Elsevier

Analytica Chimica Acta

Volume 731, 20 June 2012, Pages 68-74
Analytica Chimica Acta

Highly sensitive and selective detection of biothiols using graphene oxide-based “molecular beacon”-like fluorescent probe

https://doi.org/10.1016/j.aca.2012.04.020Get rights and content

Abstract

A fluorometric method for quantity analysis of biothiols was developed using a graphene oxide (GO)-based “molecular beacon”-like probe, which consisted of FITC labeled thymine (T)-rich single-stranded DNA (ssDNA), GO and Hg2+ ions. The labeled ssDNA containing T–T mismatches would self-hybridize to duplex in the presence of Hg2+, which can avoid its adsorption on GO and the fluorescence of this GO-based probe was recovered. The fluorescence of the probe quenched after the addition of biothiols such as glutathione (GSH) and cysteine (Cys) owing to thiol groups can selectively competitive ligation of Hg2+ ions with T–T mismatches. In the present work, the GO-based probe was used for the determination of GSH and Cys. Under the optimal conditions, a linear correlation was established between fluorescence intensity ratio I0/I and the concentration of GSH in the range of 2.0 × 10−9–5.0 × 10−7 mol L−1 with a detection limit of 1.0 × 10−9 mol L−1. The linear range for Cys is from 5.0 × 10−9 to 4.5 × 10−7 mol L−1 with a detection limit of 2.0 × 10−9 mol L−1. The proposed method was applied to the determination of GSH in human serum and cell extract samples with satisfactory results.

Highlights

► The GO-based probe consisted of FITC labeled T-rich ssDNA, GO and Hg2+ ions. ► The method was based on a competitive ligation of Hg2+ by GSH/Cys and T–T mismatches. ► Good sensitivity and selectivity were obtained for the determination of GSH/Cys. ► Biothiols in serum and cell extract samples were detected with satisfactory results.

Introduction

In recent years, great attention has been paid to biothiols sensors due to their crucial role in the physiological systems. Biothiols, the thiols-containing amino acid and peptides, participate in the process of reversible biological redox homeostasis and important cellular functions including detoxification and metabolism [1], [2], [3]. The studies on biothiols have shown that various clinical disorders relate to the level of biothiols [4], [5], so the analysis of their level in physiological fluids is very important for the early diagnosis of many diseases [6]. Homocysteine (Hcy) is a risk factor for cardiovascular and Alzheimer's disease. Cysteine (Cys) deficiency is involved in hematopoiesis decrease, leukocyte loss, skin lesions, and weakness [7], [8], [9]. Glutathione (GSH) level is biomarker of oxidative stress and the key factor for preventing serious damage to DNA, proteins, and lipid membranes. And there is an evidence that low GSH level contributes to the development of autism in children [10]. Various detection technologies were used for the determination of biothiols, including high-performance liquid chromatography (HPLC) [11], mass spectrometry [12], chemiluminescence [13], electrochemical method [14], optical spectroscopy [15] and colorimetric assays [16]. The concerns have been raised on fluorometric methods for biothiols determination owing to its simple, sensitive and efficient performance.

Graphene oxide (GO) has recently emerged as a fascinating material with one-atom-thick two-dimensional graphitic carbon system [17] and rich of oxygen-containing groups on its surface, including carboxyl, hydroxyl and epoxy groups. GO is attractive for its good water-solubility, unique DNA adsorbing ability and wide-range energy transfer properties [18], which make it a wide prospect for biological application [19], [20], [21]. GO has been applied in DNA analysis [22], protein assay [23], ion detection [24], drug delivery [25], live cell imaging [18], etc. After Yang reported a graphene platform for biomolecules sensing, which was based on fluorescence resonance energy transfer (FRET) between fluorescent dye and graphene oxide via strong adsorption of labeled DNA on graphene oxide [26], various GO-based fluorescent sensors have been developed [27].

Thymine (T) has been demonstrated to be one of the most specific ligands for Hg2+ that can form a T–Hg2+–T complex with strong affinity and high selectivity [28], [29], [30]. Scientists have developed many methods based on the T–Hg2+–T duplex for Hg2+ sensor or other detections [10], [31]. Zhang's group detected Hg2+ in aqueous solution based on mismatched DNA and a molecular light switch complex, [Ru(phen)2(dppz)]2+. Liu reported a visual method for detection of Hg2+ by using gold nanoparticles and T-rich hairpin DNA probes. Hepel developed a molecular beacon probe for the detection of glutathione and cysteine based on a competitive ligation of Hg2+ by GSH/Cys and T–T mismatches in a DNA beacon strand.

Herein, we proposed a fluorometric method for quantity analysis of biothiols via biothiols competitive binding of Hg2+ with T–T mismatches in the GO-based “molecular beacon”-like probe. We fabricated the GO-based “molecular beacon”-like probe using FITC labeled T-rich single-stranded DNA (ssDNA) as energy donor and GO as acceptor. In the presence of Hg2+, the T–T mismatches in DNA strand will self-hybridize to duplex avoiding the adsorption on GO, which can recover the fluorescence of the GO-based probe. It is well known that biothiols have detoxifying functions with high affinity to heavy metal ions, especially for Hg2+. GSH and Cys can complex with Hg2+ to form Hg2+–GSH and Hg2+–Cys adduct with high stability constants. Thus, GSH and Cys can competitively bind Hg2+ with T–T mismatches to make DNA return to single strand and the fluorescence of the GO-based probe quenched consequently.

Section snippets

Reagents and chemicals

All chemicals used were of analytical reagent grade and used without further purification. The synthetic oligonucleotide was purchased from TaKaRa Biotechnology (Dalian) Co., Ltd. Its base sequence is 5′FITC-(CH2)6-TTCTTTCTTGGGTTGTTTGTT. Glutathione reduced was purchased from Genview Chemical Co. l-Cysteine HCl, mercury(II) nitrate and graphite powders were obtained from Sinopharm Chemical Reagent Co. Ltd. (China). GO was synthesized from graphite powder according to modified Hummer's method

The GO-based “molecular beacon”-like probe

As usual, a molecular beacon forms a stable stem-loop structure with self-complementary 5′ and 3′ ends, which are functionalized by fluorophore and quencher respectively [10]. In the present work, our designed GO-based “molecular beacon”-like probe consisted of GO, FITC-labeled T-rich ssDNA and Hg2+ ions. In the GO-based “molecular beacon”-like probe, GO acts as quencher instead of normal quencher fixed at the end of DNA sequence. The principle of the GO-based sensor was depicted in Scheme 1.

Conclusions

In the present work, we developed a fluorometric method for quantity analysis of biothiols by using GO-based “molecular beacon”-like probe, which is composed of FITC labeled T-rich ssDNA, GO and Hg2+ ions. The fluorescence of FITC labeled ssDNA was quenched by GO via FRET in the absence of Hg2+, and was recovered by forming T–Hg2+–T duplex in the presence of Hg2+. The thiol groups in biothiols can competitively capture Hg2+ with T–T mismatches causing the fluorescence intensity of the GO-based

Acknowledgements

This work was financially supported by the National Natural Science Foundation of China (Nos. 20075009, 20875036 and 21075050) and the Science and Technology Development Project of Jilin Province, China (No. 20110334).

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