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Electrochemical Biosensor for the Determination of Amlodipine Besylate Based on Gelatin–Polyaniline Iron Oxide Biocomposite Film Elbahi Djaalab *, Mohamed El Hadi SAMAR, Saida Zougar and Rochdi Kherrat Laboratory of Environmental Engineering, Department of Process Engineering, Faculty of Engineering Sciences, Badji Mokhtar University-Annaba, P.O. Box 12, 23000 Annaba, Algeria; [email protected] (M.E.H.S.); [email protected] (S.Z.); [email protected] (R.K.) * Correspondence: [email protected]

Received: 19 April 2018; Accepted: 18 May 2018; Published: 4 June 2018

Abstract: In the present study, a new biosensor based on lipase from Candida rugosa (CRL) was developed for amlodipine besylate drug (AMD) with biodegradable material using a mixture of polyaniline iron oxide and gelatin. Polyaniline/Fe2 O3 (PANI@Fe2 O3 ) was prepared by a chemical polymerization method in a medium of ammonium persulfate as an oxidant and characterized by employing Scanning Electron Microscopy (SEM), Fourier Transform Infrared (FTIR), and Ultra-violet (UV) spectroscopy. The purified enzyme was entrapped in the biocomposite matrix film with the aid of a glutaraldehyde cross-linking reagent to establish the immobilization of the lipase. The principle of the biosensor is based on the electrochemical properties of amlodipine besylate (AMD), which were studied for the first time using the cyclic voltammetric method. The cathodic behavior of AMD was measured on the irreversible reduction signal at −0.185 V versus Ag/AgCl at pH 7.4 and 30 ◦ C in a phosphate alkaline buffer. Keywords: biosensors; lipase; amlodipine; polyaniline; cyclic voltammetry

1. Introduction Over the past few decades, biosensors have emerged from the laboratories into the everyday life of millions of people around the world. Like some other sensors, they were first developed for the detection of particular low-molecular species, e.g., metabolites or disease biomarkers, of importance for clinical diagnostics, pharmaceutics, and the healthcare industry [1]. Biosensors have been developed and used in a wide variety of analytical fields, including environmental monitoring, chemical, physics, and biotechnology research, etc. Conducting polymers have attracted much attention in the development of efficient biosensors. Their unique electroactive properties allow them to act as excellent substrates for the immobilization of biomolecules and rapid transfer of electrons [2–6]. Amongst the various conducting polymers, polyaniline (PANI) has been extensively studied as an important conducting material that possesses interesting electrical, electrochemical, and optical properties. The potential applications of PANI include corrosion, secondary batteries, electrochromic devices, and biosensors. In recent years, the preparation of hybrid nanocomposites with both magnetic and electrical properties has received great attention in the industrial and academic fields. Magnetic nanoparticles have been considered interesting materials for the immobilization of desired biomolecules because of their biocompatibility, strong superparamagnetic property, low toxicity, etc. [7,8]. For instance, nanocomposites based on conducting polymers and magnetic nanoparticles are some of the most widely studied materials. Enzymatic biosensors based on polymerized films constitute an important field of pharmaceutical research [9,10]. Enzymes interact specifically with some substrates, and can thus be used for the Catalysts 2018, 8, 233; doi:10.3390/catal8060233

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detection Enzymatic of these substrates. Thebased lipaseon produced by Candida is one an of the most commonly biosensors polymerized filmsrugosa constitute important field of used pharmaceutical Enzymes interact specifically with some substrates, and can thus be enzymes in organic research solvents[9,10]. owing to its high activity in hydrolysis, esterification, transesterification, used forand the biosensing detection of [11–13]. these substrates. The lipase produced by Candida rugosa is one of the most aminolysis, commonly used enzymes in organic owing to its highmedications activity in hydrolysis, esterification, Cardiovascular drugs are amongsolvents the most prescribed nowadays since various transesterification, aminolysis, and biosensing [11–13]. cardiovascular diseases are predominant in developed countries worldwide. In this group, the most Cardiovascular drugs are among the most prescribed medications nowadays since various important drugs are antihypertensives, cardiotonics, antiarrhythmics, anticoagulants, coronary cardiovascular diseases are predominant in developed countries worldwide. In this group, the most vasodilators, and hypolipemics. Since nifedipine was introduced in Germany in 1975 by Bayer important drugs are antihypertensives, cardiotonics, antiarrhythmics, anticoagulants, coronary AG [14], many other products suchSince as nicardipine, nilvadipine, nimodipine, vasodilators, and hypolipemics. nifedipine was introduced nitrendipine, in Germany in foridone, 1975 by Bayer AG benidipine, manidipine, amlodipine, felodipine, and lercanidipine have appeared on the market [15]. [14], many other products such as nicardipine, nilvadipine, nitrendipine, foridone, nimodipine, Amlodipine, chemically, 2-[(2-aminoethoxy)methyl]-4-(2-chlorophenyl)-1,4-dihydro-6-methyl-3,5benidipine, manidipine, amlodipine, felodipine, and lercanidipine have appeared on the market [15]. Amlodipine, acid, chemically, 2-[(2-aminoethoxy)methyl]-4-(2-chlorophenyl)-1,4-dihydro-6-methylpyridinedicarboxylic 3-ethyl,5-methylester, besylate (Figure 1) [15], is a dihydropyridine calcium 3,5-pyridinedicarboxylic besylate (Figure 1) is a dihydropyridine channel blocker, which actsacid, only3-ethyl,5-methylester, on the L-type channel to produce its[15], pharmacological effect [16]. channel blocker, which acts onlyitonhas thegreater L-type channel to produce its pharmacological effect than Like calcium most dihydropyridine derivatives, selectivity for vascular smooth muscle [16]. Like most dihydropyridine derivatives, it has greater selectivity for vascular smooth muscle for myocardial tissue, and therefore its main effect is vasodilatation. Amlodipine is usedthan alone or for myocardial tissue, and therefore its main effect is vasodilatation. Amlodipine is used alone or in in combination with other medicines for the treatment of chronic stable angina and certain types of combination with other medicines for the treatment of chronic stable angina and certain types of vasospastic angina, and in the management of mild to moderate essential hypertension. vasospastic angina, and in the management of mild to moderate essential hypertension. H3 C

N

NH2 O

O

O

H3C O

O

Cl CH3

Figure 1. Chemical structures of amlodipine.

Figure 1. Chemical structures of amlodipine. Several methods, based on various analytical techniques, have been described in the literature

Several methods, based on various besylate analytical the literature for the determination of amlodipine in techniques, pure form ashave wellbeen as indescribed chemistryinlaboratory formulations, pharmaceutical formulations, and in biological fluids.asThey include for the determination of amlodipine besylate pure form well as inspectrophotometric chemistry laboratory methods [17,18], spectrofluorometric methods high-performance thin-layer formulations, pharmaceutical formulations, and[19], biological fluids. They includechromatography spectrophotometric [20], high-performance liquid chromatography (HPLC) [21,22], liquid chromatography (LC) [23], gas [20], methods [17,18], spectrofluorometric methods [19], high-performance thin-layer chromatography chromatography (GC) [24], capillary electrophoresis [25], flow injection analysis [26], and enzymehigh-performance liquid chromatography (HPLC) [21,22], liquid chromatography (LC) [23], linked immunosorbent assay [27]. gas chromatography (GC) [24], capillary electrophoresis [25], flow injection analysis [26], Although these methods have high sensitivity, they are unfortunately multistep, time-consuming and enzyme-linked immunosorbent assay [27]. processes, requiring extensive pretreatment of the sample and qualification for rapid detection. In Although these highattention sensitivity, they on aredeveloping unfortunately multistep, time-consuming recent years, theremethods has beenhave increased focused electroanalytical methods for processes, requiring extensive pretreatment of the sample and qualification for carbon rapid (GC), detection. amlodipine (AMD) analysis using gold electrodes [28], pyrolytic graphite (PG) [29], glassy In recent years,paste there[30,31]. has been increasedattention attention focused on developing electroanalytical or carbon Considerable has been focused on the chemical modificationmethods of microelectrodes in analysis order tousing enhance performance. Graphene–chitosan (GC) for amlodipine (AMD) gold electroanalytical electrodes [28], pyrolytic graphite (PG) [29], glassy carbon nanocomposite [32] and a multi-walled carbon nanotube-modified carbon paste [33] have been (GC), or carbon paste [30,31]. Considerable attention has been focused on the chemical modification developed recently. addition, boron-doped diamond (BDD)performance. electrodes have been fabricated [34,35]. (GC) of microelectrodes in In order to enhance electroanalytical Graphene–chitosan In the present work, we described the synthesis of polyaniline with iron oxide composites for nanocomposite [32] and a multi-walled carbon nanotube-modified carbon paste [33] have been fabrication in the presence of a gelatin substrate for the immobilization of Candida rugosa lipase (CRL), developed recently. In addition, boron-doped diamond (BDD) electrodes have been fabricated [34,35]. and then used this modified platinum electrode in amlodipine biosensor detection. The polyaniline In present work, described the synthesis of UV-vis polyaniline with iron composites wasthe characterized withwe SEM micrography, FTIR and spectroscopy. Tooxide the best of our for fabrication in the presence of a gelatin substrate for the immobilization of Candida rugosa lipase knowledge, this is the first fabrication of polyaniline and gelatin for an amlodipine biosensor based(CRL), and then used this modified electrode in amlodipine biosensor detection. The polyaniline on Candida rugosa lipase. platinum In addition, the experimental conditions for fabrication and analytical was performance characterized with SEM micrography, FTIRelectrochemical and UV-vis methods. spectroscopy. the best of our of the biosensor were optimized with Finally, To we investigated the performance of the amlodipine biosensor, based onand pH and the temperature of the buffer solution.based knowledge, this is the first fabrication of polyaniline gelatin for an amlodipine biosensor on Candida rugosa lipase. In addition, the experimental conditions for fabrication and analytical performance of the biosensor were optimized with electrochemical methods. Finally, we investigated the performance of the amlodipine biosensor, based on pH and the temperature of the buffer solution.


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Catalysts 8, x FOR PEER REVIEW 2. Results and2018, Discussion

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2. ResultsIron andOxide Discussion 2.1. Polyaniline Characterization Catalysts 2018, 8, x FOR PEER REVIEW

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2.1. Polyaniline Iron Oxide Characterization 2.1.1. Polyaniline Iron Oxide Scanning with SEM Micrography 2. Results and Discussion 2.1.1. Polyaniline with SEMdisplaying Micrographythe surface morphology of conducting Figure 2a,b showIron theOxide SEMScanning micrographs 2.1. Polyaniline Iron Oxide Characterization PANI@Fe2 O by the chemical oxidative method, in which a uniform in the Figure 2a,b showsynthesized the SEM micrographs displaying the surface morphology of conducting 3 composites 2O3 composites synthesized by the chemical oxidative method, in which a uniform in theparticles surfacePANI@Fe of polyaniline was observed. The uniform distribution of Fe O contained uniform 2 3 2.1.1. Polyaniline Iron Oxide Scanning with SEM Micrography surface of polyaniline was observed. The uniform distribution of Fe 2O3 contained uniform particles that indicated the good dispersion of iron oxide on the rice straw surface. Figure 2a,b the SEM micrographs displaying the surface that indicated the show good dispersion of iron oxide on the rice straw surface.morphology of conducting In addition, the presence synthesized of iron oxide with good dispersion ofa Fe O on PANI@Fe 2O3 composites by nanoparticles the chemical oxidative method, in which inthe the surface In addition, the presence of iron oxide nanoparticles with good dispersion of Fe2O 3uniform on2 the3 surface of polyaniline has a strong influence on various electrical parameters of these nanocomposites. surface of polyaniline was influence observed.on The uniform distribution of Fe 2of O3these contained uniform particles of polyaniline has a strong various electrical parameters nanocomposites. that indicated the good dispersion of iron oxide on the rice straw surface. In addition, the presence of iron oxide nanoparticles with good dispersion of Fe2O3 on the surface of polyaniline has a strong influence on various electrical parameters of these nanocomposites.

(a)

(b)

2. SEM micrographs, micrographs, (a) (a) 10 μm 5 μm,(b) of the surfaceofof the conducting PANI@Fe 2O3 Figure Figure 2. SEM 10 and µm(b)and 5 µm, surface of conducting composites. PANI@Fe O composites. 2 3 (a) (b) 2. SEM micrographs, (a) 10 μm and (b) 5 μm, of the surface of conducting PANI@Fe2O3 2.1.2. Figure FTIR Spectral Characterization

2.1.2. FTIR Spectral composites.Characterization

Infrared transmission spectroscopy (FTIR) has been widely used to examine conducting

Infrared transmission spectroscopy (FTIR) has been2O widely usedatto1599 examine conducting polymers. polymers. 3a shows the FTIR spectra of PANI@Fe 3. The bands and 1498 cm−1 of PANI 2.1.2. FTIRFigure Spectral Characterization (curve a) correspond to the stretching mode2of C–C at of 1599 quinoid and benzenoid rings. The(curve a) Figure 3a shows the FTIR spectra of PANI@Fe O3C–N . Theand bands and 1498 cm−1 of PANI transmission spectroscopy (FTIR) hasstretching widely used examinering, conducting −1 are bandsInfrared at 1298 and 1242 cmmode attributed the C–C C–N mode of benzenoid the to benzenoid and thebands at correspond to the stretching of C–N to and ofbeen quinoid and rings. The −1 polymers. Figure 3a shows the FTIR spectra of PANI@Fe 2 O 3 . The bands at 1599 and 1498 cm PANI −1 signal at 1168 cm is assigned to the protonated PANI (N–H). The presence of a sharp andofstrong − 1 1298 and 1242 cm are attributed to the mode C–N of stretching mode of the benzenoid ring, and the signal (curveata)592–588 correspond the stretching C–N and stretching C–C of quinoid and benzenoid rings. The band cm−1toindicates the presence of Fe–O vibrations. This indicates the at 1168 bands cm−1atis1298 assigned to the protonated PANI (N–H). The presence of a sharp and strong −1 and 1242composites cm are attributed to the C–N stretching benzenoid ring,3b, and the band formation of polymer [36]. The FTIR spectrum of mode Fe2O3 of is the shown in Figure with − 1 −1 at 592–588 cmat 1168 indicates the presence of Fe–O stretching the formation signal cm isoriginating assigned tofrom the protonated PANI (N–H). presence of aindicates sharp strong characteristic peaks the Fe–O vibration in vibrations. theThe range of This 400–600 cm−1.and The FTIR −1 indicates the presence of Fe–O stretching vibrations. bandcomposites atof592–588 cm indicates the of polymer [36]. The FTIR spectrum of Fe2atO831 shown in−1Figure 3b, characteristic with characteristic spectra the PANI@Fe 2O 3 composites showed bands 881 cm , whichThis are 3 isand −1 . bending formation offrom polymer composites The spectrum Fe2out O3 is shown in Figure 3b, with of the peaks of PANI. The the peak at 684 vibration cm−1[36]. is usually assigned to of the of C–H of plane in PANI. peaks originating Fe–O in FTIR the range 400–600 cm The FTIR spectra characteristic peaks originating from the Fe–O vibration in the range of 400–600 cm−1. The FTIR PANI@Fe2 O3 composites showed bands at 831 and 881 cm−1 , which are characteristic peaks of PANI. spectra of the−PANI@Fe 2O3 composites showed bands at 831 and 881 cm −1, which are characteristic 1 is usually assigned to the C–H out of plane bending in PANI. The peak at 684 cm peaks of PANI. The peak at 684 cm−1 is usually assigned to the C–H out of plane bending in PANI.

(a)

(a)

Figure 3. Cont.


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(b) (b) Figure 3. FT-IR spectra of (a) PANI/Fe2O3 composites (b) pure Fe2O3.

Figure3.3.FT-IR FT-IRspectra spectraofof(a)(a)PANI/Fe PANI/Fe 2O composites(b) (b)pure pureFe Fe22O O33. Figure 2O 33composites 2.1.3. UV Spectroscopy

2.1.3.UV UVSpectroscopy Spectroscopy 2.1.3.

The UV-vis absorption spectra of PANI/Fe2O3 composite dispersions are shown in Figure 4. The

342 nm

2.0

683 nm

342 nm

683 nm

typical absorption spectrum of PANI dispersions distinct absorption peaks atshown 342 in andFigure 683Figure The UV-vis absorption spectra of 2O 3 composite dispersions areare shown 4. The The UV-vis absorption spectra ofPANI/Fe PANI/Fe O composite dispersions in 4. 2has 3 two nm after fittingspectrum (Figure 4). The peak atdispersions 343dispersions nm ariseshas from π–π* electron transition within benzenoid typical absorption of PANI two distinct absorption peaks at 342 andand 683 The typical absorption spectrum of PANI has two distinct absorption peaks at 342 segments, and the wide peak at 683 nm is related to the doping level of polyaniline with iron oxide. nmnm after fitting (Figure 4).4). The peak atat 343 within benzenoid benzenoid 683 after fitting (Figure The peak 343nm nmarises arisesfrom fromπ–π* π–π*electron electron transition within segments,and andthe thewide widepeak peakatat683 683nm nmisisrelated relatedtotothe thedoping dopinglevel levelofofpolyaniline polyanilinewith withiron ironoxide. oxide. segments,

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Figure 4. UV-vis spectra of PANI@Fe2O3 composites. 0.6 200 of the Electrode 400 600 2.2. Electrochemical Characteristics Surface

800

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The cyclic voltammograms of soluble electro active species provide a convenient tool to monitor the various stages of theFigure biosensor buildup on the of electrode. 2O3 composites. UV-vis spectra PANI@Fe Figure 4.4.UV-vis spectra ofPtPANI@Fe 2 O3 composites. Figure 5A shows the cyclic voltammograms of the 5 mM [Fe(CN) 6]4−/3− probe for the bare platinum and modified Pt electrodes in PBS, pHSurface 7.2, at a scan rate of 50 mVs−1. The Pt surface was 2.2.Electrochemical Electrochemical Characteristics ofthe theElectrode Electrode 2.2. Characteristics of Surface modified with polyaniline bio-film (PANI@Fe2O3-Ge-GA-CRL). It can be seen that, for a bare Pt electrode, a characteristic quasi-reversible redox cycle with anodicprovide and cathodic peak currents The cyclicvoltammograms voltammograms ofsoluble solubleelectro electro active species provide convenient toolwas tomonitor monitor The cyclic of active species aaconvenient tool to obtained. When the Ptbiosensor surface wasbuildup functionalized with PANI@Fe 2O3 and lipase enzymes, the electron the various stages of the on the Pt electrode. the various stages of the biosensor buildup on the Pt electrode. transfer between the redox probe and the modified surface was changed. As a4−result, an obvious 4−/3− − probe Figure 5Ashows showsthe thecyclic cyclic voltammogramsofofthe the 5 mM [Fe(CN) 6]/3 probe for the the bare bare Figure 5A 5 mM [Fe(CN) for 6 ] ΔEp and decrease of the anodic and the voltammograms cathodic peaks was observed, leading to a high indicating −1 1 . The platinum and and modified modified Pt Pt electrodes electrodes in Pt surface was platinum in PBS, PBS, pH pH 7.2, 7.2,atataascan scanrate rateofof5050mVs mVs.−The Pt surface the formation of a bioactive layer.

modified with polyaniline bio-film (PANI@Fe 2O3-Ge-GA-CRL). It can be seen that, for a bare Pt was modified with polyaniline bio-film (PANI@Fe 2 O3 -Ge-GA-CRL). It can be seen that, for a bare Pt electrode,aacharacteristic characteristicquasi-reversible quasi-reversible redox redox cycle cyclewith withanodic anodicand andcathodic cathodicpeak peakcurrents currentswas was electrode, obtained. When the Pt surface was functionalized with PANI@Fe 2O3 and lipase enzymes, the electron obtained. When the Pt surface was functionalized with PANI@Fe2 O3 and lipase enzymes, the electron transfer between between the the redox redoxprobe probeand andthe themodified modifiedsurface surfacewas waschanged. changed. As As aa result, result, an an obvious obvious transfer decreaseofofthe theanodic anodicand andthe the cathodic peaks was observed, leading a high indicating decrease cathodic peaks was observed, leading to atohigh ∆EpΔEp and and indicating the the formation of a bioactive layer. formation of a bioactive layer.

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Electrochemical impedance spectroscopy can also give detailed information on the dielectric impedance canchanges. also give detailed information on the spectra dielectric constantElectrochemical and the barrier properties of spectroscopy the deposit layer Figure 5B shows the impedance constant and the barrier properties of the deposit layer changes. Figure 5B shows the impedance of the bare and2018, the 8,modified Catalysts x FOR PEERplatinum REVIEW electrode. The bare Pt electrode reveals a very small semicircle, 5 of 13 spectra of the bare and the modified platinum The bare When Pt electrode revealsisamodified very small implying a very low electron transfer resistance (Ret)electrode. of the redox probe. the electrode semicircle, implying a very low electron transfer resistance (Ret) of the redox probe.on When the electrode Electrochemical impedance spectroscopy can also give detailed information the dielectric with PANI@Fe 2O3-Ge-GA-CRL film, the Ret increases significantly. The deposit film was defined with constantwith and the barrier properties of the deposit layer changes. Figure 5B shows the impedance spectra is modified PANI@Fe O -Ge-GA-CRL film, the Ret increases significantly. The deposit film was 2 the 3 negatively charged (COO–) of enzyme (amino acids containing carboxyl (–COOH) functional – of the bare and the modified platinum electrode. The bare Pt electrode reveals a very small semicircle, defined withacts negatively charged (COO ) of theresists enzyme containing carboxyl (–COOH) groups), which as an electrostatic barrier that the(amino [Fe(CN)acids 6]4−/3− redox probe and hinders its implying a very low electron transfer resistance (Ret) of the redox probe. the When the electrode 4−/3is −modified functional groups), which acts as an electrostatic barrier that resists probe 6 ] of theredox ability towith diffuse into the layer. This phenomenon probably results from the[Fe(CN) inhibition electron PANI@Fe2O3-Ge-GA-CRL film, the Ret increases significantly. The deposit film was defined with and hinders ability to diffuse into the layer. This phenomenon probably results from the inhibition transfer kineticsits between redox probe and the(amino surface of the modified electrode. –) of negatively chargedthe (COO the enzyme acids containing carboxyl (–COOH) functional of the electron transfer kinetics between the redox probe and the surface of the modified electrode. 4−/3− groups), which acts as an electrostatic barrier that resists the [Fe(CN)6] redox probe and hinders its ability to diffuse into the layer. This phenomenon1probably results from the inhibition of the electron 2 a : Pt electrode 0.4 a : Pt electrode b : probe Pt Modified electrode transfer kinetics between the redox and the surface of the modified electrode. b : Pt Modified electrode b

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Figure 5. (A) Voltammograms mVs−1 and (B) Diagram of Nyquist at −200 (B) mV potential, of (a) Pt (A)at 50 Figure 5. (A) Voltammograms at 50 mVs−1 and (B) Diagram of Nyquist at −200 mV potential, of (a) Pt bare electrode and (b) Pt electrode modified with an enzymatic membrane in phosphate buffered bare electrode andVoltammograms (b) Pt electrodeatmodified anDiagram enzymatic membrane in phosphate buffered Figure 5. (A) 50 mVs−1 with and (B) of Nyquist at −200 mV potential, of (a) Ptsaline 3−/4− saline (PBS) solution In the presence of(CN Fe (CN ) − .. (PBS) solution (0.1(0.1 M).M). In the presence of Fe )3−6/4 bare electrode and (b) Pt electrode modified with an enzymatic membrane in phosphate buffered 6 saline (PBS) solution (0.1 M). In the presence of Fe (CN6)3−/4−.

2.3. Cyclic Voltammetry Study of the Biosensor 2.3. Cyclic Voltammetry Study of the Biosensor

2.3. Cyclic Voltammetry Study of the Biosensor

2.3.1. Concentration Effect 2.3.1. Concentration Effect

2.3.1. Concentration Effect

TheThe electrochemical AMD was wasstudied studiedbyby performing cyclic voltammetry in a electrochemicalresponse responseof of AMD performing cyclic voltammetry in a solution The electrochemical response of AMD was studied by performing cyclic voltammetry in a −12 −6 solution containing between 10 10 and of AMD atCRL-PANI@Fe the CRL-PANI@Fe 2O3/Ge-Pt electrode. The −6 10 containing between 10−12 and M ofMAMD at the electrode. The cyclic 2 O3 /Ge-Pt solution containing between 10−12 and 10−6 M of AMD at the CRL-PANI@Fe 2O3/Ge-Pt electrode. The cyclic voltammograms recorded at this electrode shownininFigure Figure6.6.The The cyclic voltammetry voltammetry plots voltammograms recorded at this electrode areare shown plots of cyclic voltammograms recorded at this electrode are shown in Figure 6. Thecyclic cyclic voltammetry plots of AMD show a prominent cathodic peak at ≈−0.18 V, while no anodic peak is observed in the reverse AMDofshow a prominent cathodic peak at ≈− 0.18 V, while no anodic peak is observed in the reverse AMD show a prominent cathodic peak at ≈−0.18 V, while no anodic peak is observed in the reverse scan, indicating the irreversible reduction of AMD at the electrode surface. scan, scan, indicating the the irreversible reduction electrodesurface. surface. indicating irreversible reductionofofAMD AMD at at the the electrode 0.10

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C=0 C=0 -5 C=10 C=10-5 -6 C=10 -6 C=10 -7 C=10-7 C=10 -8 C=10 C=10-8 -9 C=10-9 C=10 C=10-10 -10 C=10-11 C=10 -11 C=10-12 C=10 C=10-12

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Figure 6. CV voltammograms concentrations 5 mM at pH Figure 6. CV voltammogramsofofvarious various AMD AMD concentrations inin 5 mM PBSPBS at pH 7.4. 7.4.

Figure 6. CV voltammograms of various AMD concentrations in 5 mM PBS at pH 7.4.


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A calibration curve was plotted between the magnitude of current response and logarithm of amlodipine concentration (Figure 7). A linear dependent relation was observed, which followed the equation: A calibration curve was plotted between the magnitude of current response and logarithm of

amlodipine concentration 7).× A linear dependent relation was which followed Y = 7.6 ×(Figure 10−2 + 3.5 10−3 .log (AMD concentration); R =observed, 0.993. (1) the equation: The electrochemical developed by us exhibited a wide linearity from 3 Y = 7.6amlodipine × 10−2 + 3.5lipase × 10−sensor .log (AMD concentration); R = 0.993. (1) 10−12 M to 10−5 M (on a logarithmic scale), as well as a low detection limit of 10 −12 M with a regression The electrochemical amlodipine lipase sensor developed by us exhibited a wide linearity from coefficient of 0.993. − 12 − 5 10 M to 10 M (on a logarithmic scale), as well as a low detection limit of 10−12 M with a regression coefficient of 0.993. 0.060 A 0,0759 0,00156 B 0,00353 1,77726E-4 ------------------------------------------------------------

0.055

R SD N P -----------------------------------------------------------0,99247 0,00115 8