Voltammetric determination of 5-methylcytosine at glassy carbon electrode

https://doi.org/10.1016/j.jelechem.2021.115437Get rights and content

Highlights

  • The electrochemical characterisation of 5-methylcytosine was performed for the first time on glassy carbon electrode.

  • An electrochemical oxidation mechanism for 5-methylcytosine is proposed.

  • Voltammetric determination of 5-methylcytosine using glassy carbon electrode was explored.

  • A new simple and sensitive electroanalytical method using a pre-treated glassy carbon electrode for the quantification of 5-methylcytosine is presented.

Abstract

The electrochemical oxidation of 5-methylcytosine (5-mCyt) was studied at glassy carbon electrode (GCE) in aqueous solution, over a wide pH range, using cyclic, square wave and differential pulse voltammetry, as well as compared with the anodic behaviour of other similar bases, Cyt, thymine (Thy) and uracil (Ura). The results revealed that the electrode reaction of 5-mCyt occurs in a single, one-electron one-proton, irreversible step controlled by diffusion, generating intermediate radicals that quickly react with water and/or dimerize. Moreover, in general, it was observed that the C5 methylated pyrimidines are more easily oxidized and thus are more reactive in relation to the unmethylated bases, Cyt and Ura. A mechanism for the electro-oxidation of 5-mCyt is proposed. Using differential pulse voltammetry (DPV) experimental conditions, such as the electrode size, supporting electrolyte composition, pH and influence of possible interferents (guanine, 7-methylguanine, adenine and Cyt), were also investigated for determination of 5-mCyt with low detection limit. Under the best conditions, the DPV method proposed, with a pre-treated 3.0 mm diameter GCE, provided a linear analytical curve to 5-mCyt in phosphate buffer solution (pH 7.0) in the concentration range from 3 up to 15 µmol L−1, with a detection limit of 0.11 µmol L−1 and a high correlation coefficient (r = 0.997). A new simple and sensitive electroanalytical method using an unmodified GCE for the quantification of DNA methylation is then presented.

Introduction

The methylation of DNA is an epigenetic modification, which plays crucial activities in numerous biological processes (including gene transcription, cell proliferation and embryonic development), that occurs almost exclusively by the addition of a methyl group of S-adenosyl-l-methionine (SAM) in cytosine at the C5 position, in cytosine-guanine (CpG) dinucleotides, catalyzed by enzymes called DNA methyltransferases (DNMTs), Scheme 1 [1], [2], [3], [4], [5], [6], [7], [8], [9], [10], [11]. In addition, the ten-eleven translocations (TETs) enzymes catalyse the oxidation of 5-methylcytosine (5-mCyt) for 5-hydroxymethylcytosine (5-hmCyt), 5-formylcytosine (5-fCyt) and 5-carboxylcytosine (5-caCyt), Scheme 1, since these products are important in demethylation processes, by thymine DNA glycosylase (TDG) implicated in base excision repair (BER), Scheme 1. [1], [2], [3], [4]. Thus, the methylation process of Cyt residues in DNA is complex and reversible.

On the other hand, it has been widely reported in the literature that variations in the normal levels of 5-mCpG dinucleotides may be involved in different pathological processes, such as some types of tumours [5], [6], Alzheimer's [7], multiple system atrophy [8], [9], Parkinson's [9], depression [10] and type 2 diabetes [11]. Consequently, several analytical methods have been proposed for the quantification of 5-mCyt in DNA samples [12], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25], [26], [27], [28], [29], [30], [31]. The list includes and is not limited to bisulfite conversion analysis with methylation-specific polymerase chain reaction (PCR) [13], chromatography [14], [15], [16], surface enhanced Raman spectrophotometry [17], fluorometry [18] and electrochemistry [1], [19], [20], [21], [22], [23], [24], [25], [26], [27], [28], [29], [30], [31].

The exceptional nature of electrochemical techniques, such as compounds are able to transfer electrons directly to an electrode surface, high sensitivity, speed of analysis, sample consumption and versatility, has made it one of the most important tools on studying redox chemistry of the biomolecules, such as DNA, amino acids and proteins [32], [33], [34], [35]. Moreover, the electrochemical properties of compounds have been profoundly investigated due to their relevance on developing electroanalytical methods and electrochemical biosensors for numerous applications [1], [19], [20], [21], [22], [23], [24], [25], [26], [27], [28], [29], [30], [31], [32], [33], [34], [35], [36], [37].

Carbon working electrodes, such as glassy carbon, boron doped diamond and graphite, are commonly used mainly due to their extensive potential window, low background current, chemical inertness and good reproducibility [32], [38], [39]. The glassy carbon electrode (GCE) has been widely used for investigating the oxidation mechanism of DNA and its components over several decades [32], [38], [40]. The electroanalysis of all DNA bases, guanine (Gua), adenine (Ade), thymine (Thy) and cytosine (Cyt), Scheme 2, in phosphate buffer (pH = 7.0) on GCE was investigated for the first time by Oliveira-Brett and co-workers [38], [40]. Recently, the oxidation mechanism of 7-methylguanine (7-mGua) in aqueous medium on GCE was studied by our group and an electroanalytical method for its quantification was proposed [41].

The electroanalysis of 5-mCyt has been investigated at carbon electrodes [22], [28] and mainly at modified carbon electrodes [23], [24], [25], [26], [27]. The electrochemical response of 5-mCyt in aqueous medium on screen printed graphite electrode (SPGE) was investigated in the absence and presence of free DNA bases and applied on developing an electroanalytical method for its detection [22]. High-performance liquid chromatography (HPLC) analysis with boron doped diamond electrode (BDDE) was applied for quantification of 5-mCyt in hydrolysed DNA samples [28]. At GCE, the electrochemical response of 5-mCyt was investigated, only under specific conditions, only to establish the electrocatalytic effects when its surface was modified with graphene or carbon nanotubes materials [23], [24], [25], [26], [27]. To our knowledge, however, there is no analytical data available for 5-mCyt oxidation at GCE, such as adsorption of products, limit of detection, sensitivity and the range of linear response, up to the date of our study, consequently additional work should be done to fully characterize the 5-mCyt electrochemical properties.

The main aim of this work was to develop a new simple and sensitive electroanalytical method, using an electrochemically pre-treated GCE, for the determination of 5-mCyt in hydrolysed DNA samples. Consequently, first, an electrochemical study of the oxidation of 5-mCyt on the GCE, using cyclic, square wave (SW) and differential pulse voltammetry (DPV) over a wide pH range, was performed and a mechanism for the reaction of the electrode was proposed. Second, using DPV, experimental conditions, such as electrode size, support electrolyte composition and pH, were investigated for quantification of 5-mCyt with low detection limit. Finally, to investigate the selectivity of the proposed method, the determination of 5-mCyt in the presence of possible interferences (Gua, 7-mGua, Ade and Cyt) was studied under the optimal conditions.

Section snippets

Materials and reagents

Guanine (Gua), adenine (Ade), thymine (Thy), cytosine (Cyt), uracil (Ura), 5-methylcytosine (5-mCyt) and 7-methylguanine (7-mGua) were obtained from Sigma and used without any additional purification. Stock solutions (500 µmol L−1) of Gua, Ade, Thy, Cyt, Ura, 5-mCyt and 7-mGua were prepared in supporting electrolyte (phosphate or acetate buffer) and a few drops of 0.1 mol L−1 NaOH were added.

Supporting electrolyte solutions of different pH / composition (3.5–5.5 / HAcO + NaAcO and 6.0–8.0 / NaH2

Voltammetric studies of the electro-oxidation of 5-methylcytosine

In order, to establish the electro-oxidation mechanism of 5-mCyt, cyclic, DP and SW voltammograms were recorded, under different experimental conditions, in electrolytes with different pH values, all containing 500 µmol L−1 5-mCyt, using a 1.6 mm diameter GCE, Fig. 1, Fig. 2, Fig. 3, Fig. 4.

Firstly, successive cyclic voltammograms at a scan rate of 100 mV s−1 were performed for 5-mCyt and only one single irreversible anodic peak appeared, at Epa = 1.35 V in acetate buffer, pH = 4.5, Fig. 1A,

Conclusion

The results established that the oxidation of 5-mCyt is an irreversible process that occurs in a single step, pH-dependent, with mass transport to the GCE surface controlled predominantly by diffusion. The anodic peak of 5-mCyt corresponds to the oxidation of the methyl group, with the withdrawal of one electron and one proton, generating intermediate radicals that react by numerous pathways, including dimerization and with water to form 5-hmCyt.

Voltammetric data also detected that the

CRediT authorship contribution statement

Carlos H.S. Mendes: Investigation, Methodology, Visualization, Writing - original draft, Writing - review & editing. Maycom W.F. Silva: Investigation, Visualization. Severino Carlos B. Oliveira: Conceptualization, Funding acquisition, Investigation, Methodology, Supervision, Writing - review & editing.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

Financial support from the Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco (FACEPE), PPP/FACEPE/CNPq/APQ-0535-1.06/14, Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), MCTI/CNPQ/Universal/APQ-422757/2018-7, and PIBIC/CNPq/UFRPE Grant (Maycom W.F. Silva), are gratefully acknowledged.

Data availability statement

All relevant data are within the manuscript.

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      As expected, all voltammograms detected the anodic peak 1a and showed that its peak current increased linearly with square root of scan rate, Fig. 4D, indicating that the oxidation of 3-Cl-Tyr at GCE consistent at a diffusion-controlled process [27–29]. The peak current, in amperes, for a diffusion-controlled irreversible system, is given by the Randles-Sevcik equation [27–30], Ipf = 2.99 × 105 n (αc + n’)1/2 A C0 DO1/2 ν1/2 [16,29,30], where Ipf is the peak current associated with the forward scan (A), n is the number of electrons in an overall electrochemical process, n’ is the number of electrons in the rate-determining step, αc is the transfer coefficient, A is the GCE electroactive area (cm2), DO is the diffusion coefficient (cm2 s−1), C0 is the concentration of the species in solution (mol cm−3) and ν is the scan rate (V s−1). So, by Randles-Sevcik equation by plotting Ip1a vs. ν1/2, Fig 4D, the value of D3-Cl-Tyr = 6.20 × 10−6 cm2 s−1 of 3-Cl-Tyr was calculated in phosphate buffer, pH = 7.0.

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