Self-assembled near-infrared dye nanoparticles as a selective protein sensor by activation of a dormant fluorophore

Article abstract of DOI:10.1021/ja503850b

Design of selective sensors for a specific analyte in blood serum, which contains a large number of proteins, small molecules, and ions, is important in clinical diagnostics. While metal and polymeric nanoparticle conjugates have been used as sensors, small molecular assemblies have rarely been exploited for the selective sensing of a protein in blood serum. Herein we demonstrate how a nonspecific small molecular fluorescent dye can be empowered to form a selective protein sensor as illustrated with a thiol-sensitive near-IR squaraine ( Sq) dye (λ_abs= 670 nm, λ_em= 700 nm). The dye self-assembles to form non fluorescent nanoparticles (D_h = 200 nm) which selectively respond to human serum albumin (HSA) in the presence of other thiol-containing molecules and proteins by triggering a green fluorescence. This selective response of the dye nanoparticles allowed detection and quantification of HSA in blood serum with a sensitivity limit of 3 nM. Notably, the Sq dye in solution state is nonselective and responds to any thiol-containing proteins and small molecules. The sensing mechanism involves HSA specifi c controlled disassembly of the Sq nanoparticles to the molecular dye by a noncovalent binding process and its subsequent reaction with the thiol moiety of the protein, triggering the green emission of a dormant fluorophore present in the dye. This study demonstrates the power of a self-assembled small molecular fluorophore for protein sensing and is a simple chemical tool for the clinical diagnosis of blood serum.

Full text of DOI:10.1021/ja503850b

Article
pubs.acs.org/JACS
Self-Assembled Near-Infrared Dye Nanoparticles as a Selective
Protein Sensor by Activation of a Dormant Fluorophore
Palapuravan Anees,†,‡ Sivaramapanicker Sreejith,† and Ayyappanpillai Ajayaghosh*,†,‡
†Chemical Sciences and Technology Division and ‡Academy of Scientific and Innovative Research (AcSIR), CSIR-National Institute
for Interdisciplinary Science and Technology (CSIR-NIIST), Trivandrum 695019, India
S
* Supporting Information
ABSTRACT: Design of selective sensors for a specific analyte in blood serum,
which contains a large number of proteins, small molecules, and ions, is
important in clinical diagnostics. While metal and polymeric nanoparticle
conjugates have been used as sensors, small molecular assemblies have rarely
been exploited for the selective sensing of a protein in blood serum. Herein we
demonstrate how a nonspecific small molecular fluorescent dye can be
empowered to form a selective protein sensor as illustrated with a thiol-
sensitive near-IR squaraine (Sq) dye (λabs= 670 nm, λem= 700 nm). The dye
self-assembles to form nonfluorescent nanoparticles (Dh = 200 nm) which
selectively respond to human serum albumin (HSA) in the presence of other
thiol-containing molecules and proteins by triggering a green fluorescence.
This selective response of the dye nanoparticles allowed detection and
quantification of HSA in blood serum with a sensitivity limit of 3 nM. Notably,
the Sq dye in solution state is nonselective and responds to any thiol-
containing proteins and small molecules. The sensing mechanism involves HSA specific controlled disassembly of the Sq
nanoparticles to the molecular dye by a noncovalent binding process and its subsequent reaction with the thiol moiety of the
protein, triggering the green emission of a dormant fluorophore present in the dye. This study demonstrates the power of a self-
assembled small molecular fluorophore for protein sensing and is a simple chemical tool for the clinical diagnosis of blood serum.
for protein sensing.9,10 The disassembly driven turn-on
fluorescence approach reported by Hamachi and co-workers
makes use of a ligand-protein affinity labeling which requires
INTRODUCTION
■
Over the years, molecular self-assembly has matured into a
powerful tool for the construction of soft functional materials
integration of a fluorophore with a suitable ligand through
having wide ranging applications in materials science and
elaborate synthetic procedures.11,12 Alternatively, Rotello and
biology.1 Many of these dimensionally tunable soft architec-
co-workers have reported array of green fluorescent protein
tures are enormously powerful when compared to their
(GFP) and gold nanoparticle conjugates for multiple protein
individual molecular building blocks in terms of physical or
analysis in blood serum.13 Nevertheless, selective detection of a
chemical properties.2 While metal and polymeric nanoparticles
target analyte among thousands of others present in a biofluid
provide versatile scaffolds as sensors for proteins, use of small
molecule-based nanoparticles has not been much exploited in
protein sensing applications. Herein we demonstrate that a
thiol-sensitive near-infrared (NIR) dye becomes a powerful tool
for the specific sensing of serum albumin proteins (SAP) in the
presence of other competing thiol-containing proteins and
small molecules, only when the dye self-assembles to form
using a small molecular fluorophore-based sensor continues to
be a challenge to chemists. While metal nanoparticles, polymers
and affinity ligand-based sensors have their merits and demerits,
the small molecule-based sensor described here is the simplest
and easy to operate.
Thiol-containing proteins are important in biological
functions and are present in high concentrations in blood
nanoparticles, whereas in the isotropic solution state, the dye
serum. Among different thiol-containing proteins in blood,
responds to all thiol-containing molecules, losing the selectivity.
human serum albumin (HSA) plays a key role in maintaining
Protein sensing and imaging are important in clinical
human health.14,15 Variation in HSA concentration indicates
diagnosis to detect protein biomarkers and in biology to
explore cellular processes.3 Previously, several strategies have
liver and kidney diseases or malnutrition owing to a low protein
been reported for protein sensing.4−8 These strategies are
diet. Contrarily, severe or chronic dehydration has been related
to an excess of albumin. Detection of HSA in blood serum with
mainly based on fluorophores, which lack selectivity in many
a fluorescent molecular probe is often interfered by the
cases and have the disadvantage of small-to-moderate
fluorescence response due to high background noise signals.
Recently, Thayumanavan and co-workers have extensively
studied the use of polymer nanoparticle disassembly approach
Received: April 17, 2014
Published: September 8, 2014
© 2014 American Chemical Society
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nonspecific covalent interaction with competing proteins.
Moreover, blood serum has a large number of thiol-containing
molecules present in small quantities, and hence thiol
responsive probes cannot be used for selective HSA sensing
in biofluids. This is due to the high affinity of thiol group with
the reactive site of the molecular sensors.16,17 Therefore, a
selective, sensitive, and simple fluorescent sensor with
controlled reactivity is required for the diagnosis of HSA in
blood serum. Squaraines are an important class of dyes which
are extensively used as probes for the detection of cations,18,19
neutral molecules,20 proteins,21 and for imaging of cells.22−24
However, squaraine dyes in general have the tendency to form
aggregates under aqueous conditions which is a drawback for
their use due to the strong quenching of the emission.25 In this
work, we explore this otherwise unfavorable property and prove
that the aggregation of a fluorescent squaraine dye to
nonfluorescent nanoparticles can be turned around as an
opportunity to the selective sensing of SAP in the presence of
other competing biomolecules.
the breakage of conjugation of the dye to generate a new
chromophore in situ that activates a strong green fluorescent
signal. On the other hand, a homogeneous solution of Sq dye
responds to any thiol-containing molecules, triggering
fluorescent signals without any selectivity. This remarkable
selectivity of the Sq nanoparticles when compared to the
molecular dye facilitates the use of the former as a selective
sensor for the detection of albumin content in human blood
serum in the presence of other competing biomolecules and
proteins.
RESULTS AND DISCUSSION
■
The squaraine dye Sq was synthesized by the reaction of the
strongly fluorescent styrylpyrrole with the semisquaraine
derivative of N,N-dibutyl aniline in the presence of
tributylorthoformate (TBOF) and isopropanol in a 40% yield.
The dye was purified by column chromatography (silica gel, 2%
methanol-chloroform) and characterized by H and 13C NMR
1
and mass spectral analyses (Supporting Information). A
solution of Sq in acetonitrile (2 μM) showed an absorption
maximum at 670 nm and an emission maximum at 700 nm (ΦF
= 0.083, determined using indicarbocyanine dye in methanol as
standard). Addition of phosphate buffer (pH 8.0) into an
acetonitrile solution of the dye resulted in the broadening of
the absorption spectrum in the 550−850 nm range and the
gradual quenching of the emission at 700 nm (Figures 2a and
The underlying mechanism of our sensing strategy involves
the noncovalent interaction of the protein with the nano-
particles and the consequent controlled disassembly driven
selective covalent interaction of free cysteine residue in SAP
with the dye molecule (Figure 1). Initially, the fluorescent Sq
Figure 1. A schematic representation of the Sq dye nanoparticle for
the specific sensing of SAP. The NIR emitting squaraine dye, Sq in the
monomeric state (30% acetonitrile/phosphate buffer pH 8.0) interact
nonspecifically to all thiol-containing proteins and small molecules,
whereas the nonemissive Sq nanoparticles (in phosphate buffer, pH
8.0) interact specifically with SAP to trigger a green emission at 480
nm.
Figure 2. (a) UV−vis absorption spectra of Sq in acetonitrile (red
line) and 25 mM phosphate buffer (black line). (b) TEM and (c) DLS
analyses of Sq (6 μM) spherical assemblies obtained from 25 mM
phosphate buffer at a pH of 8.0. (d) DLS analysis of Sq (6 μM) in the
presence and absence of BSA protein (12 μM). DLS data of BSA
protein alone (6 μM) are also shown.
dye undergoes aggregation and self-assembly in aqueous
medium to form nonfluorescent spherical nanoparticles in
which the dye remains chemically dormant. Therefore, the Sq
nanoparticles are found nonreactive to small molecular thiols.
However, addition of bovine serum albumin (BSA) or HSA
induces the disassembly of the dye nanoparticles that facilitates
encapsulation of the dye in the hydrophobic pockets of the
protein. This process reactivates the dye and triggers its
reaction with the cysteine residue at the 34th position of the
protein. The chemistry involves a base catalyzed nucleophilic
addition of the thiol moiety to the cyclobutene double bond of
the Sq dye at a pH of 7.0−8.0. This addition reaction results in
S1). This change in the absorption spectrum indicates the
aggregation of the dye which was further confirmed by
temperature-dependent UV−vis absorption and emission
spectroscopic studies in 15% acetonitrile/phosphate buffer
(pH 8.0) mixture (Figure S2). When the solution temperature
was increased from 25 to 70 °C, a significant increase in the
absorption intensity at 670 nm was observed, indicating the
disassembly of the aggregates to the monomeric dye.
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Atomic force microscopy (AFM) and transmission electron
microscopy (TEM) revealed the formation of spherical particles
of the dye with diameters ranging from 100 to 300 nm (Figure
S3 and Figure 2b). The nanoparticle formation was further
confirmed by dynamic light scattering (DLS) experiments of a
buffer solution containing Sq (6 μM) which showed particles
with a mean diameter of 200 nm (Figure 2c). The dynamic
correlation data show the characteristic of spherical particles
(Figure 2c, inset). In an attempt to encapsulate and stabilize the
Sq nanoparticles within a protein matrix, we added bovine
serum albumin (BSA) in a phosphate buffer (6 μM) at a pH
8.0. Surprisingly, DLS analysis after the addition of BSA showed
the disappearance of the peak at 200 nm corresponding to the
Sq nanoparticles with the appearance of a new band at 10 nm
which corresponds to the size of the globular protein (Figure
2d).26 This observation reveals that the BSA protein interacts
with the Sq nanoparticles, leading to a gradual disassembly of
the latter. Similar observation was found when HSA was added
to Sq nanoparticles. For a deeper understanding, the response
of Sq nanoparticles was investigated with the addition of a large
number of other proteins which did not show any variation in
the DLS data indicating that proteins other than BSA and HSA
do not interact with the dye nanoparticles (Figure S4).
In the light of the above findings, we studied the changes in
the absorption and emission spectral properties of the Sq
nanoparticles with SAP. For this purpose, we used BSA instead
of HSA since the former is easily available and cheaper but
structurally similar to the latter. Addition of BSA to Sq
nanoparticles (6 μM) in phosphate buffer at a pH of 8.0
resulted in the decrease of the broad absorption band 670 nm
with the formation of a new band at 380 nm (Figure 3a). The
dye nanoparticles initially had no emission at the 480 nm
region when excited at 380 nm, however addition of BSA
resulted in the formation of a band at 480 nm (Figure 3b). The
light-blue solution of Sq nanoparticles became pale yellow
(Figure 3a, inset) in color with the appearance of a green
emission upon the addition of BSA (Figure 3b, inset) in the
phosphate buffer at a pH of 8.0. Similar behavior was observed
with the addition of HSA (Figure S5). The fluorescence
intensity increased linearly with the increase in concentration of
the protein between 0−100 nM indicating a broad detection
range. From the fluorescence intensity variation (above 5% of
the initial fluorophore emission), the lower detection level of
BSA and HSA was found as 3 nM (Figure S6). Below this
concentration, no detectable fluorescence variation could be
noticed.
The selectivity of our probe toward SAP was established in
the presence of other thiol-containing proteins such as
glutathione reductase (GSSR), hemoglobin, bromelain, and
small thiol-containing molecules such as cysteine (Cys),
homocysteine (Hcy), glutathione (GSH), mercaptoethanol
(ME), dithiothreitol (DTT), thioglycolic acid (TGA), and
cysteamine. Excess (10 equiv) of these thiol-containing
molecules could not change the fluorescence intensity at the
480 nm region (Figure 4a,b) indicating that thiol group in these
molecules are nonresponsive to the Sq nanoparticles. The
selectivity could be visually observed by adding different thiol-
containing proteins and small molecules in a microwell plate
filled with the Sq nanoparticle solution. Under UV illumination,
green fluorescence was observed only for wells containing BSA
and HSA (Figure 4a inset). This observation reveals the
enhanced selectivity of Sq nanoparticles toward the free
Figure 3. (a) UV−vis absorption and (b) emission spectral changes of
Sq nanoparticles upon addition of BSA protein (0−14 μM) (λexc
@
380 nm). Arrows indicate the relative change in absorption and
emission with increase in concentration of BSA protein. Plots of the
absorption (top) emission (bottom) intensity vs BSA concentration
and photographs of Sq nanoparticles before and after addition of BSA
protein under UV illumination (365 nm) are shown in the inset.
cysteine residue in SAP in the presence of similar competing
functional groups.
In order to establish the role of Sq nanoparticles in the
selective sensing of SAP, we investigated the response of the Sq
dye in its monomeric form with various thiol-containing
molecules. For this purpose, the Sq dye solution in 30%
acetonitrile/phosphate buffer at pH 8.0 is prepared. Under this
condition, the dye exists in the monomeric state, which is clear
from the absorption maximum at 670 nm and emission
maximum at 700 nm (Figure S1). Upon addition of GSH, the
absorbance at 670 nm was decreased with the concomitant
formation of a new band at 380 nm (Figure 5a). When excited
at 380 nm followed by addition of GSH, the emission spectrum
of Sq solution exhibited a new band at 510 nm with a green
fluorescence, whereas the NIR emission intensity at 700 nm of
the Sq dye was decreased (Figures 5b and S7).
We further studied the effect of different thiol-containing
molecules such as Cys, Hcy, ME, DTT, TGA, and cysteamine
with Sq solution. These thiols exhibited fluorescence enhance-
ment at 510 nm with the concomitant decrease of the intensity
at 700 nm (Figure 5c), indicating that, though Sq in their self-
assembled state is specific to cysteine residue of the SAP, in the
monomeric state, Sq does not show selectivity toward any
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Figure 4. (a and b) Fluorescence response of Sq nanoparticles at 480
nm (λexc @ 380 nm) monitored against 1 equiv of HSA and BSA and
10 equiv of different proteins and small molecules. Inset in (a) shows
photograph of various proteins and small molecules in a microwell
plate containing Sq nanoparticles under UV illumination (365 nm).
Well A1 (GSSR), A2 (hemoglobin), A3 (bromelaine), A4 (trypsin),
B1 (pepsin), B2 (lysozyme), B3 (insulin), B4 (BSA), C1 (HSA), C2
(Cys), C3 (Hcy), C4 (GSH), D1 (ME), D2 (DTT), D3 (TGA), D4
(cysteamine). All experiments were performed using 6 μM Sq
nanoparticle solution in 25 mM phosphate buffer at pH 8.0.
specific thiols. Moreover, the fluorescence intensity of Sq
changes only with thiol-containing molecules and not with
other nonthiolated molecules (Figure S8). In order to confirm
the addition of thiol with Sq dye, we prepared a thiol adduct of
Sq with hexanethiol which displayed a peak at m/z 777.8
corresponding to the Sq-hexanethiol adduct (Figure S9).
A practical use of the Sq nanoparticles is established by the
estimation of HSA content in blood serum. Addition of aliquots
of the blood serum samples (10, 20, 30, and 40 μL) to Sq
nanoparticles (6 μM, in phosphate buffer pH 8.0) showed an
enhancement of the fluorescence intensity at 480 nm (Figure
6a). The HSA contents were estimated from the fluorescence
intensity values using a calibration graph (Figure S10). A
comparative plot of the concentrations of HSA in different
blood samples using the Sq nanoparticles and those obtained
for the same blood samples by the standard clinical test
(obtained independently from a clinical laboratory) is shown in
Figure 6b. The values obtained using our protocol were slightly
lower than those obtained from the standard clinical laboratory
analysis. The relative lower values of our method further reveal
that the free thiols present in blood serum in small quantities
do not interfere with the analysis of HSA as also independently
established by the detection of HSA in the presence of
equivalent amount of added thiols (Figure S11). Since, it is
known that the clinical method sometimes may overestimate
the albumin content, the Sq nanoparticle-based protocol
Figure 5. (a) UV−vis absorption and (b) fluorescence responses of Sq
(2 μM) upon addition of GSH (0−4 μM) (λexc @ 380 nm). (c)
Fluorescence response of Sq (2 μM) at 510 and 700 nm (λexc @ 380
nm) with different thiol-containing molecules (4 μM). These
experiments were performed in 30% acetonitrile/25 mM phosphate
buffer at pH 8.0.
described in this work can be used when a more accurate
estimation is needed (accurate up to the third decimal).27
From the above experimental observations, we hypothesized
that the protein selectivity could be due to the characteristic
tertiary structure of the SAP which may be responsible for the
disassembly of the Sq nanoparticles and the subsequent binding
with the protein. In order to prove this hypothesis, we reduced
the disulfide bonds that maintain the tertiary structure in SAP
using DTT reagent. The reduced proteins, despite of having
more numbers of thiol groups, showed relatively weak
fluorescence response to Sq nanoparticles when compared to
that of the native proteins (Figure 7a). DLS analysis of the Sq
nanoparticles in the presence of the denatured proteins at pH
8.0 did not show any change in the size distribution
corresponding to the Sq nanoparticles (Figure S12). This
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Figure 7. (a) Fluorescence response of Sq at 480 nm (λexc @ 380 nm)
upon addition of 1 equiv of native and denatured BSA and HSA
proteins. (b) Time-dependent fluorescence response of 1:10 Sq-BSA
at 480 (λexc @ 380 nm, green) and 700 nm (λexc @ 380 nm, red). All
experiments were performed using 6 μM Sq in 25 mM phosphate
buffer at pH 8.0.
Figure 6. (a) Emission intensity change of Sq nanoparticles (6 μM) in
25 mM phosphate buffer, pH 8.0 at 480 nm (λexc @ 380 nm) upon
addition of aliquots (0, 10, 20, 30, and 40 μL) of human blood serum
(HBS). (b) Validation of data obtained using Sq nanoparticles with
those obtained independently from a clinical laboratory using the
standard procedure.27
CONCLUSIONS
■
We have demonstrated the versatility of an organic dye
nanoparticle for the sensing of SAP in a pool of other
biomolecules. Sq dye in its native molecular form is reactive to
a variety of thiol-containing molecules. However, when the dye
self-assembles to form nanoparticles, only SAP could selectively
interact with the dye thereby opening the access for a thiol
attack. Thus, the dormant fluorescence moiety present in the
Sq dye gets activated latently, allowing the specific sensing of
SAP by “turn-on” green fluorescence. The fact that this
selective covalent modification of SAP is achieved only with the
self-assembled system and not with the monomeric dye does
make the Sq nanoparticles a selective supramolecular
fluorescent sensor. The enhanced selectivity of the Sq
nanoparticles allowed us to use them for the quantitative
estimation of HSA in human blood samples. The described self-
assembly approach using a small organic NIR dye having a
dormant fluorophore, which is latently activatable through a
nucleophilic attack is a model example for empowering a small
molecular fluorophore to a reaction-specific nanosensor by self-
assembly. This work is expected to encourage scientists to
design similar systems for sensing and imaging of biorelevant
molecules by exploring the unlimited potential of molecular
self-assemblies.
observation indicates that the denaturing of the tertiary
structure of the protein does not facilitate the disassembly of
the Sq nanoparticles, thus preventing the interaction of the
thiol moiety with the dye. The SAP-induced disassembly of Sq
nanoparticles may be driven by the strong affinity for the
binding of the Sq dye at the hydrophobic sites of the protein.28
Squaraine dyes are known to interact with SAP selectively by a
combination of hydrophobic, hydrogen bonding, and electro-
static interactions.21
If the addition of SAP induces the disassembly of the Sq
nanoparticles and its subsequent noncovalent binding within
the hydrophobic pocket, the native NIR fluorescence of the
molecular dye should be observed. This noncovalent
interaction pathway that occurs before the covalent mod-
ification of the protein is confirmed by a time-dependent
fluorescence monitoring of the Sq-BSA complex at 700 nm
(λexc @ 640 nm) (Figure 7b). Addition of 10 equiv of BSA
protein into the Sq nanoparticles (6 μM, phosphate buffer, pH
8.0) immediately enhanced the fluorescence intensity at 700
nm. With time, the fluorescence intensity at 700 nm decreased,
and the fluorescence intensity at 480 nm increased indicating
the covalent addition of the SAP thiol moiety to the dye. This
study confirms the initial complexation of the dye with the
protein before it undergoes covalent thiol addition which is
responsible for the selective sensing of SAP even in the
presence of free thiols.
EXPERIMENTAL SECTION
■
Materials and Reagents. Unless otherwise stated, all materials
and reagents were purchased from commercial suppliers. The solvents
were purified and dried by standard methods prior to use. Human
serum albumin (HSA), bovine serum albumin (BSA), glutathione
reductase (GSSR), hemoglobin, bromelain, trypsin, pepsin, lysozyme,
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and other amino acids were purchased from Sigma-Aldrich. All other
reagents were of analytical grade and were used without further
purification. Reactions were performed under inert atmosphere of
nitrogen unless specified otherwise. The pyrrole derivative 7 and the
semisquaraine derivative 10 were prepared as per literature
reports.29,30
AUTHOR INFORMATION
Corresponding Author
ajayaghosh@niist.res.in
■
Notes
The authors declare no competing financial interest.
Preparation of Sq Nanoparticles. A stock solution of Sq (1.2 ×
10−3 M) was prepared from acetonitrile. Fifteen μL of this solution
was injected into phosphate buffer (3 mL) maintained at pH 8.0, and
the solution (6 × 10−6 M) was kept under room temperature. The
green color of the stock solution turned blue indicating aggregation of
the dye. Nanoparticle formation was confirmed by AFM and TEM
analyses of the solution by drop casting on freshly cleaved mica surface
or carbon-coated copper grid (400 mesh), respectively, after drying in
vacuum.
Protein Sensing Experiments. The stock solutions of the
required proteins were prepared by dissolving in 25 mM sodium
dihydrogen phosphate and 10 mM sodium chloride. Concentrations of
these stock solutions were calculated from the absorbance at a
particular wavelength and molar extinction coefficient values. 0−100
μL of protein from the stock solution (4.2 × 10−4 M, phosphate buffer
at pH 8.0) was added to a stirring solution of Sq nanoparticles (6 ×
10−6 M, phosphate buffer at pH 8.0) in a glass cuvette with a path
length of 1 cm at room temperature (25 °C). The solution was kept
for 15 min, and the fluorescence intensity at 480 nm was measured
after exciting at 380 nm. For protein selectivity studies, 100 μL of
different proteins and small molecules from the stock solution (4.2 ×
10−3 M, phosphate buffer at pH 8.0) were added slowly to a stirring
solution of Sq nanoparticles (6 × 10−6 M) and kept for 1 h at room
temperature. The change of fluorescence intensity at 480 nm was
measured at an excitation wavelength of 380 nm.
Estimation of HSA in Human Blood Serum. Blood samples (3
mL each) were collected from healthy donors into a blood collecting
tube using sterilized syringe and needle. The blood samples were
allowed to clot by leaving it undisturbed at room temperature for 15−
30 min. The blood samples were centrifuged (Biofuge stratus, Heracus
instrument, Germany) at 3000 rpm for 10 min at 4 °C to separate the
serum from the red blood cells. Serum on the top portion is then
pipetted out into another vial which was used for the analysis. The
HSA content in blood serum was estimated with Sq nanoparticles by
using standard addition method. A calibration plot was prepared by
measuring the emission maximum at 480 nm (I480) upon addition of
different concentration of HSA (3 × 10−7 to 9 × 10−7 M) to the Sq
nanoparticles (6 × 10−6 M). The unknown concentration of HSA
protein in the blood serum was calculated from the calibration curve
by diluting the serum sample appropriately within the linear range.
Interaction of Sq Nanoparticles with Reduced Proteins.
Protein (4 × 10−4 M) and DTT (4 × 10−2 M) solutions dissolved in
phosphate buffer at pH 8.0 were taken in a 2 mL round-bottom flask
and was refluxed in a water bath for 4 h. After cooling, the solutions
were syringed out into a dialysis membrane of pore size 1000 and kept
for dialysis for 24 h in a 1 L beaker filled with phosphate buffer having
pH 8.0. The buffer solution was replaced each 2 h interval to remove
the excess DTT and its oxidized products. Presence of more numbers
of sulfhydryl groups in the reduced proteins were confirmed using
DTNB (5,5′-dithio-bis-(2-nitrobenzoic acid)) reagent.31 The dena-
tured protein (4 × 10 −4 M) from the dialysis membrane (45 μL) was
added to Sq nanoparticles (6 × 10−6 M), prepared in 3 mL phosphate
buffer at pH 8.0, and the emission intensity at 480 nm was recorded
after keeping for 1 h.
ACKNOWLEDGMENTS
■
We thank CSIR, Government of India, New Delhi for financial
support under NWP-023, DST and Dept. Atomic Energy,
Government of India for a DAE-SRC Outstanding Researcher
Award to A.A. P.A. and S.S. are grateful to CSIR for fellowships.
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ASSOCIATED CONTENT
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* Supporting Information
Materials, instruments, synthesis, and characterization of
compounds, experimental procedures, and supplementary
figures and schemes. This material is available free of charge
via the Internet at http://pubs.acs.org.
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