Elsevier

Biosensors and Bioelectronics

Volume 64, 15 February 2015, Pages 306-310
Biosensors and Bioelectronics

Toehold strand displacement-driven assembly of G-quadruplex DNA for enzyme-free and non-label sensitive fluorescent detection of thrombin

https://doi.org/10.1016/j.bios.2014.09.018Get rights and content

Highlights

  • Non-enzyme and label-free fluorescent detection of thrombin at the low picomolar level is realized.

  • Signal amplification is achieved by toehold strand displacement-driven assembly of multiple G-quadruplex DNA.

  • The binding of the fluorescent dye to the G-quadruplex DNA generates significantly amplified signal output.

Abstract

Based on a new signal amplification strategy by the toehold strand displacement-driven cyclic assembly of G-quadruplex DNA, the development of an enzyme-free and non-label aptamer sensing approach for sensitive fluorescent detection of thrombin is described. The target thrombin associates with the corresponding aptamer of the partial dsDNA probes and liberates single stranded initiation sequences, which trigger the toehold strand displacement assembly of two G-quadruplex containing hairpin DNAs. This toehold strand displacement reaction leads to the cyclic reuse of the initiation sequences and the production of DNA assemblies with numerous G-quadruplex structures. The fluorescent dye, N-Methyl mesoporphyrin IX, binds to these G-quadruplex structures and generates significantly amplified fluorescent signals to achieve highly sensitive detection of thrombin down to 5 pM. Besides, this method shows high selectivity towards the target thrombin against other control proteins. The developed thrombin sensing method herein avoids the modification of the probes and the involvement of any enzyme or nanomaterial labels for signal amplification. With the successful demonstration for thrombin detection, our approach can be easily adopted to monitor other target molecules in a simple, low-cost, sensitive and selective way by choosing appropriate aptamer/ligand pairs.

Introduction

Aptamers are single-stranded oligonucleotides (DNAs or RNAs) that can bind to a wide range of target molecules such as metal ions (He et al., 2005, Wang et al., 2006), small molecules (Liu and Lu, 2005, Kong et al., 2013, Xu et al., 2014), peptides (Michaud et al., 2003), proteins (Xu et al., 2014, Lou et al., 2003) and even entire cells (Fang and Tan, 2010) with high specificity and affinity (dissociation constant ranging from micromolar to picomolar). Aptamers are obtained in vitro by an evolution process named systematic evolution of ligands by exponential enrichment (SELEX) from large random-sequence nucleic acid libraries (Ellington and Szostak, 1990, Tuerk and Gold, 1990). Compared with antibodies (conventional affinity reagents for the target antigen proteins), aptamers hold many advantageous characteristics, including simple and reproducible synthesis, easy and controllable labeling, long-term stability and design flexibility (Liu et al., 2009, Fang and Tan, 2010). Due to these unique features, aptamers have been increasingly used as promising recognition elements alternative to antibodies in designing different electrochemical (Jiang et al., 2012, Wu et al., 2008), electrochemiluminescent (Chen et al., 2011), fluorescent (Kong et al., 2013, Xu et al., 2014) and colorimetric (Zhu et al., 2011, Zhou et al., 2014) biosensors for the detection of a variety of target molecules. Among these biosensing methods, the fluorescent aptasensor is one of the most widely used approaches because of the high sensitivity, homogeneity, speed and simplicity nature of the fluorescent signal transduction technique (Yang et al., 2008). In addition, the enzyme-free and non-label fluorescent aptasensing systems have received increasing interest, due to the simplicity of these sensing approaches without the involvement of any enzymes or modifications to the aptamers to generate the fluorescent signal output (Huang and Chang, 2008, Kong et al., 2009, Qin et al., 2010, Yuanboonlim et al., 2012, Zhu et al., 2013, Zhu et al., 2014).

Thrombin, a multifunctional serine protease which produces insoluble fibrin through proteolytic cleavage of a soluble fibrinogen, plays an essential role for coagulation and cardiovascular disease therapy (Zhu et al., 2006). Considering the important biological function of thrombin, many fluorescent methods for thrombin detection have been reported over the past decade (Kong et al., 2013, Tan et al., 2012, Yan et al., 2011, Zheng et al., 2012, Xue et al., 2012, Xue et al., 2010, Niu et al., 2012). Special attentions have been given to non-labeled approaches for thrombin detection due to the simple operation and reduced cost of these methods (Kong et al., 2013, Tan et al., 2012, Yan et al., 2011). Although simple, the sensitivity (commonly in the nanomolar level) of these non-labeled approaches has been comprised. To circumvent this limitation, a number of signal amplification strategies, such as nicking enzyme-assisted fluorescent signal enhancement (Zheng et al., 2012, Xue et al., 2012), magnetic microparticle based sandwich assays (Xue et al., 2010, Niu et al., 2012) and target-catalyzed hairpin assembly (Zheng et al., 2012), have been reported to achieve high sensitivity for thrombin detection by using different fluorescent labels. Indeed, these effective signal amplification approaches have significantly improved the sensitivity for thrombin detection. However, the involvement of protein enzymes and conjugation of aptamers with fluorescent labels may lead to other issues. For example, the protein enzymes are susceptible to the reaction conditions (Fu et al., 2013) and the modification of the aptamers may alter their binding affinity to the corresponding target (Choi et al., 2009). Besides, these procedures potentially increase the complexity and cost of the assay methods. Therefore, the development of fluorescent signal amplification strategies without using enzymes or modification of aptamers will undoubtedly benefit the detection of thrombin and other proteins.

To meet the demands for non-label, simple and sensitive fluorescent detection of proteins, we here report an enzyme-free and non-label approach for sensitive fluorescent detection of thrombin by coupling toehold strand displacement-driven assembly of G-quadruplex DNA with aptamer probes. Toehold strand displacement refers to the displacement of one or more pre-hybridized strands from partial or full complementary dsDNAs, which is initiated at the complementary single-stranded domains (toeholds) (Yurke et al., 2000, Zhang and Winfree, 2009, Genot et al., 2011). The toehold strand displacement process is guided by the biophysics of DNA and occurs independently without the presence of enzymes. Due to the nucleic acid nature of this process, toehold strand displacement has been demonstrated to be useful in designing new enzyme-free signal amplification approaches for detecting DNA targets (Lin et al., 2007, Niu et al., 2012). However, the application of this new strategy for protein detection has been scarcely reported (Li et al., 2013, Zheng et al., 2012, Niu et al., 2012). In our design, the presence the target thrombin triggers the toehold strand displacement reaction and the formation of DNA assemblies with multiple G-quadruplex structures. Here we show that the fluorescent dye, N-methyl mesoporphyrin IX (NMM), binds to these G-quadruplex structures and emits significantly amplified fluorescent signals for sensitive detection of thrombin down to a low pM level in a non-label fashion.

Section snippets

Materials and reagents

Thrombin, mouse immunoglobin G (IgG), lysozyme and bovine serum albumin (BSA) were purchased from Sigma (St. Louis, MO). The oligonucleotides were synthesized and purified by Shanghai Sangon Biological Engineering Technology and Services (Shanghai, China). N-methyl mesoporphyrin IX (NMM) was obtained from J&K Scientific Ltd. (Beijing, China). The as received NMM was diluted to a stock solution (5 mM) with dimethyl sulfoxide (DMSO), stored in darkness at −20 °C, and freshly diluted to the required

Principle of the proposed method

The proposed principle for enzyme-free, non-label and sensitive fluorescent detection of thrombin based on the target-induced assembly of G-quadruplex DNA is depicted in Scheme 1. The G-quadruplex forming sequences are initially locked in the hairpin structures of HP1 and HP2, and the initiation strand (IS) is partially hybridized with the thrombin binding aptamer sequence. In the absence of thrombin, no free IS can be generated and the G-quadruplex forming sequences remain locked in the

Conclusions

In summary, we have demonstrated a new enzyme-free and non-label sensitive fluorescent strategy for thrombin detection based on target-induced assembly of G-quadruplex DNA. The presence of the target thrombin triggers the toehold strand displacement cyclic assembly of multiple G-quadruplex structures to achieve significant signal amplification for thrombin detection down to 5 pM. Our method is also selective towards thrombin against other control proteins. In addition, the signal amplification

Acknowledgment

This work was supported by NSFC (Nos. 21275004, 20905062 and 21275119), the New Century Excellent Talent Program of MOE (NCET-12-0932) and Fundamental Research Funds for the Central Universities (XDJK2014A012).

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