Accepted Manuscript

Title: A study of the interactions of Hg(II) with T-T mispair

containing hairpin loops

Authors: Ajar Kamal, Zhe She, Renu Sharma,

Heinz-Bernhard Kraatz

PII: S0013-4686(17)31041-1
DOI: http://dx.doi.org/doi:10.1016/j.electacta.2017.05.058
Reference: EA 29493

To appear in: Electrochimica Acta

Received date: 16-1-2017

Revised date: 9-5-2017
Accepted date: 10-5-2017

Please cite this article as: Ajar Kamal, Zhe She, Renu Sharma, Heinz-Bernhard Kraatz,
A study of the interactions of Hg(II) with T-T mispair containing hairpin loops,
Electrochimica Actahttp://dx.doi.org/10.1016/j.electacta.2017.05.058

This is a PDF file of an unedited manuscript that has been accepted for publication.
As a service to our customers we are providing this early version of the manuscript.
The manuscript will undergo copyediting, typesetting, and review of the resulting proof
before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that
apply to the journal pertain.
A study of the interactions of Hg(II) with T-T mispair containing hairpin
loops

Ajar Kamal, Zhe She, Renu Sharma and Heinz-Bernhard Kraatz*

Department of Physical and Environmental Sciences, University of Toronto Scarborough,

Toronto M1C 1A4, Canada, e-mail:bernie.kraatz@utoronto.ca

Graphical abstract

Highlights
Increase in T-T mismatches enhances charge transfer resistance on gold electrode.
Maximum structural changes were observed for DNA III having 6 T-T mismatches.
Hairpin DNA III shows high selectivity for Hg(II) over other metal ions.
Impedimetric sensing of Hg (II) with DNA III could be useful for analytical applications.
Negative ellipticity conformational changes observed in DNAs by circular dichroism.

Abstract
This manuscript describes the result of a series of electrochemical, spectroscopic and
thermodynamic studies into oligonucleotide hairpin structures equipped with T-T mispairs and
investigates their interaction with Hg(II). For this purpose, three hairpin DNAs were designed, in
which the stem-region remains constant and loop region contains increasing T-T mispairs (5-

1
HO-(CH2)6-S-S-(CH2)6-TGTAGTTCCTTCTACA-3, 5-HO-(CH2)6-S-S-(CH2)6-
TGTAGTTTTCCTTTTCTACA-3; 5-HO-(CH2)6-S-S-(CH2)6-
TGTAGTTTTTTCCTTTTTTCTACA-3). Various techniques such as electrochemical
impedance spectroscopy (EIS), circular dichroism (CD), UV-visible and isothermal titration
calorimetry (ITC) were employed to get a systematic evaluation of the need for multiple T-T
mispairs for enhanced Hg(II) binding. EIS shows an increase in charge transfer resistance (RCT)
due to structural changes within the thin film caused by Hg(II) binding. The CD studies indicate
the Hg(II) induced changes in CD signals of hairpin DNAs from right-handed to left handed
structures. Highest cooperative binding was observed with DNA having maximum T-T mispairs
which was supplemented by detailed UV-visible thermal analysis of DNA-Hg(II) structures.
These findings were further supported by detailed measurements of the thermochemistry of the
interaction by ITC. Our studies show that the number of T-T mispairs are critical for maximizing
nucleobase-Hg(II) interactions.

Keywords: hairpin DNA; loop size; surface electrochemistry; metal-DNA interactions; mercury

1. Introduction
In recent years, the interactions between DNA and metal ions have received enormous
interest due to their potential applications in various fields. DNA probes have been widely
employed for biosensor construction in aqueous as well as solid state platform [1-3]. Metal ions
are extremely diverse in their interactions with DNA. The mercury-base pair interaction has been
extensively studied for understanding the toxicity of mercury contamination on human health
and environment [4]. Its reliable detection from aqueous solution is highly desirable. It is now
well known that Hg(II) binds strongly to T-T mispairs in DNA through formation of N-Hg(II)-N
bond (N3 of thymine), accompanied with release of two imino protons without binding to
phosphate group [5, 6]. The T-Hg(II)-T base pairs enable the single-stranded DNAs to fold into
duplexes and strengthen DNA duplexes. Based on the stable T-Hg(II)-T base pair, various types
of sensing platform such as electrochemical and optical biosensors for Hg(II) have been
developed for environmental pollution assays [7-9].
Recently, Liu and his coworkers described fluorescent, colorimetric and electrochemical
Hg(II) biosensors based on the specific interaction between Hg(II) and oligonucleotides [10]. In

2
their study authors highlighted the pros and cons of different techniques employed for Hg(II)
recognition and discussed that the oligonucleotides based Hg(II) biosensors showed immense
outlook in future environmental monitoring. In other report, single and dual hairpin DNAs
structures were synthesized using insertion approach and further employed to detect Hg(II) by
electrochemical approach [11]. Among the two DNAs, dual hairpin DNA possessed more charge
transfer resistance with low detection limit of 28 pM. Our group is also actively engaged in
developing DNA biosensors for metal ions recognition [12-14]. DNA-based label free biosensor
had been constructed for simultaneous detection of Pb(II), Ag(I) and Hg(II) which showed high
sensitivity and selectivity over diverse metal ions. Further, this sensor was successfully
employed for analysis of these metal ions in different sample matrices [7].
In addition to its bio-sensing applications, T-Hg(II)-T base pairs in DNA play an
important role in DNA based nanotechnology like electric nanowire, molecular magnet and
genetic code expansions [15-17]. However, the lack of structural information for T-Hg(II)-T base
pair prevented rationalization of various types of experiments needed for their growth. Literary
revealed that there is no report to date that studied the effect of variation in number of T-T base
pairs on Hg(II) interaction. But, it should be taken into account since the number of T-T mispairs
affects significantly the binding properties of DNA with Hg(II). Taking this view in mind, the
present work investigates the effect of increase in number of T-T mispairs in three hairpin
DNAs, designated as DNA I, DNA II and DNA III having 2, 4 and 6 T-T mispairs, respectively
on Hg(II) binding by means of electrochemical/spectroscopic studies. To the best of our
knowledge, this is the first literature report that sheds light on the effect of loop size of hairpin
DNA on their interaction with Hg(II).
In this study, DNA-Hg(II) interactions were firstly probed by using electrochemical
impedance spectroscopy (EIS) and the result is interesting as it shows the dominancy of
conformational changes over the DNA-Hg(II) mediated conductivity which has been usually not
observed in literary to date [18, 19]. EIS results were supported by other techniques employed.
The results demonstrated that DNA III having 6 T-T mispairs in loop exhibited maximum
conformational changes induced by Hg(II) due to formation of more rigid and stable DNA-
Hg(II) complex as compared to other hairpin DNAs. The number of T-T mispairs affects
sensitivity and signal intensity of Hg(II) biosensors significantly which reveals the importance of
number of T-T base pair for Hg(II) binding as explored by electrochemical and spectroscopic

3
studies. We hope that the outcomes of this work could be useful in design and development of
more accurate and efficient DNA based biosensors for Hg(II) recognition. Additionally, this
work may step ahead to understand the fundamentals of structural transformations induced due
to different number of T-T base pairs in DNA with Hg(II) which further might be useful for
rapidly growing nanotechnology based applications.

2. Experimental section
2.1 Materials. Three DNA sequences having increasing number of T-T base pair groups and
their abbreviation used throughout the manuscript are shown in Table 1 and these DNAs were
purchased from BioCorp, Canada. CHI 101 Gold disc electrodes having diameter of 2.0 mm
were used as working electrodes throughout this study. All the reagents used were of analytical
grade and deionized water (18.2 M cm) from a Millipore Milli-Q system was used for solution
preparation. Metal perchlorates, potassium ferricyanide (K3[Fe(CN)6]), potassium ferrocyanide
(K4[Fe(CN)6]), Tris-(hydroxymethyl)-aminomethane and 6-mercaptohexanol (MCH) were
purchased from Aldrich and used without further purification.

2.2 Monolayer preparation. 25M (30L) solutions of DNA I, DNA II and DNA III were
prepared in the 20 mM Tris-ClO4 buffer of pH = 7.4 containing 150 mM NaClO4. The gold
electrodes were cleaned according to the reported procedure and incubated in the DNA solutions
for 5 days. Then, the electrodes were removed, washed thoroughly with the buffer solution and
incubated in 1 mM 6-mercaptohexanol (MCH) in 150 mM Tris-ClO4 buffer for 2 hrs.
Subsequently, DNA/MCH modified gold electrodes were incubated in 10M Hg(ClO4)2 for 2
hrs. The electrodes were washed with Tris-ClO4 buffer after the immersion and mounted into an
electrochemical cell with 3-electrodes configuration. The EIS measurements were performed for
all films.

2.3 Electrochemical measurements. The electrochemical measurements (EIS, CV and DPV)

were carried out with an electrochemical work-station (CHI instruments, Austin, TX, USA)
Model 660D with a three-electrode cell, including a gold-disc modified/unmodified electrode, a
Pt wire and a Ag/AgCl as respective working, counter electrode and reference electrodes. All the
electrochemical measurements were performed in 20 mM Tris-ClO4 and 150 mM NaClO4

4
solution. For EIS studies, the AC voltage amplitude was 5 mV, and the voltage frequency was
from 100 kHz to 100 mHz. The applied potential was 240 mV vs. Ag/AgCl (formal potential of
the redox probe [Fe(CN)6]3-/4- in the buffer solution). The experimental EIS curves were
analyzed to calculate the film resistance using ZSimpWin 2.0 software. The CV and DPV studies
were conducted in the potential range of -0.4 V to 0.8 V at a scan rate of 0.05 Vs-1, pulse
amplitude of 0.05 V, pulse width of 0.05 s and pulse period of 0.08 s. All measurements were
repeated three times with separate electrodes to obtain statistically relevance.

2.4 Circular dichrosim (CD) and UV-visible measurements. CD measurements were

performed using JASCO J-815 spectrometer in the wavelength range of 230-320 nm in 10 mm
path length quartz cuvette at 0.1 nm intervals and scan speed of 100 nm/min for 1.0 M and 5.0
M samples of three DNAs at 298.15 K temperature in the absence and presence of increasing
concentrations of Hg(ClO4)2. The absorption spectra were recorded on a Cary 60 UV-visible
spectrophotometer with a quartz cuvette having a path length of 10 mm. The titrations were
performed at 298.15 K by successive additions of stock solutions of Hg(ClO4)2 into the quartz
cuvette containing 1.5 mL of 1.0 M and 5.0 M DNA solution. After every addition, the
solution was equilibrated for 5 minutes to reach thermal equilibrium. The spectra were recorded
in the range 230-290 nm.

2.4.1 Thermal transition studies. Thermal transition studies were carried out using UV-visible
measurements. UV-visible spectra of all three DNAs at low and high concentration of DNAs
were recorded in the absence and presence of Hg(II) at a temperature range of 10-90C with
interval of 5 C. In the temperature profiles, UV-visible absorbance at 260 nm and 265 nm were
plotted against temperatures for all DNAs in the absence and presence of Hg(II).

2.5 Isothermal titration calorimetry (ITC). ITC measurements were performed with TA
instruments Nano ITC Low Volume instrument (Waters Corp., Milford, MA, USA) with cell
volume of 250 L using ITC Run version 2.1.7.0 Firmware version 1.31 (TA instruments,
Waters Corp., Milford, MA, USA) as software. The reference cell was filled with deionized
water and all the solutions were degassed for 10 minutes prior to measurements. The titrations
were performed automatically by adding 2.5 L aliquots of prepared concentrated stock solution

5
of 400 M Hg(ClO4)2 into the sample cell containing 250 L of 10 M solution of DNAs with
continuous stirring of 200 rpm. The heat changes observed for the gradual addition of 400 M
Hg(ClO4)2 into the sample cell containing DNA solution were subtracted from the corresponding
heat changes of 400 M Hg (ClO4)2 solution in same buffer.

3. Results and discussion

3.1 EIS studies
A faradic impedance measurement involving a redox couple [Fe(CN)6]3-/4- as the probe
offer a high sensitivity. The charge transfer between this redox probe and gold surface occur
either by tunneling of electrons through surface self-assembly film or through the defects on the
surface. The impact of surface passivation on charge transfer serves to change the charge transfer
resistance (RCT) in EIS [20]. Therefore, the loop size effect of hairpin DNA probes having
different number of T-T mispairs on binding interaction of Hg(II) were evaluated using EIS.
The DNA films were prepared by incubating freshly cleaned gold electrodes in the DNA
solution, followed by soaking it in 1 mM mercaptohexanol (MCH) solution for backfilling the
defects and pinholes. This procedure prevents the DNA molecules from lying flat on the surface
and aligns the immobilized DNA hairpins in an upright orientation [21]. Further, the modified
gold electrodes were incubated in 10 M Hg(II) solution for 2 hours and EIS measurements were
carried out in 1.0 mM [Fe(CN)6]3-/4- as the redox probe in 20 mM Tris-ClO4 buffer solution
containing 150 mM NaClO4 at pH = 7.4 [22]. Scheme 1 illustrates the MCH modification and
binding strategy of DNA film toward the Hg(II) on gold electrode. The representative Nyquist
plots for hairpin DNAs without and with Hg(II) are shown in Fig. 1(a-c). The effect of loop size
on Hg(II) interactions was monitored using charge transfer resistance (RCT), which is the result of
resistance to charge transfer between the solution-based anionic redox probe [Fe(CN)6]3-/4- and
the gold surface of electrode. The DNA film resistance, RCT, was observed to be increasing for
all three DNAs after exposure of film to Hg(II). As shown in Table 2, the respective RCT for
DNA I, DNA II and DNA III increased from 8.3 to 9.5 k cm2, 10.2 to 12.9 k cm2 and 11.8 to
15.3 k cm2 with the corresponding RCT difference (RCT) of 1.2 k cm2, 2.6 k cm2 and 3.5
k cm2, respectively. The statistical plots shown in Fig. 1(d) illustrate an enhanced RCT
difference as the number of T-T mispairs increase. This increase in RCT values suggests the
binding of Hg(II) with T-T rich hairpin film to form intramolecular T-Hg(II)-T complex that

6
increases the film thickness due to which charge transfer of redox probe get impede resulting in
higher RCT values as shown in Scheme 1 [23, 24]. In other words, we can say that Hg(II) binding
induce conformational changes switching from non structural hairpin loop to well defined
hairpin loop (more rigid) structure. The increase in RCT values after binding of Hg(II) with
DNAs reveals the more influence of conformational changes over DNA-Hg(II) mediated
conductivity changes which are usually known to decrease RCT values [18, 19]. The EIS results
were found to be in good agreement with those obtained from cyclic voltammetry (CV) and
differential pulse voltammetry (DPV) (Fig. S1 and S2, ESI). Clearly, the RCT profile shown in
bar diagram (Fig. 1(d)) indicates the structural changes on addition of Hg(II) that follows the
order: DNA III (6 T-T) > DNA II (4 T-T) > DNA I (2 T-T). Highest RCT value for DNA III
system indicates the least penetration of redox probe into DNA III film due to more rigid
structure induced by Hg(II) as compared to other two DNAs. Therefore, EIS results indicate the
importance and influence of number of T-T base pair in loop region of hairpin DNA for Hg(II)
binding which further influence the signal intensity of Hg(II) biosensor.
Next, the impedance data were analyzed using a modified Randles equivalent circuit
(inset of Fig. 1(a)). The modeling related specific properties to resistive and capacitive
components, and the fitting results are listed in Table 2. Consistent electrochemical cell set-up
has been used throughout the experiments. The values of Rs (solution resistance) are only
dependent on the ionic strength of solution and the distance between the counter electrode and
gold modified electrodes [25]. The electrolyte concentration (20 mM Tris-ClO4 & 150 mM
NaClO4) for all measurement remained fixed to minimize variation in Rs, which ranged from 2.0
to 2.5 cm2. Cmonolayer accounts for the capacitance of the DNA film on the gold electrode. As
shown in Table 2, the film capacitance (Cmonolayer) slightly decreases after exposure of DNA to
Hg(II), which is speculated to be due to increase in film thickness that is induced by structural
alterations upon binding of Hg(II) [26]. The combination of Rx and constant phase element
(CPE) account for the behavior of 6-mercaptohexanol-diluted films on the electrode surfaces as
reported earlier [26]. Diffusion of redox probe from the solution to the DNA film was ignored in
these systems which is not important as is clear from absence of any Warburg impedance in Fig.
1(a-c).
The binding behavior of DNAs towards Hg(II) was further explored by performing EIS
titrations of these DNA films with the varying concentration of Hg(II). For these measurements,

7
the modified gold electrodes were incubated in Hg(II) solution with the concentration range from
10-5 to 10-8 M for 2 hours. On Hg(II) binding with DNA III, RCT increased to 14.1 k cm2 with
10-5 M Hg(II) as shown in Fig. 2(a). Accordingly, RCT increased to 12.6 k cm2 with 10-6 M, to
11.6 k cm2 with 10-7 M, and 10.7 k cm2 with 10-8 M Hg(II), respectively. The RCT values
(before and after addition of Hg(II)) increases gradually with the increase in Hg(II) concentration
from 10-8 to 10-5 M as shown in Fig. 2(b). The detection limit of DNA III towards Hg(II) was
found to be 7.3 nM. Under similar conditions, DNA I and DNA II modified gold electrodes were
employed to quantify the different amount of Hg(II), but we could not get any good linear
relation between RCT and Hg(II). It suggests that only DNA III can be employed for Hg(II)
recognition in various sample matrices.
Further, selectivity of DNA III containing 6 T-T mispairs towards Hg(II) was
investigated. A range of possible other metal ions, including Co (II), Ni (II), Ca (II), Cu (II), Cd
(II), Al (II), Mg (II), Zn (II), Ag (I) and Pb (II) were studied by exposing the DNA III film to
each ion at a consistent concentration of 10 M each. As shown in Fig. 2(c), DNA III exhibited
high selectivity for Hg(II) over the other metal ions. A small charge resistance change was
observed for Pb (II), but even though the change induced by Hg(II) was several times more. The
result indicates the excellent selectivity attributable to highly specific interaction of T-Hg(II)-T
which could be beneficial for its sensing applications.

3.2 Spectroscopic studies

Here, the interactions of hairpin DNAs (DNA I, DNA II and DNA III) having 2, 4 and 6
T-T mispairs with Hg(II) have been investigated by CD and UV-visible spectroscopic techniques
and discussed the effect of loop size on hairpin conformational transitions induced by Hg(II)
binding. The formation of more stable DNA-Hg(II) complexes was further characterized by UV-
visible melting curves.

3.2.1 Conformational characterization of hairpin DNA-Hg(II) interactions by CD

CD studies have been performed at 1.0 M concentration of DNAs in the region of 320-
230 nm in 20 mM Tris-ClO4 buffer containing 150 mM NaClO4 at varying mole equivalents of
Hg(II) and the corresponding spectra are shown in Fig. 3(a) and Fig. S3(a, b) (ESI),
respectively. All the DNAs show CD spectra with negative and positive bands at 250 and 280

8
nm, respectively. The gradual addition of Hg(II) to DNAs decreases the peak intensity of both
positive as well as negative bands accompanied by a bathochromic shift of 20 nm [27, 28].
Hg(II) binding to hairpin DNAs is reported to induce changes from hairpin B to Z transition, or
alternatively, to a hairpin structure of presumably left-handed geometry [29]. A CD study by
Gruenwedel and Cruikshank shows similar structural changes of DNA upon addition of Hg(II)
[30]. Liang et al. reported the changes in the ellipticity from positive to negative, along with a
red shift and enhanced negative peak intensity due to formation of hairpin-like structure after
Hg(II) binding [31]. Such changes in CD measurements confirm the formation of a highly
ordered and compact hairpin configuration that is induced by the addition of Hg(II). It is believed
that the Hg(II) is chelated between Watson-Crick T-T base pairs, forming strong bonds to
electron pairs of nitrogen atoms in a linear =N-T-N= configuration. Similar observations have
been made for the binding of Hg(II) to DNA [32-34]. On comparing the CD spectra of DNA I,
DNA II and DNA III, it was found that presence of Hg(II) induces negative ellipticity effect in
CD spectra of all DNAs and maximum negative ellipticity changes were observed with DNA III.
This probably due to more conformational changes by virtue of more number of T-T mispairs
which forms more stable and rigid Hg(II)-hairpin DNA complex. Thus, loop size of hairpin DNA
having more number of T-T greatly affects the stability of the DNA-Hg(II) complex.
Fig. 3(b) and Fig. S3(c, d) (ESI) show the calibration plot for the ellipticity changes of
three DNAs as a function of mole equivalents of Hg(II). From Fig. 3(b), it is clear that DNA III
gets saturated at mole equivalents of 6.3 and beyond that, there were no changes in the CD
spectrum observed. On the other hand, DNA II and DNA I get saturated at mole equivalents of
4.2 and 2.3 respectively, corresponding to the number of T-T base pairs present in the loop of
hairpin DNA. These results indicate the stoichiometry and the formation of intramolecular DNA-
Hg(II) complexes [35].
Similar CD titration experiments have also been performed at high (5 fold, 5.0 M)
concentration of all the three DNAs, since single stranded species are favored at low
concentration whereas duplexes or higher aggregates are favored at high concentration [35].
Similar titration profiles and calibration plots have been found with the addition of Hg(II)
(shown in Fig. S4, ESI) even at high concentration of DNAs which indicate similar binding
modes and the formation of similar DNA-Hg(II) adducts. Therefore, it is expected that no
intermolecular bonding (duplex formation) occurs and only intramolecular bonding is

9
responsible for the T-Hg(II)-T complex formation. These results are in agreement with those
reported by Kuklenyik and Marzilli, in which they concluded that hairpin DNAs having T-T
mispairs can be converted to a intrastand hairpin-Hg(II)-hairpin complex. This is affected by
both the concentration and length of the (T)n segment in any given oligonucleotide [35].

3.2.2 UV-visible measurements

UV-visible experiments have been carried out for DNA I, DNA II and DNA III at 1.0 M
in the wavelength range of 230-290 nm in 20 mM Tris-ClO4 buffer contain 150 mM NaClO4 at
varying mole equivalents of Hg(II). Fig. 4(a) and Fig. S5 (a, b) (ESI) show the absorption
spectra of DNAs in the presence of increasing concentrations of Hg(II). The UV-visible spectra
of DNAs show absorption spectra at 260 nm and with the successive addition of Hg(II), the
absorption intensity decreases along with a bathochromic shift of 5.0 nm which indicates the
formation of DNA-Hg(II) complex [36, 37]. The UV-visible spectra of DNAs show isosbestic
point at 238 nm suggesting that the T-Hg(II)-T base pairs are formed in equilibrium process on
gradual addition of Hg(II). The calibration plots for the UV-visible titrations indicate the
formation of 1:2, 1:4 and 1:6 complexes for DNA I, DNA II and DNA III respectively. DNA I,
DNA II and DNA III exhibited working concentration range of 0.2-2.4 M, 0.4-4.8 M and 0.6-
7.2 M with the detection limit of 34 nM, 89 nM and 112 nM respectively towards Hg(II). DNA
I shows lowest working concentration range with lowest detection limit as compare to DNA II
and DNA III towards Hg(II). Thus, we assume that hairpin DNA with smaller loop containing T-
T mispairs might potentially help to improve the detection limit as well as sensitivity of the
Hg(II) biosensor [38]. In addition, UV-visible titrations were also performed at 5 fold (5.0 M)
high concentrations of DNAs under identical conditions as shown in Fig. S6 (ESI). We
observed similar titration profile as we get at low DNAs concentration which show similar
binding mode of complex formation. The results extracted from UV-visible studies compliment
well with those obtained from CD measurements.
The Hill equation (equation S1) describes the degree of cooperative interactions between a
ligand binding and a macromolecule. The Hill coefficient, n, describes the degree of
cooperativity and its value can be greater than, less than or equal to 1, depending on the presence
of positive cooperative, negative cooperative or non-cooperative binding, respectively. Plaxco et
al. have reported the effect of positive cooperativity on binding of Hg(II) on two site Hg(II)

10
receptor and reported that the Hill coefficient increases with an increase in loop size from 6 to 50
bases [39]. Fitting our UV-visible data with the Hill equation, it was possible to determine the
degree of cooperativity for binding of DNA I-III to Hg(II). The Hill coefficient increases from
1.25 for DNA I to 1.54 for DNA II to 1.86 for DNA III (see Fig. S7). The increase of T-T
mispairs results in an enhanced cooperativity for Hg(II) binding, which is in agreement with
earlier results that suggested coorperative Hg(II) binding to oligonucleotides [39-41].

3.2.2.1 Thermal transition analysis

The thermal transition method has been employed to identify the strong binding of metal
ions to mispairs in base pairs of DNA molecules. When a metal ion binds tightly in mispair, it
increases the thermal stability of metallo-base pair and thus, the transition curve shifts towards
higher temperature [42-44]. In case of hairpin loop DNA, the presence of T-T mispair
destabilizes its structure depending upon the number of T-T mispairs in a loop, consequently
lowering the melting temperature (Tm). However, the addition of Hg(II) increases the stability of
these hairpin DNAs and as a result, Tm of such systems increases. Thus, in this study, we
examine the melting temperature profiles of DNA I, DNA II and DNA III at concentration of 1.0
M under same buffer conditions within temperature range of 10C to 90C. Fig. 5(a) shows the
thermally induced transition profiles of DNA III in the absence and presence of Hg(II) and the
corresponding profiles for DNA II and DNA I are given in supporting information as Fig. S8 (a,
b) (ESI). The Tm of each DNA increases significantly in the presence of Hg(II) which is
calculated from the first derivative plot as shown in Fig. 5(b) and Fig. S8 (c, d). The change in
melting temperature (Tm) was found to be 9C, 19C and 28C for DNA I, DNA II and DNA
III, respectively and this variation is shown in the bar diagram (Fig. 5(c)). Thus, maximum Tm
changes were observed for DNA III (6 T-T) which suggests the formation of more stable T-
Hg(II)-T complex due to presence of more number of T-T mispairs in comparison to DNA II and
DNA I. These findings are in agreement with reports by Tanaka and his group, which show an
increase in the melting point Tm from 25C to 54C upon Hg(II) binding, indicating an increase
in duplex stability [43].
Further, the melting temperature studies have also been performed at high concentration
(5.0 M) of DNAs (Fig. S9) to get clear picture of intramolecular bonding among Hg(II) and
DNAs. The Tm values were observed to be exactly the same even at high concentration of

11
DNAs which indicates that Hg(II) goes in the loop of hairpin DNAs by virtue of intramolecular
bonding and as a result, melting temperature increases. However, if intermolecular bonding also
participates, then we would expect the different Tm profile for DNA-Hg(II) complex at high
concentration of DNAs [35].

3.3 Effect of loop size on thermodynamics of Hg(II)-DNA interactions

Isothermal titration calorimetry (ITC) measurements have been done to evaluate the
thermodynamic parameters for the binding between Hg(II) and DNA I, DNA II and DNA III at
298.15 K temperature and pH 7.4 under similar buffer conditions. Hg(II) is known to bind
selectively to T-T mispairs rather than with phosphate and sugar groups in DNA [45, 46]. The
thermodynamic profile showing the variation of heat rate with time for DNA III, DNA II and
DNA I are shown in Fig. 6(a) and Fig. S10 (ESI), respectively. The area under each peak was
integrated and then these values were subtracted from those of heat of dilution of Hg(II) in the
same buffer done in separate experiment. The heat values thus obtained were plotted against the
molar ratio of [Hg(II)]/[DNA]. The titration plots for Hg(II)-DNA systems were found to be
nearly sigmoidal which suggests the specific binding of Hg(II) to DNAs. Exothermic heat
changes were observed for all the Hg(II)-DNA systems and the magnitude of each peak
decreases with each new injection till it approaches saturation as shown in Fig. 6(b) and Fig. S11
(ESI). Since the extent of exothermicity and endothermicity in an enthalpogram is a measure of
strength of interactions among the two participants, thus, in the present study, Hg(II)-DNA III
system shows maximum exothermic heat changes which is an indication of strong binding
between them due to bigger loop size having more number of T-T mispairs (6 T-T) in
comparison to other DNAs.
The ITC data has been fitted to multiple site model to get the values of binding constant
(Ka), stoichiometry and thermodynamic parameters i.e. Gibbs free energy change (G), entropy
change (S) and enthalpy change (H) corresponding to the interactions between Hg(II) and
DNAs which are listed in Table 3. The observed G values come from the mutual effect of
negative H and positive S values [47, 48]. Torigoe and his group reported that the binding of
Hg(II) to oligonucleotides was driven by positive dehydration entropy changes and negative
enthalpy changes [40]. The positive entropy changes occur due to conformational changes and
dehydration by the release of water molecules surrounding Hg(II) and DNA [43]. From the keen

12
analysis of the Table 3, it is clear that the higher values of Ka and G were found for DNA III-
Hg(II) system suggesting the spontaneous formation of more stable complex due to the presence
of positive cooperativity as discussed earlier in UV-visible studies [40]. These values were
higher than those reported earlier for nonspecific binding interactions between metal ions and
DNA [49, 50]. Thus, our experimental results demonstrated that, with the increase in loop size of
hairpin DNA, especially if loop contains more thymine groups, the binding constant of DNA-
Hg(II) complex enhanced considerably. The stoichiometry of binding was found to be 1:2, 1:4
and 1:6 between Hg(II) and DNA I, DNA II and DNA III respectively which corroborate well
with the results obtained from CD and UV-visible measurements.

4. Conclusions
In summary, we have analyzed the influence of increase in T-T mispairs in the loop of
hairpin DNA on the DNA-Hg(II) interaction by multi-techniques approach. The results obtained
from EIS studies show an increase in charge transfer resistance (RCT) with the increase in number
of T-T mispairs in the loop of hairpin DNA in the presence of Hg(II). This happens probably due
to more influence of conformation change over DNA-Hg(II) mediated conductivity changes
which are usually known to decrease RCT values. The DNA III having 6 T-T mispairs showed
high selectivity towards Hg(II) over other metal ions and the change in charge transfer resistance
(RCT) was linearly proportional to the concentration of Hg(II) within the range of 10-5 M to 10-
8
M with detection limit of 7.3 nM.
On addition of Hg(II) into DNAs, a bathochromic shift with decrease in intensity was
observed by UV-visible and CD measurements, suggesting the formation of DNA-Hg(II)
complex. Hg(II) induced negative ellipticity effect in CD spectra of DNAs but the maximum
ellipticity change was observed for DNA III having 6 T-T mispairs due to more conformational
changes. The UV-visible and CD titration studies performed at low as well as high
concentrations of DNAs demonstrated the formation of similar binding adducts via similar
binding modes which is due to the exclusive intramolecular (T-Hg(II)-T) complexation.
Difference in the Tm values for all DNA-Hg(II) systems reveals that the thermodynamic
stability depends significantly on number of T-T mispairs in loop of hairpin DNA. The
calculated values of Hill coefficient for DNA-Hg(II) systems reveals that the extent of positive
cooperativity depends on number of T-T mispairs present in DNA probes. This factor further

13
accounts for the more stability of the DNA-Hg(II) complex having a larger number of T-T
mispairs and these results are supported by our ITC measurements, in which the binding constant
(Ka) follows the order: DNA III > DNA II > DNA I. The binding stoichiometry follows the
expected order of 1:2 for DNA I, 1:4 for DNA II and 1:6 for DNA III.

In summary, our results demonstrate that the number of T-T mispairs in oligonucleotide
probes plays an important role for Hg(II) binding, presumably due to an increase in coorperative
binding. In light of these findings, we provide a better understanding of the design of Hg(II)
biosensor based on T-Hg(II)-T co-ordinate mechanism so as to tune its sensing properties for
particular applications. Additionally, the information about Hg(II) induced structural changes in
DNAs will be useful in potential application of Hg(II) mediated base pair in growing field of
DNA nanotechnology.

Acknowledgements
This work is supported by NSERC (RGPIN-2016-06122). We also acknowledge the
support from the University of Toronto Scarborough.

14
 References
[1] H. Yang, K.L. Metera, H.F. Sleiman, DNA modified with metal complexes: applications
in the construction of higher order metal-DNA nanostructures, Coord. Chem. Rev. 254
(2010) 2403-2413.
[2] K.S. Park, H.G. Park, Technological applications arising from the interactions of DNA
bases with metal ions, Curr. Opin. Biotechnol. 28 (2014) 17-24.
[3] S. Ranallo, A. Amodio, A. Idili, A. Porchetta, F. Ricci, Electronic control of DNA-based
nanoswitches and nanodevices, Chem. Sci. 7 (2016) 66-71.
[4] I. Onyido, A.R. Norris, E. Buncel, Biomolecule-mercury interactions: modalities of
DNA base-mercury binding mechanisms. remediation strategies, Chem. Rev. 104 (2004)
5911-5929.
[5] E. Xu, Y. Lv, J. Liu, X. Gu, S. Zhang, An electrochemical study based on thymine-Hg-
thymine DNA base pair mediated charge transfer processes, RSC Adv. 5 (2015) 49819-
49823.
[6] R.-G. Cao, B. Zhu, J. Li, D. Xu, Oligonucleotides-based biosensors with high sensitivity
and selectivity for mercury using electrochemical impedance spectroscopy, Electrochem.
Commun. 11 (2009) 1815-1818.
[7] Z. Lin, X. Li, H.-B. Kraatz, Impedimetric immobilized DNA-based sensor for
simultaneous detection of Pb2+, Ag+, and Hg2+, Anal. Chem. 83 (2011) 6896-6901.
[8] F. Long, C. Gao, H.C. Shi, M. He, A.N. Zhu, A.M. Klibanov, A.Z. Gu, Reusable
evanescent wave DNA biosensor for rapid, highly sensitive, and selective detection of
mercury ions, Biosens. Bioelectron. 26 (2011) 4018-4023.
[9] G.H. Chen, W.Y. Yen, Y.C. Yen, C.W. Wang, H.T. Chang, C.F. Chen, Detection of
mercury(II) ions using colorimetric gold nanoparticles on paper-based analytical devices,
Anal. Chem. 86 (2014) 6843-6849.
[10] L. Li, Y. Wen, L. Xu, Q. Xu, S. Song, X. Zuo, J. Yan, W. Zhang, G. Liu, Development of
mercury (II) ion biosensors based on mercury-specific oligonucleotide probes, Biosens.
Bioelectron. 75 (2016) 433-445.
[11] J. Jing, L.Yu, G.Z. Feng, L.J. Lei, L.H. Qun, L.N. Bing, A regenerative electrochemical
biosensor for mercury(II) by using the insertion approach and dual-hairpin-based
amplification, J. Hazard. Mat. 295 (2015) 63-69.

15
[12] H.J. Sun, X.H. Li, Y.C. Li, L.Z. Fan, H.-B. Kraatz, A novel colorimetric potassium
sensor based on the subsitution of lead from G-quadruplex, Analyst 138 (2013) 856-862.
[13] I. Gao, C. Li, X. Li, H.-B. Kraatz, Electrochemical impedance study of the interaction of
metal ions with unlabeled PNA, Chem. Commun. 46 (2010) 6344-6346.
[14] X. Bin, H.-B. Kraatz, Interaction of metal ions and DNA films on gold surfaces: an
electrochemical impedance study, Analyst 134 (2009) 1309-1313.
[15] H. Urata, E. Yamaguchi, T. Funai, Y. Matsumura, S. Wada, Incorporation of thymine
nucleotides by DNA Iolymerases through THgIIT base pairing, Angew. Chem. Int. Ed.
49 (2010) 6516-6519.
[16] K.S. Park, C. Jung, H.G. Park, Illusionary polymerase activity triggered by metal ions:
use for molecular logic-gate operations, Angew. Chem. Int. Ed. 49 (2010) 9757-9760.
[17] G.H. Clever, C. Kaul, T. Carell, DNA-metal base pairs, Angew. Chem. Int. Ed. 46 (2007)
6226-6236.
[18] S.O. Kelley, J.K. Barton, DNA-mediated electron transfer from a modified base to
ethidium: -stacking as a modulator of reactivity, Chem. Biol. 5 (1998) 413-425.
[19] X. Zhu, L. Chen, Z. Lin, B. Qiu, G. Chen, A highly sensitive and selective signal-on
electrochemiluminescent biosensor for mercury, Chem. Commun. 46 (2010) 3149-3151.
[20] J. Wu, D.M. Cropek, A.C. West, S. Banta, Development of a Troponin I biosensor using
a peptide obtained through phage display, Anal. Chem. 82 (2010) 8235-8243.
[21] T.M. Herne, M.J. Tarlov, Characterization of DNA probes immobilized on gold surfaces,
J. Am. Chem. Soc. 119 (1997) 8916-8920.
[22] Z. She, K. Topping, M.H. Shamsi, N. Wang, N.W.-C. Chan, H.-B. Kraatz, Investigation
of the utility of complementary electrochemical detection techniques to examine the in
vitro affinity of bacterial flagellins for a toll-like receptor 5 biosensor, Anal. Chem. 87
(2015) 4218-4224.
[23] H. Gong, X. Li, Y-type, C-rich DNA probe for electrochemical detection of silver
ion and cysteine, Analyst 136 (2011) 2242-2246.
[24] W.B. Mefteh, H. Touzi, F. Bessueille, Y. Chevalier, R. Kalfat, N.J. Renault, An
impedimetric sensor based on a gold electrode functionalized with a thiol self-assembled
monolayer modified by terpyridine ligands for the detection of free gadolinium ions,
Electroanal. 27 (2015) 84-92.

16
[25] M. Dijksma, B.A. Boukamp, B. Kamp, W.P. van Bennekom, Effect of
hexacyanoferrate(II/III) on self-assembled monolayers of thioctic acid and 11-
mercaptoundecanoic acid on gold, Langmuir 18 (2002) 3105-3112.
[26] H. Gong, T.Y. Zhong, L. Gao, X.H. Li, L.J. Bi, H.-B. Kraatz, Unlabeled hairpin DNA
probe for electrochemical detection of single-nucleotide mispairs based on MutS-DNA
interactions, Anal. Chem. 81 (2009) 8639-8643.
[27] J. Wang, B. Lin, Highly sensitive and selective detection of Hg2+in aqueous solution
with mercury-specific DNA and Sybr Green I, Chem. Commun. 2008, 4759-4761.
[28] Y. Miyake, H. Togashi, M. Tashiro, H. Yamaguchi, S. Oda, M. Kudo, Y. Tanaka, Y.
Kondo, R. Sawa, T. Fujimoto, T. Machinami, A. Ono, MercuryII-mediated formation of
thymineHgIIthymine base pairs in DNA duplexes, J. Am. Chem. Soc. 128 (2006)
2172-2173.
[29] D.A. Heller, E.S. Jeng, T.-K. Yeung, B.M. Martinez, A.E. Moll, J.B. Gastala, M.S.
Strano, Optical detection of DNA conformational polymorphism on single-walled carbon
nanotubes, Science 311 (2006) 508-511.
[30] D.W. Gruenwedel, M.K. Cruikshank, Mercury-induced transition between right-handed
and putative left-handed forms of poly[d(A-T).d(A-T)] and poly[d(G-C)-d(G-C)],
Nucleic Acids Res 17 (1989) 9075-9086.
[31] W. Ding, M. Xu, H. Zhu, H. Liang, Mechanism of the hairpin folding transformation of
thymine-cytosine-rich oligonucleotides induced by Hg(II) and Ag(I) ions, Eur. Phys. J. E.
36 (2013) 101-108.
[32] G. Liu, Z. Li, J. Zhu, Y. Liu, Y. Zhou, J. He, Studies on the thymine-mercury-thymine
base pairing in parallel and anti-parallel DNA duplexes, New J. Chem. 39 (2015) 8752-
8762.
[33] C.-W. Liu, C.-C. Huang, H.-T. Chang, Highly Selective DNA-based sensor for lead(II)
and mercury(II) ions, Anal. Chem. 81 (2009) 2383-2387.
[34] D. W. Gruenwedel, M.K. Cruikshank, Mercury-induced DNA Iolymorphism: probing the
conformation of mercury(II)-DNA via staphylococcal nuclease digestion and circular
dichroism measurements, Biochemistry 29 (1990) 2110-2116.

17
[35] Z. Kuklenyik, L.G. Marzilli, Mercury(II) site-selective binding to a DNA hairpin.
relationship of sequence-dependent intra- and interstrand cross-linking to the hairpin-
duplex conformational transition, Inorg. Chem. 35 (1996) 5654-5662.
[36] A. Walter, G. Luck, Interactions of Hg(II) ions with DNA as revealed by CD
measurements, Nucleic Acids Res. 4 (1977) 539-550.
[37] D.W. Gruenwedel, The mercury(II) and high-salt-induced conformational BZ
transitions of poly[d(G-m5C) d(G-m5C)] as studied by non-polarized (ultraviolet) and
circularly polarized (CD) ultraviolet spectroscopy, Eur. J. Biochem. 219 (1994) 491-496.
[38] G. Wang, Q. Zhao, X. Kang, X. Guan, Probing mercury(II)-DNA interactions by
nanopore stochastic sensing, J. Phys. Chem. B 117 (2013) 4763-4769.
[39] A.J. Simon, A.V.-Belisle, F. Ricci, K.W. Plaxco, Intrinsic disorder as a generalizable
strategy for the rational design of highly responsive, allosterically cooperative receptors,
Proc. Natl. Acad. Sci. U.S.A. 111 (2014) 15048-15053.
[40] H. Torigoe, Y. Miyakawa, A. Ono, T. Kozasa, Positive cooperativity of the specific
binding between Hg2+ ion and T:T mismatched base pairs in duplex DNA, Thermochim.
Acta 532 (2012) 28-35.
[41] M.P. Waalkes, L.A. Poirier, In vitro cadmium-DNA interactions: Cooperativity of
cadmium binding and competitive antagonism by calcium, magnesium, and zinc,
Toxicol. Appl. Pharmacol. 75 (1984) 539-546.
[42] K. Petrovec, B.J. Ravoo, J. Muller, Cooperative formation of silver(I)-mediated base
pairs, Chem. Commun. 48 (2012) 11844-11846.
[43] H. Yamaguchi, J. Sebera, J. Kondo, S. Oda, T. Komuro, T. Kawamura, T. Dairaku, Y.
Kondo, I. Okamoto, A. Ono, J.V. Burda, C. Kojima, V. Sychrovsky, Y. Tanaka, The
structure of metallo-DNA with consecutive thymineHg IIthymine base pairs explains
positive entropy for the metallo base pair formation, Nucleic Acids Res. 42 (2014) 4094-
4099.
[44] X. Xue, F. Wang, X. Liu, One-step, room temperature, colorimetric detection of mercury
(Hg2+) using DNA/nanoparticle conjugates, J. Am. Chem. Soc. 130 (2008) 3244-3245.
[45] R.M. Izatt, J.J. Christensen, J.H. Rytting, Sites and thermodynamic quantities associated
with proton and metal ion interaction with ribonucleic acid, deoxyribonucleic acid, and
their constituent bases, nucleosides, and nucleotides, Chem. Rev. 71 (1971) 439-481.

18
[46] H. Torigoe, A. Ono, T. Kozasa, HgII Ion specifically binds with T:T mismatched base
pair in duplex DNA, Chem. Eur. J. 16 (2010) 13218-13225.
[47] H. Torigoe, K. Kawahashi, A. Takamori, A. Ono, Novel strategy for single nucleotide
polymorphism (SNP) genotyping by heteroduplex analysis: specific stabilization of TT
mismatch base pair by mercury (II) cation and CC mismatch base pair by silver (I) cation,
Nucleosides Nucleotides Nucleic Acids 24 (2005) 915-917.
[48] A. Ono, H. Torigoe, Y. Tanaka, I. Okamoto, Binding of metal ions by pyrimidine base
pairs in DNA duplexes, Chem. Soc. Rev. 40 (2011) 5855-5866.
[49] J. Wu, F. Du, P. Zhang, I.A. Khan, J. Chen, Y. Liang, Thermodynamics of the interaction
of aluminum ions with DNA: Implications for the biological function of aluminum, J.
Inorg. Biochem. 99 (2005) 1145-1154.
[50] E. Stellwagen, Q. Dong, N.C. Stellwagen, Quantitative analysis of monovalent
counterion binding to random-sequence, double-stranded DNA using the replacement ion
method, Biochemistry 46 (2007) 2050-2058.

19
 16 16
Cmonolayer (b)
(a)
12 Rsolution RCT 12
-Zim / k cm 2

-Zim / k cm 2
Rx CPE
8 8

4 4

0 0
0 4 8 12 16 0 4 8 2 12 16
Zre / k cm
2 Zre / k cm

16 4
(c) (d)
12 3
RCT / k cm 2
-Zim / k cm 2

8 2

4 1

0 0
0 4 8 12 16 2 4 6
2 Number of T-T mismatches in loop
Zre / k cm

Fig. 1. Representative Nyquist plots (-Zim vs. Zre) for (a) DNA I (b) DNA II (c) DNA III films
before ( ) and after incubating ( ) in buffer solution (20 mM Tris-ClO4 buffer solution
containing 150 mM NaClO4 at pH = 7.4) containing 10 M Hg(II). Experimental data is shown
as scatters with calculated solid lines fitted using the equivalent circuit shown in the inset of Fig
1(a). Rsolution, Cmonolayer, RCT, Rx and CPE represent respective elements of solution resistance,
capacitance of DNA film, resistance of DNA film, resistance and constant phase element of the
pin holes. The measurement was carried out in an electrolyte containing. 1.0 mM [Fe(CN)6]3-/4-
as the redox probe in 20 mM Tris-ClO4 buffer solution containing 150 mM NaClO4 at pH = 7.4;
(d) Variation of RCT vs. number of T-T mispairs in the loop of hairpin DNAs.

20
 8 4
(a) 4 (b) (c)
3

2
6
-Zim / k cm 2

RCT / k cm
RCT / k cm 2
3
2
4
2
1
2
1
0

+
g +
+

+
+
+

Ag +
+

+
+

Pb 2
M 2
Co 2

Cd 2
Zn 2
Cu 2
Hg 2

Ca 2

Al 3
Ni 2
0
0 3 6 9 12 15 5 6 7 8
2
Zre / k cm -log [Hg(II)]/ log [M]

Fig. 2. (a) Representative Nyquist plots (-Zim vs. Zre) for films of DNA III before ( ) and after
incubating in buffer solution (20 mM Tris-ClO4 buffer solution containing 150 mM NaClO4 at
pH = 7.4) containing Hg(II) with concentrations ranging from 10-5 ( ), 10-6 ( ), 10-7 ( ) and 10-8
M ( ). Experimental data is shown as scatters with calculated solid lines fitted using the
equivalent circuit shown in the inset of Fig 1(a); (b) Relationship between RCT and
concentration of Hg(II); (c) Selectivity test of hairpin DNA III with 6 mispairs in the loop against
Hg(II) and other possible interfering metal ions (Co2+, Ni2+, Ca2+, Cu2+, Al3+, Mg2+, Cd2+, Zn2+,
Ag+, Pb2+). The selectivity test was carried out in 10 M metal ions. Error bars are derived from
a minimum of three electrodes.

21
 4 (a) -2 (b)
Hg (II)
Ellipticity / mdeg

Ellipticity (mdeg)
2 0

0
2

-2
4
-4
240 260 280 300 320 0 2 4 6 8
Wavelength / nm Mole equivalents of Hg(II)

Fig. 3. (a) CD spectra of DNA III at 1.0 M in the absence and presence of increasing
concentration of Hg(II) from 0.5 to 8.0 molar equivalents in 20 mM Tris-ClO4 buffer solution
containing 150 mM NaClO4 at pH = 7.4. (b) Varaition of ellipticity of DNA III vs. mole
equivalents of Hg(II).

22
 (a) 0.20
0.24 (b)
Hg (II)
0.21
Absorbance

0.20

Absorbance
0.22
0.16 0.23

0.12 0.24

0.08 0.25
240 255 270 285 0 2 4 6 8
Wavelength / nm Mole equivalents of Hg(II)

Fig. 4. (a) UV-visible spectra of DNA III at 1.0 M in the absence and presence of increasing
concentration of Hg(II) from 0.5 to 8.0 molar equivalents in 20 mM Tris-ClO4 buffer solution
containing 150 mM NaClO4 at pH = 7.4. (b) Variation of absorbance of DNA III vs. mole
equivalents of Hg(II).

23
 30
1.0
(a) (b) 25
(c)
0.8 0.03
20
Absorbance

First derivative

T m / C
0.6 0.02
15
0.4
0.01 10
0.2
5
Tm
0.0 0.00
0
15 30 45 60 75 90 15 30 45 60 75 90 2 4 6
Temperature / oC Temperature / oC Number of T-T mismatches in loop

Fig. 5. (a) Normalized UV melting curves of DNA III at 1.0 M in the absence ( ) and presence
( ) of 6.0 equivalents of Hg(II) in 20 mM Tris-ClO4 buffer solution containing 150 mM NaClO4
at pH = 7.4; (b) First derivative plot of absorbance vs. temperature and the separation of the
peaks gives change in melting temperature (Tm); (c) The melting temperature difference, Tm
vs. number of T-T mispairs in the loop of hairpin DNAs. Error bars are derived from a minimum
of three electrodes.

24
 0.0 -70
(a) (b)
0.3 -80
Heat R ate (J/sec )

H / kJ/mol
0.6 -90

0.9
-100

0 1000 2000 3000 4000 0 2 4 6 8 10

Time (sec) Molar ratio

Fig. 6. (a) Variation of heat rate vs. time for DNA III in presence of Hg(II); (b) ITC profile of the
interaction between Hg(II) and DNA III in 20 mM Tris-ClO4 buffer solution containing 150 mM
NaClO4 at pH = 7.4 and 298.15 K.

25
Scheme 1. Schematic illustration of the immobilized hairpin DNA based sensor for recognition
of Hg(II). (a) A gold electrode surface modified with hairpin DNAs; (b) The DNA modified
surface was back-filled with 6-mercaptohexanol; (c) The blocked DNA was exposed to the Hg
ions. The eT refers to electron transfer between gold electrode surface and redox probe in buffer
solution (20 mM Tris-ClO4 buffer containing 150 mM NaClO4 at pH = 7.4).

26
Table 1. Sequences of hairpin DNAs and their abbreviation employed in present work.
DNA Sequences No. of T-T mispairs Abbreviation
5-HO-(CH2)6-S-S-(CH2)6- 2 DNA I
TGTAGTTCCTTCTACA-3
5-HO-(CH2)6-S-S-(CH2)6- 4 DNA II
TGTAGTTTTCCTTTTCTACA-3
5-HO-(CH2)6-S-S-(CH2)6- 6 DNA III
TGTAGTTTTTTCCTTTTTTCTACA-3

Table 2. Equivalent circuit element values for DNA films in the absence and presence of binding
to Hg(II). *represents change in charge transfer resistance (RCT) values of DNAs before and after
incubating with 10 M Hg(II). RCT = RCT (after Hg immersion) RCT (before Hg immersion).
Rs Cmonolayer RCT Rx CPE n RCT*
(.cm2) (10-8 F/cm2) (.cm2) (.cm2) (10-7 F/cm2) (.cm2)
DNA I 2.2 3.50 8312 99.0 2.49 0.98 -
DNA I+Hg(II) 2.1 2.00 9531 91.2 2.55 0.97 1219
DNA II 2.5 2.58 10210 100.7 1.85 0.95 -
DNA II+Hg(II) 2.3 1.31 12880 75.5 1.51 0.96 2670
DNA III 2.0 3.26 11810 96.3 2.43 0.98 -
DNA III+Hg(II) 2.1 2.39 15330 92.2 2.07 0.95 3520

Table 3. ITC derived thermodynamic parameters: binding constant (ka), change in Gibbs free
energy (G), enthalpy change (H) and entropy change (S) for binding between the Hg(II) and
hairpin DNAs at 298.15 K and pH 7.4.
System ka (M-1) n G (kJ mol-1) H (kJ mol-1) S (J K-1 mol-1)
DNA I + Hg(II) 0.18106 2.3 -29.94 -14.58 51.54
DNA II + Hg(II) 1.95106 4.1 -35.88 -27.45 28.29
DNA III + Hg(II) 7.50107 5.9 -44.95 -39.95 16.78

27