Exonuclease III-based and gold nanoparticle-assisted DNA detection with dual signal amplification

https://doi.org/10.1016/j.bios.2012.01.003Get rights and content

Abstract

Herein we report a sensitive electrochemical biosensor for DNA detection by making use of exonuclease III and probe DNA functionalized gold nanoparticles. While probe DNA P1 modified on a gold electrode surface can self-hybridize into a stem-loop structure with an exonuclease III-resistant 3′ overhang end, in the presence of target DNA, P1 may also hybridize with the target DNA to form a duplex region. Therefore, exonuclease III may selectively digest P1 from its 3′-hydroxyl termini until the duplex is fully consumed. Since a single target DNA can trigger exonuclease III digestion of numerous P1 strands, the first signal amplification is achieved. On the other hand, since the digested P1, exposing its complementary sequence to probe DNA P2, can further hybridize with P2 that has been previously modified on the surface of gold nanoparticles, many nanoparticles loaded with numerous DNA strands are immobilized onto the electrode surface. Consequently, large amount of electroactive molecules [Ru(NH3)6]3+ can bind with the DNA strands to produce an intense electrochemical response as the second signal amplification. Based on the studies with cyclic voltammetry (CV) and chronocoulometry (CC) techniques, the proposed biosensor can sensitively detect specific target DNA at a picomolar level with high specificity.

Highlights

► A dual signal amplification-assisted electrochemical DNA biosensor was developed. ► Dual signal amplification was based on the use of ExoIII and gold nanoparticles. ► The biosensor could sensitively detect target DNA at a picomolar level. ► The well specificity and reproduction of the detection were also demonstrated.

Introduction

Ultrasensitive detection of specific DNA sequences plays a critical role in clinical diagnostics of genetic diseases, accurate identification of pathogens and early detection of cancers (Staudt, 2001, Debouck and Goodfellow, 1999). Target amplification and signal amplification are two commonly used approaches to increase the detection sensitivity (Saiki et al., 1985). As a classical target amplification method, polymerase chain reaction (PCR) can amplify a few copies of target DNA across several orders of magnitude through thermal cyclic reaction (Heid et al., 1996). But PCR is prone to yielding false-positive results from the artificial amplification that may have effect on the detection specificity (Halford, 1999, Schweitzer and Kingsmore, 2001, Song et al., 2009). In contrast, signal amplification that directly enhances the detection signals from a single analyte-probe recognition event can efficiently improve the detection sensitivity without sacrificing the specificity. In recent years, the use of nicking endonuclease that cleaves only one strand of double-stranded DNA at specific recognition nucleotide sequences has largely facilitated the development of signal amplification (Tan et al., 2007, Xu et al., 2009). Yet, only 13 nicking endonucleases are commercially available, which extremely restricts the application of nicking endonuclease-based technology (Zhao et al., 2011).Different to the sequence-specific nicking endonuclease, exonuclease does not require a specific recognition sequence, so it may be more beneficial to the development of universal signal amplification strategies for DNA detection. Exonuclease III (ExoIII) is a kind of exonuclease catalyzing the stepwise removal of mononucleotides from 3′-hydroxyl termini of double-stranded DNA, which is not active on 3′-overhang ends of double-stranded DNA or single-stranded DNA (Cui et al., 2010). In the reported ExoIII-assisted signal amplification approaches, the fluorescent signaling probes are selectively digested by ExoIII with the target cycling, generating multiple signaling events and achieving signal amplification (Zhang et al., 2011, Zuo et al., 2010, Cui et al., 2010). These ExoIII-based fluorescent methods can sensitively detect target DNA at a picomolar level.

Fluorescent technique often suffers from several drawbacks, such as high cost, easy contamination and high complexity (Xu et al., 2009), so electrochemical technique is suggested to be an alternative due to its advantages in cost, portability and convenience (Liu et al., 2009, Xiao et al., 2008, Cao et al., 2010). Hsieh et al. have reported an electrochemical DNA detection method by using λ exonuclease-assisted signal amplification, and demonstrated 16-fold signal amplification at low target concentrations. However, the detection limit in this assay is only 10 nM, which cannot be as good as the result in the fluorescent methods (Hsieh et al., 2010).

In this work, we have designed an improved electrochemical DNA detection strategy by coupling probe DNA functionalized gold nanoparticles (Au NPs)-assisted electrochemical signal amplification with ExoIII-assisted signal amplification. DNA functionalized Au NPs have been often used as colorimetric probes for DNA detection. Combined with ExoIII, a sensitive colorimetric method for DNA detection has been proposed (Cui et al., 2011). Since Au NPs have large surface to volume ratio, huge amount of DNA strands can be loaded onto their surfaces for the hybridization with the complementary sequences immobilized at an electrode surface, thus intense electrochemical response can be achieved to realize a very high sensitive electrochemical detection (Wang et al., 2009, Miao et al., 2011a, Zhang et al., 2006, Miao et al., 2009a, Steel et al., 1998). Therefore, due to dual signal amplification, our assay can have not only the advantages of electrochemical technique but also the same high detection sensitivity as the fluorescent method.

Section snippets

Materials and chemicals

Chloroauric acid (HAuCl4) was obtained from Shanghai Sinopharm Chemical Reagent Co., Ltd. Mercaptohexanol (MCH), ethylenediamine-tetraacetic acid (EDTA), hexaammineruthenium(III) chloride ([Ru(NH3)6]Cl3), and tris(2-carboxyethyl) phosphine hydrochloride (TCEP) were from Sigma. ExoIII (specific activity, 100,000 units/mL) was purchased from New England Biolabs. Other chemicals were of analytical grade and were used without further purification. For all experiments, Milli-Q water (>18.0 MΩ) was

Results and discussion

In this work, we have chosen a short DNA sequence related to human immunodeficiency virus (HIV) as the target DNA, since the nucleic acid-based tests are most accurate for HIV detection (Weiss, 1993, Baur et al., 2010). Fig. 1 may illustrate the mechanism of the proposed method. Firstly, probe DNA P1 immobilized on the surface of the gold electrode can self-hybridize into a stem-loop structure with an ExoIII-resistant 3′ protruding termini (Fig. 1a). Nonetheless, in the presence of target DNA,

Conclusions

In conclusion, based on ExoIII and Au NPs-assisted dual signal amplification, we have proposed a very sensitive and simple, convenient, easily-operated electrochemical approach for DNA detection with improved sensitivity and high specificity. The recyclable use of target DNA promotes the efficiency of the digestion of P1 on the electrode surface, and the employment of DNA functionalized Au NPs can further enlarge the obtained electrochemical responses. Moreover, the enzymatic digestion reaction

Acknowledgments

This work is supported by the National Science Fund for Distinguished Young Scholars (grant no. 20925520), the Fundamental Research Funds for the Central Universities (grant no. 1116020801), and the Leading Academic Discipline Project of Shanghai Municipal Education Commission (J50108).

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