Naked Eye DNA detection: Synthesis, characterization and DNA binding studies of a novel azo-guanidine

Article abstract of DOI:10.1016/j.saa.2012.12.020

A novel class of azo-guanidine compounds is introduced in this article. The novel compound 2-(2-hydroxyphenyl)-1-(phenylamino)-3-(phenylimino)guanidine (AG) was synthesized and well characterized by using different analytical instrumental techniques like elemental analysis, FTIR, ¹H and ¹³C NMR, UV-Visible spectroscopy and cyclic voltammetry. The new compound was found interacting with DNA and shows clear color change in the solution. The AG-DNA complex was qualitatively and quantitatively characterized with UV-Visible spectroscopy and cyclic voltammetry. Electrostatic mode of interaction, clear color change and moderate binding constant (K_b = 10⁴) indicate its potential use as DNA staining agent.

Full text of DOI:10.1016/j.saa.2012.12.020

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 105 (2013) 165–170
Contents lists available at SciVerse ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Naked Eye DNA detection: Synthesis, characterization and DNA binding
studies of a novel azo-guanidine
b,
b
b
Muhammad Jamil a, Ataf Ali Altaf b,c, , Amin Badshah , Shafiqullah , Ibrar Ahmad ,
⇑
⇑
Muhammad Zubair a, Saqib Kemal b, Muhammad Irshad Ali b
a Department of Chemistry, Govt. College University Faisalabad, Pakistan
b Department of Chemistry, Quaid-i-Azam University, Islamabad 45321, Pakistan
c Department of Chemistry, University of Malakand, Chakdara, Dir (lower), Pakistan
g r a p h i c a l a b s t r a c t
h i g h l i g h t s
UV–Visible absorption spectra of 70
ous ethanol (30% H2O:70% ethanol) buffer: 0.1 M phosphate buffer (pH 6.8), inset the visible picture of
70 M AG in the absence (Left) and presence (Right) of 140 M DNA in 30% aqueous ethanol (30%
lM AG in the absence and presence of 20–140 lM DNA in 30% aque-
"
"
"
"
"
A novel class of azo-guanidine
compounds is introduced in this
article.
The compound (AG) interacts with
DNA and gives clear color change in
70% ethanol.
The AG–DNA complex was
characterized with CV and UV–
Visible spectroscopy.
Electrostatic mode of interaction,
and moderate binding constant
(Kb = 104).
l
l
H2O:70% ethanol) buffer: 0.1 M phosphate buffer (pH 6.8).
Results indicate its potential use as
DNA staining agent.
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 16 August 2012
Received in revised form 29 November 2012
Accepted 6 December 2012
Available online 20 December 2012
A novel class of azo-guanidine compounds is introduced in this article. The novel compound 2-(2-
hydroxyphenyl)-1-(phenylamino)-3-(phenylimino)guanidine (AG) was synthesized and well character-
ized by using different analytical instrumental techniques like elemental analysis, FTIR, 1H and 13C
NMR, UV–Visible spectroscopy and cyclic voltammetry. The new compound was found interacting with
DNA and shows clear color change in the solution. The AG–DNA complex was qualitatively and quanti-
tatively characterized with UV–Visible spectroscopy and cyclic voltammetry. Electrostatic mode of inter-
action, clear color change and moderate binding constant (Kb = 104) indicate its potential use as DNA
staining agent.
Keywords:
Azo-guanidine
DNA detection
DNA binding
Ó 2012 Elsevier B.V. All rights reserved.
Naked Eye DNA detection
Introduction
Huge numbers of chemical and/or instrumental methods are avail-
able for the DNA detection. The phenanthridium dye, ethidium
The science of DNA sensing is always an open field of interest. It
is important in exploring the inherited secrete of the nature of life.
bromide (EtB), is the stain that is most commonly used for directly
detecting nucleic acids in electrophoretic gels [1]. This popularity
⇑
Corresponding authors. Address: Department of Chemistry, Quaid-i-Azam University, Islamabad 45321, Pakistan (A. Badshah.). Tel.: +92 5190642131; fax: +92
512873869.
E-mail addresses: atafali_altaf@yahoo.com (A.A. Altaf), aminbadshah@yahoo.com (A. Badshah).
1386-1425/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.saa.2012.12.020

166
M. Jamil et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 105 (2013) 165–170
is due in part to the fact that, although unbound EtB has significant
intrinsic fluorescence, the dye displays a 20–25-fold increase in
fluorescence upon intercalation into double-stranded (ds) regions
of nucleic acids [2]. EtB is used mainly in molecular biology labo-
ratories. It is a suspected reproductive toxicant and exposure to
it has the potential to negatively affect the human reproductive
system. The severity and nature of the adverse effect is variable
and can be influenced by factors such as sex, level of exposure
and individual sensitivity to the chemical. It is thought to act as
a mutagen because it intercalates ds-DNA, deforming the DNA
and could affect DNA biological processes, like DNA replication
and transcription [3–7]. The search for the alternatives is the keen
interest of biochemist to avoid the use of EtB. Many other dyes
have been reported in the past years to replace EtB and its adverse
side affects for example SYBR, TOTO, YOYO families of dyes [8–10].
Among these dyes SYBR Green-I is considered to be the best and
safe [1]. But in recent years, SYBR Green-I was found to be more
mutagenic than EtB to the bacterial cells exposed to UV (which
is used to visualize either dye) [7]. The more toxicity of SYBR Green
I might be due to its stronger binding with DNA than EtB [11]. We
consider the hypothesis, ‘‘weaker binding dyes with more observa-
ble change in any property would be the best and safe DNA sensing
agent’’. In the present study we have synthesized a novel azo-gua-
nidine dye and characterized its DNA binding behavior and binding
constant using normally used instrumental techniques like cyclic
voltammetry and UV–Visible spectroscopy [12–14].
icant yield of AG shows the success of the protocol employed for
the first time to synthesize such novel class of compounds (may
be named as azo-guanidine). The synthesized compound was char-
acterized in solid state by using FT-IR and elemental analysis as
well as in solution phase using NMR and UV–Visible spectroscopy.
The elemental analysis data shows that compound is highly
pure and confined to the purposed structure in solid phase as the
experimental elemental composition is in close agreement with
calculated values. In infra-red spectroscopic characterization a
sharp band at 3229 cmꢀ1 was observed that indicates the presence
of free hydrogen attached to nitrogen or oxygen (i.e. non H-bonded
NH and OH groups). N@N of AG gives stretching band at
2358 cmꢀ1, C@N appears at 1736 cmꢀ1. These N@N and C@N bands
shifted towards higher frequency than their normal absorption val-
ues might be originated from the extended conjugation in the
structure (Fig. 1). While the other CAH, CAN and CAC bands ap-
pear in their characteristic regions.
Solution chemistry
AG is well characterized in solution phase (NMR, UV–Visible
spectroscopy and electrochemical characterization with cyclic vol-
tammetery) along with solid phase (elemental analysis and infra
red spectroscopic) characterization. For solution phase character-
ization 1H and 13C NMR spectra were recorded in CDCl3 and the
data obtained is recorded in the experimental section. Signals at
2.904, 2.977 and 9.381 ppm may well be assigned to two NH and
an OH proton while the other multiplet signal is originated from
aromatic protons in the range of 7.142–7.806 ppm. In the 13CNMR
all the magnetically different carbons appeared at different posi-
tions. CN3 carbon appears as most de-shielded carbon at
166.6 ppm and the carbon attached to the OH group appeared at
151.2 ppm.
Results and discussion
Synthesis
2-(2-Hydroxyphenyl)-1-(phenylamino)-3-(phenylimino)guani-
dine (AG) was synthesized by a well known literature reported
guanylation method, our group have been reported many similar
compounds using the same protocol [15–20]. AG was synthesized
by the coupling of o-aminophenol and dithizone in the presence of
Hg(II) and triethylamine in DMF (Scheme 1). Hg(II) reacts there to
abstract sulfur while triethylamine capture HCl formed. The signif-
In UV–Visible spectrum (Fig. 2), AG has three strong bands with
high molar absorption coefficient (
11200 Mꢀ1 cmꢀ1 at 222, 264 and 498 nm, a relatively low absorp-
tivity (
= 5728 Mꢀ1 cmꢀ1) band appeared at 570 nm as a shoulder.
The high absorptivity bands in the UV-region of spectrum may
confidently be assigned to the aromatic phenyl ring based
transitions, and N@N, C@N based
pꢁ transitions.[21] In the visi-
ble region strong band at 498 nm with absorptivity
= 10571 Mꢀ1 cmꢀ1) can be attributed to the pꢁ transitions in
e) in the range of 10500–
e
p–
pꢁ
p
–
(e
p–
the azo (N@N) group with highly extended conjugation over the
seventeen atoms as shown in Fig. 1 and the shoulder at 570 may
originated from n – pꢁ transitions in the azo (N@N) group [22].
The electrochemical properties of the present compound was
investigated by CV on a glassy carbon electrode in 30% aqueous
ethanol (30% H2O:70% ethanol) 100 mV sꢀ1, supporting electrolyte:
0.1 M TBAP, in the concentration of 1 ꢂ 10ꢀ3 M vs. Standard Calo-
mel Electrode (SCE). In the cathodic direction from +0.4 V to
+0.65 V at scan rate of 100 mV sꢀ1, the CV of AG is characterized
by one cathodic waves (at about 0.58 V) and flat line (no waves)
in the anodic direction as depicted by cyclic voltammograms given
in Fig. 2. No redox couple was observed in the voltamogram only a
single oxidation peak observed in the CV graph as like most of the
organic compounds. This oxidation peak may be originated from
the oxidation of azo (N@N) group. The oxidation potential value
is in agreement with the literature reported values of azo (N@N)
group.[23,24] The appearance of only one oxidation peak indicates
the irreversible electrochemical process [25].
Scheme 1. Synthesis of 2-(2-hydroxyphenyl)-1-(phenylamino)-3-(phenylimino)
guanidine (AG).
HO
N
H
N
N
H
N
N
DNA binding studies
Cyclic voltammeteric titration
The cyclic voltammetric behavior of AG in the absence and pres-
Fig. 1. The extended resonance in the AG structure.
ence of 40 lM DNA at bare GCE is shown in Fig. 3. The voltammo-

M. Jamil et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 105 (2013) 165–170
167
Fig. 2. (Left) UV–Visible absorption spectra of 70
l
M AG in 30% aqueous ethanol (30% H2O:70% ethanol); (Right) Cyclic voltammogram of 100
l
M AG on a polished GC
electrode in 30% aqueous ethanol (30% H2O:70% ethanol) 100 mV sꢀ1, supporting electrolyte: 0.1 M TBAP.
gram without DNA (Fig. 2b) featured a well defined stable oxida-
tion peak at the potential value of 0.578 V. By the addition of
40 lM DNA into 100 lM AG (Fig. 3) the cathodic peak potential
(appeared at 0.536 V) was shifted by 42 mV in the negative going
direction and Ipa was dropped by 21.4%. The substantial diminution
in peak current is attributed to the formation of slowly diffusing
AG–DNA supramolecular complex due to which the concentration
of the free compound (mainly responsible for the transfer of cur-
rent) is lowered. The mode of drug-DNA interaction can be judged
from the variation in peak potential.
In general the positive shift (anodic shift) in formal potential is
caused by the intercalation of the drug into the double helical
structure of DNA, while negative shift is observed for the electro-
static interaction of the cationic drug DNA backbone [26]. So the
obvious negative peak potential shift (cathodic shift) in the CV
behavior of AG by the addition of DNA is attributable to the elec-
trostatic interaction (H-bonding) between AG and the DNA. The
cathodic peak potential shift further indicates that AG left over is
easier to oxidize in the presence of negative environment of
DNA. Because oxidized (AG+) form is bound weakly to DNA than
the reduced (neutral) form. As the ability of hydrogen bonding de-
creased on positively charging the compound in AG+ state. Further
addition of DNA diminishes the oxidation peak which supports the
participation of AG in hydrogen bonding with DNA, and not let
electrons to go out of the system (Fig. 4).
Fig. 4. Cyclic voltammograms of 100
100
lM AG in the absence and presence of 20–
l
M DNA on a polished GC electrode in 30% aqueous ethanol (30% H2O:70%
ethanol) 100 mV sꢀ1, buffer: 0.1 M phosphate buffer (pH 6.8), supporting electro-
lyte: 0.1 M TBAP.
AG–DNA complex formation were calculated according to the fol-
lowing Eq. (1) [25]:
I2 ¼ ðI2 ꢀ I2Þ1=Kb½DNAꢃ þ I2 ꢀ ½DNAꢃ
ð1Þ
o
o
where Kb is the binding constant, I and Io are the peak currents with
and without DNA. A plot of I2 vs. (I2o ꢀ I2)/[DNA] gave a straight line
(Fig. 5a) with a slope equal to the reciprocal of binding constant,
Kb = 1.0 ꢂ 104 which is less than the binding constant of EtB (KEB
more than 105 Mꢀ1) with DNA as reported in previous reports
[27,28].
Based upon the decrease in peak current of AG by the addition
of different concentration of DNA, the binding constants, Kb for the
Fig. 3. Cyclic voltammograms of 100 lM AG in the absence and presence of 40 lM
DNA on a polished GC electrode in 30% aqueous ethanol (30% H2O:70% ethanol)
100 mV sꢀ1, buffer: 0.1 M phosphate buffer (pH 6.8), supporting electrolyte: 0.1 M
TBAP.
Fig. 5a. The plot of I2 versus (Io2 ꢀ I2)/[DNA] for the calculation of DNA binding
constant from Cyclic voltammetric titration.

168
M. Jamil et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 105 (2013) 165–170
Scheme 2. Photoisomerization in AG many authors reported such kind photoiso-
merization in similar compounds [31–33].
interaction and binding strength. AG interacts with DNA and gives
a clear color change visible with Naked Eye as shown in Fig. 6. UV–
Visible spectroscopy is an effective tool for quantification of bind-
ing strength of DNA with small molecules. The effect of different
Fig. 5b. Plot of Cb/Cf versus DNA concentration for the calculation of the size of
binding site.
concentration of DNA (20–140
spectrum of 70 M AG is shown in Fig. 6.
Different
pꢁ and n–pꢁ transitions appeared in the UV–Visible
lM) on the electronic absorption
l
For the determination of binding site size the following equa-
tion was used [29]:
p–
spectrum of the compound, as discussed earlier. Upon interaction
with DNA, AG reveals characteristic changes in the UV–Visible
spectrum. In the spectral response of AG different type of changes
occur after the addition of DNA. Both hypo and hyper chromic
changes observed along with hypsochromic shift at 498 nm. There
exist three isosbestic points (at 292, 402 and 526 nm) in the spec-
tral picture of the AG–DNA reaction. The region below 300 nm is
characteristic region of DNA where DNA nucleotides absorbs
strongly, so the isosbestic point at 292 nm appears due to the
increasing concentration of DNA which reveals the increasing
absorbance in this region. The DNA interaction causes hypochro-
mism above 526 nm and below 402 nm. The shoulder above
526 nm appears due to the azo based n–pꢁ transition which shows
hypochromism after DNA interaction. This hypochromism may
arise due to the hydrogen bonding of azo nitrogen with DNA and
lone pair nitrogen becomes busy in the H-bonding and n–pꢁ tran-
sition depressed. As result of hydrogen bonding the molecule may
become hindered molecule which increased the planarity of the
molecule and stop the isomerism process shown in scheme 2
[31–33].
Cb=Cf ¼ Kb ðfree base pairsÞ=s
ð2Þ
where s is the binding site size in terms of base pairs (bp). Mea-
suring the concentration of DNA in terms of base pairs concentra-
tion, the concentration of the base pairs can be expressed as [DNA]/
2. So Eq. (2) can be written as:
Cb=Cf ¼ Kb½DNAꢃ=2s
ð3Þ
Cf and Cb denote the concentration of the free and DNA bound spe-
cies respectively. The Cb/Cf ratio was determined by the Eq. (4) given
below [30]:
Cb=Cf ¼ ðIo ꢀ IÞ=I
ð4Þ
where Io and I stands for the peak current of AG in the absence and
presence of DNA, Putting the value of Kb as calculated according to
above Eq. (1), the binding site size was obtained from the plot of Cb/
Cf versus [DNA] (Fig. 5b). Putting the value of K = 1.0 ꢂ 104 Mꢀ1 as
calculated according to Eq. (1), the binding site size of 0.6 was ob-
tained from the plot (Fig. 5b) of Cb/Cf versus [DNA]. The small value
of s indicates electrostatic interaction of AG with DNA. Such an
interaction may induce perturbation in the normal functioning of
DNA which could presumably culminate in the prevention of repli-
cation and ultimate cell death [26].
This planarity helps the penetration of AG into DNA strained
structure which results in the expansion of helical structure of
DNA. The planarity of the molecule also increase the chances of
p
-stacking which ultimately cause the hypsochromic shift of
10 nm in
p–
pꢁ transition. The maximum absorption of AG at this
UV–Visible spectrometric titration
The interaction of AG with DNA was also studied by UV–Visible
absorption titration for getting further clues about the mode of
transition exhibited slight hypsochromic shift and pronounce
hyperchromic shifts by the incremental addition of DNA. The
expansion in the structure of either the compound alone and/or
DNA after the formation of compound-DNA complex may result
in hyperchromism. Hyperchromism of the same kind has also been
reported by many other authors.[34–36] Based upon the decrease
in absorbance at 570 nm and increase in absorbance at 498 nm the
binding constant was calculated according to the following host
guest Eq. (5) [37]:
Ao
eG
ꢀ
eG
eG eHꢀG
1
¼
þ
ð5Þ
A ꢀ Ao eHꢀG
ꢀ
eG K½DNAꢃ
where Ao and A are the absorbance of free compound and com-
pound-DNA complex respectively, eG and eHꢀG is the molar extinc-
tion coefficients of free compound and compound-DNA complex
respectively. The slope to intercept ratio of the plot Ao/(A ꢀ Ao) ver-
sus 1/[DNA] yielded the binding constant Fig. 7, Kb = 1.4 ꢂ 104 Mꢀ1
,
which is close to the value of Kb (1 ꢂ 104 Mꢀ1) obtained from CV.
The moderate binding constant is indicative of electrostatic interac-
Fig. 6. UV–Visible absorption spectra of 70
20–140 M DNA in 30% aqueous ethanol (30% H2O:70% ethanol) buffer: 0.1 M
phosphate buffer (pH 6.8), inset the visible picture of 70 M AG in the absence (Left)
and presence (Right) of 140 DNA in 30% aqueous ethanol (30% H2O:70%
ethanol) buffer: 0.1 M phosphate buffer (pH 6.8).
lM AG in the absence and presence of
tion. The Gibbs energy change (
D
G = ꢀRT ln Kb) of approximately
l
ꢀ23.7 kJ/mol at 25 °C signifies the spontaneity of AG–DNA interac-
tion. The binding behavior comparative data of AG, EtB and some
other dyes given in Table 1 indicates that AG having the least bind-
l
lM

M. Jamil et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 105 (2013) 165–170
169
Experimental
Materials
Mercuric chloride, dithizone, triethylamine, o-aminophenol,
potassium dihydrogen phosphate, sodium hydroxide and Tetrabu-
tylammoniumperchlorate were purchased from Sigma Aldrich.
Deoxyribonucleic acid Sodium salt was purchased from acros
organics. Solvents such as ethanol, DMF and methanol were puri-
fied before use according to the standard reported protocols
[60,61]. Melting points were measured with BIO COTE Model
SMP10 melting point apparatus. FT-IR and NMR spectra were ob-
tained with Thermo Scientific Nicolet-6700 FT-IR and BRUKER
AVANCE NMR spectrometer respectively.
Fig. 7. The plot of Ao/(A ꢀ Ao) versus 1/[DNA] for the calculation of DNA binding
constant from UV–Visible spectroscopic titration.
Table 1
Synthesis of 2-(2-hydroxyphenyl)-1-(phenylamino)-3-
(phenylimino)guanidine (AG)
Comparative data of some normally used DNA staining agents and AG.
Sr.
No.
Dye
DNA
interaction
mode
Binding
Detection
limit [40–
44]
Ref.
constant
To the 50 ml DMF solution of 100 mmol dithizone, 100 mmol
mercuric chloride were added with constant stirring in a round
bottom flask fitted with reflux condenser, magnetic stirrer and
hot plate. A 25 ml DMF solution of o-aminophenol (100 mmol)
and triethylamine (200 mmol) were added drop wise to the dithiz-
one and mercuric chloride solution. After complete addition the
reaction mixture were refluxed for 6 h and extant of reaction were
monitored on TLC. The reaction was completed with the formation
black precipitates. The reaction mixture was filtered using sintered
glass crucible. The solid crude product was then collected by pour-
ing filtrate in ice cold water. The crude product was re-crystallized
from hot methanol as crimson red fibrous crystals. Yield 78%; m.p
240–243 °C; Anal. Calc for C19H17N5O: C, 68.87; H, 5.17; N, 21.13,
(Mꢀ1
104
)
1
AG
Electrostatic
15 ng/
l
Lb
This
work
[27,28]
[1,45]
2
3
EtB
SYBR
Green I
TOTO-I
YOYO-I
Intercalation
>105
1 ng/
60 pg/
l
l
L
Groove binding >106
L
4
5
Intercalation
Intercalation
10ꢀ9a
20 pg/
0.5 ng/
l
L
L
[46]
[47–
49]
>1010
l
6
Methylene
blue
Intercalation/
groove binding
>104
5 ng/l
L
[27,50]
a
The dissociation constant value.
b
The detection limit calculated by the absorbance change at 484 and 604 nm
wavelength.
Found C, 68.84; H, 5.15; N, 21.17%. FTIR (
t
, cmꢀ1); 3229, 3048,
2358, 1736, 1654, 1592, 1524, 1494, 1455, 1434, 1349, 1269,
1189, 1132, 1069, 745, 676; UV–Visible kmax (nm, (
e
) Mꢀ1cmꢀ1
)
ing constant and electrostatic mode of interaction might be the
least toxic and the potential DNA detecting agent. As electrostatic
interacting agents can cause just conformational changes in DNA
and are less mutagenic than the intercalating agents [38,39].
222 (11171), 264 (10685), 498 (10571), 570 (5728); 1HNMR
(300 MHz, ppm, d); 2.904(bs, 1H), 2.977 (bs, 1H), 7.142–7.806 (m,
14H), 9.381 (bs, 1H); 13CNMR (75 MHz, ppm, d); 117.6 (2C),
122.3, 124.3, 126.1 (2C), 127.3, 128.0 (2C), 129.3, 129.5 (2C),
131.5, 136.4, 137.2, 138.1, 144.1, 151.2, 166.6 (CN3).
Conclusion
Methods
In this paper, the synthesis, characterization and the DNA inter-
action of a noval azo-guanidine dye was comprehensively studied
by spectrometry and electrochemical methods. The obvious hypo-
chromic effect in absorption spectra and negative cathodic peak
currant shifting suggested electrostatic binding mode between
AG and DNA. The presence of isosbestic points in the UV–Visible
spectroscopic behavior provides the evidence of the availability
of two sites of interactions in the system. The strong color change
in the visible picture of the reaction between AG and DNA provides
the Naked Eye detection of DNA under the studied reaction condi-
tions. These observations suggest the potential of AG as DNA stain-
ing agent. The binding constant data also suggesting, AG might be
the better staining agent over normally used EtB due to weaker
binding ability and clear color change [51]. Furthermore, the
DNA staining agents like EtB cannot be used for the detection of
DNA in ethanolic solution [52–55] whereas the novel AG will
potentially do this job. Also the AG will be helpful for the quantifi-
cation of DNA, as in the protocols used for the extraction of DNA,
cold ethanol is used for the precipitation of DNA [56–59]. So the
novel AG will be helpful for estimation of DNA move to the ethanol
solution during precipitation. In future we are interested in the
experimental toxicity, protein interaction and the other evolutions
related to the use of AG as replacement of EtB.
DNA binding studies
Sodium salt of DNA was used as available from Acros Organics.
Its stock solution was prepared by dissolving small amount in dou-
bly distilled water. Concentration of DNA was measured spectro-
photometerically by using 6600 Mꢀ1 cmꢀ1 molar absorption
coefficient at 260 nm. The DNA used was sufficiently pure and free
from proteins evidenced from the ratio of absorbance at 260 and
280 nm (A260/A280 = 1.85) [62]. The system was buffered at
pH = 7 by phosphate buffer (0.1 M KH2PO4 + 0.1 M NaOH). Stock
solution was stored below 4 °C and used with in 4 days.
UV–Visible spectroscopy
Absorption spectra were measured on a UV–Visible spectropho-
tometer; Shimadzu 1800. The experiments involving the interac-
tion of the compound AG with DNA were carried out in doubly
distilled buffer (0.1 M KH2PO4 + 0.1 M NaOH, pH = 7) using 20%
aqueous ethanol. The electronic spectrum of a known concentra-
tion of the AG was obtained without DNA. The spectroscopic re-
sponse of the same amount of the AG was then monitored by
varying the conc. of DNA. All of the samples were allowed to equil-
ibrate for 5 min prior to every spectroscopic measurement [37].

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Voltammetric experiments were performed using EZstat-Pro
Potential/ Galvanostatic of Nuvant System, Inc.USA. Measurements
were carried out in a conventional three electrode cell which con-
sisted of saturated calomel electrode (SCE) as a reference electrode,
a thin Pt wire of thickness 0.5 mm with an exposed end of 10 mm
as the counter electrode and a bare glassy carbon electrode (GCE)
with a geometric area of 0.071 cm2 as the working electrode. For
electrochemical measurements TBAP was used as supporting elec-
trolyte and the test solution was kept in an electrochemical cell
(model K64 PARC) under constant temperature. The procedure
was repeated for systems with constant concentration of AG and
varying concentration of DNA at constant pH = 7. The working elec-
trode was cleaned after every electrochemical assay.
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