The effect of temperature on electrochemically driven denaturation monitored by SERS
Graphical abstract
Introduction
Detection and identification of specific DNA sequences are critical in clinical diagnostics [1], drug screening [2] and forensic analysis [3]. Optical and electrochemical DNA-biosensors have attracted particular attention due to their sensitivity, robustness, low cost and ability to carry out multi-target analysis. Both types of DNA biosensors rely for their specificity on DNA hybridization. The most common optical biosensors usually utilise techniques such as fluorescence [4], [5], [6], fluorescence resonance energy transfer (FRET) [7], [8] or surface Plasmon resonance (SPR) [9], [10]. Fluorescence based techniques measure the emission intensity of a dye-labelled probe DNA or an intercalating dye after hybridization whereas surface plasmon resonance methods utilise the change in the refractive index at the SPR sensor surface upon hybridization with a surface bound probe. On the other hand, electrochemical biosensors use electrical measurement and can be based either on changes in impedance [11], [12] or on the measurement of redox currents [13].
Surface-enhanced Raman spectroscopy (SERS) is an alternative technique that has attracted interest in the development of DNA sensors due to the high sensitivity of the technique and the specificity provided by the use of vibrational spectroscopy. Label free approaches that directly detect the distinctive SERS spectra from DNA bases have been reported [14], [15], [16], [17], in particular the discrimination of single base-mismatches within short single stranded oligonucleotides has been shown [14]. Although these approaches do not require a hybridization event to discriminate DNA sequences, they have currently been restricted to studies of short (< 25 base pairs (bp)) synthetic DNA samples and no data have been published that show discrimination of long (> 100 bp) DNA strands generated from biological samples. Attaching Raman dye-labelled oligonucleotides to SERS substrates [18], [19], [20] or utilising dye-coded nanoparticles [21], [22] generates strong, specific Raman signals, permitting identification of a large number of oligonucleotides without the need for separation. These techniques have been demonstrated to be useful for the analysis of clinical samples. However they are usually hybridization-based, thus they are not sensitive enough to very small differences in the DNA such as single base mutations.
Alternatively, DNA discrimination can be achieved using techniques that exploit the denaturation of DNA rather than the hybridization. The most popular examples are real-time polymerase chain reaction (PCR) in conjunction with absorption/fluorescence melting analysis [23] and denaturating gradient gel electrophoresis that uses a gel to separate DNA fragments according to their melting characteristics (DGGE) [24], [25]. Both techniques require expensive instrumentation. Electrochemical techniques can also be used to monitor the denaturation. This “electrochemical melting” [26], [27] is distinct from the process described here as the melting of the dsDNA is brought about thermally but detected electrochemically. Thus, for example in early work in this area Paleček used polarography to study conformational changes in DNA with temperature [28]. More recently techniques such as cyclic voltammetry [29], square wave voltammetry [30], differential pulse voltammetry [26], [27], [31] and AC voltammetry [32] have all been used to monitor the thermally driven melting of dsDNA at electrode surfaces. In contrast we have developed an alternative denaturation-based approach to discriminate DNA sequences in which we use electrochemically driven denaturation (E-melting) of double-stranded (ds) DNA immobilised at a nanostructured gold electrode where the DNA melting is monitored by SERS. Electrochemically driven melting has been reported by several authors. Early studies by Brabec and Palecek observed melting of dsDNA adsorbed on mercury electrodes [33], and Sosnowski et al. [34] described electrochemically driven denaturation of dsDNA bound to an agarose gel up to 1 μm above a Pt electrode surface. In addition, Hassmann et al. [35], [36] described studies in which they covalently attached nucleic acid probes to conducting polycarbonate/carbon fibre electrodes and showed that complementary oligonucleotides were enriched at the electrodes by cyclic inversion of an electrochemical potential between − 0.2 and − 1.5 V vs. Ag/AgCl. In our work a DNA target labelled with a fluorophore is hybridised to a surface bound probe, followed by the application of a suitably negative potential so that the dsDNA melts [37]. E-melting curves, constructed by plotting the SERS intensity of the fluorophore Raman signal against the electrode potential, are then used to discriminate DNA sequences based on differences in the stability of the dsDNA [38]. This technique relies on the fact that the less stable DNA duplexes e.g. duplexes containing a single base mutation, will have less negative denaturation (melting) potentials. Sphere segment void (SSV) Au surfaces [39] are used as the SERS substrates due to their large and reproducible SERS enhancements, well defined surface areas and good electrochemical stability [40], [41], [42], [43].
We have demonstrated that this methodology is sensitive, robust and suitable for analysis of DNA generated from biological samples. More precisely, we showed discrimination of genetic markers and pathogenic bacteria by targeting short tandem repeats [44] and single nucleotide polymorphism [37], [38], [45] with detection limits of 0.3 μM without the need for prior-purification of the DNA samples. In addition, electrochemical potentials were found to be insensitive to changes in the local pH over the range 6–8 [46]. Most significantly, we have recently demonstrated the discrimination of nucleotide polymorphism in long (> 250 bp) PCR amplicons which allowed us to distinguish DNA generated from Yersinia pestis, the causative agent of plague, from the closely related species Y. pseudotuberculosis [45]. In this case the discrimination was significantly improved when the experiments were conducted at lower temperature due to the slope of the wild type amplicon melting profile being steeper when compared to that recorded at room temperature.
In the current study we expand this work to examine in greater detail the effect of the experimental temperature on the electrochemical melting potential and the sharpness of the melting curves. E-melting experiments were carried out for two different DNA duplexes, 20 and 21 bp, over a temperature range from 10 °C to 28 °C. The results clearly show a shift in the melting potential to more negative values coupled with sharper melting curves when the temperature is ≤ 18 °C. The results are of interest since E-melting experiments can be combined with reduced temperature to directly address more complicated problems for real life applications.
Section snippets
Material and methods
All reagents used were of analytical grade and obtained from Sigma-Aldrich unless otherwise stated. Oligonucleotide synthesis was performed using standard methods by ATDBio Southampton, United Kingdom. The DNA probes (Table 1) had a three di-thiol linker at the 5′ end (Fig. S1). The DNA target sequences were fully complementary to the probes and labelled with Texas Red.
DNA preparation and design
To study the effect of the experimental temperature on the E-melting, two different 21 and 22-bp double stranded DNA (dsDNA) sequences were used. The two sequences possess similar thermodynamic stabilities which can be seen from their calculated melting temperatures: 56.6 °C for DNA-1 and 57.2 °C for DNA-2 (both calculated using IDT OligoAnalyzer [47]). Similar conditions to the Raman experiments were used to calculate the melting temperatures (10 mM phosphate buffer, 0.1 M NaCl). The probe
Discussion
Early studies on the melting of short oligonucleotides using NMR showed that the terminal base pairs are unstable due to the tendency of the inter-base hydrogen bonds at the ends of the duplex to exchange with H-bonds to water (the solvent) [49], [50], [51]. Thus the terminal base pairs can break before those located inside the duplex. This effect is referred to as terminal end-fraying and leads to melting of DNA through a non-cooperative mechanism (the transition from duplex to single strands
Conclusions
E-melting experiments for two different short DNA duplexes (20 and 21 bp) possessing similar melting temperatures were carried out between 10 and 28 °C. A significant temperature dependence for both the melting potentials, Em, and the steepness of the melting curves, described by dE, was found over the range 10 to 18 °C. The results are consistent with greater stability of the dsDNA at the lower temperature together with greater cooperativity in the melting, approaching a two state transition from
Acknowledgements
E.P. acknowledges the Defence Science and Technology Laboratory (contract number DSTLX 1000061240) for funding this research. We thank Y-C. Lin for help with preliminary E-melting experiments. PNB gratefully acknowledges receipt of a Wolfson Research Merit award.
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