This article was downloaded by: [Duke University Libraries] On: 19 August 2012, At: 06:14 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK Analytical Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lanl20 Glycerol Dehydrogenase Based Amperometric Biosensor for Monitoring of Glycerol in Alcoholic Beverages Mihaela Niculescu a , Svetlana Sigina b & Elisabeth Csöregi a a Department of Biotechnology, Lund University, Lund, Sweden b Department of Chemistry, Moscow State University, Moscow, Russia Version of record first published: 02 Feb 2007 To cite this article: Mihaela Niculescu, Svetlana Sigina & Elisabeth Csöregi (2003): Glycerol Dehydrogenase Based Amperometric Biosensor for Monitoring of Glycerol in Alcoholic Beverages, Analytical Letters, 36:9, 1721-1737 To link to this article: http://dx.doi.org/10.1081/AL-120023611 PLEASE SCROLL DOWN FOR ARTICLE Full terms and conditions of use: http://www.tandfonline.com/page/terms-and-conditions This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. The publisher does not give any warranty express or implied or make any representation that the contents will be complete or accurate or up to date. The accuracy of any instructions, formulae, and drug doses should be independently verified with primary sources. The publisher shall not be liable for any loss, actions, claims, proceedings, demand, or costs or damages whatsoever or howsoever caused arising directly or indirectly in connection with or arising out of the use of this material.
Transcript
This article was downloaded by: [Duke University Libraries]On: 19 August 2012, At: 06:14Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House,37-41 Mortimer Street, London W1T 3JH, UK
Analytical LettersPublication details, including instructions for authors and subscription information:http://www.tandfonline.com/loi/lanl20
Glycerol Dehydrogenase Based Amperometric Biosensorfor Monitoring of Glycerol in Alcoholic BeveragesMihaela Niculescu a , Svetlana Sigina b & Elisabeth Csöregi aa Department of Biotechnology, Lund University, Lund, Swedenb Department of Chemistry, Moscow State University, Moscow, Russia
Version of record first published: 02 Feb 2007
To cite this article: Mihaela Niculescu, Svetlana Sigina & Elisabeth Csöregi (2003): Glycerol Dehydrogenase BasedAmperometric Biosensor for Monitoring of Glycerol in Alcoholic Beverages, Analytical Letters, 36:9, 1721-1737
To link to this article: http://dx.doi.org/10.1081/AL-120023611
PLEASE SCROLL DOWN FOR ARTICLE
Full terms and conditions of use: http://www.tandfonline.com/page/terms-and-conditions
This article may be used for research, teaching, and private study purposes. Any substantial or systematicreproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form toanyone is expressly forbidden.
The publisher does not give any warranty express or implied or make any representation that the contentswill be complete or accurate or up to date. The accuracy of any instructions, formulae, and drug doses shouldbe independently verified with primary sources. The publisher shall not be liable for any loss, actions, claims,proceedings, demand, or costs or damages whatsoever or howsoever caused arising directly or indirectly inconnection with or arising out of the use of this material.
Glycerol is, besides ethanol, an important component of wine, con-tributing to its viscosity and smoothness, with an accentuated effect on itstaste. The amount of glycerol formed by yeast during the fermentationprocess is normally between 1:10 to 1:15 of that of alcohol, with finalconcentrations varying from 1 to 10 g/L.[1] High glycerol–ethanol ratiosindicate an addition of glycerol, which is a fraudulent action[2]; low ratioscan be the result of addition of ethanol or microbial degradation ofglycerol. The faster an undesirable fermentation can be detected, thebetter both for the quality of wine, and possible economical damages.Therefore, the need of a rapid, sensitive, reliable and at the same timesimple and cheap, method is obvious.
Classical methods for monitoring glycerol in wines are based onoxidation with periodic acid to formaldehyde, prior to extraction withacetone[3,4] or liquid chromatography.[5,6] Several enzymatic methodshave also been developed for the analysis of glycerol, combining theselectivity of the involved enzymes with the simplicity of different detec-tion methods. In these methods, glycerol was quantitatively related to theenzymatically produced NADH, which was monitored spectrophotomet-rically,[7] chemiluminometrically,[8] fluorimetrically,[9,10] or electrochemi-cally.[11–13] Measurement of NADH using different electrodes made ofcarbon, Pt, or Au was usually performed at potentials as high as 0.4, 0.7,or 1V vs. saturated calomel electrode. This high operating potentialcould be improved by using together with GlDH another enzyme, suchas diaphorase[14] or a suitable mediator e.g., K4Fe(CN)6, Meldola Blue
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(MB), Toluidine Blue (TB), N-methylphenazinium (NMP), MethyleneBlue (MeB), phenoxazine and phenothiazine,[15,16] phenazine metho-sulphate (PMS),[17] or different Os polymers.[18,19]
Another often used method for glycerol determination is based onthe enzymatic reaction catalyzed by glycerol kinase (GK), wherebyglycerol is converted to 1-glycerophosphate, which is next oxidized byoxygen in the presence of glycerol phosphate oxidase (GPO), producingdihydroxyacetone phosphate and hydrogen peroxide. Subsequently, theconcentration of H2O2 was measured amperometrically (Pt electrode,650mV)[1,20] or spectrophotometrically.[21] A main drawback of theabove-mentioned method is the high overpotential necessary for H2O2
oxidation, when many electrochemically active compounds present inwine (e.g., phenols and polyphenols), can simultaneously be detectedyielding a bias signal.
In this work, we present a flow injection analysis system withamperometric detection of glycerol in alcoholic beverages. All sensorelements (the enzyme i.e., NADþ dependent-glycerol dehydrogenase,PMS or Os polymer and PEGDGE), except the enzyme cofactor,NADþ, were integrated on the surface of graphite electrode. The devel-oped integrated biosensor configuration is more robust than the onesdescribed in the Lit.[1,12,20] Moreover, the working potential is consider-ably reduced (þ0.3V vs. Ag/AgCl) in comparison to the one used for thedirect detection of biogenerated H2O2
[1,20] or NADH,[12,22] which makesthe hereby described biosensors less prone to interfering reactions.
Finally, the two integrated biosensors were successfully used to deter-mine the glycerol content in wine, with a recovery of 98.6–103.3% whencompared with a reference spectrophotometric method.
EXPERIMENTAL
Chemicals
b-Nicotinamide adenine dinucleotide approximately 99% was pur-chased from Sigma (St. Louis, MO, USA). The NADþ-dependent gly-cerol dehydrogenase from Cellulomonas sp. (EC. 1.1.1.6) and the enzymesubstrate, glycerol, were also from Sigma.
PVI13dmeOs was prepared by complexing poly(1-vinylimidazole)with [osmium(4,40-dimethylbipyridine)2Cl]
þ/2þ, as described elsewhere.[23]
Poly(ethylene glycol) (400) diglycidyl ether (PEGDGE, Polysciences,Warrington, PA, USA) was used for crosslinking the enzyme to theosmium complexed polycation.
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Zero point one molar potassium phosphate buffer, pH¼ 8.5 contai-ning 30mM ammonium sulfate was used as the supporting electrolyte.[14]
The buffer chemicals were obtained from Merck (Darmstadt, Germany).All aqueous solutions were prepared using water purified with a Milli-Qsystem (Millipore, Bedford, MA, USA).
Preparation of Enzyme Electrodes
The graphite electrodes were prepared using rods of solid spectro-scopic graphite (SGL Carbon, Werke Ringsdorff, Bonn, Germany, TypeRW001) with a diameter of 3.05mm. The rods were cut, polished on wetfine emery paper (Tufback, Durite P1200, Allar, Sterling Heights, MI,USA) and thoroughly washed with de-ionized water. The pretreatedgraphite electrodes were modified by placing 5 mL of a mixture containingthe enzyme and the mediator (Type I-GlDH/PMS) or the enzyme, theOs redox polymer and the cross-linking agent (Type II-GlDH/PVI13dmeOs/PEGDGE). The droplet was allowed to dry at room tem-perature for 2 h. The enzyme electrodes were thoroughly rinsed withwater before use. All presented results are the mean of three equallyprepared electrodes if not otherwise mentioned.
Cyclic Voltammetry
Cyclic voltammetric measurements were performed using a BAS100W Electrochemical Analyzer (Bioanalytical Systems, West Lafayette,IN, USA). A three-electrode cell was used equipped with the workingelectrode and an Ag/AgCl as the reference- and a platinum wire as theauxiliary electrode, respectively. The electrolyte solution was phosphatebuffer (100mM, pH 8.5) containing 30mM ammonium sulfate.
Amperometric Measurements
To perform amperometric measurements, the glycerol sensor wasfitted into a Teflon holder and inserted into an in-house built flow-through wall-jet cell. The enzyme electrode was used as the workingelectrode, an Ag/AgCl (0.1M KCl) electrode as the reference-, and aplatinum wire as the auxiliary electrode, respectively. The electrodeswere connected to a three-electrode potentiostat (Zata Elektronik,Hoor, Sweden), controlling the applied potential of the working
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electrode, and the current was recorded on a strip-chart recorder (Kippand Zonen, Delft, The Netherlands). The electrochemical cell was con-nected to a single line flow injection system, in which the carrier flow wasmaintained with a peristaltic pump (ALITEA, Stockholm, Sweden). Thesamples were injected into the carrier flow using an electrically controlledsix-port valve with an injection loop volume of 50 mL (Valco InstrumentsCo. Inc., Houston, TX, USA). The carrier was phosphate buffer, whichwas de-gassed and filtered through a 0.45 mm pore membrane (Millipore)prior to use.
Sample Preparation
Wine samples (Dunavar, Hungary, 1998) from commercial sourceswere analyzed in the flow injection system using the developed biosen-sors. Taking into account that the concentration of the interested com-pounds in wine is far outside the working range of the sensors, a dilutionof the sample was necessary prior to analysis in order to adjust the sampleconcentration to the linear range of the developed biosensors.
Spectrophotometric Experiments
The method used for the validation of the obtained results was basedon the following reaction:
GlycerolþNADþ���!GlDH
DihydroxyacetoneþNADHþHþ
The absorbance change caused by the produced NADH allows thespectrophotometric quantification of the analyte at 340 nm using anUltraspec 1000, UV/Visible Spectrophotometer (Pharmacia Biotech,Boule Nordic AB, Huddinge, Sweden).
RESULTS AND DISCUSSION
Electrocatalysis of NADH Oxidation at
Graphite Modified Electrodes
Efficient and reversible recycling of NADH is of particular interest inthe construction of dehydrogenase-based amperometric biosensors
Monitoring of Glycerol in Alcoholic Beverages 1725
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because more than 450 enzymes require NAD(P)þ as cofactors.[24] Froman analytical point of view mainly the electrochemical oxidation ofNADH is of interest as the formal potential, E0
0
, at pH 7 is �560mVvs. SCE.[15] Studies of the direct electrochemical oxidation of NADH onbiosensors were carried out under different conditions, which are opti-mum for the particular enzymes knowing that most of the commonlyused (proton acceptor) mediators are inactive in alkaline media(pH>8).[25]
The present work targeted both the improvement of NADH deter-mination generated by GlDH (an enzyme with an optimal pH of catalysisin alkaline media) and the application of the optimized biosensors formonitoring the glycerol content in wine.
Several electrochemical mediators (e.g., PMS, PVI13dmeOs, dichlo-roindophenol, 1,2-naphtaquinone, thionin, brilliant cresyl blue ALD,brilliant alizarin blue G, ferrocene carboxaldehyde) were tested bycyclic voltammetry experiments in order to find the most efficient onefor the conversion of NADH back to NADþ. The only mediators thatproved to be efficient, generating a catalytical current for NADH oxida-tion were PMS (see Fig. 1) and PVI13dmeOs (results not shown) underthe studied conditions of 0.1M potassium phosphate buffer, pH¼ 8.5containing 30mM ammonium sulfate, conditions which are optimumfor glycerol dehydrogenase.[14] The other mediators showed lower sensi-tivity (e.g., dichloroindophenol, 1,2-naphtaquinone, thionin, brilliantcresyl blue ALD) or almost no signal at all (e.g., ferrocene carbox-aldehyde, brilliant alizarin blue G).
The biocatalytic oxidation of glycerol by the glycerol dehydrogenase,in the presence of the coenzyme NADþ, can be represented as shown inFig. 2a.
In the presence of a mediatorM, the enzymatically-produced NADHis re-oxidized and the generated MRed is monitored amperometrically.The glycerol concentration is proportional to the yield of NADHformation, the product of the enzymatic reaction, according to thescheme depicted in Fig. 2b.
Optimization
Effect of Electrode Composition
The electrode composition was optimized with regard to theenzyme/mediator and enzyme/mediator/crosslinker ratio. As shown in
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Table 1, the electrodes with a mixture containing 75% GlDH and 25%PMS, and the ones with 22% GlDH, 22% PVI13dmeOs and 56%PEGDGE, respectively, were found to give the best sensitivity andthese ratios have been chosen as optimal for all further experiments.
-2
0
2
4
6(b)
0 500 1000 1500 2000 2500 3000
Time (s)
Cur
rent
(µA
)
Figure 1. (a) Cyclic voltammograms obtained in buffer solution with or without
5mM NADH using an electrode modified with GlDH/PMS as the working elec-
trode. Scan rate: 2mV/s; (b) Chronoamperometric studies for type I electrodes
modified with GlDH/PMS and naked electrodes. Each step represents the addi-
tion of 100mM NADH solution. Applied potential þ300mV (vs. Ag/AgCl).
-20
0
20
40
60(a)
-400 -200 0 200 400Potential (mV)
Cur
rent
(µA
)
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Effect of NADþ Concentration
Attempts to integrate NADþ into the sensor configuration failedsince no signals were obtained. Therefore, NADþ was added in a nextstep in the carrier buffer. However, as NADþ is a rather expensivereagent, NADþ has been included only in the injected sample in a follow-ing strategy. Since the obtained results were similar to the ones obtainedwith NADþ in the carrier, the amount of NADþ added to the injectedsample was optimized. The response increased with increasing NADþ
concentration reaching a plateau, then slowly decreasing (results notshown). The concentration chosen as optimal was thus, 3mM NADþ
for all further experiments for both types of biosensors.
Effect of Applied Potential
The optimum operating potential of the amperometric glycerol mea-surements was determined by studying the dependence of the glycerolresponse on the potential applied at Type I and II electrodes. Figure 3displays the hydrodynamic voltammograms in the �250 to þ450mVpotential range. The amperometric response of glycerol increased withincreasing oxidation potential and reached a limiting value at a potentialof þ300mV for both biosensor types. After this value, the current raisedsharply with the potential, due to the direct oxidation of the NADH and
e- GlDHELECTRODEE=300 mV
vs. Ag/ AgCl
C3 H 8 O 3
Glycerol
C3 H 6O 3Dihydroxyacetone
NADH
NAD +
a)
b)
e -GlDH
MOx
MRed
ELECTRODEE=300 mV
vs. Ag/ AgCl
C3 H 8O 3
Glycerol
C3 H 6 O3
Dihydroxyacetone
NADH
NAD +
Figure 2. Reaction scheme of the amperometric glycerol biosensor in the (a)
absence and in the (b) presence of an electrochemical mediator.
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maybe of glycerol at the electrode. Therefore, this potential value wasselected for further constant potential experiments.
Effect of Flow Rate
The variation of the biosensor response to 0.5mM glycerol with theflow rate was studied over the range 0.2–1.3mL/min. The responsedecreased slowly (results not shown) with increasing flow rates, whichwas attributed to the slow kinetics of the enzymatic reaction. The highestsensitivity of the Type I biosensor response was obtained at 0.5mL/min,a flow rate further used in all experiments. The same flow rate was chosenfor the Type II of biosensor as a compromise between the response timeand the sensitivity of the electrode.
Effect of Solution pH
The effect of solution pH on the analytical performance of the bio-sensor was also studied following the glycerol signal in buffer solutionswith pH values between 7 and 10. Figure 4 shows the resulting responsecurrents vs. pH for the two biosensor types, both exhibiting a signalincrease over the studied range and a leveling off tendency. At pH
0
100
200
300
400
500
-300 -100 100 300 500
Potential (mV)
Cur
rent
(nA
)
Figure 3. Hydrodynamic voltammograms obtained at (�) GlDH/PMS and (g)
GlDH/PVI13dmeOs/PEGDGE electrodes for 0.5mM glycerol in 0.1mM phos-
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values below 8, outside the optimal range for the enzymatic activity, thesignals measured were low. At pH 10 a slight difference can be observedbetween the two electrode types, Type I, showing a decreasing tendency,whereas Type II a leveling off of the signal. This effect might be due to thestructural differences between the two mediators, leading to differentelectrocatalytical mechanisms (i.e., the Os-based polymer can only shuttleelectrons between NADH and the electrode, whereas PMS is both elec-tron and proton acceptor/donor). A pH of 8.5 was chosen for furtherinvestigations for both electrode configurations.
Glycerol Monitoring
Calibration curves for glycerol obtained for Type I and II electrodes,in the absence and in the presence of the mediator, are presented inFigs. 5a and 5b, respectively. As seen, in both cases the mediator mod-ified electrodes displayed an increased biocatalytic activity, the sensitivitybeing approximately three times higher for Type II electrodes than forType I ones. Both calibration graphs exhibit a good linearity up to 1mM,with a correlation coefficient of 0.998.
The sensors based on GlDH/PMS were characterized by a glycerolsensitivity of 0.134� 0.026 mA/mM, a detection limit of 0.9 mM, and a
0
50
100
150
200
250
300
6 7 8 9 10 11
pH
Cur
rent
(nA
)
Figure 4. Effect of solution pH on the response of (�) GlDH/PMS and (g)
GlDH/PVI13dmeOs/PEGDGE biosensors at a flow rate of 0.5mL/min.
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dynamic range of 0.01–1mM. Type II sensors based onGlDH/PVI13dmeOs/PEGDGE were characterized by a higher sensitivityi.e., 0.350� 0.016 mA/mM, a detection limit of 0.1 mM and a similardynamic range (0.01 and 1mM).
Type I electrodes were found to operate continuously at roomtemperature for 15 h when about 50% decrease of the initial signal was
0
1000
2000
3000
4000
5000
6000(b)
0 2 0 4 0 6 0 8 0 100Glycerol (mM)
Cur
rent
(nA
)
y = 346.43x + 106.98
y = 62.602x + 8.2162
0
250
500
0 0.5 1
Figure 5. Glycerol calibration curves obtained for (a) GlDH/PMS and (b)
GlDH/PVI13dmeOs/PEGDGE biosensors both (i or g) in the presence and
(^) in the absence of the electrochemical mediator. Conditions: 0.1mM phos-
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noticed. After the first 2 h of operation the response of the biosensordecreased quickly reaching 80% of the initial signal after which thedecrease was much slower. Type II electrodes were more stable thanType I, 20% decrease of the initial signal being obtained after 15 h(results not shown).
Measurement of Glycerol in Wine
Wine samples purchased from a local supermarket were first dilutedwith the buffer solution, and then analyzed as described above. Theresulting amperometric response was used to construct a graph represent-ing the current signal vs. inverse of wine dilution (see Fig. 6a). Theglycerol content of wine was calculated as the ratio between the slopeof the graph in Fig. 6a ( y¼ 10,943xþ 6.44) and the slope of the glycerolcalibration curve in Fig. 6b ( y¼ 138.65þ 28.12) in the linear range of thesensor (see Ref.[26] for details).
The same commercially available wine was also analyzed by a spec-trophotometric method, demonstrating that the developed biosensors canbe reliably used for determination of glycerol in alcoholic beverages (i.e.,the value obtained by biosensor measurement represented 98.6–103.3%of the reference method, see Table 2).
CONCLUSIONS
This work describes the steps involved in the development of twointegrated biosensors for glycerol monitoring based on GlDH, anelectrochemical mediator (PMS or PVI13dmeOs) and a graphite rod asthe physical transducer. The second type of biosensor, where the enzymeand the Os mediator were components of a redox hydrogel has theadvantage of providing higher sensitivity and a better operationalstability when compared with the first type of biosensor, where the
Table 2. Monitoring of glycerol content in wine (Dunavar,
Hungary, 1998) by different methods.
Biosensor
Glycerol content
electrochemical
(mM)
Glycerol content
spectrophotometrical
(mM)
Type I 79� 6 80� 6
Type II 83� 4
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mediator was PMS. For both types, the response current was found tochange linearly with glycerol concentration over the range 10 mM–1mMand a detection limit in the mM range was obtained. These studies illus-trate the successful use of biosensors for the detection of glycerol at arelatively low potential of þ300mV vs. Ag/AgCl both in model and inreal samples.
0
30
60
90
120(b)
0 0 .1 0.2 0 .3 0.4 0.5Glycerol (mM)
Cur
rent
(nA
)
Figure 6. Determination of glycerol concentration in real samples (Dunavar,
Hungary, 1998) as the ratio between the slopes of the graphs (a) current vs.
inverse of sample dilution and; (b) current vs. glycerol concentration. Biosensor
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ACKNOWLEDGMENTS
The authors are grateful to the European Commission (ContractsCRAFT-1999-70884 and HPRN-CT-2002-00186) and INTAS 00-751for financial support.
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Received March 7, 2003Accepted April 7, 2003
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