Development of a DNA Sensor Based on Alkanethiol Self- Assembled Monolayer-Modified Electrodes

An electrochemical DNA biosensor based on recognition of double or single stranded DNA (ds-DNA/ss-DNA) immobilised on a self-assembled modified gold electrode is presented for denaturalisation and hybridisation detection. DNA is covalently bond on a self assembled 3-mercaptopropionic acid monolayer by using water soluble N-3-(dimethylaminopropyl)-N prime;ethylcarbodiimide hydrochloride (EDC) and N-hydroxisulfosuccinimide (NHSS) as linkers. The interaction between the immobilised DNA and methylene blue (MB) is investigated using square wave voltammetry (SWV). The increase or diminution of peak currents of the MB upon the hybridisation or denaturalisation event at the modified electrode surface is studied.


Introduction
In recent years there has been a considerable interest in the development of DNA sensors, due to its theoretical and practical significance in many fields. The detection of DNA hybridisation is of central importance in the diagnosis and treatment of genetic diseases, detection of infectious agents, industrial processing and reliable forensic analysis [1][2][3][4][5].
The analysis of specific gene sequences in the diagnostic laboratory is usually based on DNA hybridisation. Here, the target gene sequence is identified by a DNA probe able to form a doublestranded (ds) hybrid with its complementary nucleic acid with high efficiency and specificity [1].
Recently, a lot of progress has been made in the development of electrochemical DNAhybridisation biosensors (or genosensors). These biosensors rely on the conversion of the base-pair recognition event into a useful electrical signal. The sensing events on the electronic transducers are designed to alter the interfacial properties at the transducer/solution interface. Changes in the interfacial charge, capacitance, resistance, or mass thickness can occur upon the hybridisation of the analyte DNA with a probe nucleic acid and upon stimulation of the amplification route. Thus, various electronic transduction methods that follow such interfacial changes have been employed to probe DNA recognition events. These include electrochemical transduction means such as amperometry, faradaic impedance spectroscopy or chronopotentiometry, and microgravimetric, quartz crystal microbalance (QCM) measurements [6].
The DNA immobilization procedure on the electrode surface is a very important aspect since it influences the characterisation of the DNA probe, the sensor response, and its performance [7]. Thus, a key issue with a DNA biosensor is the accessibility and molecular orientation of the probe DNA, which requires a high degree of control over the immobilisation of the probe oligonucleotides. Selfassembled monolayers (SAMs) of alkanethiols, which have shown to provide molecular level control over the immobilisation of several types of biomolecules, have been used as active films on which DNA segments can be attached using covalent linkers [8]. Electrochemical DNA sensors based on the amperometric or voltammetric transduction of the formation of double stranded (ds) oligonucleotide-DNA complexes have been reported by following the direct electrical response of the ds-assembly [9], and the electrical response of the ds-assembly of the transition metal complexes [10] or dyes [11] that are intercalated or electrostatically attracted to the double stranded assembly. Many electroactive indicators such as cationic metal complexes [12], anticancer drugs [13] or organic dyes [14] have, thus, been employed in DNA hybridisation biosensors.
The electrochemical response of these labels or indicators changes upon DNA hybridisation, usually increasing when the hybridisation process occurs due to an increase of the indicator concentration at the electrode surface.
Methylene blue (MB), an organic dye that belongs to the phenothiazine family, is a redox indicator, which, due to its interaction with the guanine bases in DNA, displays significantly different voltammetric signals in the presence of ss-DNA or ds-DNA modified electrodes [15]. The interaction between MB and DNA has, in fact, been studied by means of spectrophotometric and electrochemical methods [8,16,17]. So, Kelley et al [17,18] investigated the charge transfer mechanism of Au electrodes covered with ds-DNA in the presence of MB concluding that MB show high affinity for immobilized DNA (K≈4x10 6 M -1 ) and that MB binding sites are localized mainly to the solutionaccessible periphery of the monolayer. It has also been reported that the peak potential of the MB square wave voltammetric signals at thiol terminated probe-modified AuEs was 10-15 mV more positive than the one at thiol terminated hybrid-modified AuEs [19].
When electrochemical genosensors are based on the use of commercially available Au disk or wire electrodes, an important drawback is found in the need of regeneration of a new, bare electrode surface after each measurement. Mechanical and chemical procedures used are tedious and time consuming. Thus, the main objective of this article is the development of a DNA electrochemical biosensor for detecting the hybridisation and denaturalisation events occurring at the surface of a gold electrode modified with a SAM of 3-mercaptopropionic acid (MPA) derivatised with EDC/ NHSS. The possibility of re-using the ss-DNA modified electrode for successive hybridisation/denaturalisation cycles will be checked.

Apparatus and electrodes
Voltammetric measurements were carried out with an ECO Chemie Autolab PSTAT 10 potentiostat using the software package GPES 4.9 (General Purpose Electrochemical System). A P-Selecta Digiterm 100 thermostatic bath, a P-Selecta ultrasonic bath, a IKAMAG ® RET heating plate and a P-Selecta Agimatic magnetic stirrer were also used. Scanning electron micrographs were obtained with a JEOL JSM-6400 scanning microscope.
A Metrohm 6.1204.020 gold disk electrode (3-mm φ) was used as electrode substrate to be coated with the modified MPA-SAM. A BAS MF-2063 Ag⏐AgCl⏐KCl 3 M reference electrode and a Pt wire counter electrode were also employed. A 10-mL glass electrochemical cell was used.

Reagents and solutions
Methylene blue (MB) was purchased from Sigma.  All chemicals used were of analytical-reagent grade, and water was obtained from a Millipore Milli-Q purification system.

Pretreatment of the gold electrode (AuE)
Before carrying out the deposition of the SAM, the gold disk electrode (AuE) was pretreated as follows. The AuE was polished with 3-µm diamond powder (BAS MF-2059) for 1 min. Then, it was sonicated in deionized water for 1 min, immersed in an aqueous solution of 0.5 M H 2 SO 4 where the potential was cycled between 0.0 and +1.7 V (versus Ag/AgCl) at a scan rate of 100 mV s -1 for 10 consecutive scans [20]; then the electrode was rinsed with water and immersed in a 0.1 M NaOH solution for 15 min while applying a potential of -0.8 V [21], rinsed with water, and then it was immersed for 1 h in a hot 2 M KOH solution. Next, the electrode was rinsed with water, immersed in concentrated H 2 SO 4 for 10 min, rinsed with water, immersed in concentrated HNO 3 (Scharlau) for 10 min, and rinsed again with deionized water. water. Under these conditions, a surface coverage of θ = 0.95 was calculated by electrochemical impedance spectroscopy, indicating a high electrode coverage by the alkanethiol SAM.

DNA immobilization
The MPA-modified AuE was activated by immersion for 1 h at room temperature (in dark condition) in a solution formed with 2 mM EDC and 5 mM NHSS. Subsequently, a 2.5 µL drop of a 1000 mg L -1 ss-DNA or ds-DNA solution was deposited on the linker/MPA/AuE, let to dry at ambient temperature for at least 24 h, and finally soaked in water for 2 h to remove unbound ss-DNA or ds-DNA.

MB accumulation onto the DNA/Linker/MPA/AuE
The DNA-modified electrode was stirred in a 50 mM Tris-HCl and 20 mM NaCl buffer solution (pH 7.2) containing 75 µM MB for 1 min at open circuit.

Voltammetric transduction
The DNA-modified electrode was then subsequently washed with the supporting electrolyte for 5 s and transferred into the blank buffer solution (MB-free 50 mM Tris-HCl + 20 mM NaCl, pH 7.2) for the voltammetric measurements. The oxidation signal of the accumulated MB was measured by using square wave stripping voltammetry (SWSV) by scanning from -0.50 to +0.10 V with an amplitude of 30 mV and a step potential of 5 mV at 100 Hz, and by differential pulse stripping voltammetry (dp-SV) performed with an initial potential of -0.5 V and an amplitude of 20 mV at 20 The electrode was electrochemically cleaned in-between stripping voltammetric measurements to assure the complete stripping of the previously accumulated redox label. This was accomplished by applying successive potential SW scans between -0.5 and 0.1 V during 5 min in the blank buffer solution.

Denaturation
For the denaturation of the ds-DNA immobilized onto the linker/MPA/AuE, the sensor was immersed in 5 mL of a 10 mN Tris-HCl and 1 mM EDTA buffer solution (pH 8.0) at 100 ºC for 5 min, followed by cooling in an ice bath [22].

Hybridisation
The hybridisation protocol was performed at 70 ºC by immersion of the denaturalised ds-DNA-EDC-NHSS/MPA-AuE in 100 mg L -1 ss-DNA in the hybridisation buffer. The hybridisation time was 1 h. Then the electrode was rinsed with deionized water to remove the unrecognised ss-DNA.

Results and discussion
Preliminary studies were carried out in order to select the optimum conditions for the detection of the structure (ss or ds) of DNA immobilised atop the electrode. The EDC/NHSS-MPA-AuE was prepared in all cases following the conditions described previously by our research group [23]. Then, 2.0 µg of ss-DNA were immobilised atop the activated electrode as described in the Experimental Section.

Optimisation of the redox label voltammetric measurement conditions
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Firstly, the accumulation process of the redox indicator MB (Figure 1) on the ss-DNA modified electrode was studied. Differential pulse (dp) and square wave (SW) voltammetric stripping signals for 20 µM MB were compared (Figure 2), a much higher peak current being obtained when the stripping step was performed by SWV using a square wave amplitude of 30 mV with a step potential of 5 mV and a frequency of 100 Hz (scan rate, 500 mV s -1 ).

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MB SW stripping currents obtained in both anodic and cathodic direction were also compared.
In the conditions specified above, an oxidation peak current of (2.6±0.6)x10 -5 A (n=5) at a peak potential of -0.212 V, and a reduction peak current of (3±1)x10 -      produced an increase of the MB peak current [24], there are several authors stating that MB binds to the free guanine bases in DNA resulting in a higher MB accumulation at ss-DNA modified electrode surfaces [8,25,26]. Results displayed in Figure 5 show that the MB stripping signal was significantly lower at the ds-DNA-modified electrode than at the ss-DNA-modified electrode. These results indicate that although there is a voltammetric signal due to MB intercalation in ds-DNA, this is smaller when   These results demonstrated that the fabrication procedure of the DNA-EDC/NHSS-MPA-AuEs was reliable, thus allowing reproducible electroanalytical responses to be obtained with different sensors.

Denaturation and hybridization of immobilized DNA: preliminary studies
The detection of denaturation and hybridization events was accomplished by using the oxidation signals obtained after the accumulation process of methylene blue at the DNA sensors. This was possible because, as demonstrated before ( Figure 5), the electroactivity of this label led to discrimination of ss-DNA or ds-DNA.
Firstly, the experimental conditions for the denaturation process of ds-DNA were optimized.
Most of ds-DNA denaturation procedures found in literature are carried out in solution, then immobilizing the obtained ss-DNA at the electrochemical sensor. Three denaturation procedures were compared. The first one, based on that used by Gu et al. [24], consisted on immersing a 1000 ppm ds-DNA stock solution in TE buffer in a water bath, heating at 100 ºC for 10 min, and then rapidly cooling in an ice-water bath. When the thus obtained single stranded DNA was immobilized at an activated MPA-AuE under the optimized conditions commented above, no increase was observed for the MB peak current if compared to that obtained with a ds-DNA-EDC/NHSS-MPA-AuE, even when the heat-denaturation process was carried out for 1 h. The second denaturation procedure tested was based on that followed by He et al. [27] and consisted on adding 75 µL of a 2 M NaOH and 1. at 100 ºC for 5 min. An increase in the MB peak current of (350±10) % was obtained at the denaturalised-ds-DNA-EDC/NHSS-MPA-AuE. This increase was shown to be reproducible as the denaturation process was repeated, and it kept after storing the denaturalised electrode at 4 ºC in dry conditions for 24 h. Furthermore, when temperatures lower than 100 ºC were used, denaturation was not reproducible, and the voltammetric signal continuously decreased, probably as a consequence of re-hybridisation at the electrode surface. Therefore, it could be concluded that no significant rehybridisation occurred after application of the denaturation procedure.
On the other hand, the possibility of SAM removal from the electrode when the denaturation procedure was applied was also checked. MPA-modified electrodes subjected to this procedure showed no significant increase for the MB response when compared to the untreated modified electrode, thus indicating that no SAM peeling off occurred.
Regarding the hybridization process, two procedures were also tested. The first one consisted on pippetting onto the denaturalized-ds-DNA-EDC/NHSS-MPA-AuE 20 µL of 20 mM TE buffer solution containing 100 ppm of ss-DNA and air-drying for 30 min [8], while in the second procedure the denaturalized-ds-DNA-EDC/NHSS-MPA-AuE was further hybridized by immersion in a 100 ppm solution of ss-DNA in 0.3 M NaCl and 30 mM sodium citrate buffer (2xSSC buffer, pH 7.0) with stirring for 1 h at 70 ºC and then cooling gradually to room temperature [24]. The observation of the decrease in the MB stripping peak current was only possible by using the latter procedure, thus demonstrating the suitability of the developed sensor for the detection of the hybridization event.
Currently, studies on the optimization of the hybridization process and detection of point mutations toward the possibility of constructing sensors to detect pathogen microorganisms, based on the reported design, are being carried out in our lab.