Electrochemical Biosensor for Detection of Perfluorooctane Sulfonate Basedon Inhibition Biocatalysis of Enzymatic Fuel CellTingting ZHANG, Huimin ZHAO,* Ayue LEI, and Xie QUAN
Key Laboratory of Industrial Ecology and Environmental Engineering (Ministry of Education, Dalian 116024, China),School of Environmental Science and Technology, Dalian University of Technology, Dalian 116024, China
ABSTRACTAn electrochemical biosensor for sensitive detection of perfluorooctane sulfonate (PFOS) was developed based onthe PFOS inhibition influence on the biocatalysis process of enzymatic biofuel cell (BFC). The one-compartment BFCwas prepared used multi-walled carbon nanohorns (MWNHs) modified glassy carbon electrodes (GCE) as thesubstrates for both bioanode and biocathode, and glutamic dehydrogenase (GLDH) and bilirubin oxidase (BOD)acted as biocatalysts of bioanode and biocathode, respectively. The BFC generated an open circuit potential (Voc) of30.65mVand amaximum power density of 27.5µW/cm2. After interaction times of 20min, a wide linear range from5 to 500nmol/L between ΔVoc and PFOS concentration was achieved with good correlation R2 = 0.976 and numberof measurements is three times (n = 3), and the detection limit was 1.6nmol/L. Furthermore, we choose 4 kindsof perfluorinated chemicals, whose structures are similar with PFOS, including perfluorooctanoic acid (PFOA),nonafluorobutanesulfonic acid potassium (PFBSK), perfluorooctanesulfonamide (PFOSA) and heptadecafluorono-nanoic acid (PFNA), and 2 kinds of chemicals (SMNBS and SDS), which co-exist with PFOS in micro-polluted waterand could possibly disturb the PFOS detection. The electrochemical biosensor exhibited good selectivity for PFOSagainst these chemicals. High precision with relative standard deviation (RSD) (n = 3) from 3.6 to 7.7% for PFOSdetection in real water samples was also demonstrated by the standard addition method. Results obtained in thisstudy indicated that this electrochemical biosensor could be successfully used for selective detection of PFOS in realmicro-polluted water.
Perfluorinated chemicals (PFCs) are synthetic fluorinatedsurfactants composed of a carbon backbone and a chargedfunctional group. As one of the most common PFCs, perfluoro-octane sulfonate (PFOS) was widely used as polymer additives,lubricants, fire retardants, suppressants, and surfactants.1 Manyresearches about PFOS showed that PFOS is harmful to thenervous systems,2,3 cardiovascular system,4 reproductive system,5
immunological system,6 embryo7,8 and postnatal growth.9 PFOS,being defined as a persistent organic pollutant, is physicochemicalstability and detectable in the serums and livers of numerousspecies of wild birds, fish, and mammals in many countries,10
even in the serums of non-occupational exposure humans.11 Itis widely distributed in the environment and be detected in thevarious kinds of micro-polluted water on a global scope,12,13 such asriver water and lake water. The conventional methods to PFOSdetermination include high performance liquid chromatography(HPLC),14 high performance liquid chromatography-mass spec-trometry (HPLC-MS),15 high performance liquid chromatography-mass spectrometry/mass spectrometry (HPLC-MS/MS)16 and gaschromatograph-mass spectrometer (GC-MS),17 these methods arewith complex pretreatment and time-consuming. It is necessary tofind a method for simple, rapid and sensitive detection of PFOS inmicro-polluted water.
Enzymatic biofuel cell is a biochemical-catalyzed system whichconverts chemical energy into electrical energy by oxidizing bio-degradable organic matter in the presence of either microorganismsor enzyme.18 In 2001, Katz et al. used an enzymatic biofuel cell(BFC) as fundamental structure for sensor.19 It was a novel sensor
device using a chemical-electrochemical energy transformationoccurring in BFC as a transducing element, which has begun toattract enormous research interest in recent years. Many researchersfocused on BFC for its operability in mild conditions and potentialapplication in vivo. For example, based on the inhibition of thecathodic enzyme by cyanide, Deng Liu et al. developed a cyanidebiosensor employing a glucose/air biofuel cell,20 in which currentdensity and power density were steadily produced in the absence ofthe analyte cyanide, while decreased in the presence of increasingconcentrations of cyanide. In 2011, Dan Wen et al. developed atrace Hg2+ biosensor employing an alcohol/air biofuel cell by wellestablished T-Hg2+-T coordination chemistry.21 It was found that thepresence of Hg2+ could result in the decrease in open-circuit voltage(Voc) of the BFC by affecting the bioactivity of biocatalysts at bothanode and cathode.
The research of Chunyang Liao indicated that PFOS couldaltered the glutamate-activated current at all doses in cultured rathippocampal neurons.22 In this work, we integrate the metaboliza-tion of glutamate into BFC, an electrochemical biosensor base on anone-compartment miniature glutamate/air BFC for PFOS detectionhas been developed, presented in Scheme 1. The multi-walledcarbon nanohorns (MWNHs) modified glassy carbon electrodes(GCE) were used as the substrates for both bioanode and bio-cathode, and glutamic dehydrogenase (GLDH) acted as biocatalystsfor bioanode. The reaction of bioanode is that the glutamate iscatalyzed by GLDH when Nicotinamide Adenine Dinucleotide(NAD+) exists. In accordance with Olson Cosford R. J.’s research,23
the reaction is as follows:
L-glutamateþ NADþ ! �-ketoglutarateþ NH3 þ NADH
Electrochemistry Received: July 16, 2013Accepted: November 8, 2013Published: February 5, 2014
The Electrochemical Society of Japan http://dx.doi.org/10.5796/electrochemistry.82.94JOI:DN/JST.JSTAGE/electrochemistry/82.94
O2 was used as the electron acceptor in the cathode and bilirubinoxidase (BOD) as biocatalysts for biocathode. The PFOS couldinhibit the bioactivity of biocatalysts, and further decrease the open-circuit voltage of the BFC. To investigate the feasibility of theelectrochemical biosensor, the real samples of micro-polluted watertaken from reservoir and river water was detected. Details of thepreparation, optimal conditions and characterization of the elec-trochemical biosensor were discussed. The experimental is shows ahighly desirable results of the PFOS detection.
2. Experimental Section
2.1 ChenicalsThe MWNHs were purchased from Shenzhen Nanotechnologies
Port Co., Ltd. GLDH (E.C. 1.4.1.3, initial activity of 100U/mg),BOD (E.C. 1.3.3.5, initial activity of 28U/mg), from Myrotheciumverrucaria, L-glutamate (98.5–100.5%), Potassium perfluorooctane-sulfonate (²98%), Perfluorooctanoic acid (²98%), N-Hydroxy-succinimide (NHS), and 1-ethyl-3-(3-dimethylaminopropyl) carbo-diimide (EDC) were obtained from Sigma and used as received.Heptadecafluorononanoic acid was purchased from Fluorochem Ltd.Nonafluorobutanesulfonic acid potassium and perfluorooctanesul-fonamide were purchased from J&K Scientific Ltd. Nicotinamideadenine dinucleotide was purchased from Beijing SuolaibaoTechnology Co., Ltd. Sodium Dodecyl Sulfonate was purchasedfrom Xilong Chemical Co., Ltd. (Shantou, China). Sodium m-nitrobenzene sulfonate was purchased from Damao ChemicalReagent Factory China. A 0.10mol/L Tris-HCl buffer solution(pH 8.0) consisting of Tris and HCl was employed as the supportingelectrolyte. All other chemicals were of analytical grade and thesolutions were prepared with the ultrapure water.
2.2 BFC assemblyThe GCE was used as the substrate for the glutamate/air BFC.
The preparation of bioanode and biocathode for the glutamate/airBFC was referred to the method in articles.24 Prior to modification,the GCE (3mm in diameter) was mildly polished with 0.5 µm and50 nm alumina slurry and then washed ultrasonically in ultrapurewater and ethanol for a few minutes, respectively. 5 µL of theMWNHs suspension was dropped on the pretreated GCE and thennaturally dried at room temperature (noted as MWNHs/GCE). TheMWNHs/GCE electrodes were carefully immersed in a solutioncontaining 10mmol/L EDC and 25mmol/L NHS at least 12 h foractivation. After the activated MWNHs/GCE was thoroughlyrinsed with ultrapure water, 5 µL of 500µg/L GLDH solution wasimmediately dropped on its surface and then incubated at least 12 hat 4°C. It was necessary to rinse the electrode with ultrapure water toremove the loosely bound enzyme molecules (noted as GLDH/
MWNHs/GCE). Finally, the obtained GLDH/MWNHs/GCE wasstored at 4°C prior to use. Similarly, BOD was adsorbed onto theMWNHs/GCE by dropping 5µL of 1mg/mL BOD solution on itssurface and also incubated at least 12 h at 4°C (noted as BOD/MWNHs/GCE). And then, a miniature BFC was constructed usingbioanode and biocathode prepared by above-mentioned methods. A0.10mol/L Tris-HCl (pH 8.0) solution containing 5mmol/L NAD+
and 25mmol/L glutamate was used as fuel solution. The modifiedelectrodes were used as the working electrode. Platinum wire andstandard calomel electrode (SCE, saturated KCl) were used as thecounter electrode and the reference electrode, respectively. Theelectrochemical behaviour of the bioelectrodes and the performanceof the BFC cell were characterized through a CHI 660 electrochem-ical workstation (Shanghai, China).
2.3 Electrochemical biosensor for PFOS detectionThe variation of Voc with PFOS concentration was investigated
for quantitative analysis by electrochemical PFOS sensor. A0.10mol/L stock solution of PFOS was prepared in a 0.10mol/LTris-HCl buffer of pH 8.0, from which PFOS solutions with variousconcentrations were prepared by serial dilutions. To detect PFOS,the sensor was immersed in 0.10mol/L Tris-HCl (pH 8.0) solutioncontaining 5mmol/L NAD+ and 25mmol/L glutamate with a seriesof different concentrations of PFOS. The BFC record was performedafter 20min.
2.4 Electrochemical biosensor for real water samplesdetection
To evaluate the applications of the proposed electrochemicalbiosensor, the detection of PFOS was carried out, and two kinds ofreal micro-polluted samples (i.e., reservoir water and river water)were collected in the locality. The real samples was filtered througha 0.45 µm membrane to removed oils and other solid impurities, andthen diluted with equal volume of 0.20mol/L Tris-HCl (pH 8.0) forthe next experiment.
3. Results and Discussion
3.1 CharacterizationFigure 1(a) shows the image of MWNHs on the surface of
the GCE electrode. The image shows that MWNHs distributesuniformly on the entire surface of the GCE electrode. It indicatesthat the MWNHs has good dispersive capacity in aqueous solutionand can form a well-distributed film on the surface of the electrode.The uniform structure can increase the effective surface of theelectrode for loading of enzyme. After enzyme was modified on thesurface of MWNHs/GCE, some small and uniformly distributedaggregated structures (as designated) appear [Fig. 1(b)]. It indicatesthat enzyme has been effectively immobilized on the surface ofMWNHs/GCE.25
Figure 2 shows cyclic voltammograms (CVs) obtained atdifferent electrodes modified with different concentrations ofWMNHs in 5mmol/L potassium ferricyanide solution. Aftermodified with WMNHs, the WMNHs/GCE shows an obviousincrescent peak current when WMNHs concentrations are 0.1, 0.2,and 0.3mg/ml, respectively. It indicates that the electron transferrate between the electrolyte solution and the electrode surface hasbeen expedited when WMNHs exist. However, it has no obviousincrescent of peak current when the concentrations of WMNHsreach 0.4 and 0.5mg/ml. It explains that the MWNHs film on thesurface of the electrode was too thick when the concentrations ofWMNHs up to 0.4mg/ml, so electron transfer rate have beenaffected. According to the results, 0.3mg/ml WMNHs was chosenfor the following experiments.
After the immobilization of GLDH onto the MWNHs/GCE, theresulting bioanode exhibits bioelectrocatalytic activity for the
Scheme 1. (Color online) Configuration of electrochemical bio-sensor.
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oxidation of glutamate [Fig. 3(a)], the GLDH/MWNHs/GCEexhibits a redox waves at ¹0.25V, which are ascribed to the redoxprocesses of glutamate catalyzed by GLDH. The electrocatalyticcurrent of glutamate at ¹0.25V at the prepared GLDH/MWNHs/GCE, is much higher than that at the prepared MWNHs/GCE.It indicates that there is an effective electrocatalytic activity toglutamate by GLDH/MWNHs modified GCE. In Fig. 3(b), electro-catalytic current reaches its plateau of 302µA at ¹0.25V for theprepared GLDH/MWNHs/GCE bioanode in 0.10mol/L Tris-HCl(pH 8.0) solution containing 25mmol/L glutamate and 5mmol/LNAD+. The prominent electrochemical catalytic property of theGLDH/MWNHs/GCE toward glutamate suggests that the electrodecould be used as bioanode for BFC.
Figure 4(a) shows the CVs of BOD/MWNHs/GCE in 0.10mol/L Tris-HCl (pH 8.0) under three different atmosphere con-ditions (O2, air, and argon atmosphere). In the presence of O2, an
increased cathodic current appears at BOD/MWNHs/GCE andthe electrocatalytic current reaches 7.54 µA at 0.47V. The electro-catalytic current reaches 6.96 µA at 0.47V under ambient air.Figure 4(b) shows the CVs of BOD/MWNHs/GCE, MWNHs/GCE, and BOD/GCE electrodes in 0.10mol/L Tris-HCl (pH 8.0)under O2 atmosphere. There is a defined peak with the apparentpotential of 0.47V at the BOD/MWNHs/GCE electrode. We cansee that O2 is reduction on the BOD/MWNHs/GCE electrode. It isproved that BOD has been effectively immobilized on the surface ofMWNHs/GCE and the BOD/MWNHs/GCE can be employed asbiocathode in the glutamate/air BFC.
Based on the above results, the MWNHs/GCE based bioanodeand biocathode show favourable electrocatalytic activity towardglutamate oxidation and O2 reduction, respectively. So it is feasibleto assemble a glutamate/air BFC with GLDH/MWNHs/GCE andBOD/MWNHs/GCE as bioanode and biocathode, respectively.Figure 5(a) shows the polarization curves of the bioelectrodesobtained in 0.10mol/L Tris-HCl (pH 8.0) containing 25mmol/Lglutamate and 5mmol/L NAD+ under air atmosphere condition.The catalytic current of direct electrocatalytic oxidation of gluta-mate at the GLDH/MWNHs/GCE reaches 38.8 µA at ¹0.25V.The catalytic current of direct electrocatalytic reduction of O2 atthe BOD/MWNHs/GCE reaches ¹4.8 µA at 0.42V. The BFCassembled with the GLDH/MWNHs/GCE and BOD/MWNHs/GCE electrodes hardly suffered from crossover, which make itpossible to assemble a one-compartment miniature glutamate/airBFC in this study. Additionally, the power and operational stabilityare two important factors to be considered in BFC. Figure 5(b)displays the power curve of the assembled glutamate/air BFC inthe above solution. The open-circuit voltage (Voc) of the BFC is
Figure 1. SEM images of MWNHs (a) and enzyme/MWNTs (b)on GCE.
Figure 2. Cyclic voltammograms (CVs) obtained at the GCEbefore and after modified with different concentrations of MWNHs.
Figure 3. (a) CVs obtained at the GLDH/MWNHs/GCE (solidline), MWNHs/GCE (dash line), and GLDH/GCE (dot line) in0.10mol/L Tris-HCl (pH 8.0) containing 5mmol/L NAD+ and 25mmol/L glutamate. (b) CVs at the GLDH/MWNHs/GCE in theabsence (solid line) and presence (short dot line) of 25mmol/Lglutamate.
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30.65mV and the maximum power density (Pmax) of 27.5 µW/cm2
is obtained at 30V. When the BFC is operated for 8 h, it still kept90% Voc of its origin. The result indicated that the BFC had acomparatively stable power output process it maintains 90% of itsoriginal Voc, indicating a comparatively stable power output process[Fig. 5(c)]. Through these test we can assurance that it is possible toassemble a one-compartment miniature glutamate/air BFC in thisstudy.
3.2 Electrochemical response of PFOSThe pH of Tris-HCl solution may affect the enzyme activity. In
order to ensure the activity of enzyme, the glutamate/air BFC wasoperated in Tris-HCl solution with different pH. Figure 6(a) showsthe Voc-t curves of the glutamate/air BFC at different pH. It showsthat the Voc of the cell rises with pH when the pH value of Tris-HClsolution is less than 8, but reversed when the pH of Tris-HClsolution is higher than 8. When the pH of Tris-HCl solution is 8, theVoc of the cell reaches the maximum, so all subsequent analyses arecarried out in pH 8.
Different interaction times might cause different response signals,so the optimum operation time was determined. Figure 6(b) showsthe changes of the Voc (¦Voc) values with interaction times. The Voc
of the cell decreases significantly when 1µmol/L PFOS is addedinto the cell as electrolyte. The result shows that ¦Voc increasedwith interaction times and reached plateau after 20min. Taking theimportance of fast response of the sensing system into consideration,all subsequent analyses use a time interval of 20min for thiselectrochemical biosensor.
Further study under the optimal conditions was carried out viathe Voc-t curve to confirm the feasibility of electrochemicalbiosensor to quantitative determination of PFOS. The electrochem-
ical biosensor was treated with different concentrations of PFOSin 0.10mol/L Tris-HCl (pH 8.0) containing 25mmol/L glutamateand 5mmol/L NAD+ under air atmosphere condition. Figure 7shows the ¦Voc of the cell vs. PFOS concentrations ranged from5 to 25000 nmol/L and linear relationship between ¦Voc andthe logarithm of PFOS concentrations. The electrochemical PFOSsensor follows a logarithmic relation: there is a good linearrelationship between ¦Voc and PFOS concentration from 5 to 500nmol/L (R2 = 0.976, n = 3). The lowest detectable concentration of1.6 nmol/L is obtained by calculation from this research and therelative standard deviation (RSD) for three repeated measurementsof 100 nM PFOS was 1.89%.
The composition of real environmental samples is complex;PFOS could co-exist with a variety of compounds in micro-pollutedwater. And some other perfluorinated chemicals with similarstructure to PFOS could affect the detection of PFOS. Therefore,to evaluate the selectivity of the electrochemical biosensor, we
Figure 4. (a) CVs at the BOD/MWNHs/GCE in 0.10mol/LTris-HCl (pH 8.0) under the O2 (solid line), Air (dash line), and Ar (shortdash line) atmosphere conditions. (b) CVs obtained at the BOD/MWNHs/GCE (soild line), MWNHs/GCE (dash dot line), andBOD/GCE (dot line) under the O2 atmosphere conditions.
Figure 5. (a) Polarization curves of the bioanode (solid line) andbiocathode (dash line). (b) Dependence of the power density on thecell voltage in 0.10mol/L Tirs-HCl (pH 8.0) under ambient aircontaining 5mmol/L NAD+ and 25mmol/L glutamate. (c) Stabilityof the Voc of the glutamate/ari BFC.
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choose 4 kinds of perfluorinated chemicals, whose structures aresimilar with PFOS, including PFOA, PFBSK, PFOSA and PFNA,and 2 kinds of chemicals (SMNBS and SDS), which co-existswith PFOS in micro-polluted water and could possibly disturb thePFOS detection. All the chemical substances mentioned abovewere detected under the same conditions with the concentrationof 1 µmol/L, the ¦Voc of PFOS is much higher than the other
chemicals, which is 14 to 46 times as much as those of the selectiveinterfering substances (Fig. 8). It is proved that the electrochemicalbiosensor have a good selectivity of PFOS against PFOA, SMNBS,SDS, PFBSK, PFOSA, and PFNA.
3.3 Analysis of real water samplesIn order to evaluated the feasibility of PFOS detection with this
method, two kinds of real water samples (i.e., reservoir water andriver water) were collected from Dalian. The real water sampleswere filtered (0.45 µm) first and then diluted with equal volume of0.20mol/L Tris-HCl (pH 8.0), and finally investigated by addingPFOS of three different concentration. The result suggests that thismethod shows a good recovery (97.3–106.5%) and the RSD forthree repeated measurements was from 3.6 to 7.7%. The results aresummarized in Table 1 as follows.
4. Conclusions
In summary, we have developed a one-compartment miniatureglutamate/air BFC using glutamic dehydrogenase and bilirubinoxidase as bioanode and biocathode biocatalysts, respectively. Theminiature BFC prepared showed a Voc of 30.65mV and maximumpower density of 27.5 µW/cm2. The power output process of theelectrochemical biosensor was comparatively stable in 8 h. Thisanalytical method showed a linear range from 5 to 500 nmol/L and adetection limit of 1.6 nmol/L for PFOS detection. It also exhibited a
Figure 6. (a) The Voc-t diagram in different pH value of electro-lyte for BFC. (b) Time-dependent signal response for 1µmol/LPFOS (the measurements were carried out in triplicate). Theincubation times are 0, 5, 10, 15, 20, and 25min, respectively.The solution of the cell operation is 0.10mol/L Tris-HCl containing5mmol/L NAD+ and 25mmol/L glutamate.
Table 1. Determination of PFOS content in real water samplesusing the electrochemical biosensor.
Watersamples
Added(nM)
Measured(nM)
Recovery(%)
RSD(%, n = 3)
Reservoir water
10 9.7 97.5 3.6
50 50.9 101.8 6.2
100 98.6 98.6 5.3
River water
10 10.6 106.5 4.5
50 48.5 97.3 7.7
100 105.4 105.4 6.2
Figure 7. Plot of ¦Voc vs PFOS concentrations: 0, 5, 10, 20, 50,100, 500, 1000, 5000, 10000, and 25000 nmol/L, respectively andlinear relationship between ¦Voc and the logarithm of PFOSconcentrations. The solution of the cell operation is 0.10mol/LTris-HCl containing 5mmol/L NAD+ and 25mmol/L glutamate. ¦Voc
represents the absolute value of difference Voc of the BFC betweenwith and without PFOS.
Figure 8. Normalized ¦Voc of the electrochemical biosensor inresponse to PFOS, PFOA, SMNBS, SDS, PFBSK, PFOSA, andPFNA (the measurements were carried out in triplicate). Theconcentration of each substance is 1 µmol/L, and the incubationtime is 20min. The solution of the cell operation is 0.10mol/L Tris-HCl containing 5mmol/L NAD+ and 25mmol/L glutamate. ¦Voc
represents the absolute value of difference Voc of the BFC betweenPFOS, PFOA, SMNBS, SDS, PFBSK, PFOSA, and PFNA.
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good selectivity for PFOS. Additionally, study on applications ofthe sensor in real water samples, reservoir water and river water,demonstrated satisfactory results. The electrochemical biosensorprovides a rapid, economical, sensitive, selective, and environmentfriendly method to PFOS detection.
Acknowledgments
This work was supported by the National Natural ScienceFoundation of China (No. 21277016).
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