A selective, long-lived deep-red emissive ruthenium(II) polypyridine complexes for the detection of BSA

Article abstract of DOI:10.1016/j.saa.2014.04.060

A selective, label free luminescence sensor for bovine serum albumin (BSA) is investigated using ruthenium(II) complexes over the other proteins. Interaction between BSA and ruthenium(II) complexes has been studied using absorption, emission, excited state lifetime and circular dichroism (CD) spectral techniques. The luminescence intensity of ruthenium(II) complexes (I and II), has enhanced at 602 and 613 nm with a large hypsochromic shift of 18 and 5 nm respectively upon addition of BSA. The mode of binding of ruthenium(II) complexes with BSA has analyzed using computational docking studies.

Full text of DOI:10.1016/j.saa.2014.04.060

Accepted Manuscript
A Selective, Long-Lived Deep-Red Emissive Ruthenium (II) Polypyridine
Complexes for the Detection of BSA
Eththilu Babu, Paulpandian Muthu Mareeswaran, Subramanian Singaravadivel,
Jayaraman Bhuvaneswari, Seenivasan Rajagopal
PII:
S1386-1425(14)00633-7
http://dx.doi.org/10.1016/j.saa.2014.04.060
SAA 12024
DOI:
Reference:
To appear in:
Spectrochimica Acta Part A: Molecular and Biomo-
lecular Spectroscopy
Received Date:
Revised Date:
Accepted Date:
14 February 2014
2 April 2014
10 April 2014
Please cite this article as: E. Babu, P. Muthu Mareeswaran, S. Singaravadivel, J. Bhuvaneswari, S. Rajagopal, A
Selective, Long-Lived Deep-Red Emissive Ruthenium (II) Polypyridine Complexes for the Detection of BSA,
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2014), doi: http://dx.doi.org/10.1016/j.saa.
2
014.04.060
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A Selective, Long-Lived Deep-Red Emissive Ruthenium (II)
Polypyridine Complexes for the Detection of BSA
1
,2
1,3
1
Eththilu Babu , Paulpandian Muthu Mareeswaran , Subramanian Singaravadivel ,
1
,4
1
Jayaraman Bhuvaneswari , Seenivasan Rajagopal *
1
Department of Physical Chemistry, School of Chemistry, Madurai Kamaraj University,
Madurai, Tamil Nadu, India.
2
Department of Chemistry, VV College of Engineering, Tisaiyanvilai, Tirunelveli, India.
3
Graduate School of EEWS (WCU), Korea Advanced Institute of Science and Technology,
Daejeon, South Korea.
4
Department of Chemistry, SRI G.V.G Visalakshi College for Women, Udumalapet, Tamilnadu,
India.
*
Corresponding author E-mail: rajagopalseenivasan@yahoo.com (S. Rajagopal)
Tel.: +914522458246, Fax: +914522459139
1

Abstract
A selective, label free luminescence sensor for bovine serum albumin (BSA) is
investigated using ruthenium (II) complexes over the other proteins. Interaction between BSA
and ruthenium (II) complexes has been studied using absorption, emission, excited state lifetime
and circular dichroism (CD) spectral techniques. The luminescence intensity of ruthenium (II)
complexes (I and II), has enhanced at 602 and 613 nm with a large hypsochromic shift of 18 and
5
nm respectively upon addition of BSA. The mode of binding of ruthenium (II) complexes with
BSA has analyzed using computational docking studies.
Keywords: Ruthenium (II) complex, luminescence, Bovine serum albumin (BSA), biosensor,
Docking
2

INTRODUCTION
Fluorescent probes are having wide range of applications in the field of biosensors and
proteomics [1-3]. Protein detection is one of the important field, since proteins playing various
roles like transporter, building blocks and pathogens in biology. Most of the molecular probes
and sensors are fluorescent organic molecules. Their photophysical properties like emission
intensity and excited state life time are used as sensing pathways. However, the use of such
probes to reveal the local biological environment is scarcely addressed in the literature [4]. Even
few reports are investigated on the mechanism of protein detection by light-up probes [4, 5] and
these light-up probes are mainly used for molecular recognition such as DNA, proteins and as
stains in gel electrophoresis [6-9].
The organic fluorophores have limitations, such as, lower excited state lifetime, poor
photostability and lower wavelength absorption [10, 11]. The fluorescence of probe molecule
should not overlap with background fluorescence from proteins. In this regard, the use of
luminescent transition-metal complexes as sensors continues to attract considerable interest
because of their long lifetimes compared to their purely organic counterparts and higher
6
wavelength absorption [12-14]. Luminescent transition metal complexes, especially those with d
electronic configuration such as ruthenium(II), osmium(II) and rhenium(I), are receiving much
interest in imaging and luminescent probe for biomolecules [15, 16]. Because of the high
photostability, low-energy absorption, and relatively long lived luminescence of ruthenium(II)-
polypyridine complexes[17], we envisage ruthenium(II)-polypyridine complexes as promising
candidates for luminescent biological probes.
3

The most abundant protein of circulatory system is serum albumins. Serum proteins play
an important role in the transportation and delivery of drug molecules in the blood [18]. Among
the serum albumins, bovine serum albumin (BSA) has a wide range of physiological functions
involving the binding, transportation and delivery of fatty acids, porphyrins, bilirubin, steroids,
etc. It is also consumed as dietary proteins. This heart shaped protein, BSA has three
homologous domains I-III and each domain is made up of two subdomains, A and B, having
unique binding properties. BSA is a single-chain 582 amino acid protein with 17 cystine cross-
linked residues [19-22]. BSA has two tryptophans, Trp-134 and Trp-212, embedded in the first
sub-domain IB and sub-domain IIA, respectively [23, 24]. Several techniques such as
electrochemistry [25], Rayleigh light scattering [26], two-photon excitation [27], seeded liquid
beam desorption mass spectrometry [28], Raman scattering [29] and optical sensor [30] have
been applied for the detection of BSA. Recently, many optical techniques have been utilized to
investigate the interaction of proteins with ligands, because these methods are sensitive and
relatively easy to use [31-33]. Among these, fluorescence technique is the most useful method to
study the biomolecular interactions since it is sensitive to low concentrations and amicable with
physiological environments [34].
Herein we report a label free biosensor using Ru(II) complexes for selective recognition
of BSA in phosphate buffer solution (pH 7.4). This approach relies on the hydrophobic
interaction with probe molecule using steady state emission signal change. The mode of binding
is also analyzed using docking studies.
EXPERIMENTAL
4

Materials
The commercial samples of RuCl
dpsphen), 4,7-diphenyl-1,10-phenanthroline (dpphen), ammonium hexaflurophosphate and
other chemicals are procured from Sigma. Bovine serum albumin (BSA), human serum albumin
HSA), lysozyme, cytochrome c, myoglobin, thrombin, platelet derived growth factor (PDGF)
3
.nH O, 4,7-diphenyl-1,10-phenanthroline disulfonic acid
2
(
(
-3
are purchased from Sigma Aldrich and used as such. The stock, 1 × 10 M, solution of all the
proteins were prepared using PBS buffer. Sodium chloride, potassium chloride, disodium
phosphate, sodium phosphate and all the solvents are purchased from Merck and used as such.
2
+
4-
The luminophores [Ru(dpphen)
reported literature methods and characterised using ESI-MS spectroscopy (Figure S1 and S2)
35, 36].
3
]
(I) and [Ru(dpsphen)
3
] (II) (Chart I) are prepared using
[
Synthesis of Tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II) chloride (I)
The [Ru(dpphen)
3
]Cl
2
is synthesized using literature method [35]. RuCl
3
.3H
2
O (1 mmol) and
4
,7-diphen-1,10-phenanthroline (3 mmol) is taken in ethylene glycol under nitrogen atmosphere
and heated to reflux for 72 h and the crude product is chromatographed using silica gel. The
solution on evaporation yielded orange red crystals. Yield = 85% ESI-MS (m/z) 548.6496 (M-
-
2
Cl doubly charged species).
Synthesis of Tetrasodium tris[1,10-phenanthrolinediyl-4,7-di(benzenesulphanato)
ruthenate(II) hexahydrate (II)
The Na
4
[Ru(dpsphen) ] is synthesized using literature method [36]. The blue solution (0.5 mmol,
3
5
mL) is prepared by refluxing 1 g of RuCl
3
.3H
2
O in 50% ethanol for 4-5 hours in a hot water
5

bath. This solution is transferred into a degassed solution of the ligand, 4,7-
diphenylphenanthroline disulphonic acid (1 g, 2 mmol), in water (20 mL) and the mixture is
heated at reflux under nitrogen for 12 h. The resultant red solution is filtered hot, and the red
filtrate evaporated to dryness to give an orange-brown solid. This is recrystallized from water-
ethanol mixture, and then dissolved in water (5 mL) and then chromatographed using water as
eluent. The red band is collected and evaporated to dryness to give a brown solid. ESI-MS (m/z):
-
2
63.6496 (6 charged species) [36].
Apparatus and Spectral Measurements
All the solutions of Ru(II) complexes and proteins are prepared using phosphate buffer at pH 7.4.
The UV-vis absorption spectra are recorded at room temperature on Analtik Jena Specord S100
diode-array spectrophotometer - equipped with 1.0 cm path length quartz cells. The UV-vis
absorption spectral studies are carried out in the wavelength range from 300 nm to 700 nm.
During the spectral measurement the concentration of the complex is maintained constant and
the concentration of BSA was varied. The emission spectra are recorded using JASCO FP6300
Spectrofluorimeter with 10 mm path length cuvette. Emission spectra are recorded in the range
of 500 to 800 nm. Titrations are done manually via a micro-syringe and the fluorescence
intensity of complex in the absence and in the presence of the BSA is measured after incubation
for 5 min. Excited state lifetime of Ru(II) complexes are determined by time dependent
luminescence decay measurements using a Czerny-Turner monochromator and analyzed by
Hamamatsu R-928 photomultiplier tube. Circular dichroism (CD) measurements are performed
on JASCO J810 spectropolarimeter at room tempreature over the wavelength 200-360 nm.
-
1
Parameters are set as follows: path length, 50 mm; resolution, 0.5 nm; scan speed, 50 nm min ;
6

band width, 1 nm; response 1 s. All the measurements are done at room temperature and repeated
three times using freshly prepared samples and the results are reported as the average.
Determination of binding constant
Scatchard equation
The binding constants between the probe and the protein have been determined from the
fluorescence intensity data using Scatchard plot. The Scatchard equation is shown in eq (1) [37,
3
8]
υ/C
In eq (1) υ – ratio of the concentration of bound ligand to total available binding sites. C
number of binding sites per protein molecule. n – binding stoichiometry. The plot of the data
υ/C vs. υ is non-linear which shows the cooperative binding.
F
= nK
a
- υK
a
(1)
F
–
F
Hills equation
Since, the Scatchard plot is non-linear we have calculated the binding constant using Hills
equation, eq (2) [39]
log[θ/(1-θ)] = n log [BSA] - log K
d
(2)
θ - number of binding sites (C in Scatchard equation) , K
F
d
– dissociation constant(the inverse of
a
K ), n – Hills coefficient of cooperativity which gives the nature of cooperativity. n > 1 positive
cooperative binding , n < 1 negative cooperative binding and n = 0 non cooperative binding.
Circular dichroism (CD)
-6
The concentration of protein is fixed at 2 × 10 M and Ru(II) complexes are varied (2 ×
-
6
-6
-6
1
0 , 5 × 10 and 10 × 10 M). The helicity of the proteins is calculated by equations (3) and
4)[40].
Mean residual elipticity (MRE) = Observed CD/ C
(
p
nl × 10
(3)
7

where, Cp – molar concentration of protein, n- amino acid residues, l – path length.
α (helix %) = (-MRE – 4000)/ (33000-4000) × 100 (4)
In eq. (4) 4000 is the MRE of the β–form and random coil conformation cross at the observed
wavelength, and 33,000 is the MRE value of a pure α–helical at the observed wavelength.
Details of docking studies
The pdb (protein data bank) files of protein molecule has been downloaded from protein
data bank (http://www.rcsb.org/pdb/home/home.do, file code: 1AO6)[41]. The final model
chosen, is minimized using DISCOVER module of Insight II with 1000 rounds of steepest
descent and 1000 rounds of conjugate minimization. The cavity of the protein model has been
picked by using Computed Atlas of Surface Topography of Proteins (CASTp) [42]. The docking
towards the luminophores of proteins such as tryptophan and tyrosine has been done using
GOLD[43]. For viewing the docked solutions and to generate pictures UCSF Chimera candidate
version 1.5.3 is used [44].
RESULTS AND DISCUSSION
The structures of the Ru(II) complexes, synthesized for the present study, are shown in Chart 1.
The absorption and emission spectra of complex I in aqueous solution containing acetonitrile/
buffer (5% /95%) and II in buffer are shown in supporting information, Figure S3 and the
spectral data of Ru(II) complexes are collected in Table 1. The absorption spectra of I and II
show strong ligand centered absorption peaks at 278 nm and less intense metal to ligand charge
transfer (MLCT) absorption on 464 nm [35]. The strong high energy absorption bands at 278 nm
are assigned to ligand centered (LC) π−π* transition and low energy absorption at 464 nm to
spin allowed MLCT transition from the Ru dπ- orbital to the π* orbital of the ligand (dπ Ru →
3
π*). The MLCT emission maximum of complexes I and II are observed at 620 nm and 618 nm
8

respectively. The lifetimes of these complexes are 2.9 and 4.0 µs and agree with the literature
values [35, 36].
Absorption and luminescence titration of Ru(II) complex with proteins
Absorption spectral technique is a simple method for exploring the structural changes and to
analyze the complex formation between the reactants [45]. The absorption spectra of Ru(II)
complexes are recorded in the presence of various concentration of BSA. The results for
complexes I and II are shown in Figure 1. The addition of BSA leads to a gradual decrease in the
absorption intensity of Ru(II) complexes at 464 nm(I and II). The above results indicate the
presence of hypsochromic effect in this system. Similar results are reported already with organic
dyes [46].
The luminescence behavior of Ru(II) complexes in the presence of proteins have also
been studied to get more details on the protein binding. The luminescence spectra of the
complexes I and II at different concentrations of BSA are shown in Figure 2. The interaction
between Ru(II) complexes (I and II) and BSA is found to produce dramatic enhancement in the
emission intensity of Ru(II) complexes. In the absence of BSA, moderate emission of Ru(II)
complexes (I and II) in aqueous buffer medium at 620 nm and 618 nm respectively are observed.
The complex I shows an enormous enhancement accompanied by a blue shift of about 18 nm in
the presence of BSA, whereas, the complex II shows comparatively lower enhancement along
with 5 nm blue shift in the luminescence maximum. This different behavior can be attributed to
the more hydrophobic nature of the complex I than the complex II (complex II is hydrophilic in
nature). The neutral and hydrophobic nature of the probe molecule affects interaction with
hydrophobic cavities of the protein (BSA) and such a shift in the luminescence maximum toward
9

blue end of the spectrum is a reflection of the sensitivity of the probe to changes in the polarity
of its surrounding microenvironment [47, 48].
The observed blue shift in the luminescence maximum of complex I (from 620 nm to 602
nm) confirms the restriction on the solvent dipolar-dipolar relaxation imposed by the constrained
environment of the probe in the hydrophobic region of the protein matrix. The increase in
intensity further indicates that the progressive binding of the probe with protein makes
nonradiative channels present in aqueous buffer medium is less operative [49]. The enhancement
of luminescence intensity of Ru(II) complexes in protein solution can be rationalized in terms of
binding of the probe, Ru(II) complexes, with the protein. The strength of the binding can be
identified through the determination of the binding constant between the probe and the protein
using Scatchard and Hills plot (Figure S4 and S5) are given in supporting information and the
4
-1
3
-1
binding constants are 3.0 × 10 M and 2.1 × 10 M for complex I and II.
Excited state lifetime studies
The luminescence lifetime is a sensitive parameter to establish the local environment around a
luminophore [50, 51]. Therefore the excited state lifetimes of the Ru(II) complexes are measured
in the absence and in the presence of BSA. Figure 3 shows the luminescence decay curve of
-6
complexes I and II (1 × 10 M) in the absence and in the presence of BSA. In the absence of
BSA, the lifetime values of Ru(II) complexes I and II are 2.9 µs and 4.0 µs respectively. In the
presence of BSA, the lifetime of complex I is increased from 2.9 µs to 5.2 µs and that of
complex II is increased from 4.0 µs to 4.9 µs. These results indicate that the Ru(II) complexes
strongly bind with BSA and the increase in the luminescence intensity as well as the excited state
lifetime are similar.
1
0

Selective binding with BSA
The selectivity of probes I and II is confirmed by binding of these complexes with a set of
extracellular proteins such as HSA, BSA, myoglobin, lysozyme, thrombin, PDGF and
cytochrome C are measured and their luminescence responses are compared (Figure 4). HSA and
BSA are inducing luminescence intensity of Ru(II) complexes I and II dramatically. The careful
observation of Figure 4 indicates that myoglobin and lysozyme interact moderately with the
probes. The interactions of other extracellular proteins are negligible. The above results can be
attributed for the hydrophobicity and the size of the proteins. HSA/BSA contains several
hydrophobic binding sites for interaction with the metal complexes [52].
Myoglobin is a small monomeric form of heme protein. Its interior is hydrophobic and
the surface is slightly hydrophilic in nature [53]. The interior and exterior of myoglobin are well-
distinguished by hydrophobic and hydrophilic side groups. The Ru(II) complexes I and II bind
via hydrophobic interaction into the interior of the myoglobin. However the lysozyme is highly
hydrophilic in nature resulting in the weak interaction with the complex I but considerable
luminescence enhancement is observed in the presence of complex II which is due to the
hydrophilic interaction of lysozyme with complex II [54]. Though the myoglobin and BSA are
hydrophobic in nature, BSA shows enormous enhancement than the myoglobin on the
luminescence intensity of Ru(II) complexes I and II. This is due to the stronger hydrophobicity
of the BSA than the myoglobin [55]. In addition the size of the protein is also in this order: BSA
-7
>
myoglobin > lysozyme [56]. The detection limit as low as 3 x 10 M.
Conformational change of BSA in the presence of Ru(II)-complex
1
1

Probe binding with protein results in change in the secondary and tertiary structures[23]. The
change in secondary and tertiary structure of BSA upon binding of complex I and II is studied
using circular dishroism technique. The peaks at 208 and 218 nm are corresponds to the alpha
and beta helical conformations of the protein [23, 57]. These peaks exhibit a decrease in intensity
on the addition of Ru(II) complexes (Figure 5).
The α–helicity and molar elipticity are calculated using equation 3 and 4. Upon addition
of Ru(II) complexes to BSA the extent of α-helicity of the protein decreases from 55% to 45 %
and 53 % for complexes I and II, respectively. According to the literature, far-UV (200-260 nm)
CD spectra is giving the quantitative information on the secondary structure and the near UV
(
260-300 nm) CD spectra is giving the information of the tertiary structure of BSA [58-60]. The
CD spectra of BSA in the absence and in the presence of Ru(II) complex are observed to be
similar in shape. There is no additional signal in the near UV region. The changes at 200-260 nm
region indicates the conformational changes in the secondary structure of BSA in the presence of
Ru(II) complexes. Therefore, the secondary structure of BSA is changed upon addition of Ru(II)
complexes with decrease in α helicity. There is no change in tertiary structure of BSA upon
binding with Ru(II) complex.
Docking studies
The results of spectroscopic studies presented above are supported by the docking studies using
GOLD software, [43] to rationalize the possible binding site and nature of interaction between
Ru(II)-complex and BSA. Since, BSA has sequence homology of 80 % over 578 residues with
HSA (PDB entry 1AO6) as template and also the complexes under investigation are selectively
sensing HSA, the crystal structure of HSA is used for docking studies. The crystal structure of
1
2

HSA comprises three domains. The three domains (I, II, III) have similar 3D structures and are
highly asymmetric in assembly with 17 disulfide bridges. Each domain can be further divided
into subdomains “A” and “B”, which are composed of six and four α-helices, respectively. A
long extended loop traverses the two subdomains to link those together [41]. Our model of the
BSA shows that the three domains are identical to those in HSA, and the binding sites for the
complexes I and II analyzed in this work have an equivalent binding site in HSA. The binding
cavity of HSA has been identified from the online tool, Computed Atlas Surface Tropology of
protein (CASTp) and we choose the large binding cavity (shown in Figure S6).
The hydrophobic binding area shown in surface view, in Figure S6, is available for Ru(II)
3
complex docking. The larger binding cavity of binding volume 3587.5 Å with six openings, is
selected for docking among 76 binding cavities, since other cavities are smaller in volume to
accommodate Ru(II) complexes and few openings for interactions. The experimental results
indicate that the Ru(II) complex strongly binds with BSA through the non-covalent hydrophobic
interaction. The possible interaction is mainly with the aromatic moiety of the protein such as
Phe, Tyr and Trp. We have selected the Tyr138 as a targeting moiety which is available in this
cavity, since it is situated deep inside of selected binding cavity. The most probable positions of
binding of complexes I and II to HSA are shown in Figure 6.
The subdomain 1B having aromatic amino acid, rotated 90º away, accommodates the
incoming ligands. Complex I preferably binds with HSA into the domain 1B moieties such as
tyrosine and phenyl alanine via the hydrophobic interaction (Figure S7). The aromatic moiety of
ligand interacts through π-π interaction with Tyr 136, Tyr 138, Phe 132 and Phe155. This rigidity
of probe through the π-π interaction increases the emission intensity with the addition of BSA.
The complex II also binds to the same domain via π-π interaction with Tyr 161 and Phe 157. The
1
3

-
hydrophilic SO group interacts with hydrophilic HSA surface (Figure S8). Hence the complex I
3
has π-π interaction with Tyr 136, Tyr 138, Phe 132 and Phe155 simultaneously but the complex
II binds with Tyr 161 and Phe 157. This binding site has 4 - 6 aromatic amino acids. The
adjacent amino acids and the distances with the complex are shown in Table 2.
The complex I accumulate on the binding packet of HSA IB subdomain, and subdomain
IB is in the inner hydrophobic pockets, and consequently it is expected to have high fluorescence
intensity. On the other hand, the complex II also binds to IB domain, which is partially exposed
-
to the solvent due the hydrophilicity of the SO
3
moiety. Both complexes have stacked π-π
interaction in the more hydrophobic site such as Try and Phe and the nearest groups are shown in
Table 2. This also supports the strong binding of complex I with BSA and accounts for the
enormous increase in the luminescence intensity compared to complex II [61].
CONCLUSION
We have demonstrated the selective BSA sensor system using the deep-red emissive Ru(II)-
complexes with the simple technique. The Ru(II)-complexes show moderate luminescence in the
absence of BSA. The luminescence intensity of probe enormously increases upon the addition of
BSA due to the strong binding of the probe with BSA through the hydrophobic interaction. The
α-helix content of BSA is found to decrease in the presence of probes (I and II). The binding
constant values indicate that the complex I shows strong binding than the complex II, which is
attributed to the hydrophobicity of complex I. The docking studies endorse the spectroscopic
results. The selectivity of BSA over other extra cellular proteins is confirmed under steady state
luminescence assay. Therefore, the complex I and II can be envisaged as efficient sensors for
BSA.
1
4

ACKNOWLEDGEMENTS
Financial support for this work from Department of Science and Technology (DST) is greatly
acknowledged. We thank Prof. S. Krishnasamy and Mr. P. Manojkumar, School of
Biotechnology, Madurai Kamaraj University, Madurai for valuable discussion.
REFERENCES
[
[
1] T. Kodadek, Chem. Biol. 8 (2001) 105-115.
2] D. Wang, X. Gong, P.S. Heeger, F. Rininsland, G.C. Bazan, A.J. Heeger, Proc. Nat. Acad.
Sci. 99 (2002) 49-53.
[
[
[
[
[
[
3] M.R. Pinto, K.S. Schanze, Proc. Nat. Acad. Sci. 101 (2004) 7505-7510.
4] A. Granzhan, H. Ihmels, G. Viola, J. Am. Chem. Soc. 129 (2007) 1254-1267.
5] P. Prentø, Biotech. Histochem. 76 (2001) 137-161.
6] L.-T. Jin, J.-K. Choi, Electrophoresis, 25 (2004) 2429-2438.
7] L. Williams, Biotech. Histochem. 76 (2001) 127-132.
8] G. Cosa, K.S. Focsaneanu, J.R.N. McLean, J.P. McNamee, J.C. Scaiano, Photochem.
Photobiol. 73 (2001) 585-599.
[
[
9] Y. Jiang, X. Fang, C. Bai, Anal. Chem. 76 (2004) 5230-5235.
10] K.K.-W. Lo, K.H.-K. Tsang, K.-S. Sze, C.-K. Chung, T.K.-M. Lee, K.Y. Zhang, W.-K. Hui,
C.-K. Li, J.S.-Y. Lau, D.C.-M. Ng, N. Zhu, Coord. Chem. Rev. 251 (2007) 2292-2310.
[
[
11] J.M. Costa-Fernández, R. Pereiro, A. Sanz-Medel, Trends Anal. Chem. 25 (2006) 207-218.
12] A.P. de Silva, H.Q.N. Gunaratne, T. Gunnlaugsson, A.J.M. Huxley, C.P. McCoy, J.T.
Rademacher, T.E. Rice, Chem. Rev. 97 (1997) 1515-1566.
13] E. Babu, P. Muthu Mareeswaran, S. Rajagopal, J. Fluoresc. 23 (2013) 137-146.
[
1
5

[
(
[
14] R. Zhang, Z. Ye, Y. Yin, G. Wang, D. Jin, J. Yuan, J.A. Piper, Bioconjugate Chem. 23
2012) 725-733.
15] V. Fernandez-Moreira, F.L. Thorp-Greenwood, M.P. Coogan, Chem. Commun. 46 (2010)
1
86-202.
[
[
[
[
[
16] P. Muthu Mareeswaran, E. Babu, S. Rajagopal, J. Fluoresc. 23 (2013) 997-1006.
17] J.R. Lakowicz, Principles of Fluorescence Spectroscopy, Springer, 2007.
18] X.M. He, D.C. Carter, Nature, 358 (1992) 209-215.
19] S. Ashoka, J. Seetharamappa, P.B. Kandagal, S.M.T. Shaikh, J. Lumin. 121 (2006) 179-186.
20] D.C. Carter, J.X. Ho, Structure of Serum Albumin, in: J.T.E.F.M.R. C.B. Anfinsen, S.E.
David (Eds.) Advances in Protein Chemistry, Academic Press, 1994, pp. 153-203.
[
[
(
[
[
(
[
(
[
[
[
21] K.F. Brown, M.J. Crooks, Biochem. Pharmacol. 25 (1976) 1175-1178.
22] K. Hirayama, S. Akashi, M. Furuya, K.-i. Fukuhara, Biochem. Biophys. Res. Commun. 173
1990) 639-646.
23] U. Kragh-Hansen, Pharmacol. Rev. 33 (1981) 17-53.
24] P. Muthu Mareeswaran, M. Prakash, V. Subramanian, S. Rajagopal, J. Phys. Org. Chem. 25
2012) 1217-1227.
25] M.W. Shao, H. Yao, M.L. Zhang, N.B. Wong, Y.Y. Shan, S.T. Lee, Applied Phys. Lett. 87
2005) 183106 1-3.
26] H. Zhong, J.-J. Xu, H.-Y. Chen, Microchim Acta, 148 (2004) 99-103.
27] U.P. Paul, Li, M.L. Lee, P.B. Anal. Chem. 77 (2005) 3690-3693.
28] A. Charvat, F. Gessler, J. Niemeyer, A. Bögehold, B. Abel, Anal. Lett. 39 (2006) 2191-
2
203.
[
29] M. Shao, L. Lu, H. Wang, S. Luo, D. Ma, Microchim Acta, 164 (2009) 157-160.
1
6

[
[
[
30] L. Zhu, C.S. Lee, D.L. DeVoe, Lab Chip, 6 (2006) 115-120.
31] P.N. Naik, S.A. Chimatadar, S.T. Nandibewoor, Spectrochim. Acta A, 73 (2009) 841-845.
32] N. Barbero, E. Barni, C. Barolo, P. Quagliotto, G. Viscardi, L. Napione, S. Pavan, F.
Bussolino, Dyes Pigments, 80 (2009) 307-313.
[
[
[
33] X. Guo, L. Zhang, X. Sun, X. Han, C. Guo, P. Kang, J. Mol. Struct. 928 (2009) 114-120.
34] A. Gong, X. Zhu, Y. Hu, S. Yu, Talanta, 73 (2007) 668-673.
35] C.T. Lin, W. Boettcher, M. Chou, C. Creutz, N. Sutin, J. Am. Chem. Soc. 98 (1976) 6536-
6
544.
36] S. Zanarini, L.D. Ciana, M. Marcaccio, E. Marzocchi, F. Paolucci, L. Prodi, J. Phys. Chem.
B, 112 (2008) 10188-10193.
37] K.K.-W. Lo, K.H.-K. Tsang, W.-K. Hui, N. Zhu, Synthesis, Inorg. Chem. 44 (2005) 6100-
110.
[
[
6
[
[
38] T.K. Dam, R. Roy, D. Pagé, C.F. Brewer, Biochemistry, 41 (2001) 1351-1358.
39] J. Bhuvaneswari, P.M. Mareeswaran, S. Shanmugasundaram, S. Rajagopal, Inorg. Chim.
Acta, 375 (2011) 205-212.
[
[
40] M. Hariharan, E. Kuruvilla, D. Ramaiah, J. Phys. Chem. Lett. 1 (2010) 834-838.
41] S. Sugio, A. Kashima, S. Mochizuki, M. Noda, K. Kobayashi, Protein Eng. 12 (1999) 439-
4
46.
[
(
[
42] J. Dundas, Z. Ouyang, J. Tseng, A. Binkowski, Y. Turpaz, J. Liang, Nucleic Acids Res. 34
2006) W116-W118.
43] M.L. Verdonk, J.C. Cole, M.J. Hartshorn, C.W. Murray, R.D. Taylor, Proteins, 52 (2003)
6
09-623.
1
7

[
44] E.F. Pettersen, T.D. Goddard, C.C. Huang, G.S. Couch, D.M. Greenblatt, E.C. Meng, T.E.
Ferrin, J. Comput. Chem., 25 (2004) 1605-1612.
[
(
[
[
[
[
[
[
45] J. Bhuvaneswari, A.K. Fathima, S. Rajagopal, J. Photochem. Photobiol. A: Chem., 227
2012) 38-44.
46] V.S. Jisha, K.T. Arun, M. Hariharan, D. Ramaiah, J. Phys. Chem. B, 114 (2010) 5912-5919.
47] O.K. Abou-Zied, O.I.K. Al-Shihi, J. Am. Chem. Soc. 130 (2008) 10793-10801.
48] S. Mahanta, R. Singh, N. Guchhait, J. Fluoresc. 19 (2009) 291-302.
49] A. Mallick, B. Haldar, N. Chattopadhyay, J. Phys. Chem. B, 109 (2005) 14683-14690.
50] F.G. Prendergast, Cur. Opin. Struct. Biol. 1 (1991) 1054-1059.
51] A. Chattopadhyay, S. Mukherjee, H. Raghuraman, J. Phys. Chem. B, 106 (2002) 13002-
1
3009.
[
[
[
[
[
52] V.G. Vaidyanathan, B.U. Nair, Eur. J. Inorg. Chem. (2005) 3756-3759.
53] Y. Li, W. Yang, Y. Bai, C. Sun, Electroanalysis, 18 (2006) 499-506.
54] T. Mishraki, D. Libster, A. Aserin, N. Garti, Colloids Surfaces B, 75 (2010) 47-56.
55] F. Torrens, G. Castellano, A. Campos, C. Abad, J. Mol. Struct. 924–926 (2009) 274-284.
56] S. Koutsopoulos, L.D. Unsworth, Y. Nagai, S. Zhang, Proc. Nat. Acad. Sci. 106 (2009)
4
623-4628.
[
[
[
[
[
57] M. Dockal, D.C. Carter, F. Rüker, J. Biol. Chem. 274 (1999) 29303-29310.
58] M. Dockal, D.C. Carter, F. Rüker, J. Biol. Chem. 275 (2000) 3042-3050.
59] N.C. Price, Biotechnol. Applied Biochem. 31 (2000) 29-40.
60] S.M. Kelly, N.C. Price, Biochim. Biophys. Acta, 1338 (1997) 161-185.
61] M. Bardhan, J. Chowdhury, T. Ganguly, J. Photochem. Photobiol. B: Biol. 102 (2011) 11-
1
9.
1
8

Chart 1. Structure of complexes I and II
a
b
Figure 1. UV-vis absorption spectral changes of complex (A) I and (B) II in the presence of
-6
different concentration of BSA. The concentration of Ru(II) complex and BSA are 1×10 M,
→ 167 µM respectively.
0
1
9

Figure 2. Luminescence spectra of complex (a) I and (b) II in the presence of various
-6
concentration of BSA. The concentration of Ru(II) complexes and BSA are 1×10 M, 0 → 167
µM respectively. Excitation maximum: 460 nm
a
b
Figure 3. Llifetime decay of (a) complex I and (b) complex II in the presence of BSA.
2
0

a
b
Figure 4. Selectivity of the sensor system towards BSA (a) complex I and (b) complex II. The
-6
concentration of each proteins are170 µM and the Ru(II) complexes are 1×10 M.
a
b
Figure 5. CD spectra of BSA in the presence of (a) complex I and (b) complex II. The
-6
-6
-6
-6
concentration of BSA and Ru(II) complexes are 10x10 , 1×10 M, 2×10 M, 5×10 M.
2
1

a
b
c
d
Figure 6. (a) Docking area of complex I (b) Interaction of complex I with amino acid residues in
HSA (c) Docking area of complex II (d) Interaction of complex II with amino acid residues in
HSA.
2
2

Table 1. Photophysical properties of complexes I and II.
a
b
a
b
a
b
-1
Complex
λabs, nm
λabs, nm
λem, nm
λ
em, nm τ, µs
τ , µs
K
a
, M
n
4
I
463
464
471
468
620
618
602
613
2.9±0.01 5.2±0.02 3.0 × 10
0.566
0.622
3
II
4.0±0.02 4.7±0.03 2.1× 10
a - Complex alone in buffer; b - Complex with BSA
Table 2. The distance between the probe and amino acid residues.
Complex I Complex II
Amino acid
Distance (Å)
Amino acid
Tyr 161
Phe 157
Glu 141
Asp 187
Leu 179
Met 123
Distance (Å)
Tyr 136
Phe 132
Val 118
Tyr 138
Phe 155
Arg 143
Asp 181
3.9
5.8
5.3
5.4
3.9
3.4
5.7
2.6
3.1
4.2
4.4
4.4
2.5
2
3

A Selective, Long-Lived Deep-Red Emissive Ru(II) Polypyridine Complexes for the
Detection of BSA
1
,2
1,3
1
Eththilu Babu , Paulpandian Muthu Mareeswaran , Subramanian Singaravadivel , Jayaraman
1
Bhuvaneswari , Seenivasan Rajagopal *
1
1
Department of Physical Chemistry, School of Chemistry, Madurai Kamaraj University, Madurai-625 021,
Tamil Nadu, India
2
Department of Chemistry, VV College of Engineering, Tisaiyanvilai, Tirunelveli-627 657, India.
3
Department of Chemical and Biomolecular Engineering, Korea Advanced Institute of Science and
Technology, Daejeon, South Korea.
Graphical Abstract
A novel luminescence turn-on detection method for BSA have been developed using Ru(II) complexes,
via non-covalent interactions.
*
To whom correspondence should be addressed.
Tel: +91–452–2458246; E-mail:rajagopalseenivasan@gmail.com
2
4

A Selective, Long-Lived Deep-Red Emissive Ru(II) Polypyridine
Complexes for the Detection of BSA
1
,2
1,3
1
Eththilu Babu , Paulpandian Muthu Mareeswaran , Subramanian Singaravadivel , Jayaraman
1
Bhuvaneswari , Seenivasan Rajagopal *
1
1
Department of Physical Chemistry, School of Chemistry, Madurai Kamaraj University, Madurai, Tamil
Nadu, India-625 021
2
Department of Chemistry, VV College of Engineering, Tisaiyanvilai, Tirunelveli-627 657, India.
3
Department of Chemical and Biomolecular Engineering, Korea Advanced Institute of Science and
Technology, Daejeon, South Korea.
Research Highlights
ꢀ
ꢀ
ꢀ
ꢀ
Binding of ruthenium(II) complexes is efficient with BSA.
Sensing of ruthenium(II) complexes with BSA and HSA is higher than other proteins.
The α helicity of BSA has decreased with the addition of ruthenium(II) complexes.
Mode of binding is established by docking studies.
2
5