ORIGINAL PAPER

Electrochemical biosensor for sensitively simultaneousdetermination of dopamine, uric acid, guanine, and adeninebased on poly-melamine and nano Ag hybridizedfilm-modified electrode

Hongying Li & Xueliang Wang & Zhangyu Yu

Received: 6 July 2013 /Revised: 22 August 2013 /Accepted: 24 August 2013 /Published online: 12 September 2013# Springer-Verlag Berlin Heidelberg 2013

Abstract An electrochemical sensor for simultaneous deter-mination of dopamine (DA), uric acid (UA), guanine (G), andadenine (A) has been constructed by copolymerizing mela-mine monomer and Ag ions on a glassy carbon electrode(GCE) with cyclic voltammetry. The poly-melamine and nanoAg formed a hybridized film on the surface of the GCE. Themorphology of the film was characterized by scanning elec-tron microscope. The electrochemical and electrocatalyticproperties of this film were characterized by cyclicvoltammetry, linear sweep voltammetry, and square wavevoltammetry (SWV). In 0.1 M phosphate buffer solution(pH 4.5), the modified electrode resolved the electrochemicalresponse of DA, UA, G, and A into four well-definedvoltammetric oxidation peaks by SWV; the oxidation peakcurrent of DA, UA, G, and A increased 13-, 6-, 7-, and 9-fold,respectively, compared with those at the bare GCE and theSWV peak currents of DA, UA, G, and Awith linear concen-trations in the ranges of 0.1–50, 0.1–50, 0.1–50, and 0.1–60μM, respectively. Based on this, a method for simultaneousdetermination of these species in mixture was setup. Thedetection limits were 10 nM for DA, 100 nM for UA, 8 nMfor G, and 8 nM for A.

Keywords Electrochemical biosensor . Simultaneousdetermination . Poly-melanine . Silver . Hybridizedmembrane

Introduction

Dopamine (DA) is one of the most significant catecholamines.It belongs to the family of excitatory chemical neurotransmit-ter and plays a significant role in functioning of the centralnervous, renal, and hormonal systems [1]. Extreme abnormal-ities of the DA levels cause symptoms of several diseases suchas Parkinsonism, schizophrenia, and Huntington's chorea [2].Therefore, it is of great clinical importance to measure the DAlevel in the extracellular fluid in order to monitor neurotrans-mission process and diagnose related diseases [3]. Thus, thereis a continuing interest in developing simple, sensitive, andreliable method for determination of DA. Generally, the de-termination of DA is performed with high-performance liquidchromatography [4], ion chromatography [5], and spectropho-tometry [6]. Meanwhile, DA can be determined by electro-chemical methods because it is an electrochemically activecompound. However, the major problem encountered in theelectrochemical detection of DA in real samples is thecoexisted interferences, such as uric acid (UA) which has anoverlapped oxidation potential with DA on unmodified elec-trodes [7]. Uric acid is the terminative oxidation product ofpurine degradation metabolism in human beings. Its concen-tration level in body fluids, such as human serum and urine, isa marker of many clinical conditions, including hyperurice-mia, gout, and Lesch–Nyan disease [8, 9]. Therefore, thedevelopment of a selective and sensitive method for its simul-taneous determination is highly desirable for analytical anddiagnostic applications. In recent years, chemically modified

Electronic supplementary material The online version of this article(doi:10.1007/s10008-013-2242-9) contains supplementary material,which is available to authorized users.

H. Li :X. Wang : Z. Yu (*)Department of Chemistry and Chemical Engineering, HezeUniversity, Heze 274015, People’s Republic of Chinae-mail: yuzywxl@163.com

H. Lie-mail: hxxlhy@163.com

X. Wange-mail: qust-1977wxl@163.com

J Solid State Electrochem (2014) 18:105–113DOI 10.1007/s10008-013-2242-9

electrodes have become important electrochemical detectionmethods for the determination of biologically important com-pounds because of their good sensitivity, selectivity, and sta-bility [10–12]. Khoo et al. fabricated a modified electrode forthe simultaneous determination of ascorbic and uric acids byimmobilizing methylene blue in a methyltrimethoxysilanesol–gel ceramic film [13]. Hasoň et al. studied the determina-tion of uric acid, adenine, and guanine in a mixture usingmechanically grinded carbon/graphite electrodes; the detec-tion limits were 2, 5, and 18 nM, respectively [14]. Hu et al.studied the selective determination of dopamine at a multiwallcarbon nanotube–Nafion film-coated glassy carbon electrode(GCE) with the detection limit of 2.5 nM [15].

Guanine (G) and adenine (A) are two important buildingblocks of both DNA and RNA and play a vital role in geneticinformation storage and involve in processes such as energytransduction, metabolic cofactors, and cell signaling [16].Indeed, G and A in physiological fluids, tissues, and cellsare related to the catabolism of nucleic acids, enzymaticdegradation of tissues, dietary habits, and various salvagepathways, so detection of elevated levels of these substancescould indicate certain disease. The electrochemical sensors forthe determination of G and A have received great interest inthe fields of pharmacological and clinical analysis due to theirfast response, high sensitivity, excellent selectivity, and real-time detection in situ condition [17–19]. However, G and Aexhibit slow direct electron transfer and irreversible absorp-tion on the electrode surface. Therefore, considerable effortshave been paid for improving its electrochemical sensingperformance. For example, Yang et al. [20] prepared TiO2–graphene nanocomposite modified glassy carbon electrode;the results showed that the incorporation of TiO2 nanoparticleswith graphene significantly improved the electrocatalytic ac-tivity and voltammetric response of these two species.Shahrokhian et al. [21] fabricated an electrochemical biosen-sor based on Fe3O4NPs/multiwalled carbon nanotubes nano-composite for sensitive simultaneous determination of traceamounts of A and John et al. [22] prepared a self-assembledmonolayer of 1, 8, 15, 22-tetraaminophthalocyanatonickel (II)modified glassy carbon electrode for selective determinationof guanine. Liu et al. [23] prepared a biosensor for simulta-neous determination of epinephrine, G, and A based on poly-melamine-modified electrode, and the biosensor just simulta-neously determined three compounds. Niu et al. [24] fabricat-ed a biosensor based on nano-Au/DNA/nano-Au/polycomposite-modified electrode and employed it to simulta-neous determination of DA, UA, G, and A. Nanosilver, as akind of important metal nanomaterial, has several advantagessuch as unique property to accelerate electron transfer, goodbiocompatibility, electrocatalytic activity and chemical stabil-ity, and has been assembled on electrodes to be used aselectrocatalyst for various reactions [25–27]. However, theimmobilization of metal nanomaterials is relatively difficult

and limits their use in electrochemical sensors and biosensorsbecause the metal nanomaterials easily fall-out from electrodesurface. Incorporating metal nanomaterials into conductingpolymer membrane is an extensively used effective methodin electronics, sensors, and catalysis [28, 29] because thesemembranes have synergistic chemical and physical propertiesbased on the constituent polymer and metal. However, to thebest of our knowledge, there have been no reports about apoly-melamine and nano Ag hybridized film-modified elec-trode being used for sensitive simultaneous determination ofDA, UA, G, and A.

In this study, an electrochemical biosensor was fabricatedby electropolymerizing Ag ions and melamine monomer onthe surface of glassy carbon electrode (Ag-PMel/GCE) for thefirst time and employed to simultaneous determination of DA,UA, G, and A. The electrochemical oxidative behaviors ofDA, UA, G, and A were investigated by linear sweepvoltammetry (LSV) and square wave voltammetry (SWV).This Ag-PMel/GCE proved as a promising biosensor forsimultaneous determination of DA, UA, G, and A due to itshigh sensitivity, wide detection range, and low detection limit.

Experimental

Reagents

DA, UA, and ascorbic acid were purchased from AladdinChemical Reagent Co., Ltd. (Shanghai, China). EP, G, A,glucose, sucrose, melamine, and L -glutamic acid wereobtained from Sinopharm Chemical Reagent Co., Ltd.(Shanghai, China). All chemicals were of analytical grade,and triple-distilled water was used throughout. All experi-ments were performed in 0.1 M phosphate-buffered saline(PBS) with different pH values adjusted with Na2HPO4,NaH2PO4, NaOH, and H3PO4.

Apparatus

Electrochemical experiments were performed on a CHI 660Delectrochemical workstation (Shanghai Chenhua Co., Ltd.,China) with a conventional three-electrode system consistingof amodified GCE (Φ =3.0mm)working electrode, a Ag/AgClreference electrode(just employed to electropolymerizationprocess) or a saturated calomel reference electrode, and aplatinum foil counter electrode. Scanning electron micrographsmeasurements were carried out on scanning electron micro-scope (JSM-6700 F, 15.0 kV, Japan). Cyclic voltammetry wasconducted in a solution containing 1.0 mM K4[Fe(CN)6]/K3[Fe(CN)6] (1:1) and 0.1 M KCl. To record cyclicvoltammograms, the following instrumental parameters wereused: initial potential, −0.2; final potential, 0.6; scan rate,100 mV·s−1; sample interval, 1 mV; quiet time, 8 s; sensitivity,

106 J Solid State Electrochem (2014) 18:105–113

1×10−5. The SWV measurements and LSV measurementswere conducted in 0.1 M PBS (pH 4.5). Instrumental parame-ters of SWV were as follows: step potential, 4 mV; modulationamplitude, 25 mV; and frequency, 15 Hz. Instrumental param-eters of LSV were as follows: sample interval, 1 mV; quiettime, 8 s; sensitivity, 1×10−5.

Fabrication of the Ag-PMel/GCE

Before modification, GCE was polished on a chamois leatherwith 0.05 μm alumina powder and then washed successivelywith 1:1 aqueous HNO3 (v /v ), ethanol, and triple-distilledwater in ultrasonic bath and allowed to dry at room tempera-ture. Finally, the Ag-PMel/GCE was prepared by cyclicvoltammetry using GCE as working electrode in 10 mL mix-ture containing 0.1 M HNO3, 0.1 M KNO3, 1.0 mM mela-mine, and 1.0 mM AgNO3 in the potential range of 0–1.6 Vfor 18 cycles at a scan rate of 100 mV s−1.

Results and discussion

The morphology of the Ag-PMel/GCE

The morphology of the Ag-PMel film was characterized byscanning electron microscope (Fig. 1). It can be seen that thenano Ag particles aggregated to form irregular large particles.The PMel film was assembled on the GCE surface to form apattern of grassland (Electronic supplementary material(ESM) Fig. S1). The grassland provides a platform for thedeposition of nano Ag particles. The PMel and nano Agparticles hybridized membrane provided large active surfacewhich is advantageous to electron transfer and electrochemi-cal sensing [30].

The electrochemical properties of the Ag-PMel/GCE

To investigate the electrochemical properties of the bare GCE,Ag/GCE, PMel/GCE, and Ag-PMel/GCE, the cyclicvoltammetry was conducted in aqueous solution containing1.0 mM K4[Fe(CN)6]/K3[Fe(CN)6] (1:1) and 0.1 M KCl(Fig. 2). On the bare GCE, a definite reversible redox peakswere obtained (curve a). When a layer of Ag was covered onthe GCE (Ag/GCE, curve b), the peak current was larger thanthat on the bare GCE, but the peak-to-peak separation had nosignificant change in comparison to curve a, which could beascribed to the good conductivity of the nano Ag particles. Onthe PMel/GCE (curve c), the redox peak current was evensmaller than that on the bare GCE although its electroactivesurface was larger than the bare GCE, indicating that the PMelwas a nonconductive film and its main effect in determinationwas electrocatalyzing oxidation analytes. With the incorpora-tion of nano Ag particles and poly-melamine film, thevoltammetric response of the Ag-PMel/GCEwas significantlyimproved (curve d). However, its peak-to-peak separation waslarger compared with that of curves a and b, which showedthat the larger electroactive surface of Ag-PMel played animportant effect.

The effective area (A ) of the Ag-PMel film-modified elec-trode was calculated using 1.0 mM K4Fe(CN)6 probeaccording to the Randles–Sevick Eq. [31]:

ip ¼ 2:69� 105n3=2AC0D01=2v1=2

where D0, C0, ip, A , n , and v are the diffusion coefficient (insquare meter per second), the bulk concentration (in mole percubic centimeter), the peak current (A), the effective area ofmodified electrode (in square centimeter), the number of

Fig. 1 Scanning electron micrographs of Ag-PMel film with 20 K×magnification

-0.2 0.0 0.2 0.4 0.6-50

-40

-30

-20

-10

0

10

20

30

40

50

60

I/A

E/V(vs.SCE)

a

c

d

b

Fig. 2 Cyclic voltammograms of 1 mMK4[Fe(CN)6]/K3[Fe(CN)6] (1:1)contained 0.1 M KCl at GCE (a), Ag/GCE (b), PMel/GCE (c), and Ag-PMel/GCE (d), respectively

J Solid State Electrochem (2014) 18:105–113 107

0.1 0.2 0.3 0.4 0.5 0.60

10

20

30

40

I /µ

A

E/V(vs.SCE)

DA

d

c

a

b

(A) e

UA

0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.30

5

10

15

20

25

30

35

I/µ

A

E/V(vs.SCE)

e

d

a

c

b

G

A

(B)Fig. 3 a SWVof 0.1 M PBS(pH 4.5) in the absence ofanalytes at Ag-PMel/GCE (a),20 μM DA and 20 μM UA in0.1 M PBS (pH 4.5) at the bareGCE (b), PMel/GCE (c), Ag/GCE (d), and Ag-PMel/GCE (e).b SWVof 0.1 M PBS (pH 4.5)in the absence of analytes atAg-PMel/GCE (a), 20 μM Gand 20 μM A in 0.1 M PBS(pH 4.5) at the bare GCE (b),PMel/GCE (c), Ag/GCE (d),and Ag-PMel/GCE (e)

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

-2

0

2

4

6

8

10

12

14

16

I /µ

A

(A)

0 100 2000

3

6

9

I pa/

A

scan rate/mV s-1

a

f

0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

2

4

6

8

10

12

14

16

0 100 200

0

3

6

I pa/

A

scan rate / mV s-1

I/

µA

E/V (vs.SCE)E/V (vs.SCE)

a

i

(B)

0.7 0.8 0.9 1.0 1.1 1.2 1.3

0

5

10

15

20

25

30

0 70 1400

6

12

I pa/

A

scan rate / mV s-1

I/

µA

E/V (vs.SCE)

(C)

a

g

1.0 1.1 1.2 1.3 1.4 1.5

0

10

20

30

40

50

60

70

60 120 180

7

14

21

I pa/

A

scan rate / mVs-1

I/µ

A

E/V (vs.SCE)

a

g

(D)

Fig. 4 a LSVof 20 μM DA atvarious scan rates (from a to f : 10,40, 80, 120, 160, and 200mV s−1)in 0.1 M PBS (pH 4.5) at Ag-PMel/GCE. The inset shows thelinear relationship between peakcurrent and scan rate. b LSVof20 μM UA at various scan rates(from a to i : 10, 30, 50, 70, 90,120, 160, 200, and 220mV s−1) in0.1 M PBS (pH 4.5) at Ag-PMel/GCE. The inset shows the linearrelationship between peak currentand scan rate. c LSVof 20 μM Gat various scan rates (from a to g :10, 30, 50, 70, 90, 120, and140 mV s−1) in 0.1 M PBS(pH 4.5) at Ag-PMel/GCE. Theinset shows the linear relationshipbetween peak current and scanrate. d LSVof 20μMA at variousscan rates (from a to g : 50, 70, 90,120, 140, 160, and 180mV s−1) in0.1 M PBS (pH 4.5) at Ag-PMel/GCE. The inset shows the linearrelationship between peak currentand scan rate

108 J Solid State Electrochem (2014) 18:105–113

transferred electrons, and the scan rate (in volt per second),respectively. The slope of the plot of ip versus v 1/2 wasobtained by varying the scan rate to obtain a series of ip.Then, the effective area of 0.098 cm2 for this film-modifiedelectrode was calculated with D0=7.6×10

−6 cm2 s−1, C0=1.0 mM, and n =1 [31].

Electrocatalytic oxidation of DA, UA, G, and Aon the modified electrode

The electrocatalytic activity of Ag-PMel/GCE, Ag/GCE,PMel/GCE, and bare GCE for DA, UA, G, andAwas recordedby SWV in 0.1 M PBS (pH 4.5) containing 20 μM DA and20 μMUA (Fig. 3a), 20 μMG, and 20 μMA (Fig. 3b). It canbe seen that there was no peak at the Ag-PMel/GCE (curve a)in the 0.1 M PBS (pH 4.5) in the absence of DA, UA, G, andA, suggesting that the blank solution was nonelectroactive inthe potential range of 0.1–1.4 V. There were four peaksobtained on the bare GCE (curve b), PMel/GCE (curve c),Ag/GCE (curve d), and Ag-PMel/GCE (curve e) correspond-ing to the oxidation of DA, UA, G, and A. These four peaks onthe different electrodes increased with the sequence of bareGCE, PMel/GCE, Ag/GCE, and Ag-PMel/GCE, indicatingthat PMel has a good catalytic effect and the Ag and PMel

played synergistic effects for the oxidation of these four com-pounds. Furthermore, the most noticeable improvement ofpeak currents were 13-, 6-, 7-, and 9-fold on Ag-PMel/GCE(curve e) compared with those on the bare GCE. These resultsindicated that Ag-PMel/GCE had the best effect for oxidationof DA, UA, G, and A among Ag-PMel/GCE, Ag/GCE, PMel/GCE, and bare GCE.

The influence of scan rates on the electrochemical behaviorsof DA, UA, G, and A

The effect of scan rates on the electrochemical behaviors of20 μM DA, 20 μM UA, 20 μM G, and 20 μM A at the Ag-PMel/GCE was investigated by linear sweep voltammetry inpH 4.5 PBS; the results were shown in Fig. 4a–d. The oxida-tion peak currents of DA, UA, G, andA increased linearly withthe scan rates in the range of 10–200, 10–220, 10–140, and 50–180 mV s−1, respectively, and the relationships between theoxidation peak currents and the scan rates were expressed asIpa=0.0370 ν +0.424 (R 2=0.971) for DA, Ipa=0.0285ν +0.193 (R2=0.987) for UA, Ipa=0.112ν +0.531 (R2=0.974)for G, and Ipa=0.108ν +3.11 (R

2=0.991) for A. This indicatesthat the electro-oxidation reactions of DA, UA, G, and A at theAg-PMel/GCE are all adsorption-controlled processes.

0.2 0.4 0.6 0.8

2

3

4

5

6

7

3 4 5

0.36

0.39

0.42

Ep

a

pH

I/A

E/V (vs.SCE)

E/V (vs.SCE) E/V (vs.SCE)

E/V (vs.SCE)

3.0

3.5

4.0

4.5

5.0

(A)

0.6 0.8 1.0 1.20

5

10

15

20

25

3 4 5

0.87

0.90

0.93

Epa

pH

I/A

(C)

3.0

3.5

4.0

4.5

5.0

1.0 1.2 1.40

5

10

15

20

25

30

35

40

45

50

3 4 5

1.14

1.20

1.26

I pa

pH

I/A

(D)

3.0

3.5

4.04.5

5.0

0.3 0.4 0.5 0.6 0.7 0.82

3

4

5

6

7

8

9

10

3 4 5

0.49

0.56

Epa

pH

I/A

3.0

3.5

4.0

4.5

5.0

(B)Fig. 5 Effect of pH value on theelectrochemistry behavior of10 μM DA (a), 50 μM UA (b),20 μM G (c), and 20 μM A (d)at Ag-PMel/GCE. The insetsshowed the linear relationshipbetween oxidation peakpotential (Epa) and pH

J Solid State Electrochem (2014) 18:105–113 109

The effect of accumulation time on the response of DA, UA,G, and A

The accumulation time is an important factor for the response ofDA, UA, G, and A in the adsorption processes. To study theeffect of accumulation time on the level of their adsorption at theelectrode surface, we investigated the LSV signals of the 20 μMof DA, UA, G, and A by varying the accumulation time be-tween 20 and 140 s. The peak current increase as accumulationtime increases and reaches its maximum value at 100 s. There-fore, 100 s was used as the accumulation time in this study.

The influence of pH values on the electrochemical behaviorsof DA, UA, G, and A

The electrochemical behaviors of DA, UA, G, and A at theAg-PMel/GCE in 0.1 M PBS with various pH were investi-gated by SWV. The kinds of peak currents with respect to pHrange from 3.0 to 5.0 were shown in ESM Fig. S2. It could beseen that the anodic peak currents of both DA and G increasedgradually with the increase of pH from 3.0 to 4.0 then de-creased slightly in higher pH value PBS; the peak currentsreached a maximum value at pH 4.0. However, the oxidationpeak current of UA and A gained a maximum value at pH 4.5.Due to the weaker current responses of UA and A than theother two compounds, pH 4.5 was chosen as the optimum pHfor simultaneous determination of these four compounds.Furthermore, the oxidation potentials of DA, UA, G, and Ashifted negatively with the increase of pH (Fig. 5a–d)manifesting that the protons has taken part in their electrodereaction processes. The relationships between oxidation po-tentials (Ep) and pH can be expressed by the equations:Ep (V)=−0.0532 pH+0.594 (R2=0.997);Ep (V)=−0.0672 pH+0.774 (R2=0.990); Ep (V)=−0.0432 pH+1.03 (R2=0.984); Ep

(V)=−0.0488 pH+1.36 (R2=0.982), respectively. According tothe Nernst equation [32], Ep (V )=−0.059 (m /n ) pH+E0, theresults was obtained in that the number of protons (m ) wasequal to the number of the transferred electrons (n ) in theprocesses. These conclusions are in accordance with themechanisms of DA, UA, G, and A electrochemical reactionsas shown in Scheme 1 [33–35].

Sensitive determination of DA, UA, G, and A

The electrochemical sensing performance of Ag-PMel/GCEfor sensitive simultaneous determination of DA and UA, G,and A was studied by SWV in 0.1 M PBS (pH 4.5) (ESMFig. S3a,b). The linear ranges of both DA and UAwere 0.1–50 μM with R2=0.990. The detection limits were 1×10−8 MDA and 1×10−7 M UA. Figures S3c,d exhibited selectivedetermination of G and A at the Ag-PMel/GCE, respectively.The linear ranges were 0.1–50 μM for G with R2=0.989 and0.1–60μM for AwithR2=0.992. The detection limits of these

two analytes were 8×10−9 M G and 8×10−9 M A. Thecomparison of this sensor with other chemically modifiedelectrodes [3, 23, 36–44] was listed in Table 1 which demon-strated that the proposed method has a comparably low detec-tion limits and wide linear ranges.

ESMFig. S4a,b illustrated the square wave voltammogramsobtained when the concentrations of DA, UA, G, and A

NH2HO

HO

H2O

NH2O

O

+ 2H+ +2e-

(A)

(B)

(C)

(D)

NH

NH

NH

HN

O

O

O2H2O NH

NH

NH

HN

O

O

O + 2H+ + 2e-

HO

HO

NH

NNH

N

O

NH2

H2ONH

NN

N

O

NH2

O + 4H+ + 4e-

N

NNH

N

NH2

H2O N

NH

N

N

NH2

O

O

+ 4H+ + 4e-

Scheme 1 Electrochemical oxidation mechanisms of DA (a), UA (b), G(c), and A (d)

-0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.40

10

20

30

40

50

I/ µ

A

E /V(vs.SCE)

a

b

c

DA

UA

G

A

Fig. 6 Square wave voltammograms of the Ag-PMel/GCE at differentconcentrations of DA, UA, G and A (a-c: 5, 10 and 20 μM)

110 J Solid State Electrochem (2014) 18:105–113

increased simultaneously at the Ag-PMel/GCE under opti-mized conditions, respectively. Figure 6 showed that the squarewave voltammograms of DA, UA, G, and A increased simul-taneously at the Ag-PMel/GCE under optimized conditions.As can be seen, DA, UA, G, and A could be oxidized at thedetached potentials from 0 to 1.4 V and the oxidation peakcurrents for these four compounds increased linearly withincrease in their concentrations. Thus, this modified electrodehas the ability to detect these four analytes simultaneouslyfrom a mixture in large concentration domains without

interfering with each other. This remarkable electrochemicalsensing performance can be attributed to good adsorption,antifouling property, and high electron transfer kinetics of theAg-PMel film which provided an efficient microenvironmentfor electrochemical reaction of DA, UA, G, and A [45].

Interference of coexistences

The selectivity of the Ag-PMel/GCE for sensitive determina-tion of DA, UA, G, and A was analyzed and several com-pounds such as important biological substances and somemetal ions were checked as potential interfering substances.The effects of these interferents were examined by carryingout the determination of 20 μM DA, 20 μM UA, 20 μM G,and 20 μM A in 0.1 M PBS (pH 4.5) in the presence of

Table 2 The influences of some metal ions and important biologicalsubstances on the peak currents of 20 μMDA, 20 μMUA, 20 μMG, and20 μM A in 0.1 M PBS (pH 4.5) at the Ag-PMel/GCE

Interferents Concentration(μM)

Signal change

DA (%) UA (%) G (%) A (%)

K+ 1600 3.01 −1.08 2.02 1.34

Ca2+ 400 −2.02 −3.56 −1.58 −2.08Zn2+ 400 4.21 2.82 3.46 2.19

Mg2+ 400 3.86 2.75 2.45 3.68

Glucose 200 4.30 3.72 1.65 2.84

Sucrose 200 2.12 1.97 1.86 4.38

Ascorbic acid 30 −5.50 50.86 4.66 4.89

L-glutamic acid 20 8.79 −10.43 3.09 2.58

Epinephrine 10 40.75 42.32 −3.23 −6.56Tyrosine 30 −2.30 −1.00 50.60 −5.48

Table 3 The results of recovery test

Samples Standardsolution (μM)

Detectionresults (μM)

Recovery(%)

RSD%(n =5)

DA1# 10 9.9 99.0 1.28

DA2# 20 21.4 107.0 2.56

DA3# 30 32 106.7 2.83

UA1# 10 10.6 106.0 4.22

UA2# 20 22.2 111.0 2.18

UA3# 30 32.4 108.0 3.56

G1# 10 9.8 98.0 1.17

G2# 20 20.6 103.0 2.12

G3# 30 30.4 101.3 2.08

A1# 10 10.6 106.0 2.98

A2# 20 21.3 106.5 3.21

A3# 30 29.8 99.3 2.16

Table 1 Comparison of major characteristics at different modified elec-trodes for the detection of DA, UA, G, and A

Electrodes Analytes Dynamicrange (μM)

Detectionlimit(μM)

References

Poly(DBA)/GCEa DA 0.1–100 0.06 [36]

NiHCF-PNH/Aub DA 0.1–9.6 0.031 [37]

Nafion/SDBS/GCEc DA 0.4–80 0.05 [3]UA 4.0–800 0.4

LaFeO3/GCE DA 0.15–800 0.03 [38]

MB-MWNTs/GCEd DA 0.4–10 0.2 [39]UA 2.0–200 1.0

Poly(rhodamine B)/CPEe DA 6–1,000 3.99 [40]

Ag-PMel/GCE DA 0.1–50 0.01 This workUA 0.1–50 0.1

GS/IL/CS/GCEf G 2.5–150 0.75 [41]A 1.5–350 0.45

PSSA–ssDNA/GCEg G 0.065–1.1 0.022 [42]A 0.065–1.1 0.022

AgNPs–Pdop@Gr/GCEh G 0.04–50 0.004 [43]A 0.02–40 0.002

Nano-Au/MB/GCEi G 0.88–21 0.18 [44]A 0.98–20 0.25

PMel/GCE G 0.1–50 0.08 [23]A 0.1–60 0.07

Ag-PMel/GCE G 0.1–50 0.008 This workA 0.1–60 0.008

a Poly(3,5-dihydroxy benzoic acid) film-modified glassy carbon electrodebNickel hexacyanoferrate and poly(1-naphthol) hybrid film-modified Auelectrodec Nafion/sodium dodecylbenzenesulfonate composite film-modifiedglassy carbon electrodedMultiwalled carbon nanotubes with methylene blue composite film-modified electrodee Poly(rhodamine B) modified carbon paste electrodef Graphene–ionic liquid–chitosan composite film-modified glassy carbonelectrodeg Single-stranded DNA–poly(sulfosalicylic acid) composite film-modi-fied electrodeh Silver nanoparticles–polydopamine–grapheme nanocomposite modi-fied electrodei Gold nanoparticles and methylene blue on thiourea modified glassycarbon electrode

J Solid State Electrochem (2014) 18:105–113 111

different concentrations of the interferents. Tolerance limitwas defined as the maximum concentration of the foreignsubstances that caused an approximately ±5 % relative errorin the detection. Table 2 shows that K+, Ca2+, Zn2+, Mg2+,glucose, and sucrose did not interfere significantly, while L-glutamic acid had an effect on the examinations of DA andUA to some degree. Moreover, ascorbic acid, epinephrine,and tyrosine had apparent influences on the determination ofUA, DA, and G, respectively, which were attributed to thepeak potentials of ascorbic acid, epinephrine, and tyrosineclose to those of UA, DA, and G, respectively.

Stability, repeatability, and reproducibility of Ag-PMel/GCE

The stability of the Ag-PMel/GCE sensor was evaluated byexamining the current responses of DA, UA, G, and A duringstorage in a refrigerator at 4 °C. The peak currents of DA, UA,G, and A decreased to 90.2, 89.8, 94.6, and 93.5 % of theinitial values after 2 weeks, respectively, indicating that theAg-PMel/GCE is a very stable and long-life voltammetricsensor.

To characterize the repeatability of the modified electrode,successive determinations of these four analytes were carriedout using the same electrode. The results of 10 repetitivemeasurements displayed the relative standard deviations of2.5, 3.5, 2.8, and 3.0 % for DA, UA, G, and A, respectively.Furthermore, the relative standard deviations for determina-tion of DA, UA, G, and A were 4.5, 5.0, 3.0, and 3.5 %,respectively, when three parallel-modified electrodes wereemployed in the same solution. The results suggested thatthe electrochemical sensor has good repeatability and repro-ducibility for response of these four compounds.

Recovery test

The practical application of the modified electrode was inves-tigated as follows: a certain volume of standard solution ofDA (pH 4.0), UA (pH 4.5), G (pH 4.0), and A (pH 4.5) werediluted to 10 mL by three different healthy person's urinewhich were not further purified. These solutions were usedfor recovery tests. The amounts of DA, UA, G, and A werecalculated from the calibration equation and the results arelisted in Table 3.

Conclusions

A biosensor for DA, UA, G, and A was prepared byelectropolymerization with cyclic voltammetry. The biosensorhas excellent electrocatalytic activity for the oxidation of DA,UA, G, and A. The method provided a good example forapplying the harmful substance of life (melamine) in electrodemodification for detection of life substances. In addition, the

effects of potential interfering substances were studied; theexperimental procedure was free from interferences from themost common interfering ions and organic compounds. Com-pared with the reported chemically modified electrodes, thedesigned sensor displayed good performances of wider linearrange and lower detection limit, which may make the strategypromising for sensitive determination of these four compo-nents and demonstrate the significant role that Ag-PMel willplay in further study of electrode modifier.

Acknowledgments The authors gratefully acknowledge the financialsupport of the National Natural Science Foundation of China (no.21105023), the Shandong Province High School Science and TechnologyPlanning Project (no. J13LD51), and the Heze University FoundationProject (nos. XY12KJ10 and XY10BS01).

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