Highly sensitive determination of uric acid in the presence of major interferents using a conducting polymer film modified electrode

Bioelectrochemistry 88 (2012) 22–29

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Highly sensitive determination of uric acid in the presence of major interferents using a conducting polymer film modified electrode S. Brillians Revin, S. Abraham John ⁎ Centre for Nanoscience & Nanotechnology, Department of Chemistry, Gandhigram Rural Institute, Gandhigram — 624 302, Dindigul, Tamilnadu, India

a r t i c l e

i n f o

Article history: Received 7 February 2012 Received in revised form 17 May 2012 Accepted 21 May 2012 Available online 30 May 2012 Keywords: 3-Amino-5-mercapto-1,2,4-triazole Uric acid Ascorbic acid Dopamine Tyrosine Methionine

a b s t r a c t This paper describes the sensitive and selective determination of uric acid (UA) in the presence of important interferences, ascorbic acid (AA), dopamine (DA), tyrosine (Tyr) and methionine (Met) at physiological pH using an electropolymerized film of 3-amino-5-mercapto-1,2,4-triazole on glassy carbon (p-AMTa) electrode. The pAMTa electrode shows an excellent electrocatalytic activity towards UA. This was understood from the observed higher oxidation current and heterogeneous rate constant (3.24 × 10− 5 m s− 1) for UA when compared to bare GC electrode (4.63 × 10− 6 m s− 1). The selective determination of UA in the presence of 1000-fold excess of AA was achieved using p-AMTa electrode. Further, the p-AMTa electrode was successfully used for the simultaneous and selective determination of UA in the presence of important interferences, DA, Tyr and Met. Using amperometric method, 40 nM UA was detected for the first time. The current response of UA was increased linearly while increasing its concentration from 40 nM to 0.1 mM and a detection limit was found to be 0.52 nM (S/ N = 3). Finally, the practical application of the present method was demonstrated by determining UA in human urine and blood serum samples. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Uric acid (UA, 2,6,8-trihydroxypurine) is the main end product of purine nucleotide catabolism in human body. It is well known that UA was present in human blood serum, plasma, urine and saliva [1]. Serum UA was a risk factor for the oxidative stress [2], coronary heart disease [3] and closely linked to vascular nitric oxide activity [4]. Elevated serum UA was associated with a risk of cardio-vascular disease [5], peripheral arterial disease [6], chronic kidney disease [7], non-alcoholic fatty liver disease [8] and silent brain infarction [9]. Increased consumption of serum UA can act as a scavenger of radicals and thus preventing from Parkinson's disease (PD) [10]. However, low UA levels in plasma and urine associate with worse cognitive performance in PD [11]. UA is a pathogenic factor in pre-eclampsia for pregnant women [12,13]. It acts as a role of insulin resistance in older [14] and pregnant women [15]. Reduced level of plasma UA leads to Schizophrenia [16] and blood UA levels contributed with sleep-disordered breathing [17]. Low concentration of UA associated with multiple sclerosis [18] and also altered UA level are associated with sex and age interaction [19]. Urinary UA is related with risk of Down syndrome for children and adults [20]. Normally, UA levels in serum range from 240 to 520 μM and in urinary excretion it ranges from 1.4 to 4.4 mM [21]. Ascorbic acid (AA) coexists with UA [21,22] and hence it is a main interference for UA determination in human fluids. Therefore, an accurate determination of UA is essential

⁎ Corresponding author. Tel.: + 91 451 245 2371; fax: + 91 451 245 3031. E-mail address: [email protected] (S.A. John). 1567-5394/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.bioelechem.2012.05.005

in the presence of AA in human fluids to secure the human health. Since both of them oxidized at the same potential in physiological pH, it is a challenging task for the analytical chemists to determine UA in the presence of large excess of AA. Thus, the aim of the present study is to determine UA in the presence of much higher concentration of AA. Few papers were published in the literature for the determination of AA/UA ratio of more than 1000 [23–26]. However, the electrodes used in these papers have several drawbacks including tedious procedure involved in the modification of the electrode, reproducibility of the electrode modification was uncertain and a more time consuming process. For example, Hasan and co-workers have followed a very tedious procedure for the modification of the electrode; (i) mechanically grinded the GC or graphite electrode with struers silicon carbide (SiC) paper of 240-, 500-, 1200-, 2400- and 4000-grit and (ii) grinded by SiC with successively decreasing diameter and (iii) finally fine polished with 1-μm diamond paste [23–26]. Kang and Lin have prepared the RNA modified GC electrode for the determination of UA in the presence of AA and DA by applying a stationary deposition potential in a solution containing RNA for 30 min followed by cycling in a potential window from −0.2 V to +0.8 V for 4 cycles [24]. On the other hand, Li and Lin have used a tedious procedure for the fabrication of gold nanocluster modified overoxidized pyrrole electrode for the determination of UA in the presence of a 1000-fold higher concentration of AA. For the fabrication of the modified electrode, they first cycled the bare GC electrode in the potential window from −0.35 V to +0.85 V in a solution containing pyrrole and sodium dodecyl sulfate and then transferred the electrode into NaOH solution followed by overoxidation at a constant potential of +1.0 V for several minutes and finally electrochemically deposit the gold nanoclusters on the modified electrode by

S.B. Revin, S.A. John / Bioelectrochemistry 88 (2012) 22–29

scanning from +0.2 V to −1.0 V in HAuCl4 solution for 15 cycles [25]. Suresh and co-workers have used layer-by-layer method for the fabrication of electrode, which involves very tedious experimental conditions (24 h maintained with 50 °C temperature) [26]. Further, the reproducibility of the electrode was unsure. In the present work, we have attempted to determine UA in the presence of 1000-fold excess AA using the electropolymerized film of 3-amino-5-mercapto-1,2,4-triazole modified GC (p-AMTa) electrode. It was found that the p-AMTa electrode successfully determined UA in the presence of 1000-fold higher concentration of AA. When compared to the above modified electrodes [23–26], the procedure for the fabrication of p-AMTa electrode is very easy, less time consuming (~20 min), highly stable and reproducible. Recently, we have successfully utilized thiadiazole based polymer electrode, poly-5-amino-2-mercapto-1,3,4thiadiazole film modified GC (p-AMT) electrode to determine variety of biologically important molecules including L-cysteine [27], homocysteine [28], xanthine [29] and norepinephrine [30]. Hence, we have examined the performance of p-AMT electrode for the simultaneous

23

determination of AA and UA and compared with p-AMTa electrode. Since both p-AMT and p-AMTa films contain positively charged backbone, the electrostatic interaction between polymer films and AA and UA is possible. Additionally, p-AMTa film has –NH– moiety in the heterocyclic ring and this may help the hydrogen bonding interactions between p-AMTa film and AA and UA [31]. On the contrary, this interaction was absent in the case of p-AMT film. Hence, the oxidation currents obtained for AA and UA at p-AMTa electrode were higher than that of p-AMT electrode. Further, we have also successfully determined UA in the presence of other important interferences, dopamine (DA), tyrosine (Tyr) and methionine (Met) because they also coexist with UA in biological fluids [32–34]. Furthermore, we have achieved the detection of 40 nM UA at p-AMTa electrode using amperometric method for the first time. Finally, p-AMTa electrode was used to determine UA concentration in human urine and blood serum samples.

A

c

S

30

A

b

30

NH

N

N

N N

O N

N

20

NH

HN

H N

NH

e

10

f

d

10

ab

O

UA

S

Ι / µΑ

Ι / μΑ

20

d

O

H N

c

a

0

e

0

0

0.5

E / V (vs. Ag/AgCl) 0

0.5

B

E / V (vs. Ag/AgCl)

3.2 3.0 2.8

k

2.2 2.0

A

1.6

0 20 40 60 80 100 120 140 160 180 200

[UA] /µM

AA

Ι / µΑ

I/μA

160 140 120 100 80 60 40 20

5

Ι/ μΑ

2.4

1.8

4

100

I/µA

I/µA

B

2.6

10

15

20

25

30

(ν / mV.s-1)1/2

3.4 3.2 3.0 2.8 2.6 2.4 2.2 2.0 1.8 1.6 1.4

B 0

10 20 30 40 50 60 70

[UA] /µM

UA

m

35

a

2

a

0 –0.2

0

0.2

0.4

E / V (vs. Ag/AgCl) –0.2

0

0.2

0.4

0.6

E / V (vs. Ag/AgCl) Fig. 1. (A) CVs obtained for 0.5 mM (a) UA at bare GC electrode; (b) UA at p-AMTa electrode; (c) AA at bare GC electrode; (d) AA at p-AMTa electrode and (e) together blank at p-AMTa electrode in 0.2 M PB solution (pH 7.2) at a scan rate of 50 mV s− 1. (B) CVs obtained for 0.5 mM UA at p-AMTa electrode in 0.2 M PB solution (pH 7.2) at different scan rates: (a) 50 (b) 100 (c) 200 (d) 300 (e) 400 (f) 500 (g) 600 (h) 700 (i) 800 (j) 900 and (k) 1000 mV s− 1. Inset: Plot of the anodic peak current vs. square root of scan rate. 1/2 I/μA = (5.15 ± 0.7)(υUA )/(μA(mV.s− 1)− 1/2) − (9.22 ± 0.03)/μA. (R2 = 0.9993).

Fig. 2. (A) CVs obtained for a mixture of 0.5 mM each AA and UA at (a) 1st and (b) after 6 cycles at bare GC electrode, (c) 1st and (d) after 6 cycles at p-AMTa electrode and (e) 1st and (f) after 6 cycles at p-AMT electrode in 0.2 M PB solution (pH 7.2) at a scan rate of 50 mV s− 1. Inset: Schematic representation of possible electrostatic and hydrogen bonding interactions between p-AMTa film and UA. (B) DPVs obtained for each increment of 15 μM AA and 5 μM UA (curves a–m) at p-AMTa electrode in 0.2 M PB solution (pH 7.2). Pulse width = 0.06 s, amplitude = 0.05 V, sample period= 0.02 s and pulse period= 0.20 s. Inset (A): Plot of concentration of AA vs. current. I/μA = (0.008± 0.0003) [AA]/(μA μM− 1) + (1.61 ± 0.02)/μA. (R2 = 0.9934). Inset (B): Plot of concentration of UA vs. current. I/μA = (0.029± 0.004) [UA]/(μA μM− 1) + (1.47 ± 0.03)/μA. (R2 = 0.9983).

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S.B. Revin, S.A. John / Bioelectrochemistry 88 (2012) 22–29

10

I/μA

AA

8

carried out under nitrogen atmosphere. Atomic force microscopy (AFM) measurements were recorded using a Nanoscope (IV) instrument (Vecco). Indium tin oxide (ITO) was purchased from Asahi Beer Optical Ltd., Japan and was used as a substrate for AFM measurements.

6.0 5.5 5.0 4.5 4.0 3.5 3.0

2.3. Fabrication of p-AMTa modified GC electrode

Ι / μΑ

0

2

4

6

8 10 12 14

[UA] / μM

UA

6

The GC electrode was polished with 0.05 μm alumina slurry and rinsed thoroughly with water. Then, the electrode was sonicated in water for 5 min to remove any adsorbed alumina particles. Fig. S1 in the Supporting Information shows the electropolymerization of AMTa on GC electrode by 15 successive potential sweeps between −0.20 V and + 1.70 V at a scan rate of 50 mV s − 1 in 1 mM AMTa containing 0.1 M H2SO4 [31]. Identical conditions were followed for the electropolymerization of AMT.

m

4

a –0.2

0

0.2

3. Results and discussion

0.4

E / V (vs. Ag/AgCl)

3.1. Characterization of p-AMTa film by AFM

Fig. 3. DPVs obtained for each increment of 1 μM UA into 1 mM AA (curves a–m) at pAMTa electrode in 0.2 M PB solution (pH 7.2). Pulse width = 0.06 s, amplitude = 0.05 V, sample period = 0.02 s and pulse period = 0.20 s. Inset: Plot of concentration of UA vs. current. I/μA = (0.19 ± 0.01) [UA]/(μA μM− 1) + (3.03 ± 0.05)/μA. (R2 = 0.9972).

2. Experimental

The size and morphology of the p-AMTa film were investigated by tapping mode AFM using ITO substrate [35]. The AFM image shows a spherical like structure with a thickness of ~ 30 nm and the diameter of each particle was found to be 20–30 nm [35]. 3.2. Electrochemical behavior of UA at bare GC and p-AMTa electrodes

2.1. Chemicals AMTa, AMT, UA, AA, DA, Tyr and Met were purchased from Aldrich and were used as received. All other chemicals used in this investigation were of analytical grade. pH 7.2 phosphate buffer (PB) was prepared using Na2HPO4 and NaH2PO4. Double distilled water was used to prepare the solutions in the present investigation. 2.2. Instrumentation Electrochemical measurements were performed in a conventional two compartment three electrode cell with a mirror polished 3 mm GC electrode as a working electrode, Pt wire as counter electrode and a NaCl saturated Ag/AgCl as reference electrode. All the electrochemical measurements were carried out with CHI model 634B (Austin, TX, USA) Electrochemical Workstation. For differential pulse voltammetry (DPV) measurements, pulse width of 0.06 s, amplitude of 0.05 V, sample period of 0.02 s and pulse period of 0.20 s were used. For chronoamperometric measurements, pulse width of 0.25 s and potential step of one were used. All the electrochemical measurements were

Physiological pH is preferable for clinical analysis because human fluids are mostly stable at this pH [36]. The present modified electrode showed higher oxidation current for UA at pH 7.2 and hence this pH was chosen for all the electrochemical measurements. Fig. 1A shows the cyclic voltammograms (CVs) obtained for 0.5 mM UA at bare GC and p-AMTa electrodes in 0.2 M PB solution (pH 7.2) at a scan rate of 50 mV s− 1. Bare GC electrode shows a broad UA oxidation wave at 0.39 (±0.03) V in the first cycle (curve a). In the subsequent cycles, the UA oxidation peak was shifted to a more positive potential. In contrast, p-AMTa electrode shows the oxidation peak for UA at 0.28 (±0.01) V in the first cycle (curve b), which is 0.11 (±0.03) V less positive potential than at bare GC electrode. In the subsequent cycles, UA oxidation peak was highly stable at p-AMTa electrode. Bare GC electrode shows a broad oxidation wave at 0.33 (±0.04) V for AA (curve c). However, p-AMTa electrode oxidizes AA at 0.18 V (±0.02) (curve d), which is 0.15 (±0.03) V less positive than at bare GC electrode. The p-AMTa electrode does not show electrochemical response in 0.2 M PB solution (pH 7.2) in the absence of analytes (curve e). The effect of scan rate (ν) on the oxidation of UA at p-AMTa electrode was studied to understand the oxidation process at the modified electrode.

Table 1 Comparison of different modified electrodes for AA/UA concentration ratio with p-AMTa electrode. Electrode

Measurement solution, pH

Mechanically grinded carbon electrode

0.1 M acetate buffer, pH AA and DA 4.8 0.1 M PB solution + 50 mM AA and DA KCl, pH 7.0 0.1 M PB, pH 7.0 AA and epinephrine (EP) 0.1 M PB solution + 0.1 M AA and DA KCl, pH 7.0 Acetate buffer, pH 5.0 AA and DA 0.1 M 0.1 M 0.1 M 0.2 M

RNA grafted modified GCE Gold nanoclusters with overoxidized polypyrrole modified GCE Polystyrene sulfonate wrapped multi-walled carbon nanotube modified graphite electrode Poly(3,4-ethylenedioxythiophene-co-(5-amino-2naphthalenesulfonic acid)) modified GCE Single-walled carbon nanohorn modified GCE Quercetin-modified wax-impregnated graphite electrode Dopamine modified pyrolytic graphite electrode Poly(3-amino-5-mercapto-1,2,4-triazole) modified GCE

PB, pH PB, pH PB, pH PB, pH

7.0 6.0 6.5 7.2

Interferences

AA and DA AA AA and DA AA, DA, Tyr and Met

Fixed AA concentration Lowest UA AA/UA Reference (mM) addition (μM) (ratio) 0.1

0.01

10,000 [23]

2

0.74

2703 [24]

0.1

0.05

2000 [25]

1

1.5

1500 [26]

1

1

1000 [40]

0.04 0.5 1 1

0.06 1 2.5 1

667 500 400 1000

[41] [42] [43] This work

S.B. Revin, S.A. John / Bioelectrochemistry 88 (2012) 22–29

25

A

A

0.03

Ι / μΑ

50

25

Ι / μΑ

a c

40 35

I/nA

b

0.02

0.01

30 25 20 15 10 5

0

0 100 200 300 400 500 600

[UA ] / μM

0

0.5

1

400

1.5

600

800

E / V (vs. Ag/AgCl)

B

B

DA

10

8

Met

6

4 2 0

4

0

0 15 30 45 60 75 90

Ι / μΑ

Ι/ μΑ

[UA] / μM

j

d 5

a b

20

40

60

80 100

[UA] / μM

c

UA Tyr

10

k

6

8

I/μA

I/μA

10

20

1000

t/s

j

i gh ef

a 0 0

0.5

1

200

1.5

Fig. 5. (A) Amperometric i–t curve for the determination of UA at p-AMTa electrode in 0.2 M PB solution (pH 7.2). Each addition increases the concentration of 40 nM of UA at a regular interval of 50 s. Eapp = + 0.3 V. Inset: plot of concentration of UA vs. current. I/μA = (0.06 ± 0.003) [UA]/(μA μM− 1) + (5.83 ± 0.08)/μA. (R2 = 0.9996). (B) Amperometric i–t curve for the determination of UA at p-AMTa electrode in 0.2 M PB solution (pH 7.2). Each addition increases the concentrations of (a) 0.04 (b) 0.1 (c) 0.2 (d) 0.4 (e) 0.8 (f) 2 (g) 4 (h) 8 (i) 20 (j) 40 and (k) 100 μM at a regular interval of 50 s. Eapp = + 0.3 V. Inset: Plot of concentration of UA vs. current. I/μA = (0.13 ± 0.01) [UA]/(μA μM− 1) + (0.05 ± 0.002)/μA. (R2 = 0.9996).

C

Met

Ι/ μΑ

DA

The CVs obtained for UA at scan rates from 50 mV s − 1 to 1000 mV s− 1 in 0.2 M PB solution at pH 7.2 are shown in Fig. 1B. A good linearity was obtained while plotting the current against square root of scan rate with a correlation coefficient of 0.9993 (Fig. 1B, inset), indicating that the oxidation of UA was diffusion controlled process. Further, we have calculated the standard heterogeneous rate constant (ks) values for UA at bare GC and p-AMTa electrodes using Velasco equation [37] as shown below.

Tyr

10

UA

0

0.5

600

t/s

E / V (vs. Ag/AgCl)

20

400

1

1.5

E / V (vs. Ag/AgCl) Fig. 4. (A) CVs obtained for 0.5 mM (a) DA (b) Tyr and (c) Met at p-AMTa electrode in 0.2 M PB solution (pH 7.2) at a scan rate of 50 mV s− 1. (B) DPVs obtained for each increment of 10 μM DA, 8 μM UA, 30 μM Tyr and 50 μM Met (curves a–j) at p-AMTa electrode in 0.2 M PB solution (pH 7.2). Inset: Plot of concentration of UA vs. current. I/μA = (0.05 ± 0.002) [UA]/(μA μM− 1) + (4.10 ± 0.07)/μA. (R2 = 0.9902). (C) DPV obtained for the 10 μM UA in the presence of 100 μM DA, 300 μM Tyr and 500 μM Met at p-AMTa electrode in 0.2 M PB solution (pH 7.2). Pulse width = 0.06 s, amplitude = 0.05 V, sample period= 0.02 s and pulse period = 0.20 s.

ks ¼ 1:11Do

1=2

 −1=2 1=2 Ep –Ep=2 ν

ð1Þ

where ks is standard heterogeneous rate constant, Do is apparent diffusion coefficient, Ep is oxidation peak potential, Ep/2 is half-wave oxidation peak potential and ν is scan rate. The Do value was determined using Cottrell slope obtained by single potential chronoamperometry technique. It is known that two electrons and two protons are involved in UA oxidation process [38]. The Do of 2.49× 10− 11 m 2 s− 1 and

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S.B. Revin, S.A. John / Bioelectrochemistry 88 (2012) 22–29

5.24 × 10− 10 m 2 s− 1 was obtained for UA at bare GC and p-AMTa electrodes, respectively. The estimated ks values for the oxidation of UA at bare GC and p-AMTa electrodes were found to be 4.63 × 10− 6 m s− 1 and 3.24 × 10− 5 m s− 1, respectively. The obtained higher ks value for UA at p-AMTa electrode indicates that the oxidation of UA was faster at the p-AMTa electrode than at bare GC electrode.

simultaneous determination of AA and UA at p-AMTa electrode in 0.2 M PB solution (pH.7.2) are shown in Fig. 2B. A well-defined signal was observed for 15 μM AA and 5 μM UA (curve a). When the concentration of AA was increased from 15 μM to 195 μM and UA was increased from 5 μM to 65 μM (curves a–m), the peak currents of the respective analytes were increased linearly with the correlation coefficient of 0.9934 for AA (Fig. 2B, inset A) and 0.9983 for UA (Fig. 2B, inset B).

3.3. Simultaneous determination of AA and UA Since AA is one of the main interferents for the determination of UA, it is essential to determine UA in the presence of AA. Fig. 2A shows the CVs obtained for a mixture of 0.5 mM each AA and UA at bare GC and pAMTa electrodes in 0.2 M PB solution (pH 7.2). Bare GC electrode fails to resolve the voltammetric signals of AA and UA in a mixture. It shows a broad oxidation wave at 0.42 (±0.03) V in the first cycle (curve a). After 6 cycles, the obtained oxidation wave was shifted to a more positive potential (curve b). On the other hand, p-AMTa electrode not only resolved the oxidation peaks of AA and UA but also enhanced their oxidation currents (curve c) compared to bare GC electrode. The p-AMTa electrode shows the oxidation peak at 0.18 (±0.01) V for AA and 0.32 (±0.01) V for UA. The oxidation peaks of AA and UA were very stable even after 6 cycles (curve d) indicating that the simultaneous determination of AA and UA is possible using p-AMTa electrode. For comparison, we have investigated the CV response for a mixture of 0.5 mM each AA and UA in 0.2 M PB solution at p-AMT electrode (curve e). Although the p-AMT electrode successfully resolved the voltammetric signals of AA and UA (curve e) when compared to bare GC electrode peak separation between AA and UA and their oxidation currents were less than that at p-AMTa electrode (curve c). Further, the oxidation peaks of both AA and UA were not stable after 6 cycles (curve f). The obtained higher oxidation currents for AA and UA at p-AMTa electrode in contrast to p-AMT electrode were explained on the basis of the respective polymer structures. Both p-AMT and p-AMTa films contain positively charged backbone. Therefore, electrostatic interaction between the polymer films and the negatively charged AA and UA molecules [39] is possible. In addition to electrostatic interaction, hydrogen bonding interaction is also possible between p-AMTa film and AA and UA due to the presence of –NH– moiety in the ring. This interaction was absent in the case of p-AMT film. Thus, the oxidation currents obtained for AA and UA at p-AMTa electrode were higher than that of p-AMT film. The different interactions between p-AMTa electrode and UA are schematically shown in inset of Fig. 2A. The DPVs obtained for the

3.4. Selective determination of UA in the presence of a very high concentration of AA High concentration of AA coexists with UA in human fluids. Therefore, determination of UA in the presence of high concentration of AA is very important. Fig. 3 shows DPVs obtained for the increment of 1 μM UA in the presence of 1000 μM AA in 0.2 M PB solution (pH 7.2) at p-AMTa electrode. A clear voltammetric signal was obtained for 1 μM UA even in the presence of 1000 μM AA (curve a), which revealed that the detection of very low concentration of UA is possible in the presence of 1000-fold excess of AA. While adding each increment of 1 μM UA to 1000 μM AA, the oxidation current of UA was increased linearly (curves a–m) with a correlation coefficient of 0.9972 (Fig. 3, inset) without affecting the potential of AA. The obtained AA/ UA concentration ratio at p-AMTa electrode was compared with other modified electrodes and was given in Table 1 [23–26,40–43]. When compared to the modified electrodes shown in Table 1 [23–26], the procedure for the fabrication of p-AMTa electrode is very easy, less time consuming, highly stable and reproducible. Further, the present modified electrode can determine AA/UA ratio of 1000 and hence it can be used for practical applications. 3.5. Simultaneous and selective determination of UA in the presence of DA, Tyr and Met Since DA, Tyr and Met also coexist with UA in human fluids [32–34], it is necessary to determine UA simultaneously and selectively in the presence of these interferences. Hence, we have investigated the simultaneous determination of UA in the presence of these analytes using p-AMTa electrode. Fig. 4A shows the CVs obtained for DA, Tyr and Met at p-AMTa electrode in 0.2 M PB solution (pH 7.2) at a scan rate of 50 mV s − 1. The modified electrode shows the oxidation peak for DA at 0.14 (±0.01) V (curve a), Tyr at 0.65 (±0.02) V

Table 2 Comparison of different modified electrodes for the detection limit of UA with p-AMTa electrode. Electrode Poly(5-amino-2-mercapto-1,3,4-thiadiazole) modified GCE

Measurement Interferences solution, pH

0.2 M PB, pH 5.0 Copper nanoparticles incorporated polypyrrole modified GCE 0.1 M PB, pH 7.0 Electrochemically treated pencil graphite electrode 0.1 M PB, pH 2.0 Poly(3,3′-bis[N,N-bis(carboxymethyl)aminomethyl]-o-cresolsulfonephthalein) 0.25 M PB, pH modified GCE 4.0 Copper modified GCE 0.1 M PB, pH 7.2 Poly(3-(5-chloro-2-hydroxyphenylazo)-4,5-dihydroxynaphthalene-2,7-disulfonic 0.2 M PB, pH acid) modified GCE 4.0 DNA and poly(p-aminobenzensulfonic acid) composite bi-layer modified GCE 0.1 M PB, pH 7.0 2,2′-[1,2-Ethanediylbis (nitriloethylidyne)]-bis-hydroquinone with carbon 0.1 M PB, pH nanotube paste electrode 7.0 Functionalized-graphene modified graphite electrode 0.1 M PB, pH 7.0 Poly(3-amino-5-mercapto-1,2,4-triazole) modified GCE 0.2 M PB, pH 7.2

AA and UA

Linear range of UA (M)

Detection limit of Reference UA (M)

2.0 × 10− 7–8.0 × 10− 5 5.7 × 10− 10 1.0 × 10

AA and DA

5.0 × 10

−8

AA and EP

2.0 × 10− 8–2.0 × 10− 3 9.0 × 10−9

[47]

AA and homovanillic acid AA and DA

2.0 × 10− 5–5.0 × 10− 5 1.0 × 10− 8

[48]

DA

–1.0 × 10

−5

–1.0 × 10

−5

[44]

−9

1.5 × 10

[46]

1.0 × 10

AA and DA

4.0 × 10

−7

DA

2.0 × 10− 5–6.5 × 10− 4 1.5 × 10− 5

[51]

AA and DA

1.0 × 10− 5–1.8 × 10− 4 1.0 × 10− 5

[52]

AA, DA, Tyr and Met

4.0 × 10

–2.3 × 10

−5

[45]

−9

−7

−8

–1.8 × 10

−5

8.0 × 10

− 10

–1.0 × 10

−4

1.6 × 10

−8

[49]

1.9 × 10

−7

[50]

5.2 × 10

− 10

This work

S.B. Revin, S.A. John / Bioelectrochemistry 88 (2012) 22–29

27

A

(curve b) and Met at 1.1 (±0.01) V (curve c). Fig. 4B shows DPVs obtained for the simultaneous determination of DA, UA, Tyr and Met at p-AMTa electrode in 0.2 M PB solution (pH.7.2). Very clear voltammetric signals were observed for 10 μM DA, 8 μM UA, 30 μM Tyr and 50 μM Met (curve a) with peak separations of 0.13 (±0.02) V between DA and UA, 0.35 (±0.04) V between UA and Tyr and 0.59 (±0.02) V between Tyr and Met. When the concentrations of DA were increased from 10 μM to 100 μM, UA was increased from 8 μM to 80 μM, Tyr was increased from 30 μM to 300 μM and Met was increased from 50 μM to 500 μM (curves a–j), the peak currents of the respective analytes were increased linearly without affecting the potentials of them. The oxidation current of UA increased linearly and showed a correlation coefficient value of 0.9902 (inset, Fig. 4B). Further, we have investigated the selective determination of UA in the presence of high concentrations of DA, Tyr and Met. Fig. 4C shows DPV obtained for 10 μM UA in the presence of 100 μM DA, 300 μM Tyr and 500 μM Met in 0.2 M PB solution at pH 7.2. A voltammetric signal was obtained for UA in the presence of 10-fold DA, 30-fold Tyr and 50-fold Met, which revealed that the detection of low concentration of UA is possible in the presence of high concentration of these interferences.

b 4

Ι/ μΑ

a 3

2

0

0.2

0.4

E / V (vs. Ag/AgCl)

B

3.6. Amperometric determination of UA

3.7. Determination of UA in human urine and blood serum samples The practical application of the p-AMTa electrode was demonstrated by measuring the concentration of UA in human urine and blood serum samples. The standard addition technique was used for the determination of UA. The human urine samples were collected from the laboratory co-workers and were diluted to 25 times with 0.2 M PB solution (pH 7.2) without any treatment. DPV of urine in PB solution (PH 7.2) shows an oxidation peak at 0.22 (±0.01) V (curve a; Fig. 6A). To confirm that the observed oxidation peak is due to UA, we have added the known concentration of commercial 10 μM UA to the urine sample. The enhancement of the oxidation current at 0.22 (±0.01) V (curve b) indicates that the obtained peak in urine sample was due to the oxidation of UA. The human blood serum samples were collected from clinical laboratories and diluted 25 times with 0.2 M PB solution (pH 7.2) without any treatment.

1.2

b Ι/ μΑ

Amperometric method was used to examine the sensitivity of pAMTa electrode towards the detection of UA. Fig. 5A shows the amperometric i–t curve obtained for UA at p-AMTa electrode in a homogeneously stirred 0.2 M PB solution (pH 7.2) by applying a potential of + 0.3 V. The p-AMTa electrode shows the current response for each addition of 40 nM UA in every 50 s interval. The current response increases and the steady state current response was attained within 3 s for further addition of 40 nM UA in each step. The dependence of current response with respect to concentration of UA was linear from 40 nM to 560 nM at p-AMTa electrode with a correlation coefficient of 0.9996 (Fig. 5A, inset). The amperometric i–t curve for each addition of 40 nM UA showed a linear current increase without noise. Further, we have also investigated the detection of UA in a wide range of concentrations using p-AMTa electrode. The amperometric current was increased linearly with increasing concentration of UA from 40 nM to 0.1 mM (Fig. 5B) with a correlation coefficient of 0.9996 (Fig. 5B, inset) by applying a potential of +0.3 V. The lowest detection limit was found to be 0.52 nM for UA at p-AMTa electrode (S/N =3). The observed linear range and the detection limit for UA at p-AMTa electrode are worth comparing with the reported modified electrodes [44–52] and are given in Table 2. As can be seen from Table 2, the present modified electrode showed the lowest detection limit for UA with a wide range of concentration compared to the reported modified electrodes [44–52].

a 1

0.8 0

0.2

0.4

E / V (vs. Ag/AgCl) Fig. 6. (A) DPVs obtained for (a) human urine and (b) after spiked with commercial 10 μM UA at p-AMTa electrode in 0.2 M PB solution (pH 7.2). (B) DPVs obtained for (a) human blood serum and (b) after spiked with commercial 10 μM UA at p-AMTa electrode in 0.2 M PB solution (pH 7.2). Pulse width = 0.06 s, amplitude = 0.05 V, sample period = 0.02 s and pulse period = 0.20 s.

DPV of blood serum in PB solution (pH 7.2) shows an oxidation peak at 0.22 (±0.01) V (curve a; Fig. 6B). To confirm that the observed oxidation peak is due to UA, we have added the known concentration of commercial 10 μM UA to the serum sample. The enhancement of oxidation current at the same potential (curve b) suggests that the obtained peak in serum sample was due to the oxidation of UA. The recovery results for the different additions of UA in human urine and blood serum samples are summarized in Table 3. The recovery results were satisfactory for the spiked UA in human Table 3 Determination of UA in human urine and blood serum samples using p-AMTa electrode.a,b Samples

Original (μM)

Added (μM)

Found (μM)

Recoveries (%)

Urine 1 Urine 2 Blood serum 1 Blood serum 2

7.800 8.040 4.180 4.320

5 10 5 10

12.779 18.040 9.154 14.320

99.84 100.00 99.72 100.00

a b

Three replicate measurements were made on the sample. 96% confidence intervals.

28

S.B. Revin, S.A. John / Bioelectrochemistry 88 (2012) 22–29

urine and blood serum samples. The UA concentrations were found in the range of 234–241.2 μM in human urine samples and 125.4– 129.6 μM in human serum samples which are in good agreement with the previous reports [46,53]. The above results indicated that p-AMTa electrode could be efficiently used for the determination of UA in practical applications. 3.8. Anti-interference ability, stability and reproducibility of the p-AMTa electrode The anti-interference ability of the p-AMTa electrode was tested towards the detection of UA in the presence of various common ions such as Na +, K +, NH4+, Mg 2+, Ca 2+, Cl −, F −, CO32− and SO42− and some physiological interferents such as glucose, urea and oxalate by amperometric method. No change in the amperometric current response was observed for 40 nM UA in the presence of 40 μM of MgSO4, CaCl2, NaCl, K2CO3, NaF, NH4Cl, urea, glucose and oxalate indicating that the present p-AMTa electrode is highly selective towards UA in the presence of 1000-fold excess of these interferents. In order to investigate the stability of the p-AMTa electrode, the CVs for 0.5 mM UA in 0.2 M PB solution were recorded for every 5 min interval. It was found that the oxidation peak current of UA remains the same with a relative standard deviation of 1.5 (±0.05) % for 20 times repetitive measurements indicating that this electrode has good reproducibility. The p-AMTa electrode was kept in pH 7.2 PB solution at room temperature after all voltammetric measurements. The current response was decreased about 1.8 (±0.04) % in one week and 4.9 (±0.03) % in two weeks. To find out the reproducibility of the results, three different GC electrodes were modified with p-AMTa film and their responses towards a mixture of 0.5 mM each AA and UA were recorded by 15 repeated measurements. The separation between the voltammetric peaks of AA–UA was the same at all the three electrodes. The peak current obtained in the 15 repeated measurements of three independent electrodes showed a relative standard deviation of 2.08 (±0.06) %, confirming that the results are reproducible. The above results showed that the present modified electrode was very much stable and reproducible towards UA. 4. Conclusions In this paper, we have demonstrated the determination 1 μM UA in the presence of 1000-fold concentration of AA using the p-AMTa electrode. The p-AMTa electrode also successfully determined UA in the presence of important interferences, DA, Tyr and Met. Using amperometric method, 40 nM detection of UA was successfully achieved without any noise which is superior than the previous reports. The amperometric current response was increased linearly from 40 nM to 0.1 mM and the lowest detection limit was found to be 0.52 nM (S/N = 3). The practical application of the present modified electrode was successfully demonstrated by determining the concentration of UA in human urine and human blood serum samples. Supplementary data related to this article can be found online at http://dx.doi.org/10.1016/j.bioelechem.2012.05.005. Acknowledgment Financial support from the Department of Science and Technology, New Delhi under Nano Mission (No. SR/NM/NS-28/2008) is gratefully acknowledged. We thank UGC, New Delhi under SAP (No. F540/4/ DRS/2009 (SAP-I)) for the partial financial support. References [1] K. Inoue, T. Namiki, Y. Iwasaki, Y. Yoshimura, H. Nakazawa, Determination of uric acid in human saliva by high-performance liquid chromatography with amperometric electrochemical detection, J. Chromatogr. B 785 (2003) 57–63.

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S. Brillians Revin received his B.Sc. degree from Manonmaniam Sundaranar University in 2004 and M.Sc. degree from the University of Madras in 2007. He is now working for his Ph.D. in the Department of Chemistry, Gandhigram Rural Institute. His research interests are synthesis of conducting polymers by chemical and electropolymerization and gold nanoparticles and application of the polymeric materials for biosensor applications.

S. Abraham John is working as an Associate Professor in the Department of Chemistry, Gandhigram Rural Institute. He received his Ph.D. degree from Madurai Kamaraj University, Madurai and Doctor of Engineering in Electrochemistry from Tokyo Institute of Technology, Japan. He worked as a post-doctoral fellow in the Department of Applied Chemistry, Tokyo University of Agriculture and Technology, Japan under JSPS program from 1999 to 2001. He also visited as an Invitation Fellow for Foreign Researchers under JSPS program to the Department of Applied Chemistry, Nagasaki University, Japan in 2006. His research interests are fabrication of thin films of porphyrins, phthalocyanines, conducting polymers and gold nanoparticles for toxic gas, chemical and biochemical sensors by both electrochemical and optochemical methods.