Laccase–MWCNT–chitosan biosensor—A new tool for total polyphenolic content evaluation from in vitro cultivated plants

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Laccase–MWCNT–chitosan biosensor—A new tool for total polyphenolic content evaluation from in vitro cultivated plants

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  Sensors and Actuators B 145 (2010) 800–806 Contents lists available at ScienceDirect SensorsandActuatorsB:Chemical  journal homepage: www.elsevier.com/locate/snb Laccase–MWCNT–chitosan biosensor—A new tool for total polyphenolic contentevaluation from  in vitro  cultivated plants Mirela Diaconu ∗ , Simona Carmen Litescu, Gabriel Lucian Radu Center of Bioanalysis, National Institute for Biological Sciences, PO Box 17-16, Bucharest, Romania a r t i c l e i n f o  Article history: Received 2 December 2009Received in revised form 22 January 2010Accepted 26 January 2010 Available online 4 February 2010 Keywords: Laccase biosensorTotal polyphenolic contentElectrodepositionNanocomposite filmChitosanMultiwall carbon nanotubes a b s t r a c t Laccase from  Trametes versicolor  , an enzyme with broad substrate specificity for the phenolic substrateswasemployedasabiorecognitionelementinordertodevelopabiosensorfortotalcontentevaluationof phenolicsecondarymetabolitesfromtwo“ invitro ”cultivatedplants: Salviaofficinalis and Menthapiperita .Theenzymeimmobilizationwascarriedoutbyentrapmentintothenanocompositefilmduringelectrode-positionprocessfrommultiwallcarbonnanotubes(MWCNT)–chitosan(CS)solutioncontaining25U/mL laccase.OptimumconditionsforMWCNT–CSfilmdeposition(2mgMWCNT/mLchitosan1%solutionpre-pared in 1% acetic acid) on gold support using a − 1.5V  vs . Ag/AgCl controlled potential V for 5min wereestablished taking into consideration the layer capacity value. FTIR studies were performed to obtaininformation about the secondary structure of enzyme entrapped into the MWCNT–CS nanocompositefilm. Calibration of the laccase biosensor was performed on four phenolic acids (caffeic acid, chloro-genic acid, gallic acids and rosmarinic acid) as substrates at − 0.2V  vs . Ag/AgCl reference electrode. Thedeveloped biosensor was sensitive to micromolar concentration of the tested polyphenols. The perfor-mance characteristics of the biosensor for rosmarinic acid were: limit of detection 2.33 × 10 − 7 molL  − 1 ,response linear range 9.1 × 10 − 7 –1.21 × 10 − 5 molL  − 1 and sensitivity 846  A/mmol. The obtained valuesof the Km app for all tested substrates proved that nanocomposite film provides a proper environmentfor enzyme immobilization, preserving enzyme catalytic specificity. The functionality of the developedbiosensor was tested to evaluate the total polyphenolic content from real samples ( S. officinalis  and  M. piperita  extracts), results being expressed in equivalent rosmarinic acid. © 2010 Elsevier B.V. All rights reserved. 1. Introduction Polyphenols, one of the most important classes of naturalantioxidants, widely occurring in fruit, vegetables and medicinalplants have been found to have a protective role against manychronic human diseases associated with oxidative stress, like can-cer or cardiovascular disease [1,2]. They have also been employed as markers in taxonomic studies and food quality control [3].Polyphenols are divided into three large groups: phenolic acids(hydroxybenzoic acids and hydroxycinnamic acids), flavonoids(anthocyanidins, flavonols, flavonones, flavones, isoflavones andchalcones) and tannins (hydrolyzable tannins and condensed tan-nins) [4]. Due to the importance of this class of compounds, manyanalyticalstrategiestoevaluatethetotalpolyphenoliccontentfromplant extract and to establish phenolic profile have been reportedintheliterature.Liquidchromatography(LC)isthemostusedtech-nique for polyphenolic compounds separation and quantification.LC with different types of detectors have been employed, among ∗ Corresponding author. Tel.: +40 212200900; fax: +40 212200900. E-mail address:  midi ro@yahoo.com (M. Diaconu). which are the photodiode array detector (DAD) [5,6], mass spec- trometer (MS) [7,8], fluorescence detector [6] or electrochemical detector [4]. Gas Chromatography (GC) coupled to Flame Ioniza-tion or Mass Spectrometer detectors are techniques also usedfor separation, identification and quantification of volatile phe-nolic compounds or non-volatile compounds readily derivatized[9,10]. However these techniques do not easily allow continu-ous monitoring in real samples. Moreover, there are expensive,time-consuming, need skilled operators, and sometimes requirepreconcentrationandextractionstepsthatincreasetheriskofsam-ple loss.Another strategy to assess total polyphenolic content isthe Folin-Ciocalteu (F-C) method [11,12]. However this method requiresmandatorystepsandconditionstoobtainreliableresults:(1) proper volume ratio of alkali and F-C reagent; (2) optimal reac-tion time and temperature for color development; (3) monitoringof the absorbance at 765nm; (4) use of gallic acid as the referencestandard phenol; (5) correction for interfering substances [13].Considering all these tedious steps of analysis, the develop-ment of a simplest procedure capable to provide reliable datawith respect to total polyphenolic content determination appearasnecessaryanduseful.Differentenzymaticbiosensorswithredox 0925-4005/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2010.01.064  M. Diaconu et al. / Sensors and Actuators B 145 (2010) 800–806 801 enzymes have been proposed as an alternative device for totalpolyphenolic content (TPC) assessment. The main advantage of using redox enzymes in amperometric biosensor developing is thevalue of the potential applied to monitor reduction or oxidation of the species at the electrode surface, that generally occur in  − 0.2to 0V range, potential frame that allows to reach a minimum of possible electrochemical interferences [19].Various electrochemical biosensors based on immobilizedtyrosinase [14,15], laccase [16,17], peroxidase [18,19], or laccase- tyrosinase [20,21] have been developed for the TPC evaluation in wine, beer, tea, olive oil, and vegetable extract. The mechanism of polyphenols detection using these types of biosensors is based onmonitoring the reduction current of quinones resulted as conse-quence of polyphenols enzymatic oxidation [22].Immobilization of enzymes on the electrode surface is con-sidered as one of the critical steps that dictate the effectivenessof the enzymatic biosensor, different approaches being devel-oped in order to preserve enzyme specificity and to retain theirnative structure. Enzyme immobilization on electrode surfaces byphysical adsorption, covalent linking of laccase to the surfaces of carbon materials, gold or platinum via bi-functional reagents orenzyme incorporation into a polymer matrix has been described[23–28]. Chitosanisabiocompatiblepolysaccharidewhichexhibitsgoodfilm forming capacity, good adhesion to various supports, highmechanicalstrengthsandhighwaterpermeability.Thepresenceof thereactiveaminoandhydroxylfunctionalgroupsallowschitosanto be easily modified by covalent functionalization, making theresulting composites more stable and with tuned properties. Chi-tosan has been widely used as matrix for enzyme immobilizationthroughionicorcovalentcrosslinking,dip-coatingortheelectrode-positionprocesses[27–30].Electrochemicaldepositionofchitosan has been reported as an effective method for enzyme immobiliza-tion and formation of chitosan film with controllable thickness.During electrodeposition other substances such as redox media-tors [31], gold nanoparticles [32,33], or carbon nanotubes [27–30] canbeincorporatedintothechitosanfilm,leadingtobiocompositeswith enhanced electrical conductivity.In present work, laccase from  Trametes versicolor  , an enzymewith broad substrate specificity for the phenolic substrates [24]was employed as a biorecognition element in order to develop abiosensor for total content evaluation of polyphenolic secondarymetabolites from two “ in vitro ” cultivated plants:  Salvia officinalis and  Mentha piperita . The enzyme immobilization was achieved byentrapmentintothenanocompositefilmduringtheelectrodeposi-tionprocessfrommultiwallcarbonnanotubes(MWCNT)–chitosan(CS) solution.From our knowledge, there are no data in literature reportingentrapment of laccase into the CS–MWCNT layer through the onestep electrodeposition process. Fourier Transform Infrared Spec-troscopy (FTIR) studies performed to obtain information aboutthe secondary structure of laccase entrapped into the CS–MWCNTnanocomposite layer provided the proof of potential preservationof enzyme conformational specificity. 2. Experimental  2.1. Reagents and solutions Gold foil (Au) (99.9% purity, 0.25mm thickness), chitosan(CS) from crab shells (medium molecular weight, 85% deacety-lated), acetic acid, laccase (Lacc) from  T. versicolor   (25U/mgsolid), K 3 [Fe(CN) 6 ] 2,2 ′ -Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) caffeic acid, rosmarinic acid, chlorogenic acidand gallic acid were purchased from Sigma–Aldrich. Multiwallcarbon nanotubes (MWCNT) (97% purity, 10–30nm diameter,length>5  m) were provided by Nanothinx SA, Greece and theywere used as received.Stocksolutionsofthephenoliccompounds(10 − 3 molL  − 1 )wereprepared daily by dissolving appropriate amount in either buffersolutionorinethanoldependingonthephenoliccompoundssolu-bility.DilutedsolutionswerepreparedinMcIlvainebuffer(mixtureof 0.1M citric acid and 0.2M disodium phosphate) at working pH(pH 4.5) which is also used as supporting electrolyte. All reagentsused in this study were of analytical grade and all solutions wereprepared from a Millipore Milli Q system.The plant extract samples were provided by University of Pisa,Department of Pharmacological Sciences, Pisa, Italy and Instituteof Soil Science and Plant Cultivation Pulawi Poland, partners in theFP6 Nutrasnack project.  2.2. Equipment  Electrochemical measurements were performed on Auto-lab System PGSTAT 302N, at room temperature. Experimentswere carried out using a three electrode cell operatingwith an Ag/AgCl (3M KCl) as reference electrode, a plat-inum wire as counter electrode and gold sheets (Au),gold/chitosan (Au/CS), gold/chitosan–multiwall carbon nan-otubes(Au/CS–MWCNT),gold/Laccase–chitosan–multiwallcarbonnanotubes (Au/Lacc–CS–MWCNT) as working electrodes. Cyclicvoltammetry was employed to assess the electrochemical behav-ior of the modified electrodes. Performance characteristics of theAu/Lacc–CS–MWCNT were evaluated using chronoamperometry.Scanning electron microscopy images of chitosan (CS) film andCS–MWCNT nanocomposites film were recorded using the TES-CAN VEGA II LMU Scanning Electron Microscope at an acceleratingpotential of 5kV and working distance of 3.5mm.Spectra of free laccase and immobilized laccase were recordedon a Bruker Tensor 27 Fourier Transform Infrared spectrometerusing KBr pellets technique, or Variable angle reflectance (VAR-FTIR) technique, angle 45 ◦ , under dry air at 25 ◦ C. Each FTIR spectrum represents the average of 64 scans at 4cm − 1 resolution.Brucker’sOPUS6softwarewasusedtorecordandprocesssamplesspectra.  2.3. Preparation of the modified electrodes Beforeeachmodificationgoldfoilusedasworkingelectrodewaspolished with 0.05  m   -Al 2 O 3  nanopowder. After 5min of soni-cation in double distilled water, the electrodes were immersed infreshly prepared Piranha solution (30% H 2 O 2 :concentrated H 2 SO 4 3:1,v:v).After15minofPiranhatreatmentelectrodesweresubmit-ted to ultrasounds once more in double distilled water for 5min.A1%CSsolutionwaspreparedbydissolvingCSin1%aceticacidsolution with magnetic stirring for about 4h and used for prepara-tion of solution undergoing electrodeposition.MWCNT were dispersed in the CS solution by ultrasounds.Before the deposition, suspension was sonicated 6h, until ahomogenous dispersion of MWCNT was achieved. Non-covalentwrapping of the tubular surface with chitosan chain during son-ication led to a stable and homogenous dispersion of MWCNT inaqueous solution. After that Laccase was also homogenously dis-persedinthechitosansolutionduetotheirelectrostaticinteractionwith chitosan chain. It has been widely described in the literaturethat the mechanism of chitosan deposition at negative potentialsis based on their charge and solubility pH dependence [30,34,35]. At slightly acidic pH (pH<6.00) chitosan is protonated and solu-ble. When the pH nears a negative electrode surface it raises above6.3 (p K  a  of CS), due to the proton consumption, the amino groupbecomes deprotonated, leading to an insoluble hydrogel networkonto the electrode surface.  802  M. Diaconu et al. / Sensors and Actuators B 145 (2010) 800–806 Fig. 1.  Dependence of   C   with the concentration of the MWCNT in the 1% chitosansolution. CS and MWCNT–CS films were electrodeposited on goldelectrode surface using a  − 1.5V controlled potential, deposi-tion time was 5min. Optimum conditions for electrodepositionof MWCNT–CS film on a gold electrode were establishedtaking into consideration the value of the layer capacity. Lac-case immobilization was carried out by entrapment into theCS–MWCNTnanocompositefilmfrommultiwallcarbonnanotubes(MWCNT)–chitosan (CS) solution containing 25U/mL enzymeduring electrodeposition process. Such approach, of one steplaccase–chitosan–multiwall carbon nanotubes electrodeposition,was direct and facile, without complex and time-consuming man-ual process.  2.4. Plant sample preparation The samples of plant extracts (sage extracts,  S. officinalis genotypes and mint extracts) were obtained by extraction inethanol:water (7:3, v/v), allowed to evaporate under vacuumfollowed by solutions restored in measuring buffer for use in elec-trochemical analysis. 3. Results and discussions  3.1. Optimization of the MWCNT–chitosan ratio In order to optimize the electrode modification, the effect of MWCNT amount on the nanocomposite layer capacity (C) wasstudied. Cyclic voltammetry experiments were carried out using[Fe(CN) 6 ] 3 − /[Fe(CN) 6 ] 4 − as redox probe to evaluate the electro-chemicalbehaviorofthenanocompositeelectrodesandtoestimatethe values of the layer capacity, parameter defined as: C   = I  where I  istheaverageofanodicandcathodiccurrentdensityand  isthepotentialscanrate[36,37].Byplotting Ivs.  (variedbetween25 and 150mV/s) linear relationships were achieved for all inves-tigated electrodes and the values of the layer capacity ( C  ) werecalculated from the slopes of these equations.Fig. 1 presents the dependence of   C   with the concentration of the MWCNT in the chitosan 1% solution (between 0 and 3mg/mL).The behavior of   C   approached to a linear dependence with theamount of MWCNT employed up to 2mg/mL. A slight decrease of this parameter for MWCNT concentrations greater than 2mg/mL couldbeascribedtoalimiteddispersibilityofthecarbonnanotubesinchitosansolutionandtothefilmthicknessincreasing.Forfurtherexperiments,aconcentrationof2mg/mLMWCNTinchitosansolu-tion (2mg CNT dispersed in 1mL chitosan 1% solution prepared in0.5% acetic acid) was chosen as optimum to prepare the modifiedelectrodes. Fig. 2.  SEM micrograph of the electrochemically deposited chitosan (A) andCS–MWCNT nanocomposite film (B)  3.2. Electrode surface characterization The morphology of chitosan and chitosan–carbon nanotubescomposite films deposed on gold surfaces was characterizedusing SEM techniques. SEM micrograph of the electrodepositedchitosan film (Fig. 2A) showed a smooth and featureless mor-phology, whereas nanocomposites film (Fig. 2B) displayed athree-dimensionalstructurewithcarbonnanotubesuniformlydis-tributed within the film. This characteristic was ascribed to CNTwrapping with the chitosan chains.In order to evaluate the charge-transfer properties on the sur-face of the modified electrodes cyclic voltammetry technique wasemployed, using [Fe(CN) 6 ] 3 − /[Fe(CN) 6 ] 4 − as redox probe. Cyclicvoltamogramms recorded in 5 × 10 − 3 molL  − 1 K 3 Fe(CN) 6  solu-tion prepared in 10 − 1 molL  − 1 KCl are shown in Fig. 3. In thissolution the values of the peak separation (difference betweenanodic peak potential and cathodic peak potential,   E  p ) weredetermined. The obtained values on Au electrode, Au/CS modi-fied electrode and respectively Au/CS–MWCNT modified electrodewere 56mV, 68mV and respectively 60mV. The electrodepositionof MWCNT–CS film on gold electrode surface leads to a two timesincrease in current intensity as a result of the electroactive areaincreasing (see Fig. 3). The anodic-to-cathodic peak current ratiowas 0.8 for this modified electrode. The preservation of a quasi-reversibleelectrodeprocessandthelargeincreaseinpeakcurrentsforthenanocompositefilmmodifiedelectrodeprovedthatMWCNTexerted an obvious improvement effect on electrode properties, Fig. 3.  Cyclic voltammograms for 5mmolL  − 1 [Fe(CN) 6 ] 3 − in 0.1molL  − 1 KCl at Au(a), Au/CS (b) and Au/CS–MWCNT (c) modified electrodes at a scan rate of 50mV/s.  M. Diaconu et al. / Sensors and Actuators B 145 (2010) 800–806 803 Fig. 4.  VAR-FTIR spectra of CS film (A)-red spectrum and CS–Lacc film (B)-grey spectrum. Inset Lacc spectrum enhancing the conductivity and facilitating the charge-transfer of the nanocomposites.  3.3. FTIR studies FTIR studies were performed in order to obtain informationaboutthestructureoftheimmobilizedenzyme.Therecordedspec-tra of enzyme entrapped into nanocomposite film were comparedto those of free enzyme. Taking into account the structural com-plexity of these bio-macromolecules, studies were limited to theinfrared absorption range of 1500–1700cm − 1 , covering the amideI and amide II characteristics absorption bands [38,39]. The FTIR  spectrum of free laccase (Fig. 4, inset) showed two absorptionbands, corresponding to amide I and amide II groups from enzymestructure,locatedat1650and1544cm − 1 .The1650cm − 1 peakwasassigned to the stretching vibrations of the C O amide bond, the1544cm − 1 peak was assigned to the bending vibration of the N–Hbond and stretching vibrations of the C–N bond. The absorptionpeaks for amide I and amide II in the Lacc/CS–CNT film were posi-tioned at 1655 and 1546cm − 1 , suggesting that laccase entrapped Fig. 5.  Typical steady-state current response of the Lacc/MWCNT–CS electrode onsuccessiveinjectionsof20  LABTSinto5mLMcIlvainebufferpH4.5.Appliedpoten-tial +0.35V  vs . Ag/AgCl. Inset is the calibration curve of the electrocatalytic currenton the concentration of ABTS in CS film retained its native structure. The observed slight shiftof peaks wavenumbers to the right and increasing of absorptionbands indicate a possible covalent interaction between chitosanand laccase (see Fig. 4).As consequence, it could be said that FTIR studies providethe proof of potential preservation of enzyme conformationalspecificity, meaning that the conditions of a presumed goodfunctionality as biocatalyst are ensured even after entrapment innanocomposite film.  3.4. Evaluation of the biosensor analytical response Optimal response of the biosensor depends on the operationalparameters(appliedpotentialandpH)andontheselectedstandardsubstrate[40].PerformancecharacteristicsoftheLacc–CS–MWCNTbiosensorwereassessedusingfourphenolicacidsandABTSwhichis both an effective mediator and a well-known unspecific laccasesubstrate [27,26]. Fig. 6.  Influence of the applied potential on the Lacc–CS–MWCNT biosensorresponse for rosmarinic acid in the range from 0.9 to 12.1  molL  − 1 . Measurementcarried out in McIlvaine buffer pH 4.5.  804  M. Diaconu et al. / Sensors and Actuators B 145 (2010) 800–806  Table 1 Performance characteristics of Lacc–CS–MWCNT biosensor.Substrate Applied potential (V/s) Linearity range (molL  − 1 )  R  Sensitivity (mA/mmol) LoD (molL  − 1 ) Km app ABTS +0.35 3.3 × 10 − 6 –4 × 10 − 5 0.9972 0.223 2.75 × 10 − 7 1.8 × 10 − 4 Caffeic acid  − 0.2 7.35 × 10 − 7 –1.05 × 10 − 5 0.9968 1.277 1.51 × 10 − 7 5.18 × 10 − 5 Rosmarinic acid 9.1 × 10 − 7 –1.21 × 10 − 5 0.9792 0.846 2.33 × 10 − 7 2.52 × 10 − 5 Chlorogenic acid 7.93 × 10 − 7 –6.71 × 10 − 6 0.9725 1.268 1.61 × 10 − 7 2.51 × 10 − 6 Gallic acid 7.9 × 10 − 7 –2.1 × 10 − 6 0.9854 3.450 – – Cyclicvoltammetryexperimentswereperformedin1mmolL  − 1 ABTS oxygen saturated solution using Lacc/CS–MWCNT workingelectrode to choose the appropriate potential for chronoampero-metric measurements. Based on the value of the peak potentialof the cathodic range process, a potential of 0.35V  vs . Ag/AgClwas selected to monitor the reduction current of ABTS solution.Steady-state current response of the Lacc/CS–MWCNT electrodeon successive addition of 20  L ABTS (1mmolL  − 1 solution) intoMcIlvaine buffer solution is presented in Fig. 5. The modified elec-trode showed a linear response up to 4 × 10 − 5 molL  − 1 as it couldbe observed from calibration curve presented in Fig. 5 (inset).The small decrease in the biosensor response at higher concen-trations can be ascribed to the saturation of the enzyme active siteby the substrate. A limit of detection (LOD) of 2.7 × 10 − 7 molL  − 1 ,calculated as the analyte concentration corresponding to a 3SD(standard deviation of the sample blank value) [41] was deter-minated. The response time of the biosensor was 2min. Theapparent Michaelis–Menten constant (Km app ) was evaluated fromthe Lineweaver–Burk plot (1/ I vs . 1/ c  ). The obtained Km app value,1.8 × 10 − 4 molL  − 1 issimilartothosereportedintheliterature[26].The optimization of biosensors applied in polyphenols deter-mination is mainly based on using certain specific polyphenoloxidases(PPOx)substrateslikephenol[42],catechol[40,20],caffeic acid[40]orevenrealsamples[44].Inourworktheoptimizationof  designedbiosensorwascarriedoutwithrespecttorosmarinicacidas laccase specific substrate because rosmarinic acid is one of themain components of Salvia species extracts [2] and mint extracts.The main optimization parameter was the value of the appliedpotential in chronoamperometric measurements. The potentialvalue was varied in a domain ensuring the reduction of corre-sponding quinone of rosmarinic acid. The applied potential rangedbetween  − 250 and 50mV and the corresponding response wasregistered. The dependence of biosensor sensitivities over thepotential range is presented in Fig. 6.The maximum Au/Lacc–CS–MWCNT biosensor response wasreached at  − 200mV  vs . Ag/AgCl, this value being used in allsubsequent measurements. Calibration of the biosensor was per-formedforseveralspecificsubstrates:rosmarinicacid,caffeicacid,chlorogenic acid and gallic acid. Performance characteristics of theamperometric response assessed in terms of linear concentrationrange, sensitivity, detection limit and Km app [43] are presented inTable 1.The widest linear range was obtained for caffeic acid androsmarinic acid, with a limit of detection of 1.51 × 10 − 7 molL  − 1 respectively2.33 × 10 − 7 molL  − 1 .Amongstudiedphenolicacidsthebestlinearresponse( R =0.9967)wasobtainedforcaffeicacid.Gallicacid exhibited linear response on a narrower range, with an upperlimit of 2.1 × 10 − 6 molL  − 1 in spite of the 3 times higher sensitivityon the micromolar concentration range with respect to sensitivityforotherinvestigatedcompoundsonthesameconcentrationrange.The phenomenon could be ascribed to the fouling of the biosensorsurface by the enzymatic product, which partially electropolymer-ize to polyaromatic compounds and therefore leads to reducingof the assays number [20,44]. The obtained values of the Km app for all tested substrates proved that nanocomposite layer pro-videsaproperenvironmentforenzymeimmobilization,preservingenzyme catalytic specificity against tested substrates. Operationalstability of Au/Lacc–CS–MWCNT electrode was checked for arosmarinic acid concentration level of 5  molL  − 1 , in MCIlvainebuffer, pH 4.50. Ten consecutive determinations gave a mean cur-rent of 890nA with a relative standard deviation of 5.62%. After15 measurements a 10% decrease of the registered current wasobserved.Toevaluateelectrode-to-electrodereproducibilitytheresponseof five electrodes was measured under optimum working condi-tions for a concentration of 5  molL  − 1 rosmarinic acid. The resultsshowed a RSD of 2.12%.In order to assess the performance level of the proposedbiosensor, its analytical characteristics were compared to thosereportedfordifferentamperometricbiosensorsappliedinpolyphe-nols detection. Data described in recent literature are presented inTable 2.As could be noticed, the sensitivity of the Au/Lacc–CS–MWCNTbiosensor on the micromolar concentration range was three tofive magnitude orders higher than sensitivities values reported inrecent publications [40,42,44]. This behavior was ascribed to the effect of MWCNT incorporation into the chitosan layer.Concluding, due to the high accessible surface area and goodelectrical conductivity, MWCNT drastically improved electrodesproperties,facilitatingcharge-transferofthenanocomposites.Also,  Table 2 Performance characteristics of some amperometric biosensors for polyphenols determination.Biosensor Substrate Linearity range (molL  − 1 ) Sensitivity (mA/mmol) LOD (molL  − 1 ) Km app ReferencesLacc a – Caffeic acid – 1.21 × 10 − 3 2.4 × 10 − 5 – [42]Tyr/diglycerysilanesol–gel matrixGallic acid – 3.39 × 10 − 3 5 × 10 − 5 –Nafion-Lacc/Sonogel– Caffeic acid 4 × 10 − 8 –2 × 10 − 6 9.9 × 10 − 5 6 × 10 − 8 3.29 × 10 − 5 [44]Carbon Gallic acid 10 − 8 –2.2 × 10 − 5 1.1 × 10 − 5 4.1 × 10 − 7 7.64 × 10 − 5 Lacc/polyethersulfone equimolar standard mixturesof catechin and caffeic acid2 × 10 − 6 –1.4 × 10 − 5 5.6 × 10 − 5 1 × 10 − 6 – [40]Lacc/(BMIPF 6 ) b rosmarinic acid 9.99 × 10 − 7 –6.54 × 10 − 5 – 1.8 × 10 − 7 – [45]Dinuclear Fe(III)Zn(II)mimetic complex c Rosmarinic acid 2.98 × 10 − 5 –3.83 × 10 − 4 – 2.30 × 10 − 6 – [46] a Laccase. b Ionic liquids 1- n -butyl-3-methylimidazolium hexafluorophosphate. c [Fe III Zn II (  -OH)bpbpmp-CH 3 ](ClO 4 ) 2 , containing the ligand (H 2 bpbpmp-CH 3  = { 2-[bis(2-pyridylmethyl)aminomethyl]-6-[(2-hydroxy-5-methylbenzyl) (2-pyridyl-methyl) aminomethyl]-4-methyl-phenol } ) which mimics the active site of purple acid phosphatase.
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