Novel electrochemical sensor based on functionalized graphene for simultaneous determination of adenine and guanine in DNA
Introduction
Deoxyribonucleic acid plays an important role in the storage of genetic information and protein biosynthesis. Guanine and adenine are important components found in deoxyribonucleic acid and play fundamental roles in life process [1]. They have widespread effects on coronary and cerebral circulation, control of blood flow, prevention of cardiac arrhythmias, inhibition of neurotransmitter release and modulation of adenylate cyclase activity [2]. The abnormal changes of the bases in organism suggest the deficiency and mutation of the immunity system and may indicate the presence of various diseases. Their concentration levels are considered as important parameter for diagnosis of cancers, AIDS, myocardial cellular energy status, disease progress and therapy responses [3]. Therefore, the determination of these bases has great significance to the bioscience and clinical diagnosis [4].
Many methods have been developed for the detection and quantification of purine bases in nucleic acids, such as high-performance liquid chromatography (HPLC) [5], capillary electrophoresis (CE) [6], spectroscopic methods [7], [8], chemiluminescence (CL) [9], and mass spectrometry (MS) [10]. Although these methods are sensitive, complicated instruments and time-consuming sample pretreatment are required. Compared to these methods, the electrochemical technique is attractive owing to its high sensitivity, inherent simplicity, miniaturization and low cost, and some electrochemical methods have been developed for the determination of the guanine and adenine.
Graphene, a single layer of carbon atoms in a closely packed honeycomb two-dimensional lattice, is a novel and fascinating carbon material. Recently, it has attracted considerable attention from both the experimental and theoretical scientific communities due to its many unique and excellent properties [11], [12], [13]. It exhibits extremely high thermal conductivity, good mechanical strength, high mobility of charge carriers, high specific surface area, quantum hall effect and upstanding electric conductivity [14], [15], [16]. Much research effort has been made to explore its fascinating applications in fabricating various electrical devices, such as battery [17], field-effect transistors [18], ultrasensitive sensors [19], electromechanical resonators [20] and electrochemical biosensors [21]. Some works have demonstrated that graphene possesses excellent electrochemical catalytic activity, and should be a novel electrode modified material with excellent performance. However, many of the interesting and unique properties of graphene can only be realized after it is integrated into more complex assemblies [21], [22], [23], [24], [25]. A useful technique to incorporate graphene into such assemblies is through chemical functionalization of the graphene, which enables chemical covalent bonding between the graphene and the material of interest. Functionalized graphene are also typically easier to disperse in organic solvents and water, which can improve the dispersion and homogeneity of the graphene within the polymer and yield novel types of electrically conductive nanocomposites [26], [27].
In this work, functionalized graphene was used to develop a simple and sensitive method for the simultaneous determination of guanine and adenine. Graphene was firstly functionalized via chemical modification of carboxyl groups on its surface. The functionalized graphene nanosheets were easily dispersed in water and then used to modify the glassy carbon electrode by simple drop moulding procedure. The negatively charged graphen-COOH nano-film could adsorb the positive charged guanine and adenine, and this led to effectively improving the sensitivity of proposed method. Based on the unique properties of graphene-COOH, the fabricated modified electrode facilitated the electron transfer of guanine and adenine, resulting in the increase of oxidation signals. The method proved to be simple, reliable, and inexpensive for the individual or simultaneous determination of guanine and adenine in DNA at low levels.
Section snippets
Chemicals and materials
Graphite powder (99.95%, 325 mesh), hydrazine solution (50 wt%) and ammonia solution (28 wt%) were purchased from Shanghai Chemical Reagent Co., Ltd. (Shanghai, China). Guanine, adenine, herring sperm, SOCl2 and NH2(CH2)2NH2 were purchased from Sigma (Saint Louis, MO, USA). All other chemicals were of analytical grade and used without further purification. Acetate buffer solutions (ABS) were prepared by mixing of 0.1 M CH3COOH and CH3COONa and adjusting the pH with NaOH. Ultrapure water (18.2 MΩ)
FTIR characterization of functionalized graphene
The IR spectrum of the graphene oxide samples (Fig. 1a) shows the presence of –OH (3421 cm−1), CO (1722, 1626 cm−1) and phenol or alcohol or ether (1384, 1063 cm−1). Fig. 1b shows the reduction of the carboxyl groups (graphene-COOH) to hydroxymethyl (graphene-CH2OH) as indicated by the disappearance of the CO bands (at 1720 cm−1) and the appearance of bands at 2924 and 2854 cm−1 corresponding to the C–H stretch vibrations of the methylene group. Fig. 1c shows FTIR results for the graphene-COOH. The
Conclusions
In this work, a novel electrochemical sensor was fabricated based on graphene-COOH modified GCE for the sensitive determination of guanine and adenine. After optimizing the experimental parameters, guanine and adenine exhibited well separated and well-defined oxidation peaks. Remarkable enhancement effects on the oxidation peak currents were observed with the negative shift of the oxidation peak potentials. The results were attributed to the specific characteristics of graphene-COOH. The
Acknowledgments
This work was supported by the National Natural Science Foundation of China (20805040), Program for Science & Technology Innovation Talents in Universities of Henan Province (2010HASTIT025), Excellent Youth Foundation of He’nan Scientific Committee (104100510020), and Foundation of He’nan Educational Committee (2009A150023).
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