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ArticleJournal of

Nanoscience and NanotechnologyVol. 14, 8432–8438, 2014

www.aspbs.com/jnn

Enzyme-Free Glucose Sensor Based on Au NanobouquetFabricated Indium Tin Oxide Electrode

Jin-Ho Lee1�2, Waleed Ahmed El-Said3, Byung-Keun Oh1�3, and Jeong-Woo Choi1�3�∗1Department of Chemical and Biomolecular Engineering, Sogang University, 35 Baekbeom-Ro,

Mapo-Gu, Seoul 121-742, Republic of Korea2Research Institute for Basic Science, Sogang University, 35 Baekbeom-Ro,

Mapo-Gu, Seoul 121-742, Republic of Korea3Interdisciplinary Program of Integrated Biotechnology, Sogang University,

35 Baekbeom-Ro, Mapo-Gu, Seoul 121-742, Republic of Korea

In this study, we demonstrated a simple, rapid and inexpensive fabrication method to develop anovel gold nanobouquet structure fabricated indium tin oxide (GNB/ITO) electrode based on elec-trochemical deposition of gold ions onto ITO substrate. The morphology of the fabricated electrodesurface was characterized by scanning electron microscopy (SEM) to confirm the GNB formation.Enzyme-free detection of glucose using a GNB/ITO electrode was described with high sensitivityand selectivity based on cyclic voltammetry assay. The results demonstrate a linear relation withinwide concentration range (500 nM to 10 mM) of glucose, with a correlation coefficient of 0.988.The interference effect of uric acid was effectively avoided for the detection of glucose (1 �M to10 mM). Moreover, the developed sensor was applied to determine the concentration of glucose inthe presence of human serum to indicate the ability of GNB/ITO electrodes in real samples. Hence,newly developed GNB/ITO electrode has potential application in enzyme-free glucose sensor withhighly sensitivity and selectivity.

Keywords: Enzyme Free, Glucose, Nano–Pattern, Electrochemical Sensor, Nanobouquet.

1. INTRODUCTIONThe determination of glucose has drawn much attentionin the field of biotechnology for clinical diagnosis, man-agement of diabetes mellitus and in food industry. There-fore, tremendous efforts have been made to develop aglucose sensor with high sensitivity and selectivity.1�2

Early studies done by Clark and Lyons that have beenshown an enzymatic glucose sensor based on immobi-lized glucose oxidase (GOx) enzyme were drawn muchattention.3 Although, these enzyme–based glucose sensorshave shown good selectivity and sensitivity; however, thesesensors have a lot of drawbacks such as loss of enzymeactivity, chemical and thermal instabilities originated fromthe intrinsic nature of enzymes.4 Previous studies reportedthat, the environmental conditions of enzyme–based glu-cose sensors, including strong acidic conditions, basic con-ditions, or high temperatures (above 40 �C) could causefatal damage to GOx enzyme, which leads to loss of

∗Author to whom correspondence should be addressed.

sensing activity. Moreover, the activity of GOx is verysensitive to sodium dodecyl sulfate (SDS) under acidicconditions as well as to hexadecyltrimethylammonium bro-mide (CTAB) under basic conditions. In addition to pH,temperature, toxic chemicals for sterilization, and humid-ity effects of enzyme, potentially causes significant harmto the sensor activity in use as well as in storage.5 Anotherlimitation for the enzyme–based glucose sensor is thesevere interference that could be caused by endogenouselectro–activity in the whole blood samples. Instabilityof GOx upon sterilization might limit the enzymatic glu-cose sensors from being used for long term monitor-ing in humans. Therefore, the need for electrochemicalenzyme–free glucose sensors has received considerableinterest. The main advantage of enzyme–free glucose sen-sor includes high sensitivity and selectivity, in additionto the prevention of fouling by adsorbed intermediatesand some anions, such as chloride ions. All of theseissues depend on the properties of the electrode materialsbecause the electro–catalytic activity is the main factor thataffects both the sensitivity and selectivity of the glucose

8432 J. Nanosci. Nanotechnol. 2014, Vol. 14, No. 11 1533-4880/2014/14/8432/007 doi:10.1166/jnn.2014.9921



Lee et al. Enzyme-Free Glucose Sensor Based on Au Nanobouquet Fabricated Indium Tin Oxide Electrode

sensor. Therefore, numerous of studies have been devotedin the investigation and preparation of glucose enzyme–free sensors.6�7 It is reported that bare metal electrodes(platinum or gold electrodes) can act as enzyme–free sen-sors for the determination of glucose.8�9 However, theseelectrodes suffer from low sensitivity, poor selectivity andpoisoning by intermediates and chloride.10

Much efforts have been focused on developing enzyme-free glucose sensors based on the direct detectionof the glucose redox behavior on various electrodematerials including nanotubular arrayed platinum (Pt),11

gold (Au) nanoparticles,12 copper nanoparticles,13�14 Ptnanoparticles,15�16 nickel nanoparticles,17 carbon nanotubes(CNTs),18�19 mesoporous Pt,20 macroporous Pt films,21

Pt–Pb nanowire arrayed electrodes,22 three–dimensionalAu films,23 and Pt–Ru nanoparticles24 to overcome thedisadvantages of the bulk electrodes.25�26 Among allthese materials, Au nanostructures are attracted muchattention for the use in a wide range of applications,including biosensors, chemical-sensor, optical scattering,diffraction, and other applications due to their higherconductivity, inertness, biocompatibility and large surfacearea.27 The enhancement of the electrochemical conduc-tivity of the nanostructured modified electrodes comparedto that of the bare electrodes could be related to theincrease of the electrode’s active surface area. Conversely,the immersion of a nanomaterial in an electrolyte couldinduce charge on the surface regions of a material via anapplication of a potential across the electrolyte–materialinterface. However, the development of a simple, rapid,inexpensive method for fabrication of a highly sensitiveelectrical nanostructured substrate is still in demand tomonitor the electrochemical characteristics of glucose in amixture with good selectivity.In this work, we present a simple, rapid, and inexpensive

method for fabricating a uniform Au nanobouquet (GNB)modified ITO electrode. The highly sensitive GNB/ITOelectrode was used to investigate the interdependence ofthe electrochemical signals on the oxidation of wide rangeof glucose (500 nM to 10 mM) without enzymes usingthe cyclic voltammetry (CV) technique. Furthermore, theCV assay was used for the simultaneous determination ofglucose in the presence of high concentration (500 �M) ofuric acid (UA) as interference. Further, the determinationof different concentrations of glucose (1 �M to 10 mM)in the presence of human serum was used to prove theability of GNB–modified ITO electrodes for detection inreal sample. These results indicate that low detection limitsfor glucose were obtained due to the high electro-catalyticproperties of the GNB/ITO electrode.

2. EXPERIMENTAL DETAILS2.1. MaterialsGlucose, UA, human serum, SDS, and gold chloride(99.9%+) were purchased from Sigma–Aldrich (St. Louis,

MO, USA). All other solutions were prepared withdistilled Millipore (Milli–Q) water. Other chemicals thatwere used in this study were obtained commercially atreagent grades.

2.2. Fabrication of Gold NanobouquetPattern on ITO Electrode

ITO-coated glass substrates were cleaned by sonicationfor 15 min in 1% Triton X–100, deionized water (DIW),and ethanol. Then, they were treated with basic piranhasolution (1:1:5, H2O2:NH4OH:H2O) for 30 min at 80 �C.Finally, the ITO substrates were cleaned again with DIWand dried under N2 stream to obtain a clean ITO surface.GNB was electrochemically deposited onto ITO substrates(2 cm× 1 cm) using a 1 mM HAuCl4 aqueous solutioncontaining 17 �g/L of SDS as a surfactant. The potentialwas maintained at −0.9 V (vs. Ag/AgCl). The active areafor the electrochemical deposition of GNB was 1 cm×1 cm. Moreover, to remove any surfactant traces, whichmay be adsorbed onto the GNB surface, the substrateswere rinsed with DIW and sonicated for 5 min with iso-propyl alcohol. The surface morphologies of the GNBelectrode were analyzed by a scanning electron micro-scope (SEM) (ISI DS-130C, Akashi Co., Tokyo, Japan).A schematic diagram for the formation of Au nanobou-quets on ITO surface by electrochemical deposition tech-nique is depicted in Figure 1(a).

2.3. Electrochemical Measurements ofGlucose Determination

All electrochemical measurements as well as the elec-trodes modification were performed using a potentiostat(CHI–660A, CHI, USA) controlled with “general purposeelectrochemical system” software. An in–house three–electrode system comprised of GNB/ITO electrode as theworking, a platinum wire as the counter, and Ag/AgCl asreference electrodes were used at a scan rate of 50 mV/s.In order to minimize the error, all the data are the mean±standard deviation of three different experiments. All themeasurement was performed in neutral pH at RT.

3. RESULTS AND DISCUSSION3.1. Surface Morphology and Current Transient of

Nanobouquet Structured Gold FilmFigure 1(b) illustrates the current density versus time curveat a potential of −0.9 V (Ag/AgCl) for 30 s. The cur-rent density increased drastically during the first two mil-liseconds and gradually decreased to a stationary valueat approximately 20 ms. This gradual decrease was dueto limited AuCl−4 diffusion to the ITO surface, whichmost likely resulted from nucleation and growth of the Aunanostructures as indicated in the current transient profile,which demonstrates the initial nucleation and growth pro-cess during metal deposition.28 SDS as an ionic surfactantwas added to modify the interfacial properties of both the

J. Nanosci. Nanotechnol. 14, 8432–8438, 2014 8433



Enzyme-Free Glucose Sensor Based on Au Nanobouquet Fabricated Indium Tin Oxide Electrode Lee et al.

Figure 1. Fabrication of GNB–modified ITO electrode surface.(A) Schematic diagram for fabrication of GNB structures modified ITOelectrode based on electrochemical deposition technique. (B) Current–versus–time profile for Au electrochemical deposition onto ITO electrodeat a potential of −0.9 V (vs. Ag/AgCl) for 30 s at 25 �C. (C) SEM imageof GNB–modified ITO electrode surface, scale bar 500 nm.

particles and the electrode to control the morphology ofthe aggregates. The SEM image of electrodeposited GNBnanostructures on an ITO surface at 30 s is shown inFigure 1(c), which clearly demonstrates that these electro-chemical deposition conditions including concentration ofAu3+ (1 mM), concentration of surfactant (17 �g/L), andtemperature (25 �C), time (30 s) and voltage (−0.9 V, vs.Ag/AgCl) of the deposition are results in the formationof uniformed distributed Au bouquet nanostructures overa large electrode surface area. The nanobouquet structures

were observed to be in the range of 400 nm to 600 nm indiameter.

3.2. Electrochemical Behavior of Glucose onBare ITO and GNB Electrodes

The general oxidation pathway for the glucose can beexplained: two hemiacetal–types of glucose (�– and�–glucose) are converted to each other through acid–catalyzed hydrolysis via aldehyde–type glucose. The ratioof two hemiacetal–types of glucose would be �:�= 11:89,if it were not for the influence of the anomeric effect.29

A schematic of the general reaction pathway is illustratedin reaction Scheme 1 for �– and �–glucose, the hydrogenatom tethered to carbon is activated due to the strongeracidity of the hemiacetalic OH group (pKa = 12�3) com-pare to alcoholic OH group (pKa = 16). Thus, the prod-uct of electrochemical oxidation of �– and �–glucose isglucono–�–lactone, which is the final stable product oftwo–electron oxidation of glucose.30

Figure 2(a) shows the cyclic voltammogram behaviorof the direct oxidation of 1 �M of glucose at bare ITOelectrode. From this result, no significant redox currentpeaks could be observed, which may be related to slowkinetic electron transfer at the bare ITO electrode in addi-tion to surface fouling due to the adsorption of interme-diates. On the other hand, large background was observedfor a GNB–modified ITO electrode in compare to thatof the bare ITO electrode (Fig. 2(a)) indicates the higherbackground charging current. This could be related to thelarger surface area of the GNB–modified ITO electrode.Therefore, the GNB–modified ITO electrode displays anadvantage for providing better electron-transfer kinetics ascompared with the bare ITO electrode.The CV for the glucose (1 �M) at the GNB–modified

ITO electrodes in the potential range from +0.9 to −0.2 V(versus Ag/AgCl) at scan rate 50 mV/s (Fig. 2(a)) showsan anodic and cathodic current peak at potential 510 mV

Scheme 1. Reaction scheme for different glucose forms: Schematic dia-gram of the equilibrium between �–glucose and �–glucose forms in anaqueous solution and the oxidation pathway of �–and �–glucose intoglucose lactone.

8434 J. Nanosci. Nanotechnol. 14, 8432–8438, 2014




Figure 2. (A) Electrochemical behavior of glucose at bare ITO andGNB–modified ITO electrode: (a) Cyclic voltammograms of glucose ona bare ITO, (b) background signal of a GNB–modified ITO electrode,and (c) CV of glucose on a GNB–modified ITO electrode. (B) Elec-trochemical behavior of glucose at different pH range from (a) 4 to(b) 9. (C) Electrochemical behavior of glucose at different temperatures(a)10 �C, (b) 24 �C and (c) 37 �C, respectively.

and 200 mV, respectively. The separation between thepotential peaks �Epc −Epa� exceeded 59 mV, which wasindicative of a distinct quasi-reversible character of theglucose at GNB/ITO electrode process. The enhancementfactor for the electrochemical activity at GNB/ITO elec-trode is mainly due to its larger surface to volume arearatio derived by three dimensional gold nanobouquet struc-tures. Moreover, these results might be related to moderateelectrocatalytic activity of the GNB/ITO electrode, whichis obtained by modifying poorly electrocatalytic elec-trode (ITO) with a highly electrocatalytic material “Au.”This might enable the electrode to obtain high signal–to–background ratios compared to those of Au and Ptelectrodes.31 Therefore, the GNB/ITO electrode displays

advantages related to providing better electron–transferkinetics than that of bare ITO electrodes, and the catalyticproperties of Au nanoparticles might advance the oxida-tion of glucose at the GNB/ITO electrode. These resultsdemonstrate the sensitivity of the GNB/ITO electrode.Moreover, the effect of pH was determined using dif-

ferent solutions with pH in the range of 4 to 9. In theacidic pH or basic pH solutions, glucose was convertedto another anomer (mannose or fructose) via the anomer-ization process, which shifts the oxidation peak potentialof glucose (Fig. 2(b)). In addition, the effect of temper-ature also was determined by studying glucose oxidationbehavior at different temperatures (10 �C–37 �C). As tem-perature changes, the oxidation peak potential changes itssignal (Fig. 2(c)). Based on these results, we selected neu-tral solution and RT as the optimized conditions for furtherexperiments.

3.3. Cyclic Voltammetry for Detection ofDifferent Concentrations of Glucoseon GNB–Modified ITO Electrodes

Figure 3(a) shows the cyclic voltammograms for differentconcentrations of glucose (from 500 nM to 10 mM) at theGNB/ITO electrode. Upon addition of glucose, the anodiccurrent peak increased with increasing concentration ofglucose. The lowest concentration measured in this systemis 500 nM, which is lower than that obtained by previ-ous approaches, such as CNT composite electrodes,32�33

Au nanoparticles,12 etc. (Table I). The anodic current peaksfound to be linearly increased with the glucose concen-trations. However, no change in anodic peak current wasobserved when the concentration of glucose was more than10 mM which could be related to the saturated GNB/ITOelectrode. The calibration plot for glucose determinationshows a linear relation in a wide range from 500 nM to1 mM with a correlation coefficient of 0.984 (Fig. 3(b)).These results suggest that the GNB/ITO electrode could beused to develop a highly sensitive biosensor for determina-tion of low glucose concentrations. This indicates that theGNB/ITO electrode exhibited good electrocatalytic perfor-mance for oxidation of glucose.

3.4. Cyclic Voltammetry for the Detection ofGlucose in a Mixture with UA onGNB–Modified ITO Electrodes

A major challenge in the electrochemical determination ofglucose is the coexistence of interfering materials, such asuric acid (UA), which are commonly found in the humanblood. The presence of UA in physiological solutionscauses the greatest interference for direct electrochemi-cal oxidation of glucose on various electrodes, especiallyenzyme–free sensors.24 Therefore, the ability of the GNB–modified ITO electrode to monitor different concentrationsof glucose in the presence of high concentration of UAwas investigated.





Figure 3. (A) Cyclic voltammograms of varying glucose concentrations (a) 500 nM, (b) 1 �M, (c) 10 �M, (d) 100 �M, (e) 1 mM, (f) 10 mMat GNB–modified ITO electrode. (B) Linear plot of anodic current peak as a function of glucose concentration (−Ip�X� = �0�14237± 0�01154�X+�1�49989± 0�05265�, R = 0�984). (C) Cyclic voltammograms of varying concentrations of UA (a) 100 �M, (b) 200 �M, (c) 300 �M, (d) 400 �M,(e) 500 �M on a GNB–modified ITO electrode. (D) Linear plot of anodic current peak as a function of UA concentration (−Ip�X� = �4�94046±0�11392�X+ �−5�14671±0�46856�, R= 0�996).

Figure 3(c) shows the cyclic voltammetric behavior ofUA on the GNB–modified ITO electrode, which showsan irreversible behavior with an anodic current peak atapproximately 720 mV. Cyclic voltammograms for vari-ous concentrations of UA (100 �M to 500 �M) on theGNB–modified ITO electrode demonstrate an increase of

Table I. Comparison of different electrode matrix for linear range andreal sample detection of enzyme free glucose sensors along with thosereported in literature.

Electrode matrix [Ref.] Linear range (mM)

Pt-nanotube arrays11 2–14Au nanoparticle12 0–8Copper/MWCNT13 0.7–3.5∗

Cu nanoparticles14 0.001–5∗

Pt nanoparticles/CNT15 1–26.5PtPbNP/MWCNT16 Up to 11Ni powder17 0.5–5∗

MWCNT18 0.002–11Multiple-branching CNT forest19 1–11Mesoporous Pt20 0–10Macroporouse Pt film21 0.001–10Pt-Pb nanowire array22 0.008–113-D Au film23 0.005–10CNT supported PtRu nanoparticles24 1–15Boron-doped CNT33 0.05–0.3MnO2/MWNTs nanocomposite34 Up to 28∗GNB modified ITO electrode (Present work) 0.0005–10∗

Note: ∗Real sample (Blood serum) tested.

anodic current peak with an increasing concentration ofUA (Fig. 3(c)), and this increase was almost linear asshown in Figure 3(d). The calibration plot for glucosedetermination shows a linear relation over range from100 �M to 500 �M with a correlation coefficient of 0.996(Fig. 3(d)).UA was used as an interference agent and its effect

on the determination of glucose was examined and pre-sented in Figure 4. The cyclic voltammograms for differ-ent concentrations of glucose (1 �M to 10 mM) in thepresence of UA (500 �M) were shown in Figure 4(a).Compared to pure glucose solution, the addition of UAto glucose solution reduce the cathodic current peaks,while the oxidation current peak for glucose in a mix-ture was observed at nearly the same potential (approx-imately 510 mV). Moreover, different concentrations ofglucose (1 �M to 10 mM) were detected in the pres-ence of a constant concentration of UA (500 �M) atGNB/ITO electrode. The oxidation current peaks of glu-cose in a UA mixture showed a linear relation to the con-centrations of glucose with a correlation coefficient of 0.98(Fig. 4(b)). Moreover, it was observed that, the presenceof UA (500 �M) did not affect the detection of glucoseat the GNB–modified electrode within the concentrationsranging from 1 �M to 10 mM. These results indicate thatthis GNB–modified ITO electrode could be used for selec-tively detecting various concentrations of glucose in thepresence of an interfering material (high concentrated UA:500 �M).34





Figure 4. (A) Cyclic voltammograms of varying concentrations of glu-cose (a) 1 �M, (b) 10 �M, (c) 100 �M, (d) 1 mM, (e) 10 mM ona GNB–modified ITO electrode in presence of fixed UA concentration(500 �M). (B) Linear plot of anodic current peak as a function of glu-cose concentration and fixed UA concentration (500 �M) (−Ip�X� =�2�4496±0�27948�X+�31�53788±1�39076�, R= 0�98). (C) Anodic cur-rent peak corresponding to oxidation of varying concentrations of glucose(1 �M to 10 mM) in both (�) distilled water and (�) human bloodserum.

Furthermore, to prove the ability of GNB–modifiedITO electrodes to detect glucose in real samples, differentconcentrations of glucose (1 �M to 10 mM) were dis-solved in human serum (1%). The anodic peak currentcorresponding to the oxidation of different concentrationsof glucose and in human serum exhibited nearly identicalanodic peak currents (Fig. 4(c)). These results indicatedthat a GNB–modified ITO electrode is suitable for thedetermination of glucose in real samples with a detectionlimit of 1 �M.

4. CONCLUSIONSHere, a free–enzymatic glucose sensor based on GNBarray modified ITO electrode was developed that demon-strated several advantages, such as good analytical per-formance and simple preparation process. It exhibitedhigh sensitivity with good potentiometric response, a lowdetection limits and a wide linear range. The GNB arraywas prepared by using electrochemical deposition method.Electrochemical results showed that the GNB–modifiedITO electrode had large active surface, a good electrontransfer rate, larger current response and high electro–catalytic activity for glucose oxidation in neutral solu-tions than a bare ITO electrode, with a detection limit of500 nM glucose. Moreover, GNB–modified ITO electrodedemonstrated an efficient determination of glucose in thepresence of UA (500 �M) with good selectivity and withsensitivity up to 1 �M. In addition, the sensor has potentialapplications in glucose concentrations detection in humanserum samples without interferences; therefore, it couldbe suitable for the determination of glucose in real sam-ples. It is expected that with such electronic and structuralproperties, the GNB–modified ITO electrode could be apromising electrode in electro–analytical and biosensingapplications. High sensitivity and selectivity and surfacerenewal make this sensor ideal for detection of glucose inreal samples.

Acknowledgments: This work was supported by theLeading Foreign Research Institute Recruitment Programthrough the National Research Foundation of Korea(NRF)funded by the Ministry of Science, ICT and FuturePlanning(MSIP) (2013K1A4A3055268), by a NationalResearch Foundation of Korea (NRF) grant funded bythe Korea government (MEST) (2009-0080860) and bythe Sogang University Research Grant of 2013 (SRF-201314003.01).

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Received: 29 March 2013. Accepted: 15 January 2014.


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