Elsevier

Biosensors and Bioelectronics

Volume 32, Issue 1, 15 February 2012, Pages 133-140
Biosensors and Bioelectronics

Electrochemically amplified molecular beacon biosensor for ultrasensitive DNA sequence-specific detection of Legionella sp.

https://doi.org/10.1016/j.bios.2011.11.046Get rights and content

Abstract

An electrochemically amplified molecular beacon (EAMB) biosensor is constructed using thiolated hairpin DNA-ferrocene probes on gold electrode. The switching from “on” to “off” states of individual probes in the presence of complementary DNA target influences the electrode potential, besides the current, owing to changes in surface density of the electroactive hairpin DNA-ferrocene probes. The EAMB biosensor demonstrates linear range over 8 orders of magnitude with ultrasensitive detection limit of 2.3 × 10−14 M for the quantification of a 21-mer DNA sequence. Its applicability is tested against PCR amplicons derived from genomic DNA of live Legionella pneumophila. Excellent specificity down to one and three nucleotides mismatches in another strain of L. pneumophila and a different bacterium species, respectively, is demonstrated.

Introduction

Methods for the identification of specific nucleic acid sequences have attracted great interest because of the urgent needs to identify and study disease-causing microbes (Lai et al., 2007, Ling et al., 2010, Mishra et al., 2006) contaminated sources including water and food, besides human diseases owing to gene variations (Wang et al., 1998). One method for rapid identification of specific nucleic acid sequences uses direct specific probe-target hybridization (Walker and Rapley, 2000). When coupled to techniques such as piezoelectric (Chen et al., 2010) and surface plasmon resonance (Mrksich et al., 1995), complementary binding of target to surface bound nucleic acids are especially useful because of minimal sample preparation steps. However, these lab-based methods cannot be readily applied at sources of contamination or points-of-care because of bulky equipment and need for stringent laboratory environment. In contrast, electrochemical DNA biosensors have attracted considerable attention because of simple instrumentation, low cost, portability, fast response time, in addition to high specificity and sensitivity which are highly attractive for such on-site analyses (Bini et al., 2007, Tlili et al., 2005, Zhang et al., 2009), with potential for applications in molecular sensing devices (Bergren and McCreery, 2011).

Among the electrochemical detection methods, reported strategies include detection of redox labels physically or covalently attached to the nucleic acid probe or target, such as enzymes (Liu et al., 2008), nano-particles (Wang et al., 2009), metal ions (Wang and Kawde, 2002) and intercalators (Wang et al., 2002) achieved detection of single-base mismatch to some degree. Other approaches include measurement of changes in electrical impedance property of surface film thickness after hybridization (Brett et al., 1999, Oliveira-Brett et al., 2002). Recently, electrochemical hairpin DNA probes show very promising performance in high specificity electrochemical detection down to single-base mismatch using impedance measurement and voltammetric methods (Fan et al., 2003, Gong et al., 2009, Jin et al., 2007, Miranda-Castro et al., 2007). However, the detection limit and sensitivity are considerably poorer than fluorescence molecular beacon probes, which can give high intensity signal arising from rapid turnover of excited and ground states of fluorophore label and a continuous excitation source, thus achieve high signal-to-noise ratio.

In this report, we construct an ultrasensitive electrochemically amplified molecular beacon (EAMB) biosensor by appending reversible ferrocene0/+1 redox species at 5′-terminal of hairpin DNA thiolated to gold electrode surface at the 3′-end. Sacrificial redox reagent Fe(CN)64− in bulk solution achieves turnover of the oxidation state of surface immobilized hairpin DNA-ferrocene (electrochemical molecular beacon), which amplifies the electrochemical signal output (Scheme 1). This amplification strategy using simple addition of commonly available Fe(CN)64− has not been reported in electrochemical hairpin DNA-based biosensors. The EAMB biosensor gives significantly lower detection limit (10−14 M) by 1–5 orders of magnitude than non-amplified electrochemical hairpin DNA-based biosensors (Xiao et al., 2006, Xiao et al., 2007, Fan et al., 2003, Long et al., 2004, Gong et al., 2009, Sun et al., 2010) and 6 orders of magnitude lower than electrochemical non-hairpin DNA sensors (Wu et al., 2009).

Herein, the biosensor achieves high specificity differentiation of a 21-mer base sequence from similar sequences with one and three base-pair mismatches. Furthermore, specific detection of label-free genomic sequence of bacterium Legionella pneumophila is demonstrated, which presently contaminates ∼4% of all potable water sources, causing a serious form of pneumonia known as Legionnaires’ disease or legionellosis. Complete elimination of Legionella bacteria is extremely difficult owing to widespread intracellular growth in protozoa of biofilms capable of surviving extreme conditions. Thus, the development of a rapid and accurate identification method will be extremely useful in monitoring quality of water sources with high risks of contamination by Legionella bacteria, especially during epidemic situations.

Section snippets

Reagents

3′-Thiolated hairpin DNA probe sequence of L. pneumophila (3′-HS-(CH2)3GCAACT TGT TTT CCC CGC CCC TCTCATAGTT(CH2)6NH2-5′), non-thiolated complementary target sequence (5′-ACA AAA GGG GCG GGG AGA GTA-3′), single nucleotide mismatch target sequence (5′-ACA AAA GGAGCG GGG AGA GTA-3′) and three nucleotide mismatch target sequence (-5′-GCA AAA GGG GCG GGG AGA GGG-3′) were obtained from Sigma–Aldrich. 6-Mercapto-1-hexanol, 1.0 M tris (2-carboxy-ethyl) phosphine hydrochloride (Tris buffer) of pH 7.0,

The EAMB biosensor

In this work, we first construct surface tethered-hairpin DNA probes tagged with redox active ferrocene, on gold electrode surface. The signal response of the EAMB biosensor is amplified using Fe(CN)64− as a sacrificial redox species to regenerate the reduced state of the DNA-ferrocene probe (Scheme 1). In general, such redox cycling scheme relies on rapid homogeneous electron transfer between the regenerating agent and ferrocene, slow heterogeneous reaction of regenerating agent and

Conclusions

We report a new electrochemically amplified molecular beacon biosensor, made from surface attached thiolated DNA hairpin probes appended to redox active ferrocene species, with electrochemical regeneration by sacrificial Fe(CN)64− reagent. Ultrasensitive detection of a label-free 21-mer complementary DNA sequence of L. pneumophila at (∼20) fM level can be achieved. The EAMB biosensor selectively differentiates between L. pneumophila and two other species and shows exceptionally wide linear range

Acknowledgements

We thankfully acknowledge financial support from NTU grant and Ministry of Education scholarship for VR.

References (39)

  • A.J. Bergren et al.

    J. Electroanal. Chem.

    (2005)
  • C.M.A. Brett et al.

    Electrochim. Acta

    (1999)
  • Q. Chen et al.

    Biosens. Bioelectron.

    (2010)
  • Y. Jin et al.

    Biosens. Bioelectron.

    (2007)
  • G. Koh et al.

    Electrochim. Acta

    (2007)
  • S. Mishra et al.

    Trans. R. Soc. Trop. Med. Hyg.

    (2006)
  • A.M. Oliveira-Brett et al.

    Bioelectrochemistry

    (2002)
  • C. Sun et al.

    Biosens. Bioelectron.

    (2010)
  • C. Tlili et al.

    Talanta

    (2005)
  • J. Wang et al.

    Biosens. Bioelectron.

    (2009)
  • J. Wu et al.

    Electrochem. Commun

    (2009)
  • A. Bini et al.

    Anal. Chem.

    (2007)
  • A.J. Bard et al.
  • A.J. Bergren et al.

    Annu. Rev. Anal. Chem.

    (2011)
  • B. Bockisch et al.

    Nucleic Acids Res.

    (2005)
  • J. Chattopadhyaya et al.

    Biochemistry

    (2005)
  • H. Du et al.

    J. Am. Chem. Soc

    (2003)
  • C. Fan et al.

    Proc. Natl. Acad. Sci. U.S.A.

    (2003)
  • H. Gong et al.

    Anal. Chem.

    (2009)
  • Cited by (0)

    View full text