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Graphitized mesoporous carbon modified glassy carbon electrode for selective

sensing of xanthine, hypoxanthine and uric acidRajendiran Thangaraj and Annamalai Senthil Kumar*

Received 10th January 2012, Accepted 19th April 2012

DOI: 10.1039/c2ay25029b

An efficient electrochemical sensor for simultaneous electrochemical sensing of three purine

compounds, uric acid (UA), xanthine (X) and hypoxanthine (HX), using a graphitized mesoporous

carbon (GMC) modified glassy carbon electrode (GCE/GMC) has been demonstrated in pH

7 phosphate buffer solution without any enzyme, prior to electrode activation, surfactant and sample

pre-concentration step. Electrochemical investigation of the GCE/GMC with [Fe(CN)6]3 indicates

metallic conductor like surface features of the modified electrode. A diffusion controlled reaction

mechanism was identified for the electro-oxidation of the three purine compounds with an

electrocatalytic pathway, except for the UA, where it shows a surface area effect with a mixed-diffusion

and adsorption controlled mechanism at higher scan rates (>70 mV s1). Calculated full-width of the

half maximum values for the simultaneous detection of the three purine compounds are 42, 53 and

64 mV respectively and these are the lowest values ever reported in the literature, suggesting effective

electron-transfer behaviour of the modified electrode for the purine oxidations. Calibration plots for

the simultaneous detection of the purine compounds were linear in the concentration range of 20

400 mM, 20320 mM and 20240 mM for UA, X and HX with detection limit values of 110 nM, 388 nM,

and 351 nM respectively. Selective sensing of the purine compounds in human blood-plasma, urine and

fish samples was successfully demonstrated with recovery values $100%.

1. Introduction

Xanthine (X), hypoxanthine (HX) and uric acid (UA) are

important purine bases, where X and HX are the intermediates

and UA is an end product of the purine metabolism of the cells

of humans, animals and plants.13 When cells die, the purines in

their genetic material break down. Purine catabolism of a dead

fish is as follows: adenosine triphosphate / adenosine

diphosphate / adenosine monophosphate / inosine mono-

phosphate/ inosine/ HX/ X/ UA.13 Xanthine oxidase

(XOD) is the specific enzyme present in the biological system,

which helps to catalyze the HX and UA oxidation reactions.4

Analyses of the intermediates and the end product can give

vital information about the freshness of the fish foods.4

Forexample, muscles of the freshly dead fish contain higher

concentrations of HX and X than the end product, UA; while

for the aged and decomposed fish, the concentration of UA is

higher than that of X and HX.5 Similarly, abnormalities of the

purine compounds present in humans are the clinical symptomsfor some critical diseases. For instance, increased excretion of

the hypoxanthine and xanthine in urine is the indication of the

inherited disease, Xanthinuria.6,7 Uric acid also serves as an

antioxidant and helps to prevent damage to our blood vessel

linings.8 For healthy individuals, the concentration of the UA

present in the plasma is approximately 116500 mM and in the

range of 1.24.5 mM in urinary excretion.9 In diseases such as

gout, hyperuricemia and LeschNyhan syndrome, high levels of

the UA are found in the blood samples.10 Meanwhile, relatively

few foods contain concentrated amounts of the purines with

high-protein content. Examples are: organ meats like kidney,

fish, herring, sardines, mussels and yeast.11 Hence selective,

sensitive and simultaneous sensing of UA, X and HX in bio-logical, food and clinical samples is of significant research

interest in analytical chemistry.

Conventional analytical methods available for the purine

analysis are based on separation (HPLC or capillary electro-

phoresis) coupled with UV-Vis or fluorescence spectroscopic

techniques.1220 These methods are time consuming and require

several off-line pre-concentration steps for the real sample

analyses. Several electrochemical methods are available for the

separation-less and enzyme-less analysis of the three purine

compounds. Following are the recently reported working elec-

trodes for the simultaneous detection of the three compounds:

Environmental and Analytical Chemistry Division, School of AdvancedSciences, Vellore Institute of Technology University, Vellore-632 014,India. E-mail: [email protected]; Fax: +91-416-2243091/93;Tel: +91-416-2202754

Electronic supplementary information (ESI) available: Effect ofsolution pH on simultaneous analysis of three purine bases atGCE/GMC (Fig. S1). See DOI: 10.1039/c2ay25029b

2162 | Anal. Methods, 2012, 4, 21622171 This journal is The Royal Society of Chemistry 2012

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unmodified glassy carbon electrode (GCE),21 preanodized

carbon paste electrode22 and functionalized single walled

carbon nanotubecarbon paste electrode23 in acid pH solutions,

and Nafion modified lead-ruthenate pyrochlore,24 Ru(DM-

SO)4Cl2 electrodes,25 preanodized nontronite clay coated

screen-printed electrode,26 electropolymerized poly(bromocresol

purple) modified GCE,27 and impure multi-walled carbon

nanotube (MWCNT).28 Most of the reported procedures either

require expensive chemicals or require complicated electrodepreparation steps which include extensive pre-anodization of

the underlying or modified electrode and the use of expensive

starting materials such as Nafion, carbon nanotubes and

ruthenium.24 Few chemically modified electrodes were also

reported for the simultaneous detection of two purine

compounds (UA and X) with examples being: 2-amino-1,3,4,-

thiadiazole modified GCE,29 mesoporous SiO2graphite paste

electrode30 and purified MWCNTdihexadecyl hydrogen

phosphate surfactant modified electrode.31 Recently, a XOD

enzyme biosensor constituted with functionalized graphene

oxide + conducting polypyrrole graft copolymer + poly-

(styrenesulfonic acid-g-pyrrole) modified electrode was reported

for HX sensing in a physiological solution.32 In this work, weare reporting a commercially received graphitized mesoporous

carbon (GMC) modified GCE (designated as GCE/GME)

prepared by a much simpler method than the previously

reported procedures without any enzyme, surfactant and acti-

vation or functionalization of the electrode. The GCE/GMC is

highly useful for a sensitive, selective and simultaneous vol-

tammetric detection of the three purine compounds UA, X and

HX in a physiological solution.

Mesoporous materials in general have pores with an average

diameter size (250 nm) in between the range of micro-porous

(50 nm) materials. GMC is

a porous carbon containing surface graphitic frameworks,

prepared using certain soft polymers as a precursor and meso-porous silica materials (MSM) as a template by a high temper-

ature pyrolysis procedure (non-conventional method of

preparation).33 Structural features of the GMCs include homo-

geneity with significant graphite-like domains and stacking

heights, low amounts of surface functional groups, and low

occurrences of imperfections such as twists, non-aromatic links,

and carbon valencies.34 GMC has been demonstrated as a highly

efficient support for Pt catalyst immobilization and for the

enhanced direct methanol oxidation and oxygen reduction

reactions suitable for fuel cell applications.35,36 Some of the

conventional carbons, which are rich in carbonyl, phenolic and

alcoholic functional groups, often result in severe oxidative

corrosion during the electro-catalysis.37,38 But no such compli-cations were reported with the GMC. The GMC has been used as

a catalyst for several organic functional group transformations

including dehydrogenation of isobutane.39 The GMC material

was rarely used for chemically modified electrode preparation

and electroanalytical applications.40 Interestingly in this work,

the GCE/GMC showed an efficient sensing of the three purine

compounds without any ascorbic acid (AA) interference in

a physiological solution. Finally, the modified electrode was

successfully applied for simultaneous or selective sensing of the

purine compounds in different kinds of clinical (blood and urine)

and fish samples.

2. Experimental

2.1. Chemicals

GMC (50 nm and 99.95% purity), MWCNT (outer diameter: 10

15 nm; inner diameter: 26 nm, length 0.110 mm and $90%

purity), uric acid, xanthine and hypoxanthine and ascorbic acid

were of analytical grade and purchased from Sigma-Aldrich.

They were used as received without any further purification.

Aqueous solutions were prepared using deionized and alkaline

KMnO4 distilled water (designated as DD water). Phosphate

buffer solution (PBS, pH 7.1 0.05) of 0.1 M ionic strength was

used as a supporting electrolyte.

2.2. Measurements

Voltammetric measurements were carried out using a CHI660C

Electrochemical work station (USA) with 10 mL working

volume. The electrode system consists of a glassy carbon elec-

trode (GCE) and its chemically modified electrode (CME) as

a working electrode (0.0707 cm2), Ag/AgCl (3 M KCl) as

a reference electrode and platinum wire as a counter electrode.

The surface of the GCE was cleaned both mechanically (polished

with 0.05 micron alumina powder in a BAS polishing kit, cleaned

with acetone and washed with double distilled water) and elec-

trochemically by performing cyclic voltammetry (CV) continu-

ously for 20 cycles in a potential window of0.2 to 1.2 V vs.

Ag/AgCl in PBS. The GMC surface morphology was examined

by transmission electron microscopy (TEM, JEOL-3010). A

Bruker D8 Advanced diffractometer X-ray diffraction (XRD,

Germany) instrument with Cu Ka source (l 1.5418 A) was

used to probe the crystallographic information of the GMC.

2.3. Preparation of the GCE/GMC modified electrode

A GMCethanol suspension stock was first prepared by mixing 1mg of the GMC powder with 500 mL of absolute ethanol (99.9%)

followed by 10 minutes sonication at room temperature (T

300(2) K). For the GCE/GMC preparation, 5 mL of the stock

was drop coated on the cleaned GCE surface and air dried for 10

minutes at room temperature. The procedure allows for

preparing a near uniform layer of GMC on the GCE surface. The

as-prepared GMC layer was further electrochemically pretreated

by continuous potential cycling (n 20, n no. of cycles) in the

potential window of0.2 to 0.8 V vs. Ag/AgCl at a scan rate (v)

of 50 mV s1 in pH 7 PBS. The GCE/GMC did not show any

redox feature in the blank base electrolyte solution. Similar to the

above procedure, GCE/MWCNT was also prepared by a drop

casting methodology. Since dissolved oxygen did not influencethe electrochemical response, experiments were all carried out

without any deaeration, as a physiological solution. The differ-

ential pulse voltammetric (DPV) technique was used as a quan-

titative electrochemical tool for the simultaneous detection of the

purine bases X, HX and UA in pH 7 PBS.

2.4. Real samples preparation

Human blood plasma (#1(A)a, #1(A)b, #1B), healthy human

urine (#2#4), red blood cell (RBC) and pus cell contaminated

urine (#5#7) and fresh fish flesh (#8#11) samples were

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subjected to real sample analyses. Three healthy blood samples of

volume 10 mL, where two samples belonging to the same person

were collectedat 60 daytimeintervals(#1(A)a andb) with thehelp

of a staff nurse from the Vellore Institute of Technology (VIT)

health care centre. The three healthy human urine samples (#2

#4) and three RBC/pus cell contaminated urine samples (#5#7)

were also collectedfrom theVIT university healthcare centre. The

RBC/pus cell counts in the urine samples are 1015/510, 1015/

1015 and1520/36 respectivelyfor samples #5,#6 and#7. Priorto the analysis, the urine samples were filtered with ordinary filter

paper and diluted (dilution factor 100) using PBS. Fresh fish

samples: seer fish #8 (Scomberomorus guttatus), red snapper #9

(Himantura bleekeri), goldstripe sardinella #10 (Sardinella gib-

bosa), and carp fish #11 (semisulcatus) were purchased from

a local animal food market in the Vellore city. All these samples

were stored in a refrigerator at $5 C until use. Following is the

procedure for the blood plasma sample preparation: 10 mL of the

blood sample with a pinch of ethylenediamine tetraacetic acid

(EDTA) was first centrifuged at 1600 rpm for about 20 minutes

and the concentric plasma that appeared as a supernatant was

taken for further analysis after proper dilution with PBS (dilution

factor 5). For the fish real sample preparation, about 2 g of thefish flesh was weighed and homogenized with 10 mL of PBS using

a mortar and pestle. The homogenized solution was filtered and

further diluted with PBS (dilution factor 5). For all the real

sampleanalyses,a 10 mLaliquotwas takenand examinedwiththe

differential pulse voltammetric (DPV) technique. A standard

addition approach was adopted for the quantification of the

purine concentrations in the real samples. Since there is a weak

adsorption of xanthine on the GCE/GMC surface, after the

experiment with the analyte, the electrode was removed from the

cell and immersed in 10 mL blank PBS for 10 seconds at room

temperature. Such an experimental procedure gives easy regen-

eration of the GCE/GMC for the successive electroanalytical

measurements.

3. Results and discussion

3.1. Physicochemical characterization of the GMC

TEM images of the GMC at different magnifications are given in

Fig. 1AC. Randomly spaced empty clusters of uniform size

$53 nm (outer diameter) are seen in the photographs. The black

spots observed in the photographs are agglomerated units of the

GMC. Details of the graphitic layers could be identified from the

XRD analysis.

The XRD pattern of the GMC was compared with another

well-known crystalline carbon, MWCNT. Fig. 2 shows a well

defined and intense diffraction peak at 2q 26.05 along with

a weak peak at 43.02 that were obtained from the XRD of the

GMC powder sample. The values correspond to the hklindex of

{002} and {101} respectively for the hexagonal graphitic struc-

ture on the mesoporous carbon. The control XRD pattern of the

MWCNT material also shows similar 2q values at 26.07 and

43.13. The calculated d-spacing (d002) value for the {002} peakusing the Braggs equation, d002 nl/2sin(q), where d002

interplanar distance, lwavelength and q angle of diffraction,

is 3.41 A for both the GMC and MWCNT samples. The values

quoted in the literature for the unmodified mesoporous and

graphitized mesoporous carbon (GMC) materials are 3.71 and

3.36 A respectively.35,41 The 0.34 A reduction in the d-spacing is

an indication of the enhanced crystallinity of the GMC

compared to the MWCNT.

3.2. Electrochemical characterization of the GCE/GMC

The ferricyanide/ferrocyanide redox couple is a bench mark one

electron-transfer electrochemical system, useful to access theconductive and the electron-transfer features of a modified

electrode system. Fig. 3(A)a is the CV response of the GCE/

GMC electrode in the presence of 1 mM of potassium ferri-

cyanide at a scan rate of 10 mV s1. A well defined redox

couple with anodic (Epa) and cathodic (Epc) peak potential

values at 240 and 155 mV vs. Ag/AgCl respectively was noticed.

Fig. 1 TEM images of GMC at different magnifications.

Fig. 2 XRD patterns of graphitized mesoporous carbon and a multi-

walled carbon nanotube.


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Calculated peak-to-peak potential (DEp Epa Epc) and

apparent standard electrode potential (Eo0 Epa + Epc/2)

values are 85 mV and 198 mV vs. Ag/AgCl respectively. For

comparison, the unmodified GCE is also subjected to the

ferricyanide experiment as in Fig. 3(A)b. Corresponding DEp

and Eo0 obtained on the GCE are 86 and 200 mV vs. Ag/AgCl.The similarity in the DEp and E

o0 values between the GCE and

GCE/GMC indicates appreciable electron-transfer behavior of

the GCE/GMC closer to the metallic conductive electrode.

From the reversible redox reaction, the electrochemically

accessible surface area, Ae (cm2), value could be calculated

using the standard RandleSevcik equation: ipa or ipc 2.69

105AeD1/2n3/2v1/2C, where n is the number of electrons involved

in the redox reaction, D is the diffusion coefficient (7.6 106

cm2 s1) and C is the concentration of the redox couple. Fig. 3B

shows the plots of ipa vs. v1/2 for the ferricyanide at GCE and

GCE/GMC. The GCE shows a linear line up to v 500 mV

s1, while for the GCE/GMC the linearity is maintained up to

70 mV s1, thereafter the line tends to saturate. The calculatedslope (ipa/v

1/2) value (1.32 mA mV1/2 s1/2) is the same for the

GCE and GCE/GMC. After substitution of the slope value in

the RandlesSevcik equation, the Ae value is calculated to be

0.0562 cm2, which is equal to $80% of the geometric surface

area of the working electrode (0.0707 cm2). Note that GCE is

atomically smooth and possesses near ideal surface features,

whereas the GCE/GMC is the modified electrode with graphitic

and non-graphitic porous carbon units. Even though the GMC

has high surface area (BET surface area 900 m2 g1),33,34 in

consideration with the Ae values, it can be speculated that only

part of the total surface area of the electrodes is accessible for

the electron-transfer reaction. The nature of surface polishing

methods including the mechanical polishing of the electrodeand electrochemical pretreatments can change the surface area

of the GCE. On the other hand, it is expected that there will be

some diffusion restriction of the reduced and oxidized forms of

the ferrocyanide complex within the GMC. This observation

could be reflected by an improper (non-ideal) tail-off peak

current and non-linearity in the ipa vs. v1/2 plot at v > 70 mV s1

for the redox couple on the GCE/GMC working electrode,

unlike the GCE case (Fig. 3(A)b). Nevertheless, due to the

unique structural feature of the GMC, the electrochemical

response of the GCE/GMC is remarkable and effective over the

unmodified electrode for the purine compounds oxidation.

3.3. Electrochemical and catalytic behaviours of the GCE/

GMC

Fig. 4AC show comparative CV responses of the GCE/GMC

and unmodified GCE with UA, X and HX discretely at a scan

rate of 50 mV s1 in pH 7 PBS. As can be seen from the CV of

UA, the unmodified GCE shows a marked anodic oxidation

peak at 295 mV vs. Ag/AgCl. The GCE/GMC also showed

a similar peak potential value but with two and half timesenhancement in the anodic peak current value over the GCE.

The observation may be due to the surface area effect of the

GCE/GMC. For the case of X (Fig. 4B), the GCE/GMC

showed a profound oxidation peak at 650 mV and was three

times higher in the peak current value than the unmodified

GCE. Note that the X oxidation peak potential at GCE is

750 mV, which is 100 mV higher in over-potential than the

GCE/GMC system. For the HX case (Fig. 4C), the unmodified

electrode shows the absence of any CV oxidation peak up to

1100 mV, whereas the GCE/GMC yielded a clear oxidation

peak at 980 mV. In general, the surface area effect can be

referred as when the modified electrode showed an enhanced

oxidation/reduction current response to the analyte over itscorresponding unmodified electrode without altering the

oxidation/reduction peak potential.42 On the other hand if the

peak potential is altered, for example, the analyte oxidation

potential is significantly reduced by the modified electrode

(over-potential reduction with the symbol h) and it is then

referred to as the electrocatalytic effect. In our case, X and

HX have shown significant reductions in the h values, while for

the case of the UA there is no such reduction in the h; however,

only enhancement in the peak current value was noticed. Based

on the information it can be concluded that X and HX

oxidations are due to the electrocatalytic behaviour, while for

the UA oxidation referred, it is due to the surface area effect of

GCE/GMC.In continuation of the CV studies, DPV was also performed on

the above purine compounds comparatively with GCE and

GCE/GMC as in Fig. 4DF. All the three purine compounds

show well-defined and sharp peak currents respectively at 250,

580 and 900 mV vs. Ag/AgCl for UA, X and HX on the GCE/

GMC. Unlike the CV behaviours, DPV of the unmodified GCE

shows significant decreases in the peak current signals and high

oxidation potential values, 460 and 780 mV for the UA and X

respectively, and nil response to HX. Calculated h reductions for

the UA and X are 200 and 220 mV respectively, in the DPV. But,

no such observations were noticed in the CV (Fig. 4AC).

Presumably, the applied amplitude (50 mV) and pulse effects

(pulse width

0.2 s; pulse period

0.5 s) may have somehindrance to the electro-oxidation of the purine compounds on

the GCE, while for the case with GCE/GMC a positive effect by

the DPV parameters and hence considerable sharp peaks and

reduction in the h were observed. Hence, it is a clear advantage of

using DPV as the qualitative tool for sensitive analyses of the

compounds in this work. Note that we have optimized the DPV

parameters viz., pulse width, amplitude and pulse period with

respect to the GMC modified electrode in order to get sharp

voltammetric responses to the analytes. This is the reason why

the corresponding unmodified GCE showed broad peaks in

Fig. 4D and E.

Fig. 3 (A) CV responses of bare GCE and GCE/GMC with 1 mM of

K3[Fe(CN)6] in 0.5 M KCl solution at v 10 mV s1. (B) Plot ofipa vs. v

1/2

for the ferricyanide redox process at two different electrodes.


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In order to compare the efficiency of the electron-transfer

behaviours (and in turn to the sharp peak currents), DPV peaks

full-width of the half maximum (FWHM) value is taken as

a sensitive parameter. Calculated FWHM values are 42, 53 and

64 mV respectively for the UA, X and HX. The FWHM values

for the recently reported Ru(DMSO)4Cl2Nafion modified

electrode are 75, 75 and 100 mV respectively.25 The lowered

FWHM values with the GCE/GMC are the clear evidence for the

efficient peak current feature of the present system.

Simultaneous analysis of the UA, X and HX on a GCE/GMC

was nextexaminedby CVand DPV techniquesas inFig. 5AandBrespectively. Interestingly, the GCE/GMC shows well-defined

and well-separated oxidation peak current responses. But the

unmodified GCE displays a 13-fold decrease in the voltammetric

peak current at theoxidation potentials of 400 and800 mV for the

UAand X, andan absence of HX signal. The current signals areill

definedtoo. Appearance of these current signals maybe dueto the

response of overlapped UA, X and HX analytes. Calculated

FWHM values for the analyte peaks from the simultaneous DPV

measurements are UA 54, X 50 and HX 50 mV and these

values were considerably smaller than the previously reported

values of 59, 74, 86 mV on Ru(DMSO)4Cl2Nafion,25 and 65, 70,

140 mV on poly(bromocresol purple) modified electrodes for the

simultaneous oxidations of the three purines in a pH 7 PBS.27 All

these experimental observations evidence the superior function of

theGCE/GMC forthe simultaneousoxidations ofUA, X andHX

in a physiological solution in this work.

In order to checkthe nature of the electron-transfer mechanism,

effects of CV scan rate (v) on the discrete oxidations of the three

purine compounds were examinedas in Fig. 6AC. An increase inthe v implies a systematic increase in the peak currents for all the

three compounds. Peak current valueswere measured andplotted

against the scan rate (v) as a doublelogarithmic plot as inFig.6D.

Calculated slope (vlog ipa/vlog v) values are: UA 0.3 (v < 70 mV

s1) and 0.85 (v > 80 mV s1), X 0.30 and HX 0.30. These

values are closer to the ideal values of 0.5 and 1 corresponding to

diffusion and adsorption controlled electron-transfer mecha-

nisms.43 Since theX hassome weak adsorption effecton theGCE/

GMC (see the Experimental section), a slope value near 1 would

be expected rather than $0.5 in this work. But the measured

experimental value is $0.5, which indicates a diffusion controlled

mechanism of the adsorbed X within the GCE/GMC film. The

trend in the mechanism of the UA changes from diffusion toadsorption controlled reaction at scan rate >70 mV s1 (slope

0.85),whichresembles the trend of theferricyanide case (Fig. 3A).

Presumably, like ferricyanide, there will be some diffusion

restriction for the UA within the pores of the GMC at a high scan

rate, which in turn leads to change in the reaction mechanism.

Exactdetailsfor the occurrence of suchreactionare still unknown.

On the basis of Epa ([balog(v)]/2) + constant, for a totally

irreversible diffusion-controlled process44,45 the ba (i.e., Tafel

slope) value is measured as 10, 40 and 43 mV per decade for UA,

X and HX oxidation reactions respectively on a GCE/GMC

(Fig. 6E). Assuming one-electron transfer in the rate

Fig. 4 Discrete CV (AC) and DPV (DF) responses of the electrochemical oxidations of UA, X and HX at GCE and GCE/GMC in a pH 7 PBS. DPV

parameters are: amplitude 50 mV; increment potential 4 mV; pulse width 0.2 s; pulse period 500 s.

Fig. 5 Simultaneous CV (A) and DPV (B) responses of GCE and GCE/

GMC for a mixture of UA, X and HX dissolved in pH 7 PBS. CV scan

rate (v) 50 mV s1. Other DPV parameters are as in Fig. 4.


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determination step (i.e., na 1), since ba 2.303RT/naFaa, the

anodic-transfer coefficient (aa) can then be calculated as 5.9, 1.48

and 1.37 for the UA, X and HX respectively. Previously, Zen

et al. reported an aa value of$1 for an irreversible oxidation of

cysteine on a lead-ruthenate pyrochlore modified electrode.46

Hence, the high values obtained in the present work are an

indication of the effective catalytic performance of the GCE/

GMC to the purine oxidation reactions. Fig. 6F is a typical

illustration for the electrochemical oxidation of the three purine

compounds on the GCE/GMC.Fig. S1(A) shows the effect of solution pH (in the range pH 3

8) on the simultaneous oxidations of the three analytes by the

DPV. Plots of the respective anodic peak current and peak

potential against the solution pH are displayed in Fig. S1(B) and

S1(C) respectively. The peak current values increased positively

against the solution pH and attained the maximum at pH $7.

The peak potential values were systematically decreased against

increase in the pH values. A slope value of60 mV per pH was

calculated for all the three compounds on the GCE/GMC, sug-

gesting the proton-coupled electron-transfer mechanism with

equal number of proton/electron involvement in the reaction

pathway (Nernstian case). Usually, depending on the pKa value

of an analyte, there is a marked alteration in the trend of theoxidation peak potential value.24 Unfortunately, considering the

pKa values of UA (5.4 and 10.3), X (7.4 and 11.2) and HX (8.94

and 12.10), there were no alterations in the peak potentials with

respect to the pKa values in this work. This observation further

suggested that the protonation reaction is not a rate determining

step in the electrochemical oxidation processes.

3.4. Analytical measurements

Effects of UA, X and HX concentrations (calibration) on the

simultaneous detection of the individual analytes are next studied

by systematically increasing one analyte concentration with the

other two fixed as in Fig. 7AC. The optimal DPV parameters

used in this work are: increment 4 mV, amplitude 50 mV,

pulse width 0.2 s, pulse period 0.5 s and sampling width

0.0167 s. Interestingly, systematic variations in the peak currents

upon increases in the analyte concentrations were noticed. As

seen in Fig. 7A, DPV of the UA shows regular increases in the

peak current signals up to 400 mM with current sensitivity and

regression coefficient values of 75.3 nA mM1 and 0.996 respec-

tively. Ten repetitive measurements of 25 mM of UA result ina relative standard deviation (RSD) value of 0.88%. For the UA,

the calculated detection limit (S/N 3) value is 110 nM.

Analytical parameters for the X are: linearity up to 320 mM;

sensitivity 61.8 nA mM1; regression 0.988, RSD 1.29%

([X] 25 mM, n 10) and detection limit 388 nM. Similarly for

HX, the results are: linearity up to 240 mM; sensitivity 112.7

nA mM1; regression 0.996, RSD 1.87% ([HX] 25 mM, n

10) and detection limit 351 nM. Obtained detection limit

values in this work are comparable to or even better than those

previously reported in the literature. For instance, reported

detection limit values for UA, X and HX on a nontronite clay

coated screen-printed electrode with 2 V pre-anodization and 40

s of pre-concentration are 0.42 mM, 7.0 mM and 0.34 mM,26

poly(bromocresol purple) modified GCE; 0.20 mM, 0.06 mM and

0.12 mM,27 NafionRu(DMSO)4Cl2; 0.37 mM, 2.35 mM and

2.37 mM,25 and impure MWCNT; 0.141 mM, 0.134 mM, and 2.87

mM,28 respectively. Simple preparation method and using rela-

tively low cost carbon materials (other than the MWCNT) as

a working electrode without any enzyme, activation and pre-

concentration procedures are the superior features of the present

working electrode over the existing CMEs. Note that the

advantage of elimination of the ascorbic acid interference, unlike

the MWCNT, was achieved with the GMC material (see Section

3.5). With regard to a stable response of the GCE/GMC surface,

Fig. 6 Effect of CV scan rate from 5500 mV s

1 on the electrochemical oxidation of (A) UA, (B) X and (C) HX at GCE/GMC in a pH 7 PBS. Plots oflog(ipa) vs. log v (D) and Epa vs. log v (E) for the purine electrochemical oxidation reactions. (F) Typical illustration for the electrochemical oxidations of

the three purines on the GCE/GMC.


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we expected that micro- and nano-porous voids might exist on

the GCE surface (after the mechanical and electrochemical

pretreatments); when GMC was modified as ethanol dispersion

on the GCE surface, some of the portions of GMC might have

occupied the porous sites of the underlying surface and thereby

stabilizing the GCE/GMC. In order to check the reproducibility

of the modified electrode, four modified electrodes were

discretely prepared and subjected to DPV analysis of UA. The

calculated RSD value for the peak currents is 3.06%, which

indicates good reproducibility of preparation of the modified

electrode.

3.5. Interference study

With the aim to extend the GCE/GMC for blood plasma purines

analyses, we have examined the effect of a co-existing biochem-

ical, AA, on the simultaneous detection of the purine samples.

For a comparison, GCE/MWCNT was also subjected to the AA

interference study (Fig. 8A). In a whole blood sample, common

interferences for the UA are glucose, cholesterol, triglycerides,

proteins, sodium chloride and AA. All these compounds, except

AA, are electro-inactive. As from the clinical diagnosis data, the

concentration range of the UA and AA present in a healthy

humans blood plasma is 112506 mM (2.08.5 mg dL1) and AA

is 40 15 mM, respectively.4749 Fig. 8B is a typical DPV response

of the GCE/GMC for the simultaneous measurements of a fixed

UA, X and HX content against variable AA concentrations in

the range of 20500 mM. As can be seen, in the absence of AA,

both GCE/MWCNT and GCE/GMC showed qualitatively

similar DPV simultaneous responses. But in the presence of

dilute AA, the GCE/MWCNT shows distinctly different vol-

tammetric purine oxidation peaks. Plots of the three purinecompounds anodic peak current (ipa, base-line corrected)

against the AA concentration, [AA], are given as insets in

Fig. 8A. Unfortunately, addition of a dilute concentration of

AA 20 mM leads to about a three fold increase in the purine

oxidation peak currents and these currents get saturated after

that. With regard to the AA oxidation peak at 0.1 V vs.

Ag/AgCl, a marked peak current signal was noticed only when

[AA] > 100 mM. Below 100 mM AA, large background currents

were observed instead of any AA oxidation feature. These

observations ruled out the choice of the GCE/MWCNT for the

precise simultaneous analyses of the purine compounds in

Fig. 7 Typical DPV responses for the simultaneous detection of UA, X

and HX using a GCE/GMC in a pH 7 PBS. Plots of ipa vs. analyte

concentration, [analyte], were given as the respective insets. DPV

parameters are as in Fig. 4. Fig. 8 Effect of AA interference for the simultaneous DPV detection of

the three fixed purine compound concentrations on (A) GCE/MWCNTand (B) GCE/GMC in pH 7 PBS. Respective ipa vs. [AA] plots were given

as insets in the figure.


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the presence of dilute AA in a physiological solution. On the

other hand, the GCE/GMC shows tolerable voltammetric

current signals for the AA concentration up to 100 mM. In

addition, the GCE/GMC did not show any AA oxidation peak

current up to 100 mM (inset, Fig. 8B). Presumably the meso-

porous structure has some negative interactive effects with the

five member non-aromatic cored AA. These observations

evidence that GCE/GMC is the better choice for the detection of

uric acid in human blood plasma.

3.6. Real sample analyses

Practical applications of the GCE/GMC for the simultaneous or

individual analyses of the three purine compounds in human

blood plasma, urine and fish flesh samples were tested (Fig. 9 and

Table 1). Typical DPV responses of the GCE/GMC for healthy

humans blood plasma samples collected at sixty day time

intervals (1st day: #1(A)a and 60th day: #1(A)b), are displayed in

Fig. 9(A)a and b. Real sample analyses data are presented in

Table 1. A marked DPV peak at 0.25 V vs. Ag/AgCl was noticed

corresponding to the presence of the UA in the blood plasma

samples. Standard spikes of the UA result in a systematic

increase in the peak currents on the real sample peak. This

observation qualitatively confirms the presence of the UA in the

blood plasma samples. Based on the standard addition method,the UA concentrations in the blood samples were calculated as

35.74 and 35.66 mM respectively for #1(A)a and #1(A)b. After

correction of the dilution factor, the values turned out as 178.7

and 178.3 mM, which are in the range of the standard UA level.9

The observation of similar UA content values in those real

Fig. 9 GCE/GMC responses for the real samples: (A) healthy human blood plasma samples (A)a and (A)b from the 1st and 60th day respectively, (B)

healthy and (C) RBC/pus cell contaminated urine samples, and (D) a fresh fish sample in a pH 7 PBS. DPV parameters are as in Fig. 4.

Table 1 Various purine real sample analyses using a GCE/GMC modified electrode by the standard addition method

S. no. Real samples Analyte

Detected value

Added (mM) Found (mM) Recovery (%)Original Totalb

1 Blood plasma (#1Aa)a UA 35.7 178.5 mM 20 20.44 102.22 Blood plasma (#1Ab)a UA 35.7 178.5 mM 20 20.73 103.73 Blood plasma (#1B) UA 63.7 318.5 mM 20 20.53 102.74 Urine (#2) X 1.9 95.0 mM 20 20.16 100.8

UA 34.3 3.4 mM 30 31.15 103.85 Urine (#3) UA 37.0 3.7 mM 30 29.30 97.76 Urine (#4) UA 16.6 1.7 mM 20 19.67 98.47 Urine (#5)c UA 81.5 4.1 mM 50 49.97 99.98 Urine (#6)c UA 44.7 2.2 mM 50 50.22 100.49 Urine (#7)c UA 118.9 5.9 mM 50 49.57 99.110 Fish (#8) X 39.9 199.5 mM (760 mg g1) 30 30.30 101.011 Fish (#9) X 27.3 136.5 mM (520 mg g1) 60 62.40 104.012 Fish (#10) X 64.4 322.0 mM (1200 mg g1) 30 29.30 102.313 Fish (#11) X 34.5 172.5 mM (655 mg g1) 25 24.71 99.0

a Collected from the same person. b After dilution factor correction. c Pus cell contaminated urine samples.


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samples proves the reliability of the present electrode for prac-

tical analyses. In another case, for healthy human urine, sample

#2, two peaks at 0.34 and 0.68 V corresponding to the UA and X

were observed (Fig. 9B). The standard addition method quali-

tatively confirms the presence of UA and X in the urine samples.

Calculated UA and X concentration values are 318.7 and 96 mM

respectively. In continuation, individual detection of UA present

in two healthy humans urine samples (#3 and #4) was also

examined and the values are 3.7 and 1.66 mM respectively andthese values are within the standard UA limits only.9 Fig. 9B is

a DPV of the RBC/pus cell containing urine sample #5. Entries

79 in Table 1 are the measured UA concentration values of the

urine samples, #5#7. Since there is no precise literature avail-

able to refer the RBC/pus cell count versus UA level in urine,

based on the experimental results we are concluding here that

there is no dependence of the excreted UA level on the RBC/pus

cell values of the human urine.

Finally four fresh fish samples (#8#11) were subjected to the

DPV analyses. The samples showed two peak responses at 0.6

and 0.95 V vs. Ag/AgCl corresponding to the X and HX

compounds. All fish samples show ill defined HX peaks except

fish sample #11. The standard addition of HX does not showincrements in the peak current. We suspect some matrix effect

with the HX analysis and hence it could not be detected for some

kinds of fish samples. Nevertheless, the X analysis content in the

fish is vital to judge the quality of the fish. As seen in Table 1,

detected X contents were in the order of 1200 > 760 > 655 > 520

mg g1 for samples #10, #8, #11 and #9 respectively. The absence

of UA with the animal food suggests appreciable freshness of the

fish foods. Amongst the fish samples, sample #10 (Sardinella

gibbosa) is found to be the richest content of X and hence these

are the richest protein diet foods. Finally, calculated recovery

values for the standard spikes of the purine samples all fall at

$100%, which indicates the efficiency of the GCE/GMC in the

real sample analyses.

4. Conclusions

The GMC modified glassy carbon electrode was developed for

efficient and simultaneous electrochemical sensing of three

purine compounds: uric acid, xanthine and hypoxanthine in

a physiological solution. The GMC modified electrode shows

well defined well separated and very sharp purine oxidation

signals at around 0.3, 0.6 and 0.95 V vs. Ag/AgCl respectively for

the compounds. The purine oxidation mechanisms follow the

electrocatalytic pathway for the X and HX, while for the UA case

it was due to the surface area effect. Investigation with the

standard bench mark redox system, Fe(CN)63, showedimproper tail-off peak current behaviours due to diffusion

restriction of the redox couple within the mesopores of the

material. The effect of solution pH on the purine oxidation

reactions obeyed the Nernstian type of proton-coupled electron-

transfer reaction mechanism. Constructed calibration plots were

linear up to 400, 320 and 240 mM of UA, X and HX respectively.

Corresponding detection limit values were 110 nM, 388 nM and

351 nM. Simultaneous or selective detection of the three purine

compounds in blood-plasma, healthy urine samples and fish

samples was successfully demonstrated. A matrix effect was

noticed with some real sample analyses. Calculated recovery

values for the analyses were $100%. Overall, the novelty of the

present electrochemical sensor system lies in the simple prepa-

ration steps using relatively low cost GMC materials for the

separation-less analyses of the three purine bases without any

enzyme, the pre-concentration procedure, the pre-anodization

steps and the extendibility of the sensor to various kinds of real

samples.

Acknowledgements

The authors are grateful for the financial support from the

Department of Science and Technology (DST) under Tech-

nology System Development Programme, India. We also thank

Material Science Division, Indian Institute of Technology,

Madras for the TEM measurements.

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