A novel electrochemiluminescence biosensor for the detection of microRNAs based on a DNA functionalized nitrogen doped carbon quantum dots as signal enhancers
Introduction
Carbon quantum dots (CQDs) have attracted attention because of their particular properties and functions regarding low toxicity (Mueller et al., 2010), good biocompatibility (Pan et al., 2010), excellent light stability (Yan et al., 2010), and sturdy chemical inertness (Han et al., 2015). However, insufficient optical characteristics and the surface chemical structure of CQDs limit the range of their practical applications. Therefore, to expand the range of applications, research has focused on CQDs surface modifications with different functionalities (Lim et al., 2015, Tetsuka et al., 2012). Doping is an excellent method to modify the properties of CQDs (Wu et al., 2013). Nitrogen-doped CQDs (N-CQDs) usually have a positive surface potential stemming from nitrogenous organic precursors. The amino groups on the surface can not only improve their fluorescence, but also facilitate subsequent modification and application (Yu et al., 2013). Because of their excellent luminous properties, light stability, and photoelectric physical properties, they have been widely used in the fields of biomedical fluorescent tags (Wang et al., 2016, Jiang et al., 2015), ion detection (Zhang et al., 2014, Li et al., 2013), and photocatalysts (Martinsa et al., 2016). The inherent virtues of N-CQDs stimulate exploration into their possible applications in electrochemiluminescence (ECL), since they are superior to the traditional inorganic nanomaterials because of their low cost, easy operation, nontoxic nature, and high detection sensitivity. Moreover, although some N-CQDs have been shown to possess ECL emission properties, there are several challenges related to their low water solubility, defects of complex operating processes, and low or unstable ECL signal for N-CQDs (Kumar et al., 2016, Xiong et al., 2016).
MicroRNAs (miRNAs) are a class of small (19–25 nucleotides) noncoding RNA molecules found in eukaryotic cells that play a critical role in gene expression in most eukaryotic organisms from plants to animals (Bernstein et al., 2001, Wienholds et al., 2005). They are post-transcriptional regulators that cause translational repression or target degradation and gene silencing via binding to complementary sequences on target mRNAs (Bartel, 2009). Increasing evidence suggests that aberrant repression of miRNAs is associated with many diseases (Huang et al., 2014), including cancer (Kosaka et al., 2010, Volinia et al., 2006) and neurodegeneration (Hébert and Strooper, 2007). The expression profile of miRNAs can thus serve as a biomarker for the diagnosis and treatment of cancer and other diseases, underscoring the need to develop highly sensitive, selective, and simple methods to detect miRNAs. ECL has attracted great interest in analytical methodology because of its unique advantages of low background signal, simplified optical setup, and high sensitivity (Hu et al., 2015; Miao, 2008). The ECL properties of quantum dots have attracted attention because of their excellent optical and electrochemical properties (Hesari et al., 2015). However, most quantum dots used in ECL studies are characterized by inherent toxicity due to the existence of toxic metal ions (e.g., cadmium), which limits their applications in bioassays (Hu et al., 2013). To improve the ECL intensity and biocompatibility, graphene oxide (GO) have been use as efficient carriers due to their lager specific surface area and excellent mechanical strength. Moreover, gold nanoparticles (AuNPs) is an excellent sensing material due to its high sensitivities and excellent reliability. GO/Au nanocomposite can greatly improve the sensitivity and selectivity of the sensor.
In the present work, a highly sensitive and selective ECL biosensor for miRNA-21 was proposed with a newly designed DNA functionalized N-CQDs. Here, graphene oxide (GO)/Au nanocomposites were used for the immobilization of hairpin probe2 (HP2) and to promote electric transmission. The stable N-CQDs could produce strong ECL signals, while the hairpin probe1 (HP1), which was linked to N-CQDs via the amide bond, was responsible for the high selectivity of this method. The HP1-N-CQDs were incubated with the assistant probe and the target miRNA to form the Y junction structure. HP1 contains three functional regions: a miRNA-binding domain that is complementary to the sequence of the target miRNA-21 (the yellow region), a complementary domain of assistant probes that acts as the recognition sequence for the nicking enzyme Nb.BbvCI (the green region), and a complementary domain of HP2 (the red region). The nicking enzyme Nb.BbvCI cleaved the Y junction on the cleavage site (5′-GC↓TGAGG-3′), bringing about the release of the target miRNA, assistant DNA, and the generation of intermediate N-CQDs-DNA (S1). Meanwhile, the released target miRNA and assistant DNA hybridized with other HP1-N-CQDs to initiate another recycling process to generate abundant S1, which was promising for signal amplification. After the completion of the nicking enzyme-assisted signal amplification (NESA), these intermediate sequences were further hybridized with HP2. The stem-loop structure was opened to form dsDNA immobilized on the GO/Au composite modified GCE surface through an Au-S bond. After construction of the dsDNA junction structure, the initial ECL intensity was enhanced by the N-CQDs. Thus, the quantitative detection of target miRNAs can be achieved by adjusting the ECL signal to the concentration of target miRNA. The nicking enzyme Nb.BbvCI-induced target RNA cycling and the N-CQDs-based enhanced ECL with the co-reactant S2O82- resulted in an ECL biosensor with an ultrasensitive response to the target miRNA.
Section snippets
Materials and reagents
6-mercapto-1-hexanol (MCH), N-hydroxy succinimide (NHS), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) and tri-(2-carboxyethyl) phosphine hydrochloride (TCEP) were purchased from Sigma-Aldrich Chemical Co. (U.S.A.). Diethy pyrocarbonate (DEPC) was provided by Sangon Biotech (Shanghai, China). Nb.BbvCI nicking enzyme and 10×NE buffer (50 mM KAc, 20 mM Tris acetate buffer, 10 mM magnesium acetate, 100 μg/μL BSA, pH 7.9 at 25 °C) were obtained from New England Biolabs (USA) and
Characterizations of GO, GO/AuNP and N-CQDs
The synthesized GO and GO/AuNP hybrids were characterized using TEM. As shown in Fig. 1A, the restacked parts and wrinkles can also be seen. AuNPs were uniformly distributed on the surface of the GO (Fig. 1B), which was beneficial for HP2 attachment and improved the electronic transfer rate. The morphology and structure of the as-prepared N-CQDs are shown in Fig. 1C. The N-CQDs show uniform spherical shapes with an average size of 4.5 nm, as estimated from the TEM image (Fig. 1C), indicating
Conclusions
The proposed ECL biosensor was constructed for the detection of miRNAs based on target cycling amplification with a novel nicking enzyme, Nb.BbvCI, and DNA functionalized N-CQDs enhancement. The newly designed nicking enzyme-triggered cycling reaction achieved promising amplification intermediate sequences and good efficiency under optimal conditions. In addition, with the help of the high ECL intensity provided by the N-CQDs, the present assay can provide a wide linear range and satisfactory
Acknowledgements
This work was supported by the National Natural Science Foundation of China (Grant Nos. 21427807, 21575001), Natural Science Foundation of Anhui Province (1508085MB37).
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