Synthesis, Characterization and DNA-Binding Studies of Hydroxyl Functionalized Platinum(II) Salphen Complexes

Article abstract of DOI:10.1007/s10895-017-2035-0

The platinum(II) salphen complex N,N′-Bis-4-(hydroxysalicylidene)-phenylenediamine-platinum(II); (1) and its two derivatives containing hydroxyl functionalized side chains N,N′-bis-[4-[[1-(2-hydroxyethoxy)] salicylidene] phenylenediamine-platinum(II); (2)

Full text of DOI:10.1007/s10895-017-2035-0

J Fluoresc
DOI 10.1007/s10895-017-2035-0
ORIGINAL ARTICLE
Synthesis, Characterization and DNA-Binding Studies
of Hydroxyl Functionalized Platinum(II) Salphen Complexes
Shahratul Ain Mohd Sukri1 & Lee Yook Heng1 & Nurul Huda Abd Karim1
Received: 23 January 2016 /Accepted: 3 February 2017
#
Springer Science+Business Media New York 2017
Abstract The platinum(II) salphen complex N,N′-Bis-
4-(hydroxysalicylidene)-phenylenediamine-platinum(II); (1)
and its two derivatives containing hydroxyl functionalized
side chains N,N′-bis-[4-[[1-(2-hydroxyethoxy)] salicylidene]
phenylenediamine-platinum(II); (2) and N,N′-bis-[4-[[1-(3-
hydroxypropoxy)] salicylidene] phenylenediamine-
platinum(II); (3) were synthesized and characterized. The
be valuable for the potential use of the platinum(II) salphen
complex as an optical DNA biosensor for the detection of
porcine DNA in food products.
.
Keywords Platinum(II) salphen Phosphorescence
.
.
.
.
complex Calf thymus DNA Porcine DNA DNA binding
Optical DNA biosensor
1
structures of the complexes were confirmed by H and 13C
NMR spectroscopy, FTIR, ESI-MS and CHN elemental anal-
yses. The effects of the hydroxyl substituent on the spectral
properties and the DNA binding behaviors of the Pt(II) com-
plexes were explored. The binding mode and interactions of
these complexes with duplex DNA (calf thymus DNA and
porcine DNA) and also single-stranded DNA were studied
by UV-Vis and emission DNA titration. The complexes inter-
act with DNA by intercalation binding mode with the binding
constants in the order of magnitude (Kb = 104 M−1, CT-DNA)
and (Kb = 105 M−1, porcine DNA). The intercalation of the
complex in the DNA structure was proposed to happen by π-π
stacking due to its square-planar geometry and aromatic rings
structure. The phosphorescence emission spectral characteris-
tics of Pt(II) complexes when interacted with DNA have been
studied. Also, the application of the chosen hydroxypropoxy
side chains complex (3) as an optical DNA biosensor, specif-
ically for porcine DNA was investigated. These findings will
Introduction
Transition metal complexes have been established to have
great ability to interact non-covalently with deoxyribonucleic
acids (DNA) via different mechanism such as intercalation,
groove binding or external electrostatic binding [1]. It is very
important to conduct binding studies of small molecules to
DNA for the development of new DNA molecular probes
and therapeutic reagents [2].
Schiff base salphen ligand “N,N′-phenylenebis(salicylideneimine)”
complexes have shown such strong DNA binding proper-
ties [3–5]. The interactions of square-planar and square-
pyramidal metal-salphen complexes with duplex DNA
and human telomeric quadruplex DNA have been studied
by Vilar et al. in which the results highlighted that square-
planar metal-salphen complexes are much more excellent
quadruplex DNA stabilizer and binder due to its planar
geometry structures which can provide optimal binding
for π-π stacking with the DNA [3]. Moreover, the metal
center in salphen complexes plays important roles in “orga-
nizing” the salphen ligands into specific geometrical con-
formations ideally suited to π-π stack onto the DNA (e.g.:
square-planar and square-based pyramidal geometries) [4].
Metal center also have an important electronic role of
“pulling” electrons from coordinated aromatic salphen li-
gands, thus making them more electron-deficient and
Electronic supplementary material The online version of this article
(doi:10.1007/s10895-017-2035-0) contains supplementary material,
which is available to authorized users.
* Nurul Huda Abd Karim
nurulhuda@ukm.edu.my
1
School of Chemical Sciences and Food Technology, Faculty of
Science and Technology, Universiti Kebangsaan Malaysia,
43600 Bangi, Selangor, Malaysia

J Fluoresc
therefore more likely to be involved in π-π interactions
with the DNA nucleobases [4]. Other Schiff bases can have
different types of binding modes with duplex DNA, for
instance nickel salen [6], copper and zinc Schiff base com-
plexes [7] which can interact with the double helix surface
by means of intercalation and groove binding, respectively.
Platinum complex is highly phosphorescent [8] and widely
used in optical devices and have diverse applications in elec-
troluminescence, photovoltaics, optical limiting,
photocatalysis, and molecular probes [9]. In addition, plati-
num has recently gained much attention in materials science
and display technology since the discovery of highly efficient
phosphorescent organic light-emitting diodes (OLEDs) based
on platinum(II) complexes [10]. Moreover, d8 square-planar
platinum(II) complexes with NN or CN bidentate; NNN,
CNN or NCN terdentate; and NNNN or ONNO tetradentate
conjugated aromatic ligands have shown to be highly phos-
phorescent at room temperature and widely used in OLEDs
and in oxygen (O2) sensors [8]. It was also reported that the
platinum(II) metal-salphen complexes have square-planar co-
ordination geometry around the metal cation, which can en-
hance the π-π stacking and increase the binding affinity with
the DNA [5]. In this study, we decided to synthesis an emis-
sive metal-salphen complex that can strongly bind to DNA.
This complex will then be used as an optical indicator for
DNA biosensor. Despite the fact that the platinum metal com-
plexes’ high optical properties could further enhance spectro-
scopic studies of DNA binding, no attempts have been made
to explore the potential of the platinum(II) salphen complex as
a new optical DNA indicator sensor material for DNA biosen-
sor. Therefore, it has become our interest to investigate this
matter.
Pork is a potential adulterant in food especially in meatballs
and sausages due to its availability at cheaper prices. The
mixing of pork or its derivatives in foods is a serious matter
as it is not permissible by certain religious laws. In addition,
unconscious consumption of pork may also cause allergic
reactions in some individuals. It contains high levels of cho-
lesterol and saturated fats that can be especially harmful for
people with diabetes and cardiovascular disease [11]. Thus,
the identification of pork in foods is very important for meat
authentication and for meeting consumers’ demand for pro-
tection against falsely labeled foods. In this regard, reliable
methods for identification of porcine derivatives in food prod-
ucts are necessary for the analysis of food ingredients.
in food products such as, for instance, the gold nanoparticle
sensor recently developed by Ali et al. [13]. Moreover, metal
complex-based sensors could provide the advantage that the
measurements can easily be run using optical instruments
such as UV-Vis and fluorescence spectroscopy that are readily
available in a chemical laboratory.
DNA-intercalation spectroscopy has been a particular
interest among researchers. This is because many
intercalators-DNA binding agents have very important
functions such as anticancer drugs [15], photocleavage
agents [16], DNA structure probes [17–19] and molecular
DNA light switches [20, 21]. From the DNA binding spec-
troscopy studies, the binding mechanism and the detailed
structure of the binding behavior properties obtained can be
used to target specific sequence of DNA structures in order
to develop a potential anticancer drugs or DNA molecular
probes [15, 22–26], in which many of these functions based
on the DNA-intercalation mechanism [23–26].
The molecular DNA light switches properties of the
ruthenium(II) and cobalt(II) polypyridyl complexes were also
determined from the DNA-intercalation spectroscopy studies
of the complexes upon binding with the given DNA struc-
tures. These complexes showed a remarkable and intense lu-
minescence and fluorescence enhancement in the presence of
DNA, but lacks of luminescence and fluorescence in the ab-
sence of DNA due to its intercalation DNA binding properties,
where it can be further use as fluorescence DNA probes or
sensors [20, 21, 23, 27–29].
Moreover, DNA-intercalators binding molecules are also
widely used in the design of optical DNA biosensors. For
example, fluorescence dyes intercalators such as ethidium
bromide (EB), acridine orange (AO) and thiazole orange
(TO) and also metallointercalator such as ruthenium(II)
polypyridine complex, [Ru(bpy)2(dppz)]2+ [30–35]. These
fluorescence intercalators act as an optical DNA hybridization
indicators to detect the DNA hybridization event in the duplex
DNA formed between the two DNA strands that are compli-
mentary to each other [32, 33, 36]. The intercalators will only
produce fluorescence signals in the presence of hybridized
DNA after intercalation of the binding molecules into the con-
jugated bases of the duplex DNA has occurred [31–33, 37].
These fibre-optic DNA biosensors are based on the monitor-
ing of fluorescence signal that is emitted from the fluorescence
intercalator-DNA binding agents, to show that hybridization
of DNA has been formed between the probe DNA sequence
and the target DNA sequence [32, 33]. The complimentary
oligonucleotide that have undergo DNA hybridization will
show an enhancement of fluorescence signal while the non-
complimentary oligonucleotide without any hybridization will
not show any enhancement of fluorescence signal [36]. Since
intercalators-DNA binding molecules have such great poten-
tial as optical DNA biosensors, we are greatly interested to
study the DNA binding properties of the platinum(II) salphen
Current practice is to use the polymerase chain reaction
(PCR) method for detection of porcine DNA in food products.
This is the most common and effective method because it
offers high levels of sensitivity [12]. Unfortunately, this meth-
od is time-consuming, tedious, and can only be run by skilled
technicians [13, 14]. An alternative to this conventional PCR
method is to develop porcine DNA biosensors that can offer
an easy, rapid and simpler verification method of pork content

J Fluoresc
complexes and investigate its potential application as an opti-
cal indicator sensor material for DNA biosensor.
AGC AAT TGC CAT AGT CCA CCT A-(3′)-591, (b)
Gallus gallus cytb (chicken ssDNA) 567- (5′)-CGC AGG
TAT TAC TAT CAT CCA CCT C-(3′)-591 were obtained
from Sigma (USA). 1H and 13C NMR spectra were recorded
on a Bruker AVANCE III 600 MHz spectrometer equipped
with a cryoprobe. Infrared Spectra were recorded on a Perkin-
Elmer FTIR 1720 spectrometer as KBr discs between 4000
and 400 cm−1. Elemental analyses were recorded on a Thermo
Finnigan EA 1112 CHNS. UV-Vis spectra were recorded in
1 cm path length quartz cuvettes using a Perkin Elmer Lambda
25 UV-Vis spectrometer. Emission DNA titration were record-
ed in 1 cm path length quartz cuvettes using a Perkin Elmer
LS55 Luminescence Spectrophotometer at a scan rate of
200 nm/min. The slit width was 5 nm for both excitation
and emission. The excitation wavelength used was 370 nm.
The emitted light was collected at an angle of 90° relative to
the excitation light.
We report herein the synthesis, characterization, and DNA
binding properties of two platinum(II) salphen complexes
with the hydroxyl functionalized side chains substituent on
their salphen ligand core. The complexes’ interactions with
DNA are studied to determine whether it can act as an
intercalator agent due to its planar aromatic rings structure
[38]. This paper is focused on exploring the effects of the
hydroxyl substituents (with varying lengths of the alkoxy side
chain arms) on the spectral properties and DNA binding be-
haviors of the complexes. The need to vary different substitu-
ents on the ligand is because it can create some differences in
the space configuration and also the electron density distribu-
tion of the complexes, therefore variation in the spectral prop-
erties and different DNA binding behaviors of the complexes
can be obtained [39]. Additionally, the binding affinities and
selectivity of the platinum(II) salphen complexes towards du-
plex porcine DNA over single-stranded DNA (represented by
beef ssDNA) and CT-DNA are determined, with their duplex
and single-stranded DNA affinities reported herein. The
strong binding affinity and high selectivity of the complex
towards porcine DNA is established in this work, so that it
can be used as a potential porcine DNA biosensor. We also
show that the platinum(II) complexes in this work are emis-
sive, which inspire us to carry out optical phosphorescence
DNA selectivity study as a preliminary sensor studies for the
potential use of the complex as an optical indicator sensor
material for porcine DNA biosensor, which can be used in
future food industry analysis. Studies on the DNA binding
of these complexes are very important in the development of
new fluorescence (or phosphorescence) optical DNA sensors
and DNA molecular probes. Therefore, it is important that we
need to establish the binding mode and affinity of the potential
metal complexes to porcine DNA first, and choose the best
metal complex intercalator before we fabricate the optical in-
dicator onto solid support for the development of porcine
DNA biosensor in our future works.
General Procedure1
Synthesis of Salphen Ligand: N,N
′-Bis-4-(Hydroxysalicylidene)-Phenylenediamine
The salphen ligand was prepared using a condensation reac-
tion of o-phenylenediamine with 2,4-dihydroxybenzaldehyde
in a 1:2 M ratio in ethanol. The mixture of 2,4-
dihydroxybenzaldehyde (0.22 g, 1.60 mmol) and 1,2-
phenylenediamine (0.09 g, 0.80 mmol) in ethanol was
refluxed at 79 °C for 4 h. The ethanol was evaporated under
reduced pressure. The resulting solid was washed with diethyl
ether and water and collected by filtration to obtain the
salphen ligand as a yellow-orange precipitate. Yield: 0.12 g
(45%). IR (KBr pellet)/cm−1: 3063 (C-H aromatic), 1609
(C = N), 1574 (C = C aromatic), 1546 (C = C aromatic),
1
1502 (C = C aromatic), 1209 (C-O), 1123 (C-O). H NMR
(600 MHz, DMSO-d6): δ 13.38 (s, 1H, H8, −OH phenol),
10.26 (s, 1H, H7, −OH phenol), 8.75 (s, 1H, H3, N = C-H),
3
7.43 (d, JHH = 9 Hz, 1H, H4, Ar-H), 7.38(dd, 3JHH = 6 Hz,
3
4JHH = 3.6 Hz, 1H, H2, Ar-H), 7.32 (dd, JHH = 6 Hz,
3
Experimental
4JHH = 3 Hz, 1H, H1, Ar-H), 6.39 (dd, JHH = 8.4 Hz,
4
4JHH = 2.4 Hz, 1H, H5, Ar-H), 6.28 (d, JHH = 2.4 Hz, 1H,
Materials, Methods and Instrumentation
H6, Ar-H). 13C NMR (600 MHz, DMSO-d6): δ 163.80,
163.39, 163.09, 142.28, 134.91, 127.52, 119.95, 112.76,
108.30, 102.87 ppm. ESI-MS calculated for C20H16N2O4
(M)+: 348.36 a.m.u. Found: 349.09 a.m.u. Anal. Calc. for
C20H16N2O4: C 68.96, H 4.63, N 8.04. Found: C 67.69, H
4.58, N 8.03%.
Analytical grade reagents and chemicals were obtained from
Sigma Aldrich Chemical Co. (USA). Single-stranded porcine
DNA corresponding to the 25 nt complimentary target porcine
sequence 567-(5′)-TAC CGC CCT CGC AGC CGT ACA
TCT C-(3′)-591 of the Sus scrofa cytochrome b (cytb) gene,
its probe porcine sequence strand (3′)-ATG GCG GGA GCG
TCG GCA TGT AGA G-(5′), calf thymus-DNA (CT-DNA)
and the single-stranded DNA (non-complimentary DNA
strands) (a) Bos taurus cytb (beef ssDNA) 567- (5′)-CAT
1 Chemical shift in units of parts-per-million, ppm (δ), proton-proton coupling
constants (JHH), dimethyl sulfoxide (DMSO).

J Fluoresc
Synthesis of Platinum(II) Salphen Complex: N,N
′-Bis-4-(Hydroxysalicylidene)
-Phenylenediamine-Platinum(II) ( 1 )
H6, Ar-H), 6.47 (dd, 3JHH = 8.7 Hz, 4JHH = 2.1 Hz, 1H, H5, Ar-
H), 4.97 (s, 1H, H13, −OH), 4.08 (t, 2H, H11, −CH2), 3.76 (t,
2H, H12, −CH2). 13C NMR (600 MHz, DMSO-d6): δ 166.80,
165.44, 149.90, 144.71, 137.28, 127.62, 116.93, 116.63,
108.64, 103.03, 70.20, 59.85. ESI-MS calculated for
C24H22N2PtO6 (M + K)+: 668.49 a.m.u. Found: 668.07 a.m.u.
The synthesis of the platinum salphen complex was carried
out using the method described by Wu et al. [9] and Zhou et al.
[8] with slight modifications. The salphen ligand ( 0.28 g,
0.80 mmol) was dissolved in 30 mL acetonitrile and sodium
acetate which act as a base was added to the salphen ligand
mixture and stirred for 5 min. Potassium tetrachloroplatinate
(0.66 g, 1.60 mmol) in DMSO (2 mL) was added and stirred at
30 °C for 72 h. The solid was then filtered and washed with
diethyl ether and water to obtain the platinum(II)-salphen
complex as an orange-brownish precipitate. Yield: 0.09 g
(22%). IR (KBr pellet)/cm−1: 3368 (OH alcohol/phenol),
3066 (C-H aromatic), 1601 (C = N), 1581 (C = C aromatic),
1545 (C = C aromatic), 1493 (C = C aromatic), 1440 (C = C
Synthesis of Platinum(II) Salphen Complex Hydroxyl
Functionalized Derivative: N,N
′-Bis-[4-[[1-(3-Hydroxypropoxy)] Salicylidene]
Phenylenediamine-Platinum(II) ( 3 )
The synthesis of the platinum salphen complex hydroxyl side
chains derivative was carried out by a slight modification from
the methods described by Vilar et al. [3] The platinum(II)
salphen complex (0.27 g, 0.49 mmol) was reacted with 3-
chloro-1-propanol (206.05 μL, 2.47 mmol) and potassium
carbonate (0.55 g, 3.94 mmol). It was stirred at 90 °C for
72 h in DMF solvent (30 mL). After this period of time the
salts were removed by filtration and the DMF was evaporated
under reduced pressure. The resulting solid was washed with
water to yield a dark brown precipitate. Yield: 0.11 g (35%).
IR (KBr pellet)/cm−1: 3400 (OH alcohol/phenol), 2916 (C-H
alkane), 1603 (C = N), 1578 (C = C aromatic), 1517 (C = C
aromatic), 1463 (C = C aromatic), 1420 (C = C aromatic),
1189 (C-O), 1129 (C-O), 1024 (C-O ether). 1H NMR
(600 MHz, DMSO-d6): δ 9.27 (s, 1H, H3, N = C-H), 8.34
1
aromatic), 1180 (C-O), 1128 (C-O). H NMR (600 MHz,
DMSO-d6): δ 10.50 (s, 1H, H7, −OH phenol), 9.19 (s, 1H,
H3, N = C-H), 8.30 (dd, 3JHH = 6 Hz, 4JHH = 3.6 Hz, 1H, H2,
3
Ar-H), 7.66 (d, JHH = 8.4 Hz, 1H, H4, Ar-H), 7.34 (dd,
4
3JHH = 6.3 Hz, JHH = 3.3 Hz, 1H, H1, Ar-H), 6.44 (d,
3
4JHH = 2.4 Hz, 1H, H6, Ar-H), 6.34 (dd, JHH = 8.7 Hz,
4JHH = 2.1 Hz, 1H, H5, Ar-H). 13C NMR (600 MHz,
DMSO-d6): δ 166.93, 165.03, 149.79, 144.83, 137.81,
127.30, 116.61, 108.98, 104.76. ESI-MS calculated for
C20H14N2PtO4 (M + Na)+: 564.41 a.m.u. Found: 564.05
a.m.u. Anal. Calc. for C20H14N2PtO4 ∙ 3H2O: C 40.34, H
3.38, N 4.70. Found: C 40.40, H 3.51, N 3.48%.
3
4
(dd, JHH = 6 Hz, JHH = 3.6 Hz, 1H, H2, Ar-H), 7.71 (d,
3JHH = 8.4 Hz, 1H, H4, Ar-H), 7.37 (dd, JHH = 6.3 Hz,
3
4JHH = 3.3 Hz, 1H, H1, Ar-H), 6.58 (d, JHH = 2.4 Hz, 1H,
4
Synthesis of Platinum(II) Salphen Complex Hydroxyl
Functionalized Derivative: N,N
′-Bis-[4-[[1-(2-Hydroxyethoxy)] Salicylidene]
Phenylenediamine-Platinum (II) ( 2 )
H6, Ar-H), 6.45 (dd, 3JHH = 8.7 Hz, 4JHH = 2.1 Hz, 1H, H5, Ar-
H), 4.63 (s, 1H, H14, −OH), 4.13 (t, 2H, H11, −CH2), 3.58 (t,
2H, H13, −CH2), 1.90 (m, 2H, H12, −CH2). 13C NMR
(600 MHz, DMSO-d6): δ 166.92, 165.46, 150.03, 144.71,
137.27, 127.62, 116.93, 116.71, 108.69, 103.00, 65.32,
57.69, 32.32. ESI-MS calculated for C26H26N2PtO6 (M +
K)+: 696.54 a.m.u. Found: 696.07 a.m.u.
The synthesis of the platinum salphen complex hydroxyl side
chains derivative was carried out by a slight modification from
the methods described by Vilar et al. [3] The platinum(II)
salphen complex (0.32 g, 0.58 mmol) was reacted with 2-
chloro-1-ethanol (199.42 μL, 2.98 mmol) and potassium car-
bonate (0.66 g, 4.76 mmol). It was stirred at 90 °C for 72 h in
DMF solvent (30 mL). After this period of time the salts were
removed by filtration and the DMF was evaporated under
reduced pressure. The resulting solid was washed with water
and n-hexane to yield a dark brown precipitate. Yield: 0.05 g
(15%). IR (KBr pellet)/cm−1: 3369 (OH alcohol/phenol), 2916
(C-H alkane), 1603 (C = N), 1578 (C = C aromatic), 1521
(C = C aromatic), 1458 (C = C aromatic), 1421 (C = C aro-
matic), 1188 (C-O), 1129 (C-O), 1026 (C-O ether). 1H NMR
(600 MHz, DMSO-d6): δ 9.29 (s, 1H, H3, N = C-H), 8.34 (dd,
DNA Binding Experiments
Single-stranded porcine DNA, corresponding to the 25 nt
probe sequence (3′)-ATG GCG GGA GCG TCG GCA TGT
AGA G-(5′) of the Sus scrofa cytochrome b (cytb) gene and its
complementary strand 567-(5′)-TAC CGC CCT CGC AGC
CGTACATCT C-(3′)-591 were dissolved in the 10 mM Tris-
HCl buffer at pH 7.4 to yield a 2 mM solution. Both single-
stranded DNA fragments were annealed by heating to 90 °C
for 5 min and then cooled to room temperature overnight to
form duplex porcine DNA. The stock solution of CT-DNA
was prepared by dissolving it in the 10 mM Tris-HCl buffer
at pH 7.4. The ratio of the absorbance of CT-DNA solution at
260 nm and 280 nm was 1.9, which is more than 1.8, indicat-
ing that CT-DNA was not contaminated by proteins [40]. The
4
3JHH = 6 Hz, JHH = 3.6 Hz, 1H, H2, Ar-H), 7.73 (d,
3
3JHH = 8.4 Hz, 1H, H4, Ar-H), 7.36 (dd, JHH = 6.3 Hz,
4
4JHH = 3.3 Hz, 1H, H1, Ar-H), 6.59 (d, JHH = 2.4 Hz, 1H,

J Fluoresc
concentration of CT-DNA was calculated from its absorption
intensity at 260 nm using a molar absorption coefficient value
of 6600 M−1 cm−1 [41]. Excitation wavelengths of 370 nm
were used for DNA emission titration studies. For DNA se-
lectivity studies, the single-stranded DNA (non-
complimentary DNA strands) used are: a) Bos taurus cytb
(beef ssDNA) 567- (5′)-CAT AGC AAT TGC CAT AGT
CCA CCT A-(3′)-591 and b) Gallus gallus cytb (chicken
ssDNA) 567- (5′)-CGC AGG TAT TAC TAT CAT CCA
CCT C-(3′)-591 [13].
complex (1) displays similar resonance signals, integration
and splitting patterns to those of the ligand except for the
resonance signal at 13.38 ppm corresponding to the free OH
protons which is previously observed in the spectrum of the
free ligand, and is now absent in the spectrum of the complex.
This confirms that deprotonation of the OH groups has oc-
curred due to the coordination of oxygen atoms to the
platinum(II) metal ion center. There is also significant differ-
ence found in their chemical shifts between the spectra of the
ligand and its complex. The singlet resonance signal at
9.19 ppm corresponding to N = C-H imine proton of the
complex is shifted downfield compared to its original reso-
nance signal at 8.75 ppm of the free salphen ligand, which also
supports the coordination of the nitrogen atoms of the imine
groups from the salphen ligand to the platinum(II) metal ion
center. 13C NMR spectrum was assigned using 2D HSQC
NMR which displays the exact carbon signal that belongs to
a certain proton signal for a simple and easy assignment of
carbons. The infrared spectrum of the complex shows absorp-
tion bands of azomethine –CH = N-, C-O, C = C aromatic, C-
H aromatic and OH functional groups at 1601, 1180–1128,
1581–1440, 3066 and 3368 cm−1, respectively. The mass
spectrum of the complex shows the pseudo-molecular ion
[M + Na]+ peak at m/z 564.05. The presence of sodium ion
is expected as a result of the mass spectrometer instrument
itself. The difference between the theoretical calculation
C20H14N2PtO4 [M + Na]+ (564.41 a.m.u) and the experimen-
tal value was 0.36. Overall, these results support the coordi-
nation of four donor atoms N2O2 from the salphen ligand to
the platinum(II) metal ion center, indicating a square-planar
geometry and aromatic rings structure.
Results and Discussion
Synthesis and Characterization of Platinum(II) Salphen
Complex and Its Hydroxyl Side Chains Derivatives
The platinum(II) salphen complex was synthesized via con-
densation reaction of 2,4-dihydroxylbenzaldehyde and o-
phenylenediamine with potassium tetrachloroplatinate to ob-
tain N,N′-Bis-4-(hydroxysalicylidene)-phenylenediamine-
platinum(II); non-side chains complex (1). The reaction
schemes for the synthesis of salphen ligand and its
platinum(II) salphen complex are illustrated in Fig. 1.
The signal of the aldehyde proton at 9.92 ppm is absent in
the 1H NMR spectrum of the salphen ligand which confirms
that the aldehyde group of the starting compound 2,4-
dihydoxybenzaldehyde has been converted to N = C-H imine
group. Moreover, the 1H NMR spectrum of the salphen ligand
displays correct integration and multiplets pattern (supple-
mentary data). The resonance signal at 13.38 ppm correspond-
ing to the free OH protons is observed, which indicates that
the ligand is free from coordination with the metal ion center.
After complexation, the 1H NMR spectrum of non-side chains
The hydroxyl side chains derivatives of this complex
were prepared by adding 2-chloro-1-ethanol to obtain
N,N′-bis-[4-[[1-(2-hydroxyethoxy)] salicylidene]
Fig. 1 Reaction scheme for the
salphen ligand and the non-side
chains platinum(II) salphen
complex (1)

J Fluoresc
phenylenediamine-platinum(II); hydroxyethoxy side
chains complex (2) and 3-chloro-1-propanol to obtain
N,N′-bis-[4-[[1-(3-hydroxypropoxy)] salicylidene]
phenylenediamine-platinum(II); hydroxypropoxy side
chains complex (3) respectively. Generally, we are interest-
ed to study the effect of longer alkoxy side chains from the
hydroxyl derivatives of the platinum(II) complex on the
binding affinity to DNA. The reaction schemes for the
synthesis of the hydroxyethoxy side chains complex (2)
and hydroxypropoxy side chains complex (3) are illustrat-
ed in Fig. 2. The 1H NMR spectrum of hydroxyethoxy side
chains complex (2) displays correct integration and multi-
plets pattern. The hydroxyethoxy side chains arms were
proven to be successfully attached to the platinum(II)
salphen complex since the 1H NMR spectrum of
hydroxyethoxy side chains complex (2) displays the for-
mation of new proton signals around the alkyl region at
3.76 ppm (triplet, 2H, −CH2), and 4.08 ppm (triplet, 2H,
side chain -CH2CH2OH and –CH2CH2CH2OH respectively.
Moreover, there is also another additional new peaks absorp-
tion bands at 1026 cm−1 and 1024 cm−1 for hydroxyethoxy
side chains (2) and hydroxypropoxy side chains (3) com-
plexes respectively, which refer to C-O aryl ether stretching
-ROCH2CH2OH and –ROCH2CH2CH2OH (R = aromatic
benzene) after the addition of the hydroxyethoxy and
hydroxypropoxy side chains respectively, in which these
peaks were absent in the infrared spectrum of the non-side
chains complex (1).
The mass spectrum of the hydroxyethoxy side chains (2)
complex shows the pseudo-molecular ion [M + K]+ peak at
m/z 668.07. The presence of potassium ion is expected as a
result of the mass spectrometer instrument itself. The differ-
ence between the theoretical calculation C24H22N2PtO6 [M +
K]+ (668.49 a.m.u.) and the experimental value was 0.42.
Similarly, the mass spectrum of the hydroxypropoxy side
chains (3) complex also shows the pseudo-molecular ion
[M + K]+ peak at m/z 696.07. The difference between the
theoretical calculation C26H26N2PtO6 [M + K]+ (696.54
a.m.u.) and the experimental value was 0.47. Overall, these
results indicate that the hydroxyl side chains derivatives of the
Pt(II) salphen complex which contain hydroxyethoxy side
chains and hydroxypropoxy side chains were successfully ob-
1
−CH2). The H NMR spectrum of hydroxypropoxy side
chains complex (3) also displays correct integration and
multiplets pattern. The hydroxypropoxy side chains arms
were also proven to be successfully attached to the
platinum(II) salphen complex since the 1H NMR spectrum
of hydroxypropoxy side chains complex (3) displays the
formation of new proton signals around the alkyl region at
1.90 ppm (quantet, 2H, −CH2), 3.58 ppm (triplet, 2H,
−CH2), and 4.13 ppm (triplet, 2H, −CH2). The 13C NMR
spectra of both hydroxyethoxy side chains (2) and
hydroxypropoxy side chains (3) complexes display the al-
kyl carbons after the attachment of hydroxyethoxy side
chains arms at 59.85 and 70.20 ppm for hydroxyethoxy
side chains complex (2) and hydroxypropoxy side chains
arms at 32.32, 57.69 and 65.32 ppm for hydroxypropoxy
side chains complex (3).
1
tained. The H NMR, 13C NMR, FTIR and ESI-MS spectra
for non-side chains (1), hydroxyethoxy side chains (2) and
hydroxypropoxy side chains (3) complexes are available in
the supplementary information on the Journal’s website.
UV-Vis DNA Titration
The UV-Vis absorption spectra of the non-side chains (1),
hydroxyethoxy side chains (2) and hydroxypropoxy side
chains (3) complexes show three intense bands at 304–
316 nm, 364–368 nm and 421–431 nm (Figs. 3, 4 and 5).
The absorption bands at 304–316 nm region are assigned to
the intraligand-charge-transfer (ILCT) π-π* transition which
involves molecular orbitals localized on the C = N group and
The infrared spectra of both hydroxyethoxy side chains (2)
and hydroxypropoxy side chains (3) complexes show addi-
tional new peaks absorption bands at 2916 cm−1 which refer
to C-H (alkane) stretching or υ(CH2) after the addition of the
Fig. 2 Reaction scheme for the
hydroxyethoxy side chains
platinum(II) salphen complex (2)
and hydroxypropoxy side chains
platinum(II) salphen complex (3)

J Fluoresc
a
% Hypochromicity : 12%
Red Shift : 2.5 nm
b
% Hypochromicity : 8%
Red Shift : 1.5 nm
c
% Hypochromicity : 12%
Red Shift : 2.0 nm
Fig. 3 Absorption spectral changes of the complexes (30 μM) in Tris-
HCl buffer (pH 7.4) in the absence and presence of increasing
concentrations of CT-DNA. The inset graph in the top right corner
shows the fitting of the absorbance data that was used to obtain the
binding constant. a non-side chains (1) (CT-DNA : 2.71 × 10−5 M -
4.34 × 10−4 M), (b) hydroxyethoxy side chains (2) (CT-DNA:
4.34 × 10−6 M - 7.8 × 10−5 M), (c) hydroxypropoxy side chains (3)
(CT-DNA: 4.34 × 10−6 M - 7.8 × 10−5 M)
the benzene ring. On the other hand, absorption bands in the
region of 364–368 nm are assigned to the n-π* transition
involving molecular orbitals of the C = N chromophore and
the benzene ring, while the lowest energy band at 421–431 nm
region are attributed to the metal-to-ligand-charge-transfer
(MLCT) transition [8].

J Fluoresc
a
% Hypochromicity : 13%
Red Shift : 3.0 nm
b
% Hypochromicity : 6%
Red Shift : 1.5 nm
c
% Hypochromicity : 10%
Red Shift : 2.0 nm
Fig. 4 Absorption spectral changes of the complexes (30 μM) in Tris-
HCl buffer (pH 7.4) in the absence and presence of increasing
concentrations of porcine DNA. The inset graph in the top right corner
shows the fitting of the absorbance data that was used to obtain the
binding constant. a non-side chains (1) (porcine DNA: 2 × 10−6 M -
6 × 10−5 M), (b) hydroxyethoxy side chains (2) (porcine DNA:
2 × 10−6 M - 6 × 10−5 M), (c) hydroxypropoxy side chains (3) (porcine
DNA: 2 × 10−6 M - 6 × 10−5 M)
In the DNA binding studies, the absorbance measurements
were performed using constant complex concentration
(30 μM) while varying the concentration of DNA until
saturation. The spectrum of the complex was recorded after
each addition of the respective DNA. Generally, binding of
intercalative compounds to DNA can be characterized by

J Fluoresc
a
% Hypochromicity : 6%
Red Shift : 0 nm
b
% Hypochromicity : 6%
Red Shift : 0 nm
c
% Hypochromicity : 10%
Red Shift : 0 nm
Fig. 5 Absorption spectral changes of the complexes (30 μM) in Tris-
HCl buffer (pH 7.4) in the absence and presence of increasing
concentrations of single-stranded DNA (beef ssDNA). The inset graph
in the top right corner shows the fitting of the absorbance data that was
used to obtain the binding constant. a non-side chains (1) (beef ssDNA:
2 × 10−6 M - 2.6 × 10−5 M), (b) hydroxyethoxy side chains (2) (beef
ssDNA: 2 × 10−6 M - 1.2 × 10−5 M), (c) hydroxypropoxy side chains (3)
(beef ssDNA: 2 × 10−6 M - 3 × 10−5 M)
absorption spectral titrations where decreased absorbance
(hypochromism) and shift to longer wavelengths
(bathochromic shift or red shift) are observed [40]. The hypo-
chromic effect is the spectral characteristics of double helix

J Fluoresc
DNA due to the contraction of the helix along its axis after
insertion of intercalative compound into the DNA double he-
lix. Meanwhile, bathochromic shift or red shift occurs because
of the conformational changes of DNA which indicate a new
compound “complex-DNA” has been formed [42].
The percentage of hypochromicity can be calculated ac-
cording to Eq. (1):
side chains (1), hydroxyethoxy side chains (2) and
hydroxypropoxy side chains (3) complexes with duplex CT-
DNA are 3.21 × 104 M−1, 1.82 × 104 M−1 and 6.99 × 104 M−1
respectively (Table 1), which is in agreement to other
established metal complex intercalators in which the order of
magnitude are 104 M−1 [22, 23, 39, 45]. The results showed
that hydroxypropoxy side chains complex (3) displays the
h i g h e s t b i n d i n g a f f i n i t y w i t h C T - D N A
(Kb = 6.99 × 104 M−1) than the other two complexes.
Moreover, hydroxypropoxy side chains complex (3) also ex-
hibit the highest affinity to CT-DNA when comparing the
intrinsic binding constants with those of other DNA-
intercalative Ru(II) complexes (1.1–4.8 × 104 M−1) [23],
Co(II) complex (3.75 × 104 M−1) [22] and Ru(II) complexes
(3.73–5.91 × 104 M−1) [39]. We can deduce that the non-side
chains (1), hydroxyethoxy side chains (2) and
hydroxypropoxy side chains (3) complexes bind to CT-DNA
by intercalation and that hydroxypropoxy side chains complex
(3) binds more strongly than non-side chains (1) and
hydroxyethoxy side chains (2) complexes.
εfree−εbound
% hyperchromicity ¼
 100
ð1Þ
εfree
From the UV-Vis DNA titrations, all the complexes ex-
hibit hypochromism (6% -13%) and red shifts (1.5–3 nm)
at 364–368 nm region with increasing amounts of DNA
(Table 1). These results are in agreement to other
established intercalators (hypochromicity =3% - 39%, red
shift =1–4 nm) [43] and (hypochromicity =9% - 41%, red
shift =1–3 nm) [44].
The intrinsic binding constant Kb of platinum(II) salphen
complex–DNA was determined according to Eq. (2):
Successful porcine DNA sensor should not only interact
strongly with their target porcine DNA sequence but also ex-
hibit the highest selectivity for duplex porcine DNA versus
other duplex DNA (CT-DNA) and single-stranded DNA (rep-
resented by beef ssDNA). Therefore, for comparative pur-
poses the UV-Vis spectroscopic titrations were carried out
for all these three DNA to establish whether the complexes
have the strongest binding affinity with porcine DNA. The
binding constants of all the complexes with duplex porcine
DNA are in the order of magnitude 105 M−1 which is in agree-
ment to other metal complex intercalators (105 M−1) [46, 47].
The Kb values of the non-side chains (1), hydroxyethoxy side
chains (2) and hydroxypropoxy side chains (3) complexes
DNA DNA
1
¼
þ
ð2Þ
Δεap
Δε
Δε Â Kb
where the apparent molar extinction coefficient,
Δεap = |εA-εF|, εA = Aobserved /[Complex], Δε = |εB-εF|. εF
and εB represent molar extinction coefficients for the free
platinum complex and for the DNA bound platinum complex,
respectively. In order to elucidate the binding affinity of the
complex to DNA, the intrinsic binding constant Kb was deter-
mined by monitoring the changes of maximum absorption
bands centred at around 364–368 nm region. From the plotted
graph of [DNA]/(Δεap) versus [DNA], the y-intercept is equal
to 1/(Δεap x Kb) whereas the slope is equal to 1/Δεap. Kb
values can be determined by dividing the slope value by the
y-intercept.
with duplex porcine DNA are 2.21 × 105 M−1
,
1.38 × 105 M−1 and 3.06 × 105 M−1 respectively. Again,
hydroxypropoxy side chains complex (3) displays the highest
binding affinity with duplex porcine DNA (Kb = 3.06 × 105
M−1) than the other two complexes. Meanwhile, the Kb values
of the complexes with single-stranded beef DNA are in the
To compare quantitatively the binding affinity of the three
complexes to DNA, the intrinsic binding constants Kb of the
complexes to duplex CT-DNA were obtained with increasing
concentration of DNA. The binding constants (Kb) of non-
Table 1 Summary of binding affinity of non-side chains (1), hydroxyethoxy side chains (2) and hydroxypropoxy side chains (3) complexes with
duplex CT-DNA, duplex porcine DNA and single-stranded DNA (beef ssDNA)
Complex
CT-DNA Kb (M−1) dsDNA
PORCINE Kb (M−1) dsDNA
BEEF Kb (M−1) ssDNA
Non-Side Chains (1)
(368 nm)
Kb: 3.21 × 10 4 3.00
% Hypochromicity: 12%
Red Shift: 2.5 nm
Kb: 2.21 × 10 5 0.26
% Hypochromicity: 13%
Red Shift: 3.0 nm
Kb: 8.56 × 10 4 0.30
% Hypochromicity: 6%
Red Shift: 0 nm
Hydroxyethoxy Side Chains (2)
(364 nm)
Kb: 1.82 × 10 4 0.01
% Hypochromicity: 8%
Red Shift: 1.5 nm
Kb: 1.38 × 10 5 0.35
% Hypochromicity: 6%
Red Shift: 1.5 nm
Kb: 7.35 × 10 4 0.01
% Hypochromicity: 6%
Red Shift: 0 nm
Hydroxypropoxy Side Chains (3)
(368 nm)
Kb: 6.99 × 10 4 0.57
% Hypochromicity: 12%
Red Shift: 2.0 nm
Kb: 3.06 × 10 5 0.07
% Hypochromicity: 10%
Red Shift: 2.0 nm
Kb: 7.27 × 10 4 0.12
% Hypochromicity: 10%
Red Shift: 0 nm

J Fluoresc
order of magnitude 104 M−1. Although the Kb values with the
beef ssDNA are in the same magnitude as the duplex CT-
DNA (104 M−1), there were no red shifts for beef ssDNA
unlike those observed for duplex CT-DNA and duplex porcine
DNA. Therefore, beef ssDNA probably exhibit a binding
mode other than intercalation such as electrostatic binding or
groove binding (surface binding) [48, 49]. Best selectivity
towards duplex porcine DNA (Kb = 105 M−1) over single-
stranded DNA (beef ssDNA, Kb = 104 M−1) and duplex CT-
DNA (Kb = 104 M−1) was obtained by one order of magnitude
higher for the porcine DNA. Therefore, from the binding con-
stants obtained from the three DNA, it can be concluded that
the highest binding affinity and selectivity towards duplex
porcine DNA is established.
The observed spectroscopic characteristics suggest that the
complex interacts with duplex CT-DNA and duplex porcine
DNA through a mode that involves a π-π stacking interaction
between the aromatic rings of salphen and the base pairs of
DNA. Our results support the theory of Liu & Sadler [38],
which states that the square-planar platinum(II) complexes
containing heterocyclic aromatic ligands non-covalently bind
to DNA duplexes by intercalating between the base pairs.
Remarkably, the attachment of [Hydroxypropoxy] side chains
arms to the platinum(II) salphen complex can increase the
binding affinity of the complex to DNA and is the best
DNA binder among the other two complexes.
Emission DNA Titration
Moreover, it was observed that the addition of the increas-
ing lengths of the alkoxy side chains arms of the hydroxyl
derivatives can reduce the binding strength of the complexes
to the single-stranded DNA compared to the non-side chains
complex (1). For instance, the non-side chains complex (1)
has the strongest binding strength (Kb = 8.56 × 104 M−1) with
single-stranded beef DNA. Meanwhile, hydroxyethoxy side
chains complex (2) with the shorter hydroxyl functionalized
alkoxy side chains arms have a weaker DNA binding
(Kb = 7.35 × 104 M−1) while hydroxypropoxy side chains
complex (3) with the longer alkoxy side chains arms has the
most weakest DNA binding (Kb = 7.27 × 104 M−1), thus
making it most selective to porcine dsDNA.
From the above results in general, hydroxypropoxy side
chains complex (3) with the longer alkoxy side chains
[Hydroxypropoxy] arms seem to have the highest binding
affinity to both CT-DNA and porcine DNA. Therefore, we
can summarize that the highest degree of binding affinity of
all the complexes to both duplex porcine DNA and CT-DNA
are in the order of complex hydroxypropoxy side chains
(3) > non-side chains (1) > hydroxyethoxy side chains (2).
Moreover, hydroxypropoxy side chains complex (3) also has
the weakest binding affinity with single-stranded beef DNA
compared to the other two complexes, which succeeded our
aim to have a lower binding with single-stranded DNA as
much as possible while achieving the highest binding affinity
with target duplex porcine DNA. In the design of duplex por-
cine DNA sensor it is highly desirable to reduce the binding
affinity of the complex to single-stranded DNA so as to in-
crease the selectivity for target duplex porcine DNA versus
non-target single-stranded DNA.
All the compounds have been tested with different wavelength
with reference to the lambda maximum obtained in the UV-
VIS spectrums of the compounds. However, only the excita-
tion wavelength of 370 nm gives the highest and strongest
emission signals intensities for all the compounds.
Therefore, it was chosen as the best excitation wavelength
and this excitation wavelength was used for all the
compounds.
The same optical alignment was used for all samples
throughout the experiments. Moreover, the baseline signals
were first recorded and have been normalized. We have done
the baseline without the DNA, without the metal complex, and
found that the emission signals obtained were due to the effect
of the excitation at that certain wavelength.
The complexes emit phosphorescence emission at 570 nm
when excited at 370 nm in DMSO solvent. The intensity of the
emission in DMSO solvent for non-side chains (1),
hydroxyethoxy side chains (2) and hydroxypropoxy side
chains (3) complexes (30 μM) are 160, 130 and 130 respec-
tively (Supplementary data, Fig. S15). In order to conduct
emission DNA titration and to study the effect of the increas-
ing amount of DNA on the phosphorescence properties of the
complex, the complexes must be diluted in Tris-HCl buffer
(30% DMSO, 70% buffer). In Tris- HCl buffer, the phospho-
rescence emission intensity for non-side chains (1),
hydroxyethoxy side chains (2) and hydroxypropoxy side
chains (3) complexes are 9, 66 and 65 respectively (Fig. 6).
Non-side chains complex (1) seems to have the lowest emis-
sion and this is probably because the complex is very sensitive
to the presence and concentration of water, being almost
quenched completely (Intensity: 9) in the aqueous solution
of Tris-HCl buffer. This phenomenon is similar to the Ru(II)
complexes which showed the progressive decrease of the
emission intensity in MeCN upon the addition of H2O [23].
A similar effect of the emission properties was also observed
for [Ru(phen)2(qdppz)]2+ which was non-luminescence in
aqueous MeCN (10% H2O) [50]. Therefore, the lack of lumi-
nescence in water or Tris-HCl buffer for non-side chains
The UV-Vis DNA titration results show that there is an
interaction between the electronic states of the chromophores
in the salphen conjugates with DNA [1]. This complex can
intercalate with the conjugated DNA bases due to its planar
structures and aromatic rings structure. Square-planar geome-
try can give planar structures, hence the planarity can provide
a suitable environment for insertion between the conjugated
base pairs of the DNA double helix during intercalation [38].

J Fluoresc
of the charge-transfer (CT) state and facilitate deactivation via
metal-centered excited state [51].
a
Emission titration experiments were performed using a
constant complex concentration (30 μM) and varying the por-
cine DNA concentration (from 2 × 10−6 M to 6 × 10−5 M),
where the ratio of complex to the porcine DNA is 1:2. The
addition of increasing amounts of porcine DNA to non-side
chains (1), hydroxyethoxy side chains (2) and
hydroxypropoxy side chains (3) complexes showed a signifi-
cant marked increase in the phosphorescence intensity with
the emission enhancement value of 1.15 (15%), 1.30 (30%)
and 1.57 (57%) times larger respectively, which indicate the
interaction of the complexes with porcine DNA (Fig. 6).
Higher emission enhancements were observed for the hydrox-
yl substituent hydroxyethoxy side chains (2) and
hydroxypropoxy side chains (3) complexes compared to the
non-side chains complex (1). Moreover, the values of emis-
sion enhancement for hydroxyethoxy side chains (2) and
hydroxypropoxy side chains (3) complexes are similar to
those observed for other Ru(II) complexes DNA-
intercalators in which the emission intensities were enhanced
by 1.3–1.6 times higher [52] than those in the absence of
DNA. The emission enhancements indicate that the com-
plexes can strongly interact with DNA and can be protected
from solvent water molecules by the hydrophobic environ-
ment inside the DNA helix [45], with hydroxypropoxy side
chains complex (3) being protected more efficiently than
hydroxyethoxy side chains (2) and non-side chains (1) com-
plexes. This suggest that the complexes can insert between
DNA bases deeply and that hydroxypropoxy side chains com-
plex (3) can bind to DNA more strongly than the other two
complexes, since the hydrophobic environment inside the
DNA helix reduces the accessibility of solvent water mole-
cules to the complex and the complex mobility is restricted at
the binding site, which leads to the decrease of the vibrational
modes of relaxation [39, 52]. The binding of the compounds
to DNA leading to a significant increase of emission intensity
also agrees with those observed for other typical intercalators
[43]. Nyarko et al. also reported the same phenomenon for
Pd(II)(TMPyP)4+ and Pt(II)(TMPyP)4+ complexes where the
increased phosphorescence and fluorescence emission in the
presence of DNA was due to the shielding of the intercalated
porphyrin by the DNA thus preventing it from reacting with
molecular oxygen dissolved in water [53]. In addition, it was
reported that if the complex interacts with DNA through clas-
sical intercalation, the emission intensity increases as the com-
plex gets into a hydrophobic environment inside DNA.
Considering all of these factors, it is suggested that the com-
plex does bind to DNA by a classical intercalative mode [22,
46]. The highest phosphorescence enhancements are in the
order of complex hydroxypropoxy side chains (3)-
> hydroxyethoxy side chains (2) > non-side chains (1).
Since hydroxypropoxy side chains complex (3) can bind to
15 %
30 %
b
c
57 %
Fig. 6 Phosphorescence emission spectra of the platinum(II) salphen
complexes (30 μM) in the Tris-HCl buffer (pH 7.4) in the absence and
presence of increasing concentrations of porcine DNA (2 × 10−6 M to
6 × 10−5 M). λ excitation =370 nm. λ emission =570 nm. The slit width
was 5 nm for both excitation and emission, with a scan rate of 200 nm/
min. (a) non-side chains (1), (b) hydroxyethoxy side chains (2), (c)
hydroxypropoxy side chains (3)
complex (1) maybe due to the mechanism similar to that of the
ruthenium complex mentioned above. This observation is also
consistent with Pt(trpy)OH+ which showed no significant
emission in water due to solvent effects that elevate the energy

J Fluoresc
duplex porcine DNA more strongly than the other two com-
plexes, and also displays the highest value of emission en-
hancement, therefore hydroxypropoxy side chains complex
(3) is the best candidate chosen as an optical phosphorescent
indicator for porcine DNA biosensor among the three
complexes.
hydrophobic environment of the DNA and the subsequent
intercalation of hydroxypropoxy side chains complex (3) into
the DNA bases as mentioned previously. Any emissive DNA
intercalator will be able to show the response reported in the
main figure of the manuscript (Fig. 7). Meanwhile the
quenching effect or the decreased emission intensity for the
non-complimentary DNA (beef and chicken DNA) was prob-
ably due to self-stacking of some free bases in the compound
along DNA surface [46, 53]. The same phenomenon was also
observed for electrostatic binding mode of cobalt(II) Schiff
base complex where the emission decrease with the addition
of DNA [48]. In addition, the emission quenching could also
be due to the photoelectron transfer from the guanine base of
DNA to the excited MLCT state of the complex as observed
by [Co(bzimpy)2], [Ru(bzimpy)2]2+, [Ru(TAP)3]2+ and
Pt(trpy)OH+ complexes [49, 51, 54, 55]. The mismatch beef
and chicken DNA cannot form duplex DNA since they are not
complimentary to the probe porcine DNA, therefore no hy-
bridization and intercalation of the complex can happen and
only single-stranded DNA were present. As a consequence,
the complex cannot be protected by the hydrophobic environ-
ment from the hybridized duplex DNA, thus giving rise to
other modes of DNA binding with the single-stranded non-
complimentary DNA such as electrostatic binding or groove
binding and at the same time allowing the complex to interact
with the free guanine bases that can act as a quencher. Because
of the differences in the phosphorescence spectral characteris-
tic of the complex when interacted with DNA, this will allow
for an easy and rapid sensor method to detect and distinguish
porcine DNA among the different DNA, where the compli-
mentary (target porcine DNA) signal will be enhanced while
the non-complimentary signals will be quenched. Since the
complex showed selectivity for hybridized duplex porcine
DNA over non-hybridized single-stranded DNA, these
Preliminary DNA Selectivity Sensor Solution Study
with Porcine, Beef and Chicken DNA
The sensor solution study of the chosen hydroxypropoxy side
chains complex (3) was conducted to investigate its selectivity
to the complimentary (target porcine ssDNA) versus non-
complimentary (beef and chicken ssDNA). 1 mM of
hydroxypropoxy side chains complex (3) was prepared in
THF -Tris HCl buffer solution (30% THF, 70% buffer) and
the concentration of the probe porcine DNA and the compli-
mentary or non-complimentary DNA were both fixed at
4 μM. Three vials tubes of 1 mM of hydroxypropoxy side
chains complex (3) were prepared with each tube containing
porcine DNA probe. The vials were then added with compli-
mentary (target porcine DNA), non-complimentary (beef
DNA), and non-complimentary (chicken DNA) separately in
each tubes. All the vials tubes were annealed at 90 °C for
5 min, then cooled to 50 °C for 1 h to allow perfectly matched
annealing (hybridization) of the complimentary DNA and the
mismatched non-annealing (non-hybridization) of the non-
complimentary DNA [13].
The complimentary target porcine DNA signal displays a
phosphorescence “enhancement” (increased by 23%), while
both non-complimentary DNA signals were “quenched” for
beef DNA (decreased by 79%) and chicken DNA (decreased
by 88%) (Fig. 7). The enhanced emission signal of the com-
plimentary target porcine DNA was attributed to the
Fig. 7 Phosphorescence
Enhance
(+Porcine)
+ 23 %
emission spectra for sensor
selectivity solution study of
hydroxypropoxy side chains
complex (3) (1 mM) in THF-Tris-
HCl buffer (pH 7.4) with different
DNA (porcine, beef, chicken)
(4 μM). λ excitation =370 nm. λ
emission =574 nm. The slit width
was 5 nm for both excitation and
emission, with a scan rate of
200 nm/min
Complex (3)
Quench
(+Beef,+Chicken)
-79 %, -88 %

J Fluoresc
Acknowledgements The authors would like to acknowledge Universiti
Kebangsaan Malaysia (UKM) for providing the research facilities and
grant funding 06-01-02-SF0898, GGPM-2012-074 and FRGS/1/2013/
ST01/UKM/03/2 for financial support throughout this research.
findings will be valuable for the potential use of
hydroxypropoxy side chains platinum(II) salphen complex
(3) as an optical porcine DNA indicator sensor material for
the detection of porcine DNA in food products. Current work
on fabrication of this phosphorescence metal complex as an
optical indicator sensor material for porcine DNA biosensors
and incorporating this optical indicator into a polymer matrix,
which is then deposited on solid support sensors are undergo-
ing in our laboratory.
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Conclusions
The platinum(II) salphen complex N, N′-Bis-
4-(hydroxysalicylidene)-phenylenediamine-platinum(II);
non-side chains (1) and its two hydroxyl substituent with vary-
ing lengths of the alkoxy side chain arms N,N′-bis-[4-[[1-(2-
hydroxyethoxy)] salicylidene] phenylenediamine-
platinum(II); hydroxyethoxy side chains (2) and N,N′-
bis-[4-[[1-(3-hydroxypropoxy)] salicylidene]
phenylenediamine-platinum(II); hydroxypropoxy side chains
(3) were synthesized and characterized by spectroscopic
methods. All the complexes interact with duplex CT-DNA
and porcine DNA via the intercalation mechanism. These
platinum(II) complexes can intercalate with the conjugated
DNA bases due to its square-planar and aromatic rings struc-
ture. The UV-Vis DNA titration showed the highest binding
affinity of N,N′-bis-[4-[[1-(3-hydroxypropoxy)] salicylidene]
phenylenediamine-platinum(II); hydroxypropoxy side chains
(3) to DNA compared to the other two complexes. The degree
of binding affinity for all the platinum(II) complexes to DNA
are in the order of complex hydroxypropoxy side chains
(3) > non-side chains (1) > hydroxyethoxy side chains (2).
Higher selectivity for duplex porcine DNA over single-
stranded DNA and also CT-DNA was achieved. Moreover,
an enhancement of phosphorescence emission intensity by
57% upon the addition of porcine DNA suggests that
hydroxypropoxy side chains complex (3) has the most poten-
tial as an optical phosphorescence porcine DNA biosensor.
The sensor solution study of hydroxypropoxy side chains
complex (3) showed a good selectivity towards complimenta-
ry target porcine DNA, allowing for an easy and rapid sensor
method to detect and distinguish porcine DNA among the
different DNA. The use of this phosphorescence metal com-
plex as an optical porcine DNA indicator sensor material in
solid sensors is currently undergoing in our laboratory. These
findings will be valuable for the potential use of the
hydroxypropoxy side chains platinum(II) salphen complex
(3) as an optical indicator sensor material for porcine DNA
biosensor, contributing to an easy, simpler, and rapid alterna-
tive method to detect porcine DNA in food samples for an
improved food industry analysis method in the future.
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