Elsevier

Biosensors and Bioelectronics

Volume 24, Issue 7, 15 March 2009, Pages 2064-2070
Biosensors and Bioelectronics

Electrochemical quantification of DNA amplicons via the detection of non-hybridised guanine bases on low-density electrode arrays

https://doi.org/10.1016/j.bios.2008.10.021Get rights and content

Abstract

A new strategy for the electrochemical detection and signal amplification of DNA at gold electrodes is described. Current methodologies for DNA biosensing based on the electrochemical detection of electroactive base-specific labels such as methylene blue (MB) suffer from lengthy incubation and washing steps. Addressing these limitations, we report a novel approach for the electrochemical quantification of surface hybrid, using the control gene LTA, 107 bases long, as a model target. An array of 15 gold electrodes was used to detect the formation of hybridised duplex following interaction of non-hybridised guanine bases with MB present in solution. Upon hybridisation the number of free guanines present at the electrode surface increased from 8 to 25 due to guanine bases present in the target sequence which did not participate in hybridisation and remained free to interact directly with methylene blue. This increase in free guanines consequently concentrated MB directly at the electrode surface. We found that the MB signal recorded for 100 nM of the complementary LTA was typically 2.14 times higher than that of the non-hybridised state. Very low cross-reactivity (<7%) with a non-complementary probe was recorded. The assay was optimised with regards to methylene blue concentration, hybridisation time and regeneration. The assay was quantitative and linear in the range of 6.25–50 nM target DNA exhibiting an LOD of 17.5 nM. The assay was rapid and easy to perform, with no need for lengthy incubations with the methylene blue label or requirement for washing steps. Ongoing work addresses the impact of guanine location on the signal in order to tailor design specific signalling domains of PCR products.

Introduction

DNA sequencing and detection techniques have been steadily developing since the 1990s (Baba and Tsuhako, 1992, Coutelle, 1991, Lemieux et al., 1998) to reach a technological maturity. The development of microarray fabrication technologies has further fostered the application of DNA sensing in a variety of fields such as genomics, transcriptomics (Schena et al., 1998) as well as environmental (Miller et al., 2008) and clinical diagnostics (McShea et al., 2006). Despite the massive and rapid advancement in microarray technology to facilitate the simultaneous assessment of many thousands of genetic sequences in a high-density format, the prohibitive price of commercial DNA microarrays and associated optical readout systems has prevented DNA biosensor arrays reaching the clinician's office (Gant, 2007).

Comparatively, clinical diagnostics aimed at the provision of individual personalised care, coined as theranostics, only requires the analysis of a few tens of sequences at once, considerably reducing the complexity and the cost of the array and instrumentation required. As such, low-density arrays of lower cost targeted at specific conditions such as breast cancer, for disease identification, staging, prognosis, and the assessment of medication effectiveness are foreseen to find their way to the point of care environment (Lodes et al., 2007, Wu et al., 2007).

For such applications, electrochemical transduction of surface hybridisation presents a very attractive prospect in terms of speed of analysis, overall instrumentation simplicity, cost and miniaturisation (Lodes et al., 2007). Electrochemical readout of hybridisation events typically requires the addition of hybridisation labels to produce measurable changes in current, potential or surface impedance. These labels are usually introduced either as electroactive moieties in PCR primers during a sample pre-treatment and amplification (Brazdilova et al., 2007) or added post-hybridisation as nanoparticles (Patolsky et al., 2000), enzymes (Pividori et al., 2001), liposomes (Patolsky et al., 2001) or intercalators (Wong et al., 2004). Of all approaches, the addition of an electrochemically active intercalator or base-specific label represent an attractive compromise in terms of ease of hybrid labelling, reduced preparation steps, mismatch identification and low detection limits (Ju and Zhao, 2005, Kelley et al., 1999).

The phenothiazine organic dye methylene blue (MB) has been widely used and reported as an effective DNA electrochemical (Boon et al., 2000, Erdem et al., 2000, Kelley et al., 1997, Yang et al., 2002), as well as fluorescent (Hu and Tong, 2007, Ihmels and Otto, 2005, Ohuigin et al., 1987) label. The interaction types and kinetics between MB and single stranded DNA (ssDNA) or double stranded DNA (dsDNA) have been intensively studied and previously reported. Methylene blue interacts either via electrostatic interaction (Zhao et al., 1999), direct intercalation in the DNA double helix (Nafisi et al., 2007) or by preferential binding to free guanine bases present at ssDNA (Erdem et al., 2000, Yang et al., 2002). Based on these interaction regimes the DNA sensing methodology developed to date typically consists of the timed exposure of the sensor surface to MB in a saline solution followed by the measurement of the MB–hybrid complex present at the sensor surface in a MB-free solution (Erdem et al., 2000). A differential measurement is taken between the sensor before and after hybridisation with the perfect match, i.e. at the ssDNA probe and the corresponding dsDNA duplex, for which a lower current response is typically recorded. This approach suffers three limitations, (i) lengthy MB–DNA interaction time is required, (ii) the measurement solutions need to be changed several times and (iii) a decrease in current is measured upon hybridisation with the corresponding complementary sequence. While the first limitation might be addressed through assay optimisation, the last two are inherent to the methodology and are found problematic for further integration of the sensor into a fully functional diagnostic device where minimum fluid handling and ease of result interpretation is preferred (Von Lode, 2005).

We address these limitations in the work presented here. As a model target and towards the realisation of a low-density electrochemical DNA sensor microarray for the diagnosis of predisposition of breast cancer via the BRCA1 gene, we used the synthetic control gene amplicon sequence lymphotoxin-α (LTA), 107 nucleotides (nt) long, and a corresponding surface immobilised probe, 20 nt long. Control experiments were carried out using the BRCA1 Exon 13, 21 nt probe. All measurements could be performed in the presence of MB in low ionic strength buffer, acting as both a measurement buffer and a high-stringency washing buffer suppressing contribution from non-specific hybridisation. Upon hybridisation, the number of free Gs present at the electrode surface increases significantly from 8, in the immobilised probe, to a total of 25 after formation of the surface hybrid. Consequently, signal amplification results from the enhanced presence of Gs at the surface following hybridisation, as depicted in Fig. 1, directly measurable in a low concentration, low ionic strength MB solution. Taking advantage of the free guanines present along the non-hybridised section of the amplicon–probe complex we measured an increase in current, rather than decrease, directly proportional to the concentration of the targeted amplicon. The results presented herein are discussed with respect to ease of sensor assessment, rapidity, reliability and robustness of the assay developed.

Section snippets

Materials

All electrochemical measurements were performed with a potentiostat/galvanostat PGSTAT 12 Autolab controlled with the General Purpose Electrochemical System (GPES) software and equipped with a MUX module (Eco Chemie B.V., The Netherlands). The MUX module allows sequential addressing of up to 64 working electrodes that share the same counter and reference electrode. The measurements were carried out in a single electrochemical cell in a “three”-electrode arrangement consisting of the DNA

Initial assay development

Electrochemical detection of surface hybridisation events based on the electroactive label MB has been widely reported. The accepted procedure to date consists in the immersion of the sensor surface in a solution of MB for 5–10 min, during which MB interacts with free guanine bases on single strands or intercalates between guanine–cytosine pairs in double stranded DNA. The surface is quickly rinsed to remove any excess of MB and DPV measurements are carried out in fresh MB-free buffer in order

Conclusions

This report demonstrates a novel strategy for the exploitation of methylene blue as an electrochemical hybridisation indicator of simulated post-PCR long single stranded DNA sequences. This approach takes advantage of the non-hybridised guanine bases available for direct interaction with the electroactive indicator methylene blue and subsequent electrochemical reduction. All measurements could be carried out directly in methylene blue, leading to a limit of detection in the low nanomolar range.

Acknowledgements

This work has been carried out as part of the Commission of the European communities ICT project Integrated Microsystem for the magnetic isolation and Analysis of Single circulating tumour Cells for Oncology diagnostics and Therapy follow-up, MASCOT, FP6-2004-IST-026752.

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