Division of Immunology, State Research Institute Center for Innovative Medicine, Zygimantu 9, LT-01102 Vilnius, LithuaniaCenter of Nanotechnology and Materials Science—NanoTechnas, Faculty of Chemistry, Vilnius University, Naugarduko 24, LT-03225 Vilnius, LithuaniaLaboratory of BioNanotechnology, Division of Materials Science and Electronics, Institute of Semiconductor Physics, State Scientific Research Instituteenter for Physical Sciences and Technology, A. Gostauto 11, LT-01108 Vilnius, Lithuania
r t i c l e i n f o
rticle history:eceived 10 December 2013eceived in revised form 31 May 2014ccepted 8 June 2014vailable online 26 June 2014
A biosensor based on glucose oxidase (GOx) immobilized on gold nanoparticles (Au–NPs) electrochemi-cally predeposited on the surface of graphite rod (GR) electrode was developed (GOx/Au-NPs/GR). Mainanalytical characteristics of this biosensor were determined and compared with those determined usinga biosensor setup without Au-NP modification (GOx/GR). The highest analytical signal of GOx/Au-NPs/GRelectrode was observed after 13 nm Au-NP deposition on the electrode from 0.8 nmol L−1 solution last-ing 20 min, when cyclic voltammetry was performed in the range from 0.0 to +1.0 V vs Ag/AgCl. Thebest analytical characteristics of the developed biosensor were obtained after 25 mg mL−1 GOx immo-bilization on the Au-NPs/GR electrode. Analytical signal registered using GOx/Au-NPs/GR electrode was2.08 times higher in comparison to GOx/GR electrode. The registered currents of both electrodes were
−1
lucose oxidaselucose biosensor
linearly dependent on glucose concentration in the range of 0.1–10 mmol L . The developed GOx/Au-NPs/GR electrode was characterized by high sensitivity, which was equal to 101.02 �A mM−1 cm−2 in thelinear glucose detection range. The limit of detection was 0.083 mmol L−1 with relative standard devia-tion of 6% for GOx/Au-NPs/GR electrode. This study demonstrates a successful practical exploitation ofthe developed biosensor in a human serum sample.
Recently scientific and industrial impact of nanoscience andanotechnology in analytical electrochemistry has been growing.ome challenging bioanalytical problems, such as sensitivity, speci-city, reproducibility and reliability can be resolved by applyinganostructure-based electrochemical biosensors [1–4]. Nanotech-ological methods enable to miniaturize biosensors and makehem suitable for rapid and low cost analyte detection in low vol-me samples [5] or reliable for continuous analyte monitoring by
mplantable bioanalytical devices. Electrochemical biosensors haveeen applied in many areas, such as food industry, agriculture, mil-
tary, veterinary, clinical applications, and environment [3,4,6].Enzymes, which are mostly used in electrochemical biosen-
or design, are usually immobilized on various solid conductingupports by passive physical adsorption, covalent attachment,ncapsulation or entrapment within an ultrathin polymeric film
with an ultrahigh density [7,8]. Electrochemical biosensors withimmobilized enzymes are characterized by high selectivity, sensi-tivity, reproducibility. In addition sometimes they are suitable forcontinuous and in situ monitoring of analyte in a complex matrixes[3,9,10]. Nowadays research is focused on the improvement of sen-sor properties by new sensing approaches. A noteworthy tendencyin biosensor design is mainly based on advantages of nano-technology, which allow reducing dimensions at the nanoscale,constructing arrays for high throughput analysis with the inte-gration of microfluidics, and enhancing the performance of thebiological components by using new nanomaterials [5]. Success-ful glucose biosensors are likely to be small, not expensive andportable, to reach the interest of millions of diabetic patients, whichhave daily need to perform glucose test in a simple way. In orderto increase durability of electrochemical biosensors the biologicalcomponents should display high storage and operational stability.
Different methods for electron transfer between active site
of the redox enzyme and the surface of electrode were devel-oped, including the application of diffusional redox mediators[11–13], incorporation of electron relaying redox centers to theproteins [14,15] and the immobilization of redox proteins within
lectroactive polymers [16,17]. The application of nanomaterialsnanoparticles, nanotubes, nanofibers, nanowires and nanocom-osites) in the fabrication of biosensor allows the improvementf signal transduction. Usually nanoparticles, including metal andxide nanoparticles, semiconductor and composite nanoparticles,xhibit unique chemical, physical and electronic properties thatre different from these of bulk materials and such nanopar-icles can be used for the construction of new electrochemicalensors and biosensors [1,6,18,19]. Nanoparticles can be used inlectrochemical biosensor design as (i) substrates for biomoleculemmobilization, (ii) catalysts of electrochemical reactions, and (iii)nhancers of electron transfer [6]. Gold nanoparticles of differentiameters with relatively high monodispersity could be preparedy chemical reduction of gold salt in the presence of agents, whichinds to nanoparticle surface to impart higher stability [1,20,21].olloidal solution of gold nanoparticles has a long shelf-life, it ison-toxic and has some amazing benefits for a wide variety of appli-ations. The incorporation of nanoparticles into redox enzymesould provide a hybrid electrically active biomaterial. Au-NPs withhe appropriate dimensions and functionalization adjacent to thenzyme redox center could act as an electron relay to a macroelec-rode [22].
Among many methods, which are suitable for the deposition ofu-NPs on the electrode surface, electrochemical methods are veryttractive. Au-NPs assembled on the electrode surface via electro-tatic interaction or covalent bond formation enhance the electrodeonductivity, facilitate the electron transfer and improve the ana-ytical sensitivity, selectivity and stability of biosensors [22–24].hese systems based on directly electrochemicaly deposited bio-omposite consisting of chitosan hydrogel, glucose oxidase, andold nanoparticles exhibited a rapid response, low detection limitf glucose and high stability due to strong adsorption and pre-ention of leakage of enzyme [25]. A novel amperometric glucoseiosensor based on Pt electrode coated by polyvinylferrocenelm and Au-NPs was developed [26]. The electrocatalytic effectf Au-NPs on enzymatically generated H2O2 offers more sensi-ive and selective monitoring of glucose than that in biosensorsithout any Au-NPs. To achieve high sensitivity and the linear
urrent dependence on glucose concentration (in the range of–10 mmol L−1) electrochemical synthesis of Au-NPs on multiwallarbon nanotubes from HAuCl4 solution by potential cycling from.0 to 0.0 V was performed [27]. The electrochemical deposition ofu-silicate-GOx biocomposite could be achieved from sol of Au-Ps containing GOx by potential cycling between −0.4 and 1.0 Vs Ag/AgCl [28]. This co-entrapment of glucose oxidase in a goldanoparticle-silicate network imparts biocatalytic activity of thelm and increases operational and long-term stability of designediosensor. Another biosensor based on gold nanoparticles electro-hemically deposited from HAuCl4 solutions under the constant0.2 V vs SCE on the surface of glassy carbon electrode showedood performance in electrochemical oxidation of tryptophan [21].maller nanoparticles are more suitable for enzyme immobiliza-ion [6,29], because changes of protein structure and function upondsorption are lower on higher curvature surfaces [30]. Nanopar-icles could act as nano-scaled electrodes and therefore they areften used in the design of electrochemical biosensors [1,20].
The main aim of this study was the development of glucoseiosensors based on glucose oxidase immobilized on a graphite rodlectrode precoated by electrochemically deposited gold nanopar-icles from a colloid solution. The optimal concentrations of Au-NPs,Ox, and a redox mediator were selected, electron transfer betweenOx and electrode in the presence of a soluble redox mediator
nd immobilized gold nanoparticles was evaluated, and analyti-al characteristics of glucose biosensors were assessed. The newlyeveloped electrodes were used for determination of glucose inuman serum samples.
uators B 203 (2014) 25–34
2. Materials and methods
2.1. Materials
Glucose oxidase (EC 1.1.3.4, type VII, from Aspergillus niger,215.3 unit mg−1 protein) and N-methylphenazonium methyl sul-phate (PMS) were purchased from Fluka and Sigma-Aldrich(Buchs, Switzerland), respectively. d(+)-glucose, d(+)-saccharose,d(+)-xylose, d(+)-galactose, d(+)-mannose, d(-)-fructose, tetra-chloroauric acid (HAuCl4·3H2O) and tannic acid were obtained fromCarl Roth GmbH&Co (Karlsruhe, Germany), sodium citrate – fromPenta (Praha, Czech Republic), hydrochloric acid 37% – from ActaMedica (Hradec Kralove, Czech Republic). Before investigations glu-cose solution was stored overnight to reach equilibrium between� and � optical isomers. All other chemicals used in the presentstudy were either analytically pure or of highest quality. All solu-tions were prepared using deionized water purified with waterpurification system Millipore S.A. (Molsheim, France). The solutionof sodium acetate (SA) buffer (0.05 mol L−1 CH3COONa·3H2O) with0.1 mol L−1 KCl was prepared by mixing of sodium acetate trihy-drate and potassium chloride, which were obtained from Reanal(Budapest, Hungary) and Lachema (Neratovice, Czech Republic).Graphite rods (3 mm diameter, 99.999%, low density) were pur-chased from Sigma-Aldrich (St. Louis, USA), alumina powder (graindiameter 0.3 �m, Type N) – from Electron Microscopy Sciences(Hatfield, USA), 25% glutaraldehyde solution, l-ascorbic acid anduric acid – from Fluka Chemie GmbH (Buchs, Switzerland) andAppliChem GmbH (Darmstadt, Germany).
2.2. Synthesis of gold nanoparticles
The 13 nm diameter Au-NPs were synthesized by the reductionof HAuCl4·3H2O by sodium citrate in the presence of tannic acid aspreviously reported [19]. The concentration of 13 nm Au-NPs wascalculated according to the amount of starting material, density ofgold, the approximate diameter of nanoparticles, and assuming thatthe reaction yield is 100%. It was determined that concentrationof Au-NPs is 3.6 nmol L−1 [31]. The solution of 13 nm Au-NPs wasstored in dark glass flask at +4 ◦C.
2.3. Pre-treatment of the working electrode
The working surface area of graphite rod electrodes was0.071 cm2. Graphite rod was cut and polished on fine emery paperand then polished by slurry of alumina powder containing 0.3 �mgrains of Al2O3. After this the surface of electrodes was rinsed withdistilled water, dried at 20 ± 2 ◦C and sealed into silicone tube inorder to prevent contact of the electrode side surface with thesolution.
2.4. Optimization of electrochemical Au-NP deposition usingcyclic voltammetry (CV)
For the preparation of GOx/Au-NPs/GR electrodes the graphiterod was placed in the 0.1 nmol L−1 solution of 13 nm Au-NPs, wheregold nanoparticles were electrochemically deposited on the surfaceof electrodes using a computerized potentiostat PGSTAT 30/Auto-lab (EcoChemie, The Netherlands) with GPES 4.9 software. CV modewith scan rate of 0.05 V s−1 was applied. For the evaluation of theoptimal electrode modification time Au-NPs were deposited on GRelectrodes for different periods of time ranging from 5 to 45 minand using cycling potentials from 0.0 to +1.0 V vs Ag/AgCl. The opti-
mization of cycling potential was performed in the range from 0.0,−0.25, −0.5, −0.75, −1.0 to +1.0 V and from +0.25, +0.5, +0.75, +1.0to −1.0 V vs Ag/AgCl. After enzyme immobilization the analyticalsignal was evaluated at 1.08, 6.14 and 17.3 mmol L−1 glucose in
N. German et al. / Sensors and Act
Scheme 1. Schematic illustration of electrochemical gold nanoparticle depositionod
0P
2e
eGtitiAcobetmBc
2
spcteaaA(Eametefampe
n a graphite rod electrode followed by glucose oxidase immobilization and glucoseetermination by the developed electrochemical biosensor.
.05 mol L−1 SA buffer (pH 6.0) with 0.1 mol L−1 KCl and 2 mmol L−1
MS.
.5. Immobilization of glucose oxidase on Au-NPs modifiedlectrode
Enzyme solution (3 �L) was deposited on the Au-NP modifiedlectrode and water was evaporated at room temperature. TheOx-modified electrode was prepared in similar way by the deposi-
ion/evaporation of 3 �L GOx solution, and it was stored for 15 minn a closed vessel over 25% solution of glutaraldehyde at roomemperature. The development and optimization of such enzymemmobilization procedure was described in details previously [18].ll conditions (duration and potential cycling of Au-NPs electro-hemical deposition, concentrations of Au-NPs, GOx and PMS) wereptimized in order to achieve the maximal sensitivity of creatediosensors. Prior to all electrochemical measurements, workinglectrodes were thoroughly washed with distilled water in ordero remove non-cross-linked enzyme and/or gold nanoparticles. The
ain steps of graphite rod modification are illustrated in Scheme 1.efore the experiments all working electrodes were stored in alosed vessel over the buffer at +4 ◦C.
.6. Electrochemical measurements
The electrochemical measurements of developed biosen-ors were performed using above-mentioned computerizedotentiostat in amperometry mode at +0.3 V vs Ag/AgCl. Aonventional three-electrodes system comprising of working elec-rode (modified as was described above), Pt electrode with thelectrochemically-active-area of 2 cm2 as an counter electrodend Ag/AgCl in 3 mol L−1 KCl Metrhom (Herisau, Switzerland) as
reference was employed for all electrochemical experiments.ll experiments were performed at room temperature in stirred
120 rpm) 0.05 mol L−1 SA buffer (pH 6.0) with 0.1 mol L−1 KCl.lectrochemical detection of the analytical signal was performedt different concentrations of glucose in the presence of N-ethylphenazonium methyl sulphate (2 mmol L−1 PMS for the
valuation of optimal conditions and 6 mmol L−1 PMS for the inves-igation of analytical characteristics). The activity of GOx wasstimated by measuring the re-oxidation current of reduced PMS
orm (PMSH2) and/or H2O2 produced by the enzymatic reactiont +0.3 V potential vs. Ag/AgCl. The results of all electrochemicaleasurements were reported as the mean value of three inde-
endent experiments. The principles of enzymatic reaction andlectrochemical measurement are illustrated in Scheme 1.
uators B 203 (2014) 25–34 27
2.7. Special conditions in stability and selectivity test
For the stability test GOx/Au-NPs/GR electrode was storedbetween measurements at +4 ◦C in closed vessel hanging over0.05 mol L−1 SA buffer to maintain constant humidity. Electrochem-ical detection of analytical signal was performed similarly as it ispresented in previous section, in 10 times diluted human serumsample.
For the substrate selectivity test analytical signals were regis-tered in 10 times diluted human serum sample with 10 mmol L−1
of glucose before and after an addition of 1 mmol L−1 saccha-rose, xylose galactose, mannose or fructose. In order to evaluatethe influence of electroactive compounds the analytical signalswere registered in the serum solution with: 10 mmol L−1 glucose;10 mmol L−1 glucose and 0.01 mmol L−1 ascorbic acid; 10 mmol L−1
glucose and 0.05 mmol L−1 of ascorbic acid; 10 mmol L−1 glucoseand 0.01 mmol L−1 of uric acid.
2.8. Imaging by emission scanning electron microscopy
The surface of graphite rod electrode, which was electro-chemically modified with gold nanoparticles, was characterizedby Hitachi SU-70 field emission scanning electron microscope(FE-SEM). SU70 with additions of EDS and EBSD analysis, and turbo-sputer were used for the imaging of modified GR surfaces.
2.9. Calculations
Amperometric signals showed hyperbolic dependenceon glucose concentration and it was in agreement withMichaelis–Menten kinetics. The parameters of kinetics, themaximal current generated during enzymatic reaction (Imax) andthe apparent Michaelis constant (KM(app)) were correspondinglya and b parameters of hyperbolic function y = ax/(b + x), whichwas used for the approximation of results. The parameters ofthe enzyme-catalyzed reaction were calculated using SigmaPlotsoftware.
Calibration curves of all investigations were obtained by trip-licate measurements, and calibration curve parameters (slope,intercept, correlation coefficient) were calculated. The limit ofdetection (LOD) as the lowest concentration of analyte, which givesan analytical signal greater than the background value plus 3 ı, wasestimated.
3. Results and discussion
3.1. The evaluation of Au-NPs electrochemical depositionconditions
In the most cases the advantage of Au-NPs application is theirability to improve the analytical characteristics of electrochemicalglucose biosensors. We have shown that Au-NPs could facilitatean electron transfer processes between the GOx, soluble PMS andgraphite electrode, and could increase the rate of GOx catalyzedenzymatic reaction [18,19,32,33]. However, the efficiency of Au-NPs-based biosensors depends on the surface concentration andsome other features of immobilized nanoparticles. Therefore, infirst stage of this research, the influence of the Au-NPs depositionconditions was evaluated. The highest activity of GOx/Au-NPs/GRelectrodes was observed at pH 5.8, and this pH value was chosenfor further investigations [34].
The influence of Au-NPs electrochemical deposition by cyclingthe electrode potential between 0.0 and +1.0 V vs Ag/AgCl at scanrate of 0.05 V s−1 was evaluated varying Au-NPs deposition timefrom 5 to 45 min. Fig. 1 illustrates that the registered current is
28 N. German et al. / Sensors and Actuators B 203 (2014) 25–34
Fig. 1. The influence of Au-NP deposition duration on the registered currentof GOx/Au-NPs/GR electrode in presence of 1–1.08, 2–6.04 and 3–17.3 mmol L−1
g0
hciso
mbcotrcAtdTdf
cGisvmmtono
CAuND [nmol L-1]0 1 2 3 4
I [A
]
0
10
20
30
40
1
2
3
Δ
μ
Fig. 2. The effect of 13 nm Au-NPs solution concentration during electrochemicalAu-NP deposition on the registered current of GOx/Au-NPs/GR electrode in pres-
TT
lucose. ECV = 0.0–(+1.0) V vs Ag/AgCl. Amperometric response was measured in.05 mol L−1 SA buffer (pH 6.0) with 2 mmol L−1 PMS.
ighest after 20 min lasting deposition at all tested glucose con-entrations. Further increase of the deposition duration negativelynfluenced the analytical signal of electrochemical glucose biosen-ors. In the further experiments 20 min was selected as the mostptimal duration for Au-NPs deposition.
In the next set of experiments cyclic voltammetric measure-ents of modified electrodes were performed in order to select the
est conditions. According to already published results negativelyharged Au-NPs were electrochemically deposited separately [35]r together with GOx [28] on the surface of electrode by poten-ial cycling in the range from −0.4 to +1.0 V vs Ag/AgCl at a scanate of 0.1 V s−1. On the contrary, the electrochemical oxidation ofolloidal Au-NPs to AuCl4− was performed at constant +1.25 V vsg/AgCl potential in 0.1 M HCl solution [36]. In order to improve
he sensitivity of GOx/Au-NPs/GR electrodes potential cycling con-itions on electrochemical deposition of Au-NPs were investigated.able 1 displays the effect of potential cycling range applied for theeposition of Au-NPs on the efficiency of GOx/Au-NPs/GR electrodeor bioelectrochemical oxidation of glucose.
Under the optimal deposition time of 20 min and potentialycling from 0.0 to +1.0 V vs Ag/AgCl the current registered byOx/Au-NPs/GR electrode at 1.08, 6.04 17.3 mmol L−1 glucose
ncreased, respectively, by 3.56, 1.53 and 1.32 times in compari-on to signal registered after potential cycling from −1.0 to +1.0 Vs Ag/AgCl. Thus, such treatment was chosen as the most opti-al for the development of GOx/Au-NPs/GR electrode. Possibleechanisms of gold nanoparticle electrochemical deposition on
he surface of electrode are based on the electrostatic attraction
f negatively charged Au-NPs and electrochemical oxidation ofanoparticles at higher potential followed by reduction of gold ionsn the already immobilized Au-NPs at lower potential. Electrostatic
able 1he effect of potential cycling range applied for Au-NP deposition on the efficiency of GO
ECV (V)
1.08 mmol L−1 of glucose
−1.0–(+1.0) 0.456
−0.75–(+1.0) 0.739
−0.50–(+1.0) 0.770
−0.25–(+1.0) 1.07
0.0–(+1.0) 1.62
+0.25–(−1.0) 0.678
+0.50–(−1.0) 0.635
+0.75–(−1.0) 0.605
+1.0–(−1.0) 0.589
ence of 1–1.08, 2–6.04 and 3–17.3 mmol L−1 glucose. Amperometric response wasmeasured in 0.05 mol L−1 SA buffer (pH 6.0) with 2 mmol L−1 PMS.
attraction predominates in the deposition mechanism of negativelycharged Au-NPs at potentials lower than 0.75 V vs Ag/AgCl, while Auoxide formation was observed at potentials higher than 0.75 V (dis-tinct oxidation peak is observed at 1.1 V vs Ag/AgCl in a 0.5 mol L−1
H2SO4 solution) [35].
3.2. The influence of Au-NPs, GOx and PMS concentrations on theresponse of GOx/Au-NPs/GR electrodes
In order to achieve the highest sensitivity of biosensor the influ-ence of Au-NPs solution concentration on the response of fabricatedGOx/Au-NPs/GR electrodes was evaluated. Graphite rod electrodeswere electrochemically modified in the presence of different con-centrations of gold nanoparticles using 20 min potential cyclingfrom 0.0 to +1.0 V vs Ag/AgCl. The impact of 13 nm Au-NPs solutionconcentration on the registered current at different concentrationsof glucose is presented in Fig. 2.
Analytical signals at 1.08, 6.04 and 17.3 mmol L−1 glucoseincreased by 2.79, 2.17 and 1.84 times if concentration of Au-NPsin solution was increased up to 0.8 nmol L−1 (Fig. 2, lines 1–3).However, with the further increase of Au-NPs concentration up to1.5 nmol L−1, the registered current decreased. This phenomenonmay be related to the saturation of GR surface by adsorbed Au-NPsand formation of large Au-NPs aggregates, which are less activein comparison with separate Au-NPs. Therefore electron transferbetween the electrode surface and solution of glucose was reduced.
When 2–4 nmol L−1 solutions of Au-NPs were applied, analyticalsignals were at the same range as that for electrodes withoutAu-NP. For further research 0.8 nmol L−1 13 nm Au-NPs solution
x/Au-NPs/GR electrode for bioelectrochemical oxidation of glucose.
Fig. 3. The effect of GOx (A) and PMS (B) concentrations on the registered cur-rent of GOx/Au-NPs/GR electrodes: (A)—analytical signal in the presence of 1–1.08,2 −1 −1
t0
wG
oGtcAtNw
pg
–6.04 and 3–17.3 mmol L glucose and 2 mmol L PMS; (B)—analytical signal inhe presence of 7.21 mmol L−1 glucose. Amperometric response was measured in.05 mol L−1 SA buffer (pH 6.0).
as selected as the optimal concentration for the preparation ofOx/Au-NPs/GR electrodes.
In the next set of investigations the effect of GOx concentrationn the sensitivity of glucose biosensor was investigated. SeveralOx/Au-NPs/GR electrodes based on the same initial concentra-
ion of Au-NPs (0.8 nmol L−1) for the GR modification and differentoncentration of GOx (from 0 to 40 mg mL−1) were developed.s it is shown in Fig. 3A, after increasing GOx concentration up
o 25 mg mL−1 in the solution used for the formation of GOx/Au-Ps/GR electrodes the registered current of developed electrodes
ere significantly increased.
Analytical signal of GOx/Au-NPs/GR electrodes, which were pre-ared using 25 mg mL−1 GOx, registered in the presence of differentlucose concentrations (1.08, 6.14 and 17.3 mmol L−1) increased by
Fig. 4. FE-SEM images of GR electrodes after electrochemical depo
uators B 203 (2014) 25–34 29
9.05, 6.40 and 5.65 times, correspondingly if compared with thesignals obtained by electrodes prepared using 0.5 mg mL−1 of GOx.Further increase of enzyme’s concentration has negligible effect onthe analytical signal of GOx/Au-NPs/GR electrode toward the sameconcentrations of analyte.
An extremely important challenge in the field of biosensorsis the establishment of satisfactory charge transfer between theactive site of the enzyme and the surface of electrode [3]. Fig. 3Bdisplays the influence of redox mediator (PMS) on the registeredcurrent of GOx/Au-NPs/GR electrode for the determination of glu-cose. The increase of analytical signal up to 2.84 times was observedby increasing the concentration of redox mediator from 0.10 to5.77 mmol L−1. However, with the further increase of PMS concen-tration up to 10.9 mmol L−1, the registered current increase wasinsignificant. Therefore for the investigation of analytical charac-teristics of developed biosensors 6 mmol L−1 of PMS was used.
Analytical signal of GOx/Au-NPs/GR electrode depends on themorphology of electrode’s surface. Factors such as surface rough-ness, porosity of films and presence of defects also significantlyaffect the signal of electrode [24]. The surface of graphite rod elec-trode with electrochemically deposited Au-NPs was investigated byemission scanning electron microscopy (Fig. 4). As observed in theFE-SEM images, Au-NPs were randomly distributed on the surfaceof GR. It is visible that gold nanoparticles intended to form aggre-gates. Although gold nanoparticles on the surface of GR electrodewere electrochemically deposited from colloidal solution of 13 nmdiameter Au-NPs, the aggregates of Au-NPs of different diametersin the range of 23–120 nm were formed. The enlargement of Au-NPs approximately 3 times was observed by other authors afterelectrochemical Au-NPs deposition at potentials exceeding +0.7 V[35].
3.3. Electrochemistry of glucose oxidase immobilized on Au-NPs
modified electrode
The application of Au-NPs of different diameter with vari-ous surface functionalizations for the creation of electrochemical
sition of 13 nm Au-NPs from 0.8 nmol L−1 colloidal solution.
3 nd Actuators B 203 (2014) 25–34
ssaamwsotmGttw
tcNtENtiat
fu
k
wtitci(eEi(−s�
f1apioinaNerrN
3
u
CGlu [mmol L-1]0 30 60 90
I [A]
0
60
120
180
1
2A
CGlu [mmol L-1 ]0 3 6 9
I [A]
0
30
60
9034B
Δ
μ
Δ
μ
Fig. 5. Calibration plots of GOx/GR (1,3) and GOx/Au-NPs/GR (2,4) electrodes for0.1–100 mol L−1 (A) and 0.1–10.0 mmol L−1 (B) glucose concentrations. Amperomet-
0 N. German et al. / Sensors a
ensing devices is very promising prospect [23]. It was demon-trated that 1.4 nm Au-NPs with appropriate functionalizationdjacent to the enzyme redox centre (the reconstitution of anpoenzyme with FAD-Au-NPs) could act as an electric relaying ele-ent between the GOx redox active centre and the macroelectrodeith high electron transfer turnover rate [22]. The glucose biosen-
or could be prepared by simple dropping glucose oxidase solutionn the surface of graphite rod electrode modified by 13 nm diame-er Au-NPs [18,19,23]. The application of Au-NPs and soluble redox
ediator increased analytical signal during biocatalytic action ofOx in the presence of glucose. The electron transfer from enzyme
o electrode via a reduced form of mediator (PMSH2) may occur inwo ways: directly to the GR electrode or in combination of PMSH2ith Au-NPs.
Reversibility of oxidation/reduction of PMS at both elec-rodes GOx/Au-NPs/GR and GOx/GR were determined byycling voltammetry at scan rate of 0.02 V/s: for GOx/Au-Ps/GR electrode Epc = 0.024 V, Epa = −0.126 V, and therefore
he �E = Epc − Epa = 0.150 V; for GOx/GR electrode Epc = 0.0420 V,pa = −0.1420 V, �E = 0.184 V. Thus the �E in the case of GOx/Au-Ps/GR electrode was lower therefore it could be concluded that
he reversibility of PMS on Au-NPS modified electrode was signif-cantly better. Cyclic volatmmograms of PMS on GOx/Au-NPs/GRnd GOx/GR electrodes in the presence of glucose are presented inhe supplementary material (Fig. S1).
The heterogeneous electron transfer rate constant (k0) of PMSor GOx/Au-NPs/GR and GOx/GR electrodes has been calculatedsing Kochi’s method [37].
0 = 2.18[
˛�nFD0
RT
]1/2exp
[−˛2nF�Ep
RT
]
here: is a dimensionless parameter known as electronransfer coefficient, n is the number of electrons involvednto redox process (n = 2), D0 is the diffusion coefficient (inhis particular case D0 was 5 × 10−5 cm2 s−1), F is Faradayonstant (96,485 C mol−1), � is scan rate (� = 0.02 V s−1), Rs a gas constant (8.314 J mol−1 K−1), T is a temperature in KT = 293 K). It should be noted, that cathodic peak in the pres-nce/absence of Au-NPs has shifted by 0.018 V (�Ec = Ec(GOx/GR) −c(GOx/Au-NPs/GR) = 0.0420 V − 0.024 V = 0.018 V); anodic peakn the presence/absence of Au-NPs has shifted by 0.016 V�Ea = Ea(GOx/GR) − Ea(GOx/Au-NPs/GR) = −0.1420 V − (−0.126 V) =0.016 V). The shifts of cathodic and anodic peaks are almost
ymmetrical therefore in this particular case the value of applied was equal to 0.5.
Calculated heterogeneous electron transfer rate constantor GOx/Au-NPs/GR and GOx/GR are 2.26 × 10−6 cm s−1 and.15 × 10−6 cm s−1, respectively. The presence of Au-NPs results inlmost double increase of electron transfer rate constant. The com-arison of k0 values indicates that the electron transfer between
mmobilized GOx and electrode in the presence of PMS is fastern the electrode modified with Au-NPs. Cyclic volatmmogramsn the absence/presence of glucose (Fig. S2) show that there iso significant shift of oxidation/reduction peak potentials in thebsence/presence of glucose, therefore we can conclude that Au-Ps significantly facilitate electron transfer between PMS andlectrode. Moreover, significantly higher amperometric signal wasegistered by GOx/Au-NPs/GR electrode in comparison with thategistered by GOx/GR electrode, what is also an evidence that Au-Ps facilitate electron transfer between PMS and electrode.
.4. Analytical characteristics of developed biosensors
The analytical characteristics of two type electrodes preparednder optimized conditions were evaluated. One type of electrode
ric response was measured in 0.05 mol L−1 SA buffer (pH 6.0) with 6 mmol L−1 PMSat +0.3 V vs Ag/AgCl.
(GOx/GR) was based on GR electrode modified by 25 mg mL−1 ofGOx solution. Another type of electrode (GOx/Au-NPs/GR) wasbased on GR electrode modified with electrochemically depositedAu-NPs from 0.8 nmol L−1 solution of 13 nm Au-NPs (20 min, poten-tial cycling from 0.0 to +1.0 V, scan rate 0.05 V s−1) and immobilized25 mg mL−1 of GOx solution. Amperometric measurements wereperformed with developed electrodes in 0.05 mol L−1 SA buffer(pH 6.0) with 6 mmol L−1 PMS and different glucose concentra-tions. During enzymatic reaction electrons were transferred towardworking electrode by PMS and steady-state currents were reg-istered. The electrode reached 95% of the steady-state currentwithin 5 s. The hyperbolic dependences of analytical signals on theconcentration of glucose in the range from 0.1 to 100 mmol L−1
are presented in Fig. 5A. The current response of the electrodesincreased along with glucose concentration until a plateau cur-rent is observed. All presented hyperbolic dependences were inan agreement with Michaelis–Menten kinetics (Fig. 5). The maxi-mal current generated during enzymatic reaction and the apparentMichaelis constant are correspondingly a and b parameters ofhyperbolic function y = ax/(b + x). Results presented in Fig. 5A illus-trates that presence of Au-NPs increased the analytical signal ofdeveloped GOx/Au-NPs/GR electrode: Imax was 1.4 times higher incomparison to GOx/GR electrode. This effect is based mainly on fourfactors: (1) significantly increased electron transfer rate from GOxto GR electrode because gold nanoparticles increases effective sur-face area of electrode and facilitate the electron transfer betweenPMS and electrode [19]; (2) improved stability of GOx/Au-NPs/GRelectrode due to strong adsorption of enzyme on Au-NPs, whichprevents the leakage of GOx from the surface [5]; (3) preservedenzyme activity because globular proteins retain their structurebetter on the surfaces of higher curvature [23,38]; (4) advanced ori-entation freedom of GOx molecules, which at some extent enhancea proper enzyme orientation on electrode [23].
The current of GOx/Au-NPs/GR electrode registered at10 mmol L−1 glucose (I = 93.2 �A) was compared with that offew other electrodes at the same concentration of glucose wheredifferent surface modification by Au-NPs methods and severalredox mediators were applied in order to improve an electro-chemical signal. For electrode based on GOx immobilized ongraphite electrode modified by 13 nm diameter Au-NPs the
−1
steady-state current in the presence of 10 mmol L glucosewas about 42.0 �A [18], for GOx immobilized on glassy carbonelectrode modified by 12 nm diameter Au-NPs and Nafion film –4.5 �A [39], for GOx immobilized on 11 nm Au-NPs self-assembled
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Table 2Analytical parameters of glucose biosensors based on Au-NPs deposited on electrodes by different methods in comparison to biosensors without Au-NPs.
Surface modification LOD/sensitivity Linear detection range Transductionsystem/mediator
Reference
With gold nanoparticlesOne step electrodeposition of chitosan hydrogel, GOx, Au-NPs (17 nm) on Au electrode 0.0027 mmol L−1 0.005–2.4 mmol L−1 Amperometry Luo et al. [25]
–Covalent immobilization of GOx on Au-NPs (2.6 nm) deposited on self-assembled monolayermodified Au electrode
Immobilization of GOx/Au-NPs (12 nm) on glassy carbon electrode by Nafion film 0.034 mmol L−1 up to 6 mmol L−1 Amperometry Zhao et al. [39]6.5 �A mM−1 cm−2 Ferrocenemethanol
Multilayer films of GOx and Au-NPs (12 nm) formation on Au electrode using a cross-linker 0.008 mmol L−1 0.01–13 mmol L−1 Amperometry Yang et al. [13]5.72 �A mM−1 cm−2 Ferrocenemethanol
Immobilization of GOx on Au-NPs (11 nm) self-assembled on Au electrode modified bythree-dimensional network of silica gel
0.023 mmol L−1 Up to 6 mmol L−1 Amperometry Zhang et al. [10]8.3 �A mM−1 cm−2 Ferrocenemethanol
GOx immobilized on electrochemically deposited of Au-NPs on graphite electrode 0.083 mmol L−1 0.1–10 mmol L−1 Amperometry German et al. (recentpublication)101.02 �A mM−1 cm−2 Phenazine methosulfate
Without gold nanoparticlesElectrically contacted GOx via reconstitution of apo-enzyme with electron-relay-functionalizedFAD derivatives
n Au electrode modified by thiol-containing three-dimensionaletwork of silica gel – 2.6 �A [10], while for electrode based onOx multilayer films structures consisting of 6 layers of GOxnd Au-NPs attached to Au electrode through a cross-linker theteady-state current at the same glucose concentration was 3.3 �A13]. In biosensors where indium tin oxide electrode was modi-ed with electrochemically synthesized Au-NPs (60–100 nm) onhitosan-ionic liquids-multiwalled carbon nanotubes and GOx theegistered steady-state current was about 40 �A in the presencef 10 mmol L−1 glucose [27]. Hence high differences betweenegistered currents at the same glucose concentration might bexplained by different surface modification methodology, varyingoncentration of immobilized enzyme, different mediator, andpplied electrochemical method. The apparent Michaelis constantf GOx/Au-NPs/GR electrode (KM(app) = 19.9 mmol L−1) determinedrom the hyperbolic dependence of analytical signal on glucoseoncentration was 1.42 times higher if compared with the samearameter for GOx/GR electrode (KM(app) = 14.0 mmol L−1). Theame tendency was observed in our previous publication whereon-electrochemical method for 13 nm Au-NPs deposition on theurface was applied [18].
The linear glucose detection range for GOx/Au-NPs/GR andOx/GR electrodes can be extended at least up to 10 mmol L−1
f glucose without the intercepts on x- or y-axis (R2 are 0.9964nd 0.9956) (Fig. 5B). It is higher or almost the same as inther glucose biosensors based on Au-NPs deposited on elec-rodes by different methods (Table 2). The linear glucose detectionange for electrode based on covalently immobilized GOx on.6 nm diameter Au-NPs deposited on Au electrode pre-modifiedy self-assembled monolayer was up to 5.7 mmol L−1[8], forOx immobilized on 13 nm diameter Au-NPs modified graphitelectrode—up to 10 mmol L−1[18], for GOx/12 nm Au-NPs immo-ilized in Nafion film on glassy carbon electrode and for GOx
mmobilized on 11 nm diameter Au-NPs self-assembled on Au elec-rode modified by thiol-containing three-dimensional network ofilica gel—up to 6 mmol L−1[10,39], and for electrode based on mul-ilayer films of GOx and 12 nm Au-NPs attached to Au electrode—upo 13 mmol L−1[13]. The similar linear glucose detection range wasegistered by electrode without any Au-NPs, where the GOx wasmmobilized in a ferrocene attached poly(4-vinylpyridine) multi-ayer film deposited on gold electrode (five layers) [40]. The widestinear glucose detection range (up to 20 mmol L−1) using electrodes
odified with gold nanoparticles was registered using indiumin oxide glass electrode modified by electrodeposited 4–6 nmiameter AuNPs-silicate-GOx biocomposite and registration ofmperometric H2O2 oxidation [28]. However, biosensors basedn GOx ‘wired’ by reconstitution of apo-enzyme with electron-elay-functionalized FAD derivatives [41] or reconstituted GOx onu-electrode, which was modified by PQQ/FAD monolayer [42],nsured linear glucose detection range up to 80 mmol L−1.
The sensitivity of developed GOx/Au-NPs/GR and GOx/GRiosensors up to 10 mmol L−1 of glucose concentration is 101.02nd 83.73 �A mM−1 cm−2, respectively. In comparison, the Aulectrode modified with GOx immobilized in films of chitosan con-aining nanocomposites of graphene and 7–16 nm diameter AuNPsxhibited good electrocatalytic activity toward H2O2 and O2, andensitivity of such system was similar to that of our system –9.5 �A mM−1 cm−2 [43]. The LODs for the developed GOx/Au-Ps/GR and GOx/GR electrodes at a signal to noise ratio 3 wereetermined to be 0.083 mmol L−1 and 0.095 mmol L−1, respectively.he relative standard deviation of analytical signal at glucose con-entrations up to 1 mmol L−1 was 6% for GOx/Au-NPs/GR electrode
nd 10% for GOx/GR electrode. The LOD for the developed GOx/Au-Ps/GR is lower or similar [18,43], but in most cases it is higher
f compared to other glucose biosensors with or without goldanoparticles [40,44] (Table 2).
Amperometric response was measured in a human serum sample diluted 10 timesin 0.05 mol L−1 SA buffer (pH 6.0) containing 0.1 mol L−1 KCl, with 6 mmol L−1 PMSat +0.3 V vs Ag/AgCl.
The major advantages of newly developed biosensing systemare: (i) simple and quick (within 5 s) detection of glucose, (ii) highanalytical signal, (iii) linear range of analyte determination up to10 mmol L−1 of glucose and (iv) reliable limit of detection. The linearrange of biosensor can be extended by addition of diffusional mem-brane based on conducting polymers in order to obtain biosensormore suitable for monitoring diabetic patients. Electrodes based onAu-NP (3.5, 6 and 13 nm) and GOx in combination with enzymati-cally formed polypyrrole layer offered higher sensitivity dependingon the size of Au-NP (electrodes with smaller nanoparticles exhib-ited higher amperometric response) and up to 2.4 times higherKM(app) (after 13 h of enzymatic synthesis of polypyrrole) [19].
3.5. Detection of glucose in a human serum sample
The feasibility of developed electrochemical biosensor to detectglucose using amperometric detection was investigated in spikedhuman serum samples. Serum was diluted 10 times in 0.05 mol L−1
SA buffer (pH 6.0) containing 0.1 mol L−1 KCl and then various con-centrations of glucose were added (Table 3). The linear responseof GOx/Au-NPs/GR electrode in the serum was until 10 mmol L−1
of glucose. It was found that the recoveries were in the range of94–99%. Therefore, the developed electrochemical biosensor basedon amperometric detection is applicable for the determination ofglucose in real samples. It should be noted that developed biosen-sor (linear range up to 10 mmol L−1 of glucose) was tested in 10times diluted serum.
3.6. Stability and selectivity of developed biosensor
In the present research the stability of developed glucosebiosensor (GOx/Au-NPs/GR electrode) was investigated by mea-suring immobilized GOx activity in diluted serum sample during9-day period. As can be seen from the results presented in Fig. 6A,the hyperbolic dependences of analytical signals on the glucoseconcentration from 1.0 to 100 mmol L−1 were registered. The cur-rent of modified electrode to glucose gradually decreased during6 days, however, after this down-fall considerably lower reduc-tion of analytical signal was observed. After 7 days GOx/Au-NPs/GRelectrode retained 64.4% of the initial activity, while after 8 and 9days very similar results were obtained – 64.2 and 61.0% (Fig. 6B).The decrease of analytical signal during 7 days could be effectedby desorption enzyme, which was non-covalently immobilized ongold nanoparticles, and the denaturation of some GOx moleculesduring electrochemical measurements.
The selectivity of developed biosensor was investigated in solu-tion of 10.0 mmol L−1 glucose after the addition of 1 mmol L−1 of
saccharose, xylose, galactose, mannose or fructose (Fig. 7A). It wasdetermined that these substrates have no effect on the analyticalsignal. It is well known that some electroactive species in serum,including ascorbic and uric acids, may influence the performance
N. German et al. / Sensors and Act
Fig. 6. Calibration plots (A) and stability (B) of GOx/Au-NPs/GR electrode. (Con-ditions: 1–8 curves were registered after 0, 1, 2, 3, 6, 7, 8 and 9 days, respectively.Steady-state currents were registered after an addition of glucose into human serumat +0.3 V vs. Ag/AgCl.)
Fig. 7. Effect of interfering species on the response of the biosensor.(A)—Chronoamperogram registered the serum after addition of 10.0 mmol L−1 Glu,1 mmol L−1 saccharose, xylose, galactose, mannose or fructose. (B)—Diagram ofraa
oeasaundsafo
pphto
4
Ah
[
[
[
[
[
egistered response after addition of 10 mmol L−1 glucose (1), 10 mmol L−1 glucosend 0.01 mmol L−1 ascorbic acid (2); 10 mmol L−1 glucose and 0.05 mmol L−1 ofscorbic acid (3); 10 mmol L−1 glucose and 0.01 mmol L−1 of uric acid (4).
f electrochemical glucose biosensor. In order to evaluate the influ-nce of ascorbic and uric acid on the determination of glucose,nalytical signals at physiological concentrations of these sub-tances were registered. As can be seen in Fig. 7B the addition of 0.01nd 0.05 mmol L−1 ascorbic acid (2 and 3 columns) or 0.01 mmol L−1
ric acid (4 column) to 10 mmol L−1 glucose solution (1 column) doot have a significant effect on the biosensor performance. It wasetermined that at higher ascorbic acid concentration registeredignal increased only by 1.06 times, while in the presence of uriccid registered signal increased by 1.12 times. It suggests that ourabricated electrodes are favorable for the selective determinationf glucose in the presence of interfering species.
Despite good analytical characteristics in buffer and serum sam-le the developed electrochemical glucose biosensor due to theresence of soluble mediator still needs some improvements forome glucose monitoring of diabetic patients. Thus the modifica-ion of electrode with insoluble mediator will be the next step ofur research.
. Conclusion
A biosensor for glucose detection based on GOx immobilized onu-NPs electrochemically predeposited on the electrode surfaceas been developed. The highest analytical signal was observed
[
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after 13 nm Au-NP deposition on the electrode from 0.8 nmol L−1
solution lasting 20 min, when cyclic voltammetry was performedin the range of 0.0 to +1.0 V, and using 25 mg mL−1 GOx solution forelectrode modification. In this study it was shown that during enzy-matic reaction Au-NPs facilitated electron transfer from the activecentre of the enzyme to electrode via PMS as the redox media-tor. The reversibility of PMS on Au-NPs premodified electrode wassignificantly better in contrast to that on the Au-NPs unmodifiedelectrode. The developed biosensor is simple to operate, yields aquick response with high analytical signals and linear range up to10 mmol L−1 of glucose. The limit of detection of glucose using thenewly developed biosensor with electrochemically deposited Au-NPs was 0.083 mmol L−1, and was lower than that of the GOx/GRelectrode. The developed electrochemical biosensor retained 61.0%of initial analytical signal after electrochemical measurements dur-ing 9 days, and it is favorable for the selective determination ofglucose in the human serum in the presence of interfering species,which are mostly present in real samples.
Acknowledgments
The work was supported by Research Council of Lithuania, Sup-port to research of scientists and other researchers (Global Grant),Enzymes functionalized by polymers and biorecognition unit forselective treatment of target cells (NanoZim’s), Project Nr. VP1-3.1-SMM-07-K-02-042. The authors are thankful to Dr. S. Sakirzanovasfor FE-SEM images of electrodes.
Appendix A. Supplementary data
Supplementary material related to this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.snb.2014.06.021.
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Biographies
Dr. Natalija German is a scientist in the State ResearchInstitute Center for Innovative Medicine, in the Divisionof Immunology. Also she works in the State ResearchInstitute Centre for Physical Sciences and Technology,Institute of Semiconductor Physics, Department of Mate-rials Science and Electronics in Lithuania. She received herPh.D. degree in chemistry at Vilnius University, Faculty ofChemistry in 2007 years. Her main research interests areelectroanalytical chemistry, nanotechnology, biosensorsand conducting polymers.
Prof. Habil. Dr. Arunas Ramanavicius is a professor atVilnius University, Vilnius, Lithuania. He is head of Depart-ment of Physical Chemistry at Vilnius University andNanoTechnas—Centre of Nanotechnology and MaterialsScience. He is also heading department of NanoBioTech-nology at State Research Institute Centre for PhysicalSciences and Technology. In 1998 he received Ph.D. degreeand in 2002 doctor habilitus degree from Vilnius Univer-sity. Prof. A. Ramanavicius is serving as expert-evaluatorin EU-FP7 program coordinated by European Commissionand he is technical advisor of many foundations located inEuropean and non-European countries. He has researchinterests in various aspects of nanotechnology, bionan-
otechnology, nanomaterials, biosensorics, bioelectronics, biofuel cells and MEMSbased analytical devices. He is a national coordinator of several nanotechnologyrelated COST actions.
Assoc. Prof. Dr. Almira Ramanaviciene is head of anbiosensor reasearch groups at Nanotechnas—Centre forNanotechnology and Materials Science at the Facultyof Chemistry of Vilnius University, and State ResearchInstitute Centre for Innovative Medicine, Lithuania. Shereceived her Ph.D. degree in biomedicine from the Insti-tute of Immunology and Vilnius University in 2002.She completed habilitation procedure in Physical Sci-ences at Vilnius University in 2008. Assoc. Prof. Dr.Almira Ramanaviciene is serving as FP7 projects expert
for the European Commission and other internationaland national foundations. She has research interests inthe field of biosensors and immunosensor development
focusing on different surface modification techniques and various detection meth-ods.
