Redox cycling amplified electrochemical detection of DNA hybridization: Application to pathogen E. coli bacterial RNA

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Abstract

An electrochemical genosensor in which signal amplification is achieved using p-aminophenol (p-AP) redox cycling by nicotinamide adenine dinucleotide (NADH) is presented. An immobilized thiolated capture probe is combined with a sandwich-type hybridization assay, using biotin as a tracer in the detection probe, and streptavidin-alkaline phosphatase as reporter enzyme. The phosphatase liberates the electrochemical mediator p-AP from its electrically inactive phosphate derivative. This generated p-AP is electrooxidized at an Au electrode modified self-assembled monolayer to p-quinone imine (p-QI). In the presence of NADH, p-QI is reduced back to p-AP, which can be re-oxidized on the electrode and produce amplified signal. A detection limit of 1 pM DNA target is offered by this simple one-electrode, one-enzyme format redox cycling strategy. The redox cycling design is applied successfully to the monitoring of the 16S rRNA of E. coli pathogenic bacteria, and provides a detection limit of 250 CFU μL−1.

Introduction

In recent years, researchers have been challenged to push the sensitivity of electrochemical DNA sensor down to subnanomolar levels while keeping these procedures as simple, reliable, and cost-effective as possible. Signal amplification is the most important strategy in lowering the detection limits [1], [2]. In particular, most of the signal-amplification methods have been developed by employing different labels, such as functionalized liposomes [3], [4], multiple enzyme coated microsphere [5] or carbon nanotube [6], bio-barcode Au nanoparticals [7], arrays of Au [8] and CdS nanoparticles [9], and dendritic-like enzyme nanoarchitectures [10]. During these labels, enzymes are still the most commonly used ones for signal amplification due to their high turnover frequencies and high reaction selectivity.

Alkaline phosphatase (ALP) is one of the most used enzymatic labels for the DNA hybridization assay. For the measurement of ALP, a standard method is that ALP dephosphorylates p-aminophenyl phosphate (p-APP) enzymatically to produce electroactive species p-aminophenol (p-AP), which is detected amperometrically by substrate electrode. This approach has been widely used in the electrochemical enzyme immunoassays [11], [12], as well as DNA hybridizations [13]. However, ALP detection suffers from drawbacks ultimately related to the limited stability of p-APP and p-AP. Substrate redox cycling, which is related to the regeneration of enzyme-amplified electroactive species after their oxidation or reduction, is a well-suited means to overcome this defect [14]. Furthermore, since the redox reaction of the regenerated species provides an enhanced electrochemical signal, higher signal amplification could be achieved by the combination of enzymatic amplification with a substrate redox cycling step [15], [16], [17].

Generally, redox cycling can be achieved electrochemically [18], [19], enzymatically [20], [21], [22], [23], or chemically [15], [24], [25], [26]. In the model of electrochemical redox cycling, the electroactive species oxidized at one electrode are reduced back at the second electrode, so two working electrodes or an interdigitated array electrode are needed, while it conflicts with the requirement of simpleness and cost-effectiveness for DNA sensor. Enzymatic redox cycling provides a relatively simple way for redox cycling amplification [14], [20], [23]. However, in this model multiple enzymes are required and the redox cycling efficiency is highly dependent on the enzyme kinetics.

Recently, a new upsurge in chemical redox cycling is in the making. Hydrazine and sodium borohydride were reported to use in p-AP redox cycling and shown good performance in immunosensor. In order to get lower background currents in electrochemical detection, the use of strong reducing agents, such as hydrazine and sodium borohydride, which are easily oxidized electrochemically at low potentials, is limited on highly electrocatalytic metal electrodes, for example, Au electrode. Benefit from both the slow electrochemical oxidation on metal electrodes and fast chemical reaction, nicotinamide adenine dinucleotide (NADH) is a good substitute for strong reducing agents in redox cycling system on metal electrodes [16].

16S rRNA gene, which is universal in bacteria, is normally used for bacterial identification and phylogenetic studies due to its variable and conserved regions [27]. The goal of the present work is to design an amplified electrochemical genosensor for sensitive sensing of 16S rRNA gene with chemical redox cycling using NADH. In this approach, the cycling was applied to an electrochemical DNA sensor based on Au working electrodes. Following a sandwich-type hybridization assay, ALP enzymes are conjugated to the surface of the genosensor, and enzymatically generate the electroactive mediator p-AP from p-APP (Fig. 1). The produced p-AP is then electrooxidized at the Au working electrode to p-quinone imine (p-QI). Immediately p-QI is reduced back to p-AP by NADH, which leads to the redox cycling of the p-AP to amplify the electrical current. Such procedure provides good performance for DNA detection as well as sensitive measurements of the pathogen E. coli bacteria.

Section snippets

Reagents

6-Mercapto-1-hexanol (MCH), Trizma hydrochloride (Tris–HCl), ethylenediaminetetraacetic acid, sodium chloride, sodium hydroxide, sodium phosphate monobasic, sodium phosphate dibasic, potassium chloride, potassium phosphate dibasic, potassium phosphate monobasic, bovine serum albumin, streptavidin-alkaline phosphatase (SA-ALP), p-AP, and NADH were purchased from Sigma-Aldrich (St. Louis, MO). The blocking agent casein was obtained from Pierce (Rockford). p-APP was purchased from Biosynth

p-AP redox cycling by NADH

Redox cycling of p-AP by NADH and its use in electrochemical immunoassay has been pioneered by Kwak et al. [16]. Different from other chemical redox cycling, this system is unique in the use of metal working electrodes. However, a ferrocenyl-tethered dendrimer layer was required for electrode modification to fast the electron transfer rate. In order to investigate the redox cycling phenomenon of p-AP directly on Au electrode without extra mediator layer, we obtained the cyclic voltammograms of

Conclusions

We have described a DNA electrochemical sensor based on signal amplification by both enzymatic amplification and substrate redox cycling. The redox cycling is achieved simply by adding a reducing agent, NADH, to the detection solution, and could be applied to an electrochemical DNA sensor based on Au working electrodes. Electroactive product, p-AP, is well protected from oxidation by air in the presence of reducing regent, a long enzymatic accumulation time is possible in thus chemical redox

Acknowledgements

Financial support from the National Institutes of Health (RO1 EB002189 and U01 AI075565) and National Science Foundation (CHE 0506529) are gratefully acknowledged.

References (31)

  • J. Wang et al.

    Biosens. Bioelectron.

    (2004)
  • R.Q. Thompson et al.

    Anal. Biochem.

    (1991)
  • Y.L. Yuan et al.

    Anal. Biochem.

    (2010)
  • D. Zheng et al.

    Anal. Chim. Acta

    (2004)
  • C. Ruan et al.

    Talanta

    (2001)
  • B. Serra et al.

    Anal. Biochem.

    (2005)
  • N.L. Rosi et al.

    Chem. Rev.

    (2005)
  • J. Wang

    Small

    (2005)
  • F. Patolsky et al.

    J. Am. Chem. Soc.

    (2001)
  • F. Patolsky et al.

    Angew. Chem. Int. Ed.

    (2000)
  • J. Wang et al.

    J. Am. Chem. Soc.

    (2004)
  • H.D. Hill et al.

    Nat. Protocols

    (2006)
  • F. Patolsky et al.

    Chem. Commun.

    (2000)
  • I. Willner et al.

    Angew. Chem. Int. Ed.

    (2001)
  • F. Lucarelli et al.

    Langmuir

    (2006)
  • Cited by (0)

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