0.99910.022 0.072 0.99660.016 0.054 0.99390.019 0.063 0.9959

Table 3Inhibition percentages and the inhibitor concentration leading to 50% inhibition(IC50).

POPs Degree of inhibition (%) IC50 (ppm)

BDE-100 53.2 ± 1.9 0.407 ± 0.291PBB-1 50.8 ± 0.97 0.487 ± 0.132PCB-1 51.7 ± 1.3 0.531 ± 0.154PCB-28 52.3 ± 0.57 0.542 ± 0.113PCB-101 52.8 ± 1.2 0.506 ± 0.511

258 P.N. Nomngongo et al. / Physics and Chemistry of the Earth 50–52 (2012) 252–261

when the inhibitor competes with the target substrate for the ac-tive binding sites of the enzyme. A possible inhibition mechanismis given in Fig. 9 suggesting that BDE-100 (inhibitor, I) binds only tofree enzyme (E) active sites rather than the enzyme–substrate (ES)complex (Li et al., 2010).

Polychlorinated biphenyls and polybrominated biphenyls af-fected the activity of HRP in a different manner. The Kapp

M valuesin the absence and the presence of biphenyl were not significantlydifferent (based on statistical student t-test, n = 3, P = 0.001 < 0.05),whereas the Imax values were affected (Table 4). The decrease inImax values suggested that the inhibition mechanism is non-com-petitive (Amine et al., 2006). This observation implies that PCBsand PBBs bind to HRP active sites different from where H2O2 binds(Fig. 10). The PBBs and PCBs can be either bound to free HRP or tothe HRP–H2O2 complex (ES). This results in the conformationalchange of HRP at active sites, thus leading to a decrease in theoverall rate of the reaction Imax (Amine et al., 2006).

Fig. 8. (A) Pt/PANi/HRP biosensor response to successive additions of H2O2 in the absenplot for HRP response to H2O2 in the absence and presence of BDE-100.

Table 4Apparent Michealis–Menten constants and maximum current in the absence and the pres

POPs Kinetic parameters

KappM (mM)

Absence of inhibitor Presence of inhibit

BDE-100 1.34 ± 0.03 2.27 ± 0.09PBB-1 2.97 ± 0.13 3.01 ± 0.07PCB-1 3.03 ± 0.09 2.99 ± 0.15PCB-28 3.05 ± 0.02 3.10 ± 0.11PCB-101 3.01 ± 0.05 3.07 ± 0.06

The inhibition constant (Ki) is an absolute value dependent onlyon the inhibitor-enzyme affinity. Generally, the smaller the Ki va-lue, the tighter the binding, and hence the more effective an inhib-itor is. Apparent Ki values for the competitive and non-competitiveinhibitor were calculated from the relationship reported by Besom-bes et al. (1995) and Tanimoto de Albuquerque and Ferreira (2007)using Eqs. (2) and (3), respectively.

Ki ¼Kapp

M ½I�K 0app

M � KappM

ð2Þ

Ki ¼I0max½I�

Imax � I0max

ð3Þ

where K 0appM and I0max are Michaelis–Menten constant and maximum

current in the presence of an inhibitor, respectively.The Ki values (Table 5) suggest that the immobilised HRP en-

zyme presents high affinity to the POPs, particularly, BDE-100,which explains the high degree of inhibition of the HRP activityby POPs (Table 5).

3.4. Selectivity of Pt/PAN/HRP biosensor towards POPs

Selectivity of the biosensor is very important for the construc-tion and application of the biosensor in real landfill leachate sam-ples. Qualitative evaluation of the selectivity of the biosensor wascarried out using phenols and some selected heavy metals. Results(values not included as this was only a qualitative evaluation)

ce and presence of BDE 100, at applied potential of �0.20 V; (B) Lineweaver–Burke

ence of BDE-100 (0.407 ppm).

Imax (lA)

or Absence of inhibitor Presence of inhibitor

1.18 ± 0.02 1.21 ± 0.081.16 ± 0.03 0.984 ± 0.141.24 ± 0.05 0.978 ± 0.071.12 ± 0.06 0.991 ± 0.031.18 ± 0.10 0.986 ± 0.08

E+S ES E+P

+I KI

EI

Competitive inhibition

Fig. 9. Mechanisms for reversible competitive enzyme inhibition.

E+S ES E+P

EI+S EIS

+I +IKI KI

Inhibitor

Substrate

Fig. 10. Mechanisms for reversible non-competitive enzyme inhibition.

Table 5The Ki values in nM for inhibition of HRP by POPs using H2O2 as asubstrate.

POPs Inhibition constant, Ki (nM)

BDE-100 0.983PBB-1 11.7PCB-1 10.5PCB-28 9.22PCB-101 7.88

P.N. Nomngongo et al. / Physics and Chemistry of the Earth 50–52 (2012) 252–261 259

show that phenols did not interfere with the biosensor response.However, Cu2+, Fe2+, Cd2+ and Pb2+ do inhibit the activity of HRP.

3.5. Determination of selected brominated flame retardants (BFRs) andpolychlorinated biphenyls (PCBs) in landfill leachate samples

3.5.1. Pt/PANi/HRP biosensorLandfill leachate samples were analysed for POPs in order to

demonstrate the applicability of the Pt/PAN/HRP biosensor. Theleachate sample was first filtered using 0.45 lm pore size celluloseacetate filter in order to remove insoluble residues. Determination

160 170 180 190 200 210 220 230 240

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e Ab

unda

nce

184.0

219.0

221.0186.1183.0 218.0

197.0174.1 207.1 222.0193.0159.1 163.1 227.0213.0 241.

RT: 12.23 - 16.57

12.5 13.0 13.5 14.0 14.5

Time (min)

05

101520253035404550556065707580859095

100

Rel

ativ

e Ab

unda

nce

13.95

13.55 14.05 14.46 113.6312.26 13.01 13.4712.79 13.1812.65

Leachate sample 1 #1861-1916 RT:13.81-14.22 AV:56 NL:1.39E5T:+ c Full ms [ 150.00-536.00]

Fig. 11. Electron ionisation mass spectra of PCB

of POPs in the leachate samples was done by employing the stan-dard addition method. The sample was spiked with a POP standardto obtain a final concentration of 0.10 mg l�1 and analysed usingthe Pt/PAN/HRP biosensor. Amperometric measurements were car-ried out as discussed in Section 2.6.1, but for this experiment PBScontained 0.5 mM H2O2 instead of 0.95 mM. The concentrationsof the detected POPs (PCB-28 and PCB-101) were calculated fromthe calibration curve. Their concentrations were found to be0.28 ± 0.03 and 0.31 ± 0.02 lg l�1 for PCB-28 and PCB-101, respec-tively. The reported concentration values obtained for PCBs werefound to be within the allowed maximum contaminant level(MCL) (0.5 lg l�1) set by the United States Environmental Protec-tion Agency (Federal Register, 1991).

3.5.2. Analysis of POPs by gas chromatography–mass spectrometryThe validation of the results obtained by the proposed ampero-

metric biosensor was performed using GC–MS as the cross-checkstandard method. The presence of the target analytes detectedby GC–MS in the leachate sample was confirmed by comparingtheir mass spectra to the National Institute of Standards and Tech-nology (NIST, MS Search 2.0) 2002 library. Full-scan Electron Ioni-sation spectra of components found in the leachate sample with

250 260 270 280 290 300 310 320 330m/z

325.8

327.8

323.9

290.9254.0

256.0

288.9

292.9

329.8

258.0

294.9331.8259.0253.0 301.0288.0281.0 332.8267.0 303.01 314.9

4.56

-101. Inset: gas chromatogram of PCB-101.

150 160 170 180 190 200 210 220 230 240 250 260m/z

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rel

ativ

e Ab

unda

nce

186.1

258.0

256.0

188.0259.9

150.9

221.0185.0189.1 223.0151.9 184.1160.1 197.0 207.0 224.0 229.0172.1162.1 213.0193.1 199.1181.0 255.0232.0219.1 243.0 249.0237.1

RT: 10.04 - 11.42

10.1 10.2 10.3 10.4 10.5 10.6 10.7 10.8 10.9 11.0 1Time (min)

10

20

30

40

50

60

70

80

90

100

110

Rel

ativ

e Ab

unda

nce

10.76

10.14

10.1210.33 10.37 10.5610.51 10.8710.4510.28 10.6810.60 10.92 11.0611.03

T: + c Full ms [ 150.00 536.00]

Fig. 12. Electron ionisation mass spectra of PCB-28. Inset: gas chromatogram of PCB-28.

260 P.N. Nomngongo et al. / Physics and Chemistry of the Earth 50–52 (2012) 252–261

fragmentation indicative of PCB-101, are presented in Fig. 11. Themass spectra were assigned according to Safe and Hutzinger(1972), Medina et al. (2009) and Ramos et al. (2007). In the Elec-tron Ionisation spectrum an intense molecular ion (M+) is foundat m/z 326 ([C12H5

35Cl437Cl]+). A less intense molecular ion is

observed at m/z 324 ([C12H535Cl5]+). Loss of one (35Cl), two

(2 � 35Cl, in the case of M+ at m/z 326 and 324) and four(3 � 35Cl and 37Cl) chlorine atoms leads to formation of the majorfragment ions at m/z 291 ([C12H5

35Cl4]+), 256 ([C12H535Cl2

37Cl]+),254 ([C12H5

35Cl3]+) and 184 ([C12H535Cl]+), respectively.

In the mass spectrum of PCB-28 (Fig. 12), the molecular ion wasobserved at m/z 258 ([C12H7

35Cl237Cl]+), along with ions at m/z 256

([C12H735Cl3]+), 186 ([C12H7Cl]+) and 151([C12H7]+). The fragment

ion at m/z 186 was due to the loss of two chlorine atoms(2 � 35Cl) (in the case of M+ at m/z 256) or 35Cl37Cl (in the case ofM+ at m/z 258)). The loss of three chlorine atoms (2 � 35Cl and37Cl) led to the formation of the fragment ion at m/z 151. The typeof fragmentation obtained for both compounds was found to be inclose agreement (100%) with the data given in the NIST library.

The results obtained with the proposed biosensor corroboratedwell with those obtained by GC–MS. Both methods recognised(detected) the presence of the two PCB congeners (PCB-28 and PCB-101). However, it was necessary to spike the individual PCB cong-eners (each at a time) so as to differentiate their specific effects onthe HRP activity. Notwithstanding this limitation, the Pt/PANi/HRPbiosensor shows a high potential for routine analysis with highthroughput for rapid monitoring of POPs in the environment.

4. Conclusions

The application of Pt/PANi/HRP biosensor for the determina-tion of selected POPs has been achieved by enzyme inhibition

mechanism. The degree of inhibition was found to increase withthe increase in concentration of the inhibitor. The percentage inhi-bition of the investigated inhibitors decreases in the following or-der: 53.2%, 52.8%, 52.3%, 51.7% and 50.8% for BDE-100, PCB-101,PCB-28, PCB-1 and PBB-1, respectively. The inhibition mechanismwas found to be of the competitive type for PBDEs but non-com-petitive for PBBs and PCBs. Thus, the amperometric biosensorwas fast, sensitive and showed good linear relationship as well aslow limits of detection for the determination of selected POPs.Since the amperometric biosensor requires minimal sample prep-aration procedures, the Pt/PANi/HRP biosensor can be used as apreliminary screening method for different halogenated aromatichydrocarbons before a detailed quantitative analysis is performed.Thereafter, those that give a positive response could then be re-analysed using the GC–MS method for confirmation. This methodcould be very useful as a quick test for qualitative monitoring ofPOPs in wastewaters and leachates discharged by various landfills.Thus the technique could be used as a management tool for deter-mining the quality of leachate (water) that could have a potentialto be reused, hence an effort to integrate water resources.

Acknowledgement

Philiswa Nomngongo is grateful for NRF funding through grant-holder Prof E.I. Iwuoha of Sensor Lab at the University of WesternCape. The Authors wish to acknowledge financial support from theOrganization for Prohibition of Chemical Weapons (OPCW).

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