electrochemical study and direct determination of adenosine-5′-monophosphate coupled to...

Full Paper

Electrochemical Study and Direct Determination ofAdenosine-5’-Monophosphate Coupled to 6-Thioguanosine and aGlassy Carbon Modified Electrode with Gold NanoparticlesLaura Mora, Pedro Hernandez, Jose Vicente, Fernando Galan, Lucas Hernandez*

Analytical Chemistry Department, Autonoma University of Madrid, Spain*e-mail: [email protected]

Received: September 11, 2007Accepted: June 17, 2008

AbstractThe direct accumulation of 6-thioguanosine (6-TG) and its electrochemistry has been studied by cyclic voltammetry indifferent conditions physical and chemical. In a first moment the surface of electrode was modified with goldnanoparticles. This modification was realized by electrodeposition on the active surface of a glassy carbon electrodewith a HAuCl4 solution. The nucleotide 6-thioguanosine was deposited in this gold nanoparticles monolayer. Thestudy of accumulation of other nucleotide, adenosine 5’-monophosphate (AMP), was realized by the direct reactionwith 6-TG deposited. The conditions of the reaction and its electrochemical response were tested to fix the idealconditions of its determination. The ideal conditions of formation of the monolayer and its electrochemical responsewere studied; the possibility of preconcentration of 6-TG nucleotide in gold nanoparticles, the possibility of catalysisand limits of identification and quantification were also determined. The method proposed can be applied in directdetermination of oligonucleotides. In this respect we applied it in the determination of AMP in a commercial productof infantile nutrition.

Keywords: Adenosine-5’-monophosphate, Modified electrode, Gold nanoparticles, Direct determination,Oligonucleotides

DOI: 10.1002/elan.200704289

1. Introduction

Themodification of the surface of electrodes is an importantfield in Electroanalytical Chemistry since this modificationhas provided an improvement in electrocatalytic propertiesof electrodes; it has avoided the passivation of the surfaceand undesirable reactions that could compete with thekinetic process of electrode. This modification has alsoprovided to electrodes with a selectivity and sometimesspecificity for a certain analyte. The modification with self-autoassembledmonolayers of sulfur organic compounds hasbeen extensively studied in the last years [1] because hasbeen used as a new method to provide organized interfacesand structurally well defined. The design of an electro-chemical sensor is based in the utilization of modifiedelectrodes with an autoassembled monolayer that is in-creased the selectivity, sensibility and timeof response of thecompound in study. Taniguchi and co-authors were the firstones in use the method of chemical modification of surfacesof gold electrodes with organosulfur compounds in the fieldof the electrochemistry of proteins [2].In the last years biological molecules and its derivatives

have been used for the surface modification of electrodes.Diverse analytical technologies have been applied in studiesof interaction between DNA and some small molecules.These interactions play an important role in the replication

and transcription of DNA [3], biosensor of hybridization ofDNA [4], mutation of genes and related variations [5],mechanisms of action and determination of some drugs [6],origins of some diseases, and mechanisms of action of somechemical synthetic nucleases [7], etc.The electrochemistry isoffered great advantages compared with spectroscopicmethods such as rapidity, simplicity, low cost and availabil-ity. The interactions between complexes, antitumoral drugsand some smallmoleculeswithDNAhavebeen investigatedby electrochemical methods [8]. Since 1990 electrochemicalbiosensors have been developed for the detection of specificsequences of targets of nucleic acids. A great variety ofsystems of electrochemical biosensors of nucleic acids havebeen proposed such as the based ones on gold nanoparticles[9, 10], catalytic oxidation of guanine [11] and interperfo-rators of DNA [12].The sensitive determination and total characterization of

mechanisms involved in oxidative damages of all DNAbases is have a great interested because the products ofoxidation of DNAbases have an important role in mutationof genes, carcinogenesis, aging and diseases related to theage. The electrochemical biosensor of DNA has demon-strated be excellent tools to investigate the effects ofpossible sources of danger for the genetic material. Theelectrochemical methods are been very promising for thesestudies and investigation of mechanisms of interactions

2084

Electroanalysis 20, 2008, No. 19, 2084 – 2089 A 2008 WILEY-VCH Verlag GmbH&Co. KGaA, Weinheim

between medicines and DNA [13]. The materials with ananometer size have been applied in technological applica-tions in diverse areas such as microelectronic designs [14],photocatalysis [15], biomedical applications [16] and chem-ical processes [17]. Nanoparticles have received a specialattention in last decades due to possess unique physical andchemical properties [18]. The nanoparticles have providedcatalytic properties to glassy carbon and gold electrodesfacilitating the electrochemical processes of electronictransference.The fundamental aim of this work has been the develop-

ment and optimization of an analytical method for deter-mination and quantification of nucleotides. A glassy carbonelectrode modified with gold nanoparticles was used. Thepossibility of accumulation of a small molecule withreagents terminal groups on the gold nanoparticles-modi-fied glassy carbon electrode was studied. Themolecule usedwas 2-amine-6-mercaptopurine riboside (6-thioguanosine,6-TG). The thiol group of 6-TG served as binding site for thecovalent attachment to self-assembled gold nanoparticles ofglassy carbon electrode (6-TG-nAu-GCE). On the otherhand we have applied the 6-TG-nAu-GCE to quantitativedetermination of in infantile milk

2. Experimental

2.1. Apparatus and Electrodes

Voltammetric measurements were carried out with anAutolab PGSTAT12 potentiostat/galvanostat with a threeelectrode cell.AMetrohm6.0805.10 glassy carbonelectrode(GCE)was used asworking electrode.ABASAg/AgCl 3 Mreference electrode and a Pt wire counter electrode werealso employed. The electrochemical software was thegeneral purpose electrochemical system (GPES) (EcoChe-mie B. V.). A Metrohm consort C381 pHmeter was alsoemployed.

2.2. Reagents and Solutions

Adenosine 5’-monophosphate (AMP) (Acros Organics,99%), 2-amine-6-mercaptopurine-9-d-riboside (6-TG) (Ac-rosOrganics, 99%).Hydrochloric acid and nitric acid (CarloErba and Panreac) were used for preparation HAuCl4solution. All other solvents and chemical reagents used forpreparation a 0.1 mol L�1 phosphate buffer solution were ofanalytical reagent grade. The water used was obtained froma Millipore Milli-Q system.

2.3. Samples

The samples analyzed were commercial infantile milk inpowder purchased in a local pharmacy.

2.4. Procedures

2.4.1. Preparation of the Working Electrode

The glassy carbon electrode was polished to a mirror-likesurface with 0.3, 0.1-mm alumina from 1 min. Then it wasrinsed with ethanol and water, alternatively, three timeseach, and was dried using a nitrogen stream. After electro-chemical cleaning by several potential cycling between 0and 1.5 V versus Ag/AgCl 3.0 mol L�1 electrode in 0.1 molL�1 phosphate buffer solution of pH 5.0, the glassy carbonelectrodewas immersed in a 100 mgmL�1 stirred solution ofHAuCl4 (previously prepared dissolving metallic gold in“aqua regia” and the convenient dilution with water) and a�0.2 V potential value was applied during 1 min. [19, 20](for realize the electrodeposition of the monolayers of goldnanoparticles).The prepared electrode (nAu-GCE) was submerged in a

0.5 mol L�1 H2SO4 solution. The intensity of reduction waveof gold oxides was increased when the time of electro-deposition increased. Figure 1 is represent the logarithmicrelation between the intensity value of reduction peak ofgold oxides and the time of electrodeposition for theformation of the gold monolayer in glassy carbon electrode:ip(A)¼�0.0048 · ln t (min) – 0.0041; R2¼ 0.9897.When we increased the time of electrodeposition the

intensity of reduction wave increased because the activesurface of electrode increased, but the nanoparticles formedwere of bigger size and the multiple layers formed wereweakly retained on the surface of electrode.In the Figure 2 are represented the cyclic voltammograms

of the glassy carbon electrode modified with gold nano-particles and a commercial gold electrode with the samephysical surface in 0.5 mol L�1 H2SO4 solution. We con-firmed that the active surface of gold nanoparticles-modi-fied glassy carbon electrodewas bigger than the commercialgold electrode because the intensities of reduction andoxidationwaves fromgold oxides in the first onewere biggerthan in the second one.The potential of reduction wave was not modified when

we were changed the time of electrodeposition. We chose atime of electrodeposition of 60 s for modifier the electrode.

Fig. 1. Intensity of the reduction wave of gold oxides withrespect to the electrodeposition time of the gold nanoparticles.

2085Determination of Adenosine-5’-Monophosphate

Electroanalysis 20, 2008, No. 19, 2084 – 2089 www.electroanalysis.wiley-vch.de A 2008 WILEY-VCH Verlag GmbH&Co. KGaA, Weinheim

www.electroanalysis.wiley-vch.de

The activation of electrode formed (nAu-GC) was realizedfor successive sweeps of potential in 0.5 mol L�1 H2SO4

solution until the image obtained was the typical residualcurrent of a gold electrode.

In Figure 3a – e we show the AFM images the surface ofthe bare glassy carbon electrode and once modified withnanoparticles of gold.

Fig. 2. Cyclic voltammogram in H2SO4 0.5 M of the glassy carbon modified electrode with gold nanocrystal with modification time of60 s to �0.2 V in HAuCl4 100 mg/mL, and of a commercial bare gold electrode.

Fig. 3. AFM images of a glassy carbon electrode, bare (a and c) and modified with gold nanoparticles (b, d, e). a) Bare glassy carbonelectrode; b) modified glassy carbon electrode with gold nanoparticles; c) bare glassy carbon electrode; d) modified glassy carbonelectrode with gold nanoparticles; e) Modified glassy carbon electrode with gold nanoparticles.

2086 L. Moraet al.



2.4.2. Modification with 6-Thioguanosine (6-TG)

The nAu-GCelectrodewasmodifiedwith 6-TGbecause thesulfur atom of this molecule reacted with the gold surface.This compound is adsorbed of spontaneous form. In theFigure 4 is represented the structure of 6-TG.Avolume of 5 mL of the organo-sulfur compound (6-TG)

0.1 mol L�1 was deposited on the active surface of nAu-GCelectrode. The electrode was dried with soft current ofnitrogen and one washed it with water, the electrode wasready for the corresponding measurement.

3. Results and Discussion

3.1. Study of the Influence of pH in the Electrochemistryof AMP

The influence of pH in the measurement was studied bysquare wave voltammetry. The concentration of phosphatebuffer solutionwas fixed in 0.1 mol L�1, the concentration ofAMP was 10�3 mol L�1 and the reaction time was of3 minutes.The intensity values of the oxidation wave were obtained

to different values of pH in AMP solution. Theses values ofintensity were increased and the potential values wereconstant in an interval of 1.44 – 1.51 V that indicated us nointerchange of protons in the electrochemistry reaction ofAMP.Avalue of pH 12 was chosen like optimal value of theelectrolyte for which the maximum intensity of peak wasobtained.

3.2. Study of Accumulation Time of AMP

The reaction time of modified electrode (TG-nAu-GCelectrode) in a AMP solution was studied. When theexhibition time was increased the intensity of the wavewas also increased of exponential form. We chose anexhibition time of 3 minutes for the successive measure-ments because of an exhibition time greater implied anincrease in time of analysis and did not improve muchintensity obtained. The electrode before eachmeasurementwas previously washed with water to remove AMP physi-cally coupled to the surface.

3.3. Study of the Instrumental Variables

3.3.1. Variation of the Amplitude

The frequency (15 Hz) and the Step (4 mV) were fixed inorder to the scan rate was kept in 60 mV/s. A lineardependence was observed between both variables: ip (A)¼2� 10�6E (mV)þ 3� 10�6,R2¼ 0.9994. We chose the valueof 25 mV for the amplitude like the optimal amplitude weobtained a good resolution of sign and the semi-wave widthwas optimal in order to a better selectivity obtain.

3.3.2. Variation of the Frequency

We were changed the frequency, and the amplitude and theStep were fixed in 25 mVand 4 mV, respectively.The relation obtained between the intensity and the

square root of the frequency was linear: ip (A)¼ 7 · 10�6 f 1/2(Hz1/2)þ 3 · 10�5, R2¼ 0.9935; this relation indicated us theprocess between the modified (6-TG) and the analyte(AMP) was controlled by diffusion. And the relationbetween potential obtained with the square root of thefrequency was also linear: Ep (V)¼ 0.020 f 1/2(Hz1/2)þ 1.302,R2¼ 0.9921, which indicated us the process was irreversibleelectrochemically. The value of 15 Hz was chosen likeoptimal value of frequency for posterior studies because thewaves obtained for this frequency had a good resolution.

3.3.3. Variation of the Step

In the voltammograms obtained for the study of Step (theamplitude and the frequencywere fixed in 25 mVand 15 Hz,respectively) we observed the peak intensities and the peakpotentials were increased when the scan increment (Step)were increased.The relations between the square root of the Step and the

peak intensity and peak potential were linear: ip (A)¼12.891 Step1/2 (V1/2)þ 27.05, R2¼ 0.9957 ; Ep (V)¼ 0.0385Step1/2 (V1/2)þ 12.963, R2¼ 0.9945. The information provid-ed to us in this study was identical to that obtained in thestudy of the frequency (process controlled for diffusion andirreversible electrochemically) because both studies reliedon the scan rate. We chose an optimal value of Step of 4 mVto maintain constant scan rate (60 mV/s) and because weobtained a good resolution in the wave with a low peakpotential value.

3.4. Study of the Scan Rate for Cyclic Voltammetry

The optimal chemical conditions previously studied werefixed (10�3 mol L�1 AMP in phosphate buffer solution0.1 mol L�1 pH 12). In the Figure 5 are represented thecyclic voltammograms of the TG-nAu-GC electrode inAMP solution obtained to different scan rates.The worst curves (oxidation wave of AMP) were defined

to higher speeds, which we chose an optimal scan rate of60 mV s�1.

Fig. 4. Structure of 2-amine-6-mercaptopurine-9-d-riboside (6-thioguanosine).




The results confirmed the mechanism of the reactionobtained in the instrumental variables. The peak potentialdepended linearly on the square root of the scan rate whichconfirmed that an irreversible reaction existed between 6-TG and AMP : Ep (V)¼ 0.3506 v1/2 (V/s)1/2þ 1.3316, R2¼0.9919. And the peak intensity depended also linearly onthe square root of the scan rate: ip (A)¼ 2� 10�5 v1/2 (V/s)1/2þ 3� 10�5, R2¼ 0.9998; this relation confirmed that anmechanism controlled by diffusion existed.

3.5. Influence of the Concentration of AMP

A study of the influence of the concentration of AMP wasperformed because the chemical and instrumental variableswere previously studied and optimized.In the Figure 6 are summarized the results in which can be

seen two zones of concentration.The first straight line was associated with low concen-

trations and its equation was: i (A)¼ 5� 10�8 CAMP (mg/mL)þ 5� 10�5 withR2¼ 0.9917; and the second straight linecorresponded to high concentrations of AMP and itsequations was: i (A)¼ 8� 10�8 CAMP (mg/mL)þ 7� 10�5with R2¼ 0.9937.The limit of detection and the limit of determination were

calculated with the information obtained: 2.98 mg L�1 and16.73 mg L�1 respectively. The standard deviation relative ofthe method obtained was less of 6.92% in all cases withvalues of relative and absolute errors of 0.39% and 0.56%,respectively.

3.6. Application in Infantile Milk in Powder

The utility of the developedmethodwas verified in infantilemilk in powder, concretely in milk in powder for sucklingbabies (babies of 0 to 6 month of age) which are enriched

with nucleotides because the milk of cow from which it areprepared does not possess it.The milk was prepared following the instructions indi-

cated in the package. The serum and the proteins wereseparated for precipitation with perchloric acid (HClO4)1 mol L�1 and centrifugation to 9000 rpm during 20 min.Later it was filtered and the pH was adjusted to 12 withNaOH.Thequantitative analysis ofAMPwas realized in theabove mentioned serum by square wave voltammetry.A standard addition of AMP was realized in the serum of

the milk. The conditions chosen for the voltammetricanalysis were the values previously studied (frequency:15 Hz; amplitude: 25 mV; Step: 4 mV).A saturation of the surface electrode took place to high

concentrations of AMP and the intensity of the peak wasdecreasing. We were able to observe a linear zone ofconcentration obtained from 0 to 25 mg mL�1 of AMPadded. The concentration of AMP that we hoped to find inthe serum of the milk it would be in this interval ofconcentration. We adjusted the data by minimums square(Fig. 7) and we obtained the following equation of straightline: i(mA)¼ 0.9236 CAMP (mg/mL)þ 184.66, R2¼ 0.9796.We obtained a concentration of AMP of 2.54 mg mL�1 in

different samples with 10 independent determinations. Theconcentration of AMP specified in the package by themanufacturer was 2.67 mg mL�1 and so we got a relativedifference of 4.86%.The officialmethod for thedeterminationofAMP implies

a determination by HPLC; the method proposed by squarewave voltammetry is a direct, rapid and specificmethod thatis valid for the determination of oligonucleotides.

4. Conclusions

The modification of glassy carbon electrode with goldnanoparticles and 6-thioguanosine for posterior accumula-tion of adenosine-5’ monophosphate and use square wavevoltammetry it allows to propose a direct, rapid and specificmethod valid for the determination of oligonucleotides.

Fig. 5. Cyclic voltammograms of the TG-nAu-GC electrodesubmerged in a AMP solution 10�3 M to different scan rates.

Fig. 6. Representation of intensities obtained with TG-nAu-GCelectrode for different concentrations of AMP.

2088 L. Moraet al.



5. Acknowledgements

The authors thank the Spanish Education Ministry forfinancial support (Project CTQ2004-04142/BQU) and agrant scholarship to Laura Mora (AP2005-0266).

6. References

[1] Ch. R. Martin, D. T. Michel, Anal. Chem. 1998, 70, 322A.[2] a) I. Taniguchi, K. Toyosawa, H. Yamaguchi, K. Yasukouchi,

J. Chem. Soc. Chem. Commun. 1982, 18, 1032; b) I. Tanigu-chi, K. Toyosawa, H. Yamaguchi, K. Yasukouchi, Electro-anal. Chem. 1982, 140, 187.

[3] D. E. Nivens, J. Q. Chambers, T. R. Anderson, D. C. White,Anal. Chem. 1993, 65, 65.

[4] J. J. Gooding, Electroanalysis 2002, 14, 1149.[5] T. G. Drummond, M. G. Hill, J. K. Barton, Nat. Biotechnol.

2003, 21, 192.[6] J. Wang, Anal. Chim. Acta 2002, 469, 63.[7] D.-W. Pang, H. D. Abruna. Anal. Chem. 1998, 70, 3162.[8] E. Palecek, M. Fojta, Bioelectronics 2005, 127.[9] T. A. Taton, C. A. Mirkin, R. L. Letsinger, Science 2000, 289,

1757.[10] R. Elghanian, J. J. Storhoff, R. C. Mucic, R. L. Letsinger,

C. A. Mirkin, Science 1997, 277, 1078.[11] E. M. Boon, D. M. Ceres, T. G. Drummond, M. G. Hill, J. K.

Barton, Nat. Biotechnol. 2000, 18, 1096.[12] M. R. Gore, V. A. Szalai, P. A. Ropp, I. V. Yang, J. S.

Silverman, H. H. Thorp. Anal. Chem. 2003, 75, 65862.[13] A. M. Oliveira-Brett, M. Vivan, I. R. Fernandes, J. A. P.

Piedade, Talanta 2002, 56, 959.[14] S. Ribrioux, G. Kleymann, W. Haase, K. Heitmann, C.

Ostermeier, H. Michel, J. Histochem. Cytochem. 1996, 44,207.

[15] J. F. Hainfeld, F. R. Furuya, J. Histochem. Cytochem. 1992,40, 177.

[16] K. C. Grabar, R. G. Freeman, M. B. Hommer, M. J. Natan,Anal. Chem. 1995, 67, 735.

[17] A. Doron, E. Katz, I. Willner, Langmuir 1995, 11, 1313.[18] T. Yoshida, H. Okabayashi, K. Takahashi, I. Ueda, Biochim.

Biophys. Acta 1984, 772, 102.[19] V. Carrarelo, M. L. Mena, A. Gonzalez-Cortes, P. Yanez-

Sedeno, J. M. Pingarron, Anal. Chim. Acta 2005, 528, 1.[20] V. Carrarelo, A. Gonzalez-Cortes, P. Yanez-Sedeno, J. M.

Pingarron, Electroanalysis 2005, 17, 289.

Fig. 7. Representation of intensities obtained with TG-nAu-GCelectrode for low concentrations of AMP added to the serum ofthe milk.




electrochemical study and direct determination of adenosine-5′-monophosphate coupled to...

Documents