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Synthetic Metals 162 (2012) 688– 694

Contents lists available at SciVerse ScienceDirect

Synthetic Metals

journa l h o me pag e: www.elsev ier .com/ locate /synmet

evelopment of a novel amperometric glucose biosensor based on copolymer ofyrrole-PAMAM dendrimers

ehmet S enel ∗, Cevdet Nergiz ∗

epartment of Chemistry, Faculty of Arts and Sciences, Fatih University, B.Cekmece, Istanbul 34500, Turkey

r t i c l e i n f o

rticle history:eceived 24 June 2011eceived in revised form 1 November 2011ccepted 20 February 2012vailable online 22 March 2012

eywords:AMAM

a b s t r a c t

Glucose oxidase (GOx) was covalently immobilised onto an electrochemically prepared co-polymer com-posed of amidoamine-pyrrole dendrimers, for the construction of an amperometric glucose biosensor.First, second, and third generation amidoamine-pyrrole dendrimers with branched amine peripheryand focal pyrrole functionality were synthesised via divergent pathways. Dendronised polypyrrole wassynthesised by electrochemical copolymerisation of the aminoamine-pyrrole dendrimers from pyrrolemonomers. These copolymers have been utilised as conducting films for amperometric glucose sensing.GOx retains its bioactivity after covalent immobilisation onto dendronised pyrrole-copolymers. Amper-

yrrolelucose oxidaseiosensorovalent immobilization

ometric response was measured as a function of glucose concentration, at a fixed potential of +0.7 V vs.Ag/AgCl in a phosphate buffered saline solution (pH 7.5). The effects of pH and temperature on storageand reusability of the amperometric glucose biosensor were investigated. Our results indicate efficientimmobilisation of the enzyme onto a PAMAM type dendrimer modified surface containing a pyrrolemonomer linked to a PAMAM dendron, with high enzyme loading, and increased electrode lifetime andstability.

. Introduction

The glucose level determination is important in the food andermentation industries and in clinical chemistry, and, as such,here have been many reports on this subject. The most frequentlymployed glucose biosensors are based on the glucose oxidasenzyme (GOx). In previous studies, catalytic conversion of glu-ose to gluconolactone by GOx in the presence of dioxygen haseen shown to result in hydrogen peroxide production, which wasetected electrochemically. This problem was later overcome, by

ntroducing mediators to replace oxygen as the means of electronransfer [1–3]. Recently, due to both a fundamental interest in thelectron-transfer reaction between GOx and electrodes, and for usen the development of glucose sensors with long-term stability,everal redox-active polymers have been prepared and utilized asolymeric mediators [4–9].

The development of biosensors requires the creation ofn efficient interface between the biomolecules and the elec-ronic transducers. Conducting polymer interfaces are particularly

∗ Corresponding authors at: Fatih University, Department of Chemistry,uyukcekmece Kampusu, 34500 B.Cekmece, Istanbul, Turkey.el.: +90 2128663300; fax: +90 2128663402.

E-mail addresses: msenel@fatih.edu.tr (M. S enel),nergiz@fatih.edu.tr (C. Nergiz).

379-6779/$ – see front matter © 2012 Elsevier B.V. All rights reserved.oi:10.1016/j.synthmet.2012.02.018

© 2012 Elsevier B.V. All rights reserved.

suitable for localizing biomolecules onto surfaces [10–12]. Poly-conjugated conducting polymers were recently proposed for use inbiosensing applications, because of a number of favorable charac-teristics, such as: (1) easy and direct deposition on the electrodesurface, (2) thickness control, (3) redox conductivity and poly-electrolyte characteristics [13]. Polypyrrole fulfills all of the aboverequirements and, in addition, is easily oxidized, has high chemicalstability, and low monomer cost [14].

Dendrimers have been extensively studied over the past threedecades, due to the unique architectural and functional controlachievable during their synthesis [15–17]. Despite the utility ofenriching the structural diversity of these dendrimers, researchershave mostly focused on dendrimers with small cores. However,a breakthrough in the synthesis of dendronized polymers wasreported [18], which recognized the significance of dendron func-tionalization for the backbone conformation and overall shape ofthe resulting macromolecules. Recently, there has been increasinginterest in exploiting these dendritic macromolecules, or dendronappendages, for the chemical and surface modification of silica [19],carbon [20], chitosan [21], DNA [20] and thiophene [22].

This work reports the preparation of conducting polypyrroleswith dendritic substituents to obtain conducting polymer-modified

electrodes. First, second, and third generation amidoamine-pyrroledendrimers, with branched amine periphery and focal pyrrolefunctionality, were synthesised via divergent pathways. Copoly-mers were synthesized using electrochemical copolymerisation of

M. S enel, C. Nergiz / Synthetic Metals 162 (2012) 688– 694 689

F hanold hanol.

asFog

2

2

dNcPgda

2

e(rbN4A1(2

ig. 1. Synthetic pathway of pyrrole-PAMAM dendrimers (i) methyl acrylate in metiamine in methanol, (v) methyl acrylate in methanol, (vi) ethylenediamine in met

midoamine-pyrrole dendrimers with pyrrole monomers. Theseynthesized dendrimers and copolymers were characterized byTIR-ATR and NMR, and utilized as conducting films for amper-metric glucose sensing. The relationship between Dendroneneration and sensor properties is discussed.

. Experimental

.1. Materials

Glucose oxidase (GOx) (EC 1.1.3.4), 1-ethyl-3-(3-imethylaminopropyl) carbodiimide hydrochloride (EDC), and-hydroxy-succinimide (NHS) were obtained from Sigma Chemi-al Co. 1-Cyanoethylpyrrole monomer was obtained from Aldrich.yrrole monomer, p-toluene sulphonic acid sodium salt, andlucose were procured from Fluka. Methyl acrylate and ethylene-iamine were obtained from Merck. All other chemicals were ofnalytical grade and were used without further purification.

.2. Synthesis of N-(3-aminopropyl)-pyrrole

A solution of 1-(2-cyanoethyl)pyrrole (0.02 mol) in anhydrousther (15 mL) was added dropwise to a suspension of LiAlH40.05 mol) in anhydrous ether (150 mL), and the mixture wasefluxed for 10 h. After cooling the excess hydride was destroyedy successive addition of water (1.7 mL), a solution of 15% (w/v)aOH (1.7 mL), and water (5.1 mL). The solution was heated to0 ◦C for 2 h and filtered on Celite before evaporating to dryness.

yellow oil was obtained with a yield of 91.6%. 1H NMR � (CDCl3):.90 (m, 2H, CH2-2), 2.70 (t, 2H, CH2-3), 3.95 (t, 2H, CH2-1), 6.14d, 2H, CH-�), 6.65 (d, 2H, CH-�). 13C NMR � (CDCl3): 35.5 (CH2-), 39.7 (CH2-3). FTIR spectroscopy: the characteristic features of

, (ii) ethylenediamine in methanol, (iii) methyl acrylate in methanol, (iv) ethylene-

the N-(3-aminopropyl)pyrrole spectrum are a strong sharp peakat 3370 cm−1 with a medium shoulder at 3295 cm−1 that corre-sponds to the “free” asymmetrical and symmetrical N H stretchingvibration modes of the aliphatic primary amines.

2.3. Synthesis of amidoamine-pyrrole dendrons (G1, G2,G3)

Divergent synthesis (Fig. 1) of the amine-terminated Pyrrole-PAMAM dendrimers was carried out by initial Michael additionof methanolic solution of N-(3-aminopropyl)-pyrrole with excessmethyl acrylate (1:10 molar ratio). The reaction mixture was stirredfor three days at room temperature. The excess methylacrylatewas removed under vacuum at 40–50 ◦C temperature to afford theester-functionalized derivative G0.5. The reaction mixture was nextsubmitted to the reaction sequence leading to the next genera-tion Pyrrole-PAMAM dendrimer G1.5, consisting of the exhaustiveamidation of the ester functionalized G0.5 to ethylenediamine(1:30 molar ratio), followed by Michael addition of the result-ing amine with methylacrylate (20 equiv. of G0.5). Excess reagentswere removed under vacuum at 60–70 ◦C temperature. Repetitionof this two-step procedure ultimately leads to the next generationPyrrole-PAMAM dendrimer G3. The dendrimers G2 and G3, isolatedin 85–90% yield, were gummy in nature.

2.4. Preparation of enzyme electrodes

The preparation of the enzyme electrode is shown in Fig. 2.

Before each new measurements, the gold electrode was polishedwith 1.0, 0.5, and 0.3 �m alumina slurry, sonicated consequen-tially in distilled water and absolute ethanol for 15 min each andetched finally in 0.5 mol L−1 H2SO4 solution by cyclic-potential

690 M. S enel, C. Nergiz / Synthetic Metals 162 (2012) 688– 694

on pro

sr

3eataaTmrdawt

etpvwu

2

wr

MtAtI

paGaoo

Moreover, the intensity of these bands at 3280 and 1640 cm−1 obvi-ously increased with increasing the generation of the dendrimers.

The structure of Py-PAMAM dendrimer (G3) can be determinedfrom the detailed 1H NMR spectrum (Fig. 4). In the spectrum,

Fig. 2. Scheme of preparati

canning between −0.3 and +1.7 V until a reproducible voltammet-ic response was obtained.

The cleaned gold electrode was first immersed in 20 mmol L−1

-mercaptopropionic acid (MPA) aqueous solution for 2 h. After thelectrode was thoroughly rinsed with water to remove physicallydsorbed MPA. Py-PAMAM dendrimers were linked chemically tohe functionalized gold electrodes by promoting the creation ofmide bonds NH(CO) between the COOH ends of the MPA andmine peripheral groups (NH2) of the Py-PAMAM dendrimers.his was achieved by immersing the thiolated gold electrodes inethanolic solutions containing 5 × 10−3 mol L−1 EDC (coupling

eagent to create amide bonds) and 21 × 10−6 mol L−1 Py-PAMAMendrimers of generation 1.0 (G1), 2.0 (G2) and 3.0 (G3) for 12 ht RT. Subsequently, the dendrimer-functionalized gold surfacesere washed by dipping them in gently stirred MeOH at room

emperature.Finally, the GOx enzyme was immobilized on the modified

lectrode by electro polymerization of pyrrole on the modified elec-rode from aqueous solution containing 10 mg mL−1 GOx, 50 mMyrrole and 1.0 M p-toluene sulphonic acid sodium salt at a fixedoltage of +0.8 V vs. Ag/AgCl. The resulting electrode was washedith PBS (pH 7.5) and stored in the same PBS at 4 ◦C when not inse.

.5. Apparatus

The IR absorption spectra (4000–400 cm− 1) were recordedith a Mattson Genesis II spectrophotometer. NMR spectra were

ecorded in CDCl3 using a Bruker 400 MHz spectrometer.Electrochemical measurements were performed using a CHI

odel 842B electrochemical analyzer. A gold plate working elec-rode (1 cm2), a platinum plate counter electrode (1 cm2), ang/AgCl-saturated KCl reference electrode and a conventional

hree-electrode electrochemical cell were purchased from CHnstruments.

All amperometric measurements were carried out at room tem-erature in stirred solutions by applying the desired potential andllowing the steady state current to be reached. Once prepared, the

Ox electrodes were immersed in 10 ml pH 7.5 10 mM PBS solutionnd the amperometric response to the addition of a known amountf glucose solution was recorded. The data shown are the averagef three measurements for each electrode.

cess of the GOx biosensor.

The morphology of the modified gold surfaces were imaged byatomic force microscopy (AFM, Park systems XE-100E). Imagingwas carried out at ambient temperature in noncontact mode.

3. Results and discussion

3.1. Characterization of pyrrole-PAMAM dendritic wedges andmodified electrodes

Fig. 3 shows a comparison of the FT-IR spectra between 4000 and400 cm−1 of the Py-PAMAM dendrimers from G-0 to G-3. The peaksaround at 3280 cm−1 can be attributed to the stretching vibrationof N H, indicating the growing of the PAMAM dendrimers on thePy monomer, other characteristic bands at around 1640 cm−1 areassigned to the vibration of the amide group in PAMAM dendrimers.

Fig. 3. FT-IR spectra of Py-PAMAM dendrimers with different generations.

M. S enel, C. Nergiz / Synthetic Metals 162 (2012) 688– 694 691

Py-PA

toam

ae−eGavcmawcmcadfisdaw0

2

TC

A

Fig. 4. 1H NMR spectrum of

he peaks between 6.1 and 6.6 ppm are specific resonance signalsf pyrrole ring protons. The peaks between 2.0 and 3.4 ppm arettributed to the growing of the PAMAM dendrimers on pyrroleonomer, respectively.Cyclic voltammetry of ferrocyanide, as redox marker, is a valu-

ble and useful technique to follow the barrier of the modifiedlectrode. Cyclic voltammograms of 5 mM [Fe(CN)6]3−/4− between0.2 and 0.5 V shown in Fig. 5A were obtained at different modifiedlectrodes; (a) bare Au electrode, (b) Au/MPA, (c) Au/MPA/Py-NH2--0, (d) Au/MPA/Py-PAMAM-G-1, (e) Au/MPA/Py-PAMAM-G-2nd (f) Au/MPA/Py-PAMAM-G-3, respectively. Well-defined cyclicoltammogram, characteristic of a diffusion-controlled redox pro-ess, is observed at the bare Au electrode (Fig. 5Aa). After theodification of the Au electrode by dipping into MPA solution,

nd the covalent attachment of the Py-PAMAM dendrimers, theell-defined peaks of the bare electrode was greatly diminished,

onfirming that the surface of the Au electrode was successfullyodified. As can be seen in Fig. 5A, the CV responses of ferro-

yanide solution on the bare Au and the MPA-modified electrodere different compared to those obtained using the Py-PAMAMendrimers-modified electrodes. Consequently, the electron trans-er rate between the ferrocyanide solution and the electrode surfaces strongly dependent on the dendrimer generation [24]. Fig. 5Bhows the cyclic voltammograms of the polypyrrole film growthuring the electrolysis of the solution containing 50 mM pyrrolend 1.0 M p-toluene sulphonic acid sodium salt in phosphate buffer

ith pH 7.5. The current begins to increase markedly at around

.85 V, and then increases continuously with increasing potential.The surface morphology of the Au/MPA, Au/MPA/Py-PAMAM-G-

, and Au/MPA/Py-PAMAM-G-2/GOx electrodes was characterized

able 1omparison of the analytical performance of the different GOx electrodes.

Electrode RT (s) LR (mM)

Au/MPA/Py-NH2 7 0.5–3.0

Au/MPA/Py-PAMAM-G-1 9 0.5–5.0

Au/MPA/Py-PAMAM-G-2 5 0.5–5.5

Au/MPA/Py-PAMAM-G-3 12 0.5–4.0

u, gold electrode; RT, response time; LR, linear range; DL, detection limit.

MAM dendritic wedge G3.

by AFM in order to investigate the homogeneity of the film(Fig. 6). As shown from Fig. 6B, the surface morphology of thePy-PAMAM-G2-modified electrode is obviously different from thatof MPA-modified Au electrode. It could be observed that GOxmolecules were entrapped onto the Py-PAMAM-G-2-modifiedelectrode as an aggregated pattern in solid-like state with keep-ing its random cloud-like structure as in bulk solution. From theabove results, it could be concluded that the modification of the Auelectrode was done and enzyme was entrapped onto the modifiedelectrode.

3.2. Optimum pH and temperature

The activity of the GOx enzyme depends on the pH of themedium, and the optimum pH of the enzyme may be affected bythe immobilization method utilized. The effect of pH on the GOxenzyme electrode was studied between pH 5.0 and 9.0. As shownin Fig. 7A, the current response increased from pH 5.0 to 7.5, anddecreased from pH 7.5 to 9.0, in agreement with previous reports[25]. Therefore, pH 7.5 was chosen for use in further experimentsand for the determination of glucose levels.

The temperature of the medium is also an important param-eter for maximal GOx enzyme activity. The effect of temperatureon the amperometric response was also investigated from 20 to60 ◦C, as shown in Fig. 6B. The amperometric response increasedwith increasing temperature and reached a maximum at 40 ◦C,

followed by a decrease at temperatures >40 ◦C. This decrease inresponse (and reduction in GOx activity) might be due to tempera-ture induced GOx denaturation and/or leaching of the immobilizedenzyme.

DL (�M) Sensitivity (�A/mM) KappM

(mM)

7.7 7 7.69.6 11 5.33.4 16 3.56.3 10 9.25

692 M. S enel, C. Nergiz / Synthetic Metals 162 (2012) 688– 694

Fig. 5. (A) Cyclic voltammograms of (a) bare Au electrode, (b) Au/MPA, (c)Au/MPA/Py-NH2-G-0, (d) Au/MPA/Py-PAMAM-G-1, (e) Au/MPA/Py-PAMAM-G-2and (f) Au/MPA/Py-PAMAM-G-3 electrodes in solution 10 mM PBS solution con-taining 5 mM [Fe(CN)6]3−/4− . Scan rate is 50 mV/s. (B) Cyclic voltammogram ofpolypoyrrole film growth on the Au/MPA/Py-PAMAM-G-3 electrode from the solu-tion containing 10 mg mL−1 GOx, 50 mM pyrrole and 1.0 M p-toluene sulphonic acids

3e

1f

D

H

TesTao

Fig. 6. AFM images of (A) Au/MPA, (B) Au/MPA/Py-PAMAM-G-2 and (C) Au/MPA/Py-PAMAM-G-2/GOx.

odium salt at the scan rate 25 mV/s.

.3. Amperometric current response of glucose oxidase enzymelectrodes

Glucose oxidase enzymatically converts glucose to glucono-,5-lactone. The enzymatic reaction of glucose may be written asollows;

-glucose + H2O + O2Gox−→D-glucono-1, 5-lactone + H2O2

2O20.7 V−→ O2 + 2e−

he hydrogen peroxide generated is detected by the workinglectrode at 0.7 V versus an Ag/AgCl electrode. Fig. 8A shows steady-

tate current response curves of the prepared enzyme electrodes.he analytical performances of the different modified electrodesre summarized in Table 1. On the basis of a comparative studyf the four modified electrodes, it can be seen that the proposed

M. S enel, C. Nergiz / Synthetic Metals 162 (2012) 688– 694 693

Fig. 7. (A) Effect of the pH on the current response of Au/MPA/Py-PAMAM-G-2/GOxto 10 mM glucose at an applied potential +0.7 V; (B) effect of temperature on theamperometric response of 10 mM glucose in 10 mM PBS solution, pH 7.5 at anapplied potential of +0.7 V vs. Ag/AgCl.

blTeN1sdn

6sbMeskAt

Fig. 8. (A) Dependence of current on glucose concentration for enzyme electrodeand (B) amperometric response of Au/MPA/Py-PAMAM-G2/GOx electrode to suc-cessive addition of 0.5 mM glucose solution at an applied potential + 0.7 V in stirred10 mM PBS. The inset shows the calibration curve.

tical response curves, an activity loss of 40% was observed uponadditional use (Fig. 9A). The response of the GOx electrode to 5 mM

iosensor, the Au/MPA/Py-PAMAM-G-2/GOx, exhibits a broaderinear range and lower detection limit than the other electrodes.he sensitivity obtainable with the Au/MPA/Py-PAMAM-G-2/GOxlectrode (16 �A/cm2) is 2.3 times higher than for Au/MPA/Py-H2/GOx, 1.5 times higher than Au/MPA/Py-PAMAM-G-1/GOx, and.6 times higher than Au/MPA/Py-PAMAM-G-3/GOx. This increasedensitivity clearly demonstrates that use of the Py-PAMAM-G-2endrimer as an immobilization platform for GOx enzymes is sig-ificantly better than Py-NH2.

As shown in Fig. 8B, at glucose concentrations higher than mM, the GOx enzyme electrode response deviates from atraight line and eventually reaches a plateau. Thus, the immo-ilised GOx activity on Au/MPA/Py-PAMAM-G-2 electrodes followsichaelis–Menten kinetics, as seen for many amperometric

nzyme electrodes [26]. The apparent Michaelis–Menten con-tant (Kapp), which gives an indication of the enzyme–substrate

Minetics for the biosensor, was calculated to be 3.4 mM foru/MPA/Py-PAMAM-G-2/GOx, using the Lineweaver–Burk equa-

ion [27]. This low value of KappM means that the immobilised

enzyme on Py-PAMAM-G-2 has retained its activity, and that thereis a low-diffusion barrier. The analytical characteristics of thisGOx enzyme electrode indicate that the enzyme was effectivelyattached onto the conducting polymer film and that the bioactiv-ity of the immobilised enzyme is well preserved in context of theenzyme electrode.

3.4. Stability and reusability

The reusability of the GOx electrode was evaluated by run-ning measurements on the same day. Between each subsequentmeasurement, electrodes were stored at 4 ◦C in buffer solution for10 min. Although the first 5 measurements revealed nearly iden-

glucose was also measured for a period of 40 days. As shownin Fig. 9B, the amperometric response of the enzyme electroderemained constant for 10 days, followed by an activity loss of 45%.

694 M. S enel, C. Nergiz / Synthetic Me

Fig. 9. (A) Reusability of the GOx immobilized electrode. Each data point representsthe average of data collected by three electrodes (pH 7.5; ∼25 ◦C); (B) storage stabil-ities of GOx immobilized electrode. The amperometric responses of these enzymeelectrodes are regularly checked during 40 days (pH 7.5; ∼25 ◦C).

Fig. 10. Amperometric response of enzyme electrode to glucose (1 mM), ascorbicacid (0.1 mM), uric acid (0.1 mM) and acetaminophen (0.3 mM) in stirred 10 mM PBS(pH 7.5; ∼25 ◦C).

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tals 162 (2012) 688– 694

3.5. Interference study

The effect of interferents on the amperometric response ofthe enzyme electrode was also studied (Fig. 10). Three maininterferents: 0.1 mM uric acid, 0.1 mM ascorbic acid and 0.3 mMacetaminophen, were individually added to the reaction mix-ture in the presence of 1 mM glucose. The presence of any ofthe above interferents resulted in a relative error of less than4% in current measurements; therefore, we conclude that thisGOx enzyme electrode can detect glucose with negligible interfer-ence.

4. Conclusions

In this work, a novel amperometric glucose biosensor wasprepared using pyrrole-PAMAM dendritic wedges with differ-ent generations. Prepared dendritic wedges were covalentlyattached to the surface of an MPA-modified Au electrode,and GOx was immobilized on this host by electropolymer-ization of the pyrrole. The experimental results presented inthis work clearly demonstrate that immobilized GOx possessesexcellent catalytic ability and well-retained activity. The resultsare consistent with efficient enzyme immobilization onto aPAMAM type dendrimer modified surface containing a pyr-role monomer linked to a PAMAM dendrimer, which resultedin high enzyme loading and increased electrode lifetime andenzyme stability. The data described here demonstrates thatpyrrole-PAMAM dendrimers-modified electrodes can be useful inbio-electrochemical studies.

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