Elsevier

Biosensors and Bioelectronics

Volume 118, 30 October 2018, Pages 1-8
Biosensors and Bioelectronics

Label-free fluorescent and electrochemical biosensors based on defective G-quadruplexes

https://doi.org/10.1016/j.bios.2018.07.033Get rights and content

Highlights

  • A fluorescent and an electrochemical biosensor were developed based on defective G-quadruplex and ThT/hemin.

  • The unique recognition of the defective G-quadruplex for guanine confers excellent properties to the proposed biosensors.

  • The perfect discriminative ability of ThT and hemin against canonical G-quadruplex further increase the sensor's performance.

Abstract

Abnormal levels of guanine closely associated with plenty of diseases are usually used as a biomarker for clinical diagnosis. In order to detect guanine and its derivatives accurately, in this paper, a defective G-quadruplex (DGQ) containing a G-vacancy at one of its G-quartet layers, and two kinds of G-quadruplex specific indicators including thioflavine T (ThT) and hemin were used for constructing a fluorescent and an electrochemical biosensor, respectively. In brief, a G-rich DNA probe is designed to form either hairpin or DGQ structure. In the absence of guanine, G-rich probes prefer to maintain hairpin structure and nearly have no interaction with ThT or hemin, leading to almost negligible signals. Upon addition of guanine, the G-rich probe fold into DGQ structure and then the G-vacancy in it is filled up immediately by guanine via Hoogsteen hydrogen bonds, resulting canonical G-quadruplex formation. Accordingly, ThT or hemin can selectively combine with G-quadruplex, giving rise to distinct fluorescent or current signal changes for label-free detection of guanine. Benefiting from the perfect discriminative ability of guanine towards DGQ and ThT/hemin against standard G-quadruplex, the fluorescent and electrochemical biosensors present better sensitivity and selectivity for guanine detection with the limit of detection (LOD) as low as 18.26 and 0.36 nM, respectively. Successful attempts were also made in applying the proposed electrochemical biosensor to detect guanine in drugs and urine, obtaining satisfactory recovery rates of 99~104% and 96~106%, respectively.

Introduction

As building blocks of nucleotides, guanine is a vital element in the coding of genetic information and plays a crucial role in numerous biological processes (Neves et al., 2002, Niu et al., 2012). Recently, it is reported the abnormal concentrations of guanine and its derivatives were capable of manifesting metabolic disorders and a variety of diseases in human body, such as cancer (Chabosseau et al., 2011), aging (Burhans and Weinberger, 2007), kidney diseases (Kimura et al., 2003), gout (Boss and Seegmiller, 1982), and a few of mitochondrial pathologies (Pitceathly et al., 2012). Therefore, the detection of guanine and its derivatives is important and meaningful (Jordheim et al., 2013).

So far, different methods for the detection of guanine and its derivatives have been widely reported, including capillary electrophoresis (CE) (Chen et al., 2002), liquid chromatography-mass spectrometry (LC-MS) (Fan et al., 2006), capillary electrophoresis-mass spectrometry (CE-MS) (Cahours et al., 2000) and so on. However, these methods either possess relatively low sensitivity or require complicated apparatus and high cost. Notably, due to known merits and considerable prospect (Shen and Xia, 2014, Wu et al., 2014), some fluorescent sensing strategies were applied to detect guanine and its derivatives (Cui et al., 2014, Li et al., 2012, Lu et al., 2016, Pang et al., 2016, Yoshimoto et al., 2003). Tan's group proposed a special defective G-quadruplex (DGQ) with ultrahigh discriminative ability against guanine (Li et al., 2015). In general, the standard canonical G-quadruplex is composed of three consensus intact G-quartet layers. In each layer, four guanine bases associate with each other through eight Hoogsteen hydrogen bonds (Biffi et al., 2013, Burge et al., 2006, Zhu et al., 2015). Differently, DGQ consists of three G3 and one G2 tracts, that is, there is a G-vacancy at one corner of its G-quartet layers (Scheme 1B). Just due to such vacancy having particular requirements for Hoogsteen hydrogen bonds and planar structure to change into canonical G-quadruplex, DGQ possesses exceptional selectivity for guanine recognition. That is, only guanine and its derivatives can fill up this vacancy and fulfill the conversion from incomplete G-quadruplex to intact one. Based on this unordinary DNA structure, Tan's group established a labeled sensing strategy for ultrasensitive detection of guanine and its derivatives, in which polyethylene glycol (PEG200) was used as a crowding reagent to promote standard G-quadruplex formation (Li et al., 2016). However, PEG is non-specific to G-quadruplex, in other words, it also facilitates the DGQ formation in the absence of guanine, generating large background signals (Kan et al., 2006). Moreover, the labeling of the fluorescent dye and quencher makes the assay cost-, time- and labor-consuming. To overcome the above limitations, a label-free strategy using a G-quadruplex specific promoter for DGQ-to-G-quadruplex conversion is worth to be expected.

Surprisingly, Mohanty et al. and Sugimoto et al. proposed a G-quadruplex specific fluorescent indicator, named Thioflavin T (ThT) (Mohanty et al., 2013, Sugimoto et al., 2015). This water-soluble fluorescent dye itself bears extremely low fluorescence, while it exhibits great fluorescence enhancement if and only if it combines with G-quadruplex DNA under physiological salt conditions. That is to say, other DNA forms including single-strand and duplexes can’t drive such a change. Tan et al. and our group have reported two kinds of fluorescent biosensors based on ThT-induced G-quadruplex formation for biomolecules detection (Chen et al., 2014, Tan et al., 2014). Additionally, it is worth noting that hemin is also a G-quadruplex specific substance. It can impel G-rich nucleic acids to fold into G-quadruplex DNA structure and then combine with it to constitute stable G-quadruplex-hemin complex in the presence of a metal ion, such as K+ (Marchand and Gabelica, 2016). This complex, called DNAzyme, has horseradish peroxidase-like activity and can effectively catalyze the H2O2-mediated oxidation along with the change of current signals and the color in reaction solution (Chen et al., 2018, Li et al., 2009). Note that only G-quadruplex has this kind of especial property, instead of all other DNA structures. Moreover, G-quadruplex-hemin DNAzyme is of better stability and lower cost when compared to other DNAzymes and proteases (Ito and Hasuda, 2004, Willner et al., 2008). Gao et al. and our group have utilized the unique characteristic of hemin specific to G-quadruplex to establish label-free electrochemical biosensor for DNA or microRNA detection (Gao et al., 2017, Zhang et al., 2014).

In order to increase assay sensitivity, reduce testing costs and simplify the procedures for guanine and its derivatives detection, we herein develop two varieties of label-free biosensors by coupling DGQ with ThT (for fluorescent sensor) or hemin (for electrochemical sensor). As illustrated schematically in Scheme 1, Scheme 2, a G-rich DNA probe is designed to form either hairpin (H1 or H3) or DGQ structure containing a G-vacancy at one corner of its G-quartet layers. Initially, the probe prefers to maintain hairpin structure in the absence of free guanines. Owing to the perfect discriminative ability of ThT and hemin towards intact G-quadruplex instead of hairpin, the fluorescent and electrochemical signals are nearly negligible. Whereas, upon the addition of guanine, the G-rich probe turns into DGQ structure and then its G-vacancy is filled up immediately by guanine to form stable standard G-quadruplex, which subsequently combines with ThT or hemin leading to a dramatic increase of fluorescent or electrochemical signals. By monitoring the signal differences before and after guanine addition, the proposed biosensors can sense the existence and precise quantity of guanine with ultrahigh sensitivity and selectivity. Particularly, together with the intrinsically electrochemical advantages of short analysis time, simple operation, low cost, high sensitivity and so on, the label-free electrochemical sensor holds great potential application in point-of-care testing (POCT).

Section snippets

Reagents and apparatus

All oligonucleotides (fluorescent DNA probes: H1: 5′-TAGGGTGGGCTGGGAGGTTTTTTTTTTTCACCCTA-3′, H2: 5′-FAM-TAGGGTGGGCTGGGAGGTTTTTTTTTTTCACCCTA-BHQ1–3′; electrochemical DNA probes: H3: 5′-TAGGGTGGGCTGGGAGGTTTTTTTTTTTCACCCTA-SH-3′, H4: 5′-MB-TAGGGTGGGCTGGGAGGTTTTTTTTTTTCACCCTA-SH-3′. FAM, BHQ1 and MB represent Carboxyfluorescein, black hole quencher 1 and methylene blue, respectively) were synthesized by Sangon Biotech (Shanghai, China) Co., Ltd, and purified by high performance liquid

Experimental principle of the label-free fluorescent biosensor

As illustrated in Scheme 1, the fluorescent G-rich probe H1 is devised to exist in either hairpin or DGQ form. In the absence of guanine, H1 prefers to fold into hairpin structure via self-hybridization of the nucleotides near the two ends. On account of little response to hairpin DNA, ThT shows very weak fluorescence. With guanine addition, the probe convert to an intact G-quadruplex since guanine fills the G-vacancy in its G-quartet layers through Hoogsteen hydrogen bonds. Meanwhile, ThT

Conclusion

In conclusion, two label-free biosensors (one is fluorescent and the other is electrochemical sensor) were developed for guanine and its derivatives detection with the LOD down to 18.26 and 0.36 nM, respectively. Owing to the extraordinary recognition ability of the defective G-quadruplex for guanine and ThT/ hemin against canonical G-quadruplex, the biosensors bear excellent selectivity. Moreover, the electrochemical biosensor also shows color changes of reaction solution regarding the

Acknowledgement

The authors gratefully acknowledge the financial support of the National Science Foundation of Fujian Province (2017J07001, 2016J01042), United Fujian Provincial Health and Education Project for Tackling the Key Research PR China (WKJ2016-2-30), Fujian Science and Technology Innovation Joint Found Project (2016Y9050).

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