Motion-Induced Changes in Emission as an Effective Strategy for the Ratiometric Probing of Human Serum Albumin and Trypsin in Biological Fluids

Article abstract of DOI:10.1002/asia.201701795

Herein, we report the formation of a highly luminescent, pH-sensitive, thermoreversible nanoaggregate in pure aqueous medium through the self-agglomeration of carbazole-based amphiphiles. The self-assembly process restricted the intramolecular motion of the molecules and induced a change in its emission signal from blue to cyan, owing to an aggregation-induced emission (AIE) effect. A similar type of ratiometric response was also observed in the presence of human serum albumin (HSA). However, in this case, the molecular motion of the flexible fluorescent probe was restricted by its embedded microenvironment, owing to a motion-induced change in emission (MICE) effect, not by aggregation. Moreover, the probe showed quite high selectivity for HSA over other serum albumin proteins. Our carbazole-based fluorescent probes are a unique example of the ratiometric sensing of HSA through the sole involvement of reversible noncovalent interactions. Considering the important of HSA in clinical diagnosis, a wide range of biological fluids, such as human urine, saliva, and plasma, were screened to analyze their HSA content. In addition, this system was also employed for the detection of trypsin at subnanomolar concentrations through the digestion of HSA.

Full text of DOI:10.1002/asia.201701795

CHEMISTRY
AN ASIAN JOURNAL
www.chemasianj.org
Accepted Article
Title: Motion Induced Change in Emission as an Effective Strategy for
Ratiometric Probing of Human Serum Albumin and Trypsin in
Biological Fluids
Authors: Nilanjan Dey, Basudeb Maji, and Santanu Bhattacharya
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To be cited as: Chem. Asian J. 10.1002/asia.201701795
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Chemistry - An Asian Journal
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Motion Induced Change in Emission as an Effective Strategy for
Ratiometric Probing of Human Serum Albumin and Trypsin in
Biological Fluids
[
a]
[a]
[a], [b]
Nilanjan Dey, Basudeb Maji and Santanu Bhattacharya*
2
0,21
Abstract: We report a highly luminescent pH sensitive, thermo-
reversible nanoaggregate material in pure aqueous medium via self-
agglomeration of carbazole-based amphiphiles. The self-assembly
process restricts the intramolecular motions of the molecules and
induces a change in emission signal from blue to cyan (AIE effect). A
similar kind of ratiometric response is also observed in presence of
human serum albumin (HSA). However, here the molecular motion
of the flexible fluorescnce probe is restricted by its embdding
microenvironment, not aggregation (MICE effect). Moreover, the
probe shows fairly high selectivity towards HSA over other serum
albumin proteins. Our carbazole-based fluorescence probes are a
unique example, where ratiometric sensing of HSA is achieved
solely by involving reversible noncovalent interactions. Considering
the high significance of HSA in clinical diagnosis, a wide range of
biological fluids, such as human urine, saliva, and plasma have been
screened for analyzing HSA contents. In addition to this, the present
system is employed for detection of trypsin at sub-nanomolar
concentration via digestion of HSA.
albumin level.
Thus this time-sensitive nature can restrict the
applicability of this probe in developing high throughput
quantification assay. On the other hand, recently, fluorogenic
probes with aggregation-induced emission enhancement (AIEE)
property have become immensely popular in biomolecular
22
sensing. Presence of intramolecular rotating units makes these
probes poorly emissive in the dispersed state (non-radiative
decay). However, aggregation can restrict these intramolecular
motions and reduce the number of non-radiative decay
pathways. Thus instead of getting conventional aggregation-
induced emission quenching (AIEQ), aggregation-induced
emission enhancement (AIEE) can be observed. Though there
are several small molecules based AIEE sensors reported in the
literature for selective detection of albumins, very few of them
found to be suitable for actual clinical applications due to limited
sensitivity (detection limit >30 µg/mL). Moreover, discrimination
among different kinds of serum albumin is very difficult utilizing
optical sensors, since all of them posses highly resembled
primary structures. Till date, only a handful of sensors have
appeared in the literature, which could exhibit preferential
2
3
2
4
interaction with HSA over its structurally homologous analog
Introduction
25-29
BSA.
Therefore
careful
consideration
of
these
aforementioned points is essential to develop proper prognostic
marker of HSA in biological fluids.
Human Serum Albumin (HSA), the most abundant plasma
protein present in our circulatory system, plays a pivotal role in
maintaining plasma osmotic pressure and also transporting a
wide range of exogenous as well as endogenous
1
N
0
N
HN
HN
4
N
N
N
N
H
H
2
3
1
,2
biomolecules. The abnormal level of HSA in human body is
the symptom of many diseases, such as hepatitis, liver cirrhosis,
N
N
Restrictionin
3,4
Intramolecular Rotation
diabetes mellitus etc. Thus quantification of albumin level in
biological fluids (blood serum, urine, saliva etc.) can serve as a
major diagnostic test for the patients suffering from the above-
Compound 1
N
N
5
-8
(Flexible state)
Strategy1
(Conformationally locked state)
Temperature
mentioned illness. There are three different sensing strategies
known in the literature for the estimation of serum albumins,
such as dye-binding based optical assay, antibody-based
immunochemical method, and the LC-MS/MS-based proteomic
9
-12
technique.
Among all these strategies, the colorimetric assay
Restriction in molecular motion due to aggregate formation (AIE)
is the most user-friendly one, since it is cost-effective and less
tedious with the minimum number of sample preparation
Strategy2
HSA
13-18
steps.
The clinically approved optical methods mostly involve
19
Bromocresol green (BCG) as the color generating dye. Though
the high sensitivity of BCG towards HSA can be beneficial for
the estimation purpose, it can interact with other plasma proteins
within the 30s of incubation leads to the overestimation of
Trypsin
Conformational rigidification during protein encapsulation (MICE)
Figure 1. Structure of compound 1 with a schematic diagram showing its
stimuli-responsive emission switching behavior [formation of highly emissive
conformationally restricted state as a result of AIE (top) and MICE (below)
effect].
[
[
a]
b]
N. Dey B. Maji and Prof. S. Bhattacharya
Department of Organic Chemistry
Indian Institute of Science
Considering the present scenario, herein we have synthesized a
series of easily synthesizable carbazole-based luminescent
probes (Figure 1), which can form self-assembled luminescent
nanoaggregates in water with high thermo-reversibility and pH
Bangalore-560012
Email: sb@orgchem.iisc.ernet.in
Present Address: Indian Association of Cultivation of Science,
Kolkata 700032, India
sensitivity. The compound
1
exhibited highly selective
ratiometric response towards (change in emission color from
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Chemistry - An Asian Journal
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blue to cyan) HSA. Thus it can be considered as a unique
example of ratiometric sensing of HSA through reversible
noncovalent interactions. It is important to note that the
ratiometric sensors are often superior to the ‘turn-on’ probes
since they can provide an inbuilt correction to eliminate
interferences caused by the sudden change in the external
environment. Further, the high specificity of the probes towards
HSA was exploited to estimate albumin content in various
biological fluids, such as human urine, saliva, and blood plasma
samples. Finally, protease-induced hydrolysis of HSA was
employed as an alternative strategy for identification of trypsin at
physiological condition (pH 7.4).
allow utilizing these probes for diverse biomedical applications,
such as drug delivery or sensing.
33-36
(a) 120
(b) 10
4
3
2
1 in THF
1
0
0
At 400 nm
At 454 nm
90
1
60
1 in Water
101
30
10
0
0
350
400
450
500
550
600
d) 100
80
20
40
60
80
100
Wavelength (nm)
Time (ns)
(c) 1
(
00
At 85 o
30% glycerol
0% glycerol
C
80
6
0
0
6
0
4
40
At 15 o
C
Results and Discussion
20
0
20
0
3
50
400
450
500
550
600
350
400
450
500
550
600
Nanoaggregates formation of compound 1 at pH 6.0 in water
In non-polar solvent like THF, the fluorescence spectrum of
compound 1 showed a moderately emissive band at ~400 nm,
which experienced a strong red-shift (~70 nm) in Tris-HCl buffer
Wavelength (nm)
Wavelength (nm)
Figure 2. (a) Emission spectrum of 1 (ex  350 nm, 10 µM) both in THF and in
PBS buffer at pH 6.0 [Inset shows TEM images of 1]. (b) Time-dependent
emission decay of 1 at 400 and 454 nm at pH 6.0 in water ((ex  350 nm, 10
µM). (c) Temperature-dependent changes in emission spectra of 1 (ex  350
nm, 10 µM) at pH 6.0 in PBS. (d) Emission spectra of 1 (ex  350 nm, 10 µM)
(
(
at pH 6.0) due to the formation of close-contact aggregates
Figure 2a). To validate the possible self-assembly processes of
o
in different glycerol-water mixture media (0-30% glycerol) at pH 6.0, 37 C.
compound 1 at pH 6.0, detailed morphological transformations
were investigated alongside with dynamic light scattering studies
Interaction of compounds with plasma proteins at pH 7.4
After demonstrating the molecular dynamic controlled two
distinct photophysical properties of the probe 1, we sought to
exploit its molecular recognition property. Thus we proceeded to
investigate its interaction with serum albumins since these
macromolecular hosts are known to induce conformational
rigidification in the amphiphilic guest molecules upon
encapsulation. To eliminate the influence of temperature and pH
on the sensing stuides, we fixed the pH of the medium as 7.4
(
Figure S1). The time-dependent emission studies indicated that
in pure THF, compound followed single-exponential decay
presumably due to the presence of uniformly-dispersed
monomeric units. However, the formation of long-lived excited
state species with multi-exponential decay channel was
observed at pH 6.0 (Figure 2b & S2). In dispersed state, the
probe molecules experience high conformational flexibility,
which enhances the possibility of non-radiative relaxation
o
(
smaller decay constant/ faster decay). On the contrary, in
and temperature at 37 C for all the subsequent studies. Though
aggregated state the intramolecular motions becomes restricted,
which stabilizes the excited state in greater extent (higher decay
the compound showed monomeric emission (em  400 nm) at
this condition due to the presence of uniformly dispersed
building units, an addition of human serum albumin (HSA) could
rapidly change the emission color from blue to cyan by
producing a red-shifted emission band at 454 nm. However, no
shift in emission maxima was observed when the compound
was allowed to interact with other non-targeted enzymes and
proteins (Figure 3a). Further, the selectivity of 1 towards HSA
was investigated in presence of various biologically relevant
ionic analytes, neutral compounds, and thiol-containing amino
acids. As shown in Fig. S6, only HSA could elicit a dramatic
increase in the fluorescence intensity at 454 nm, while other
analytes led to negligible changes in fluorescence intensity
(Figure S6). Concentration-dependent emission studies with
HSA indicated that at a lower concentration of HSA (0-25 mg/L),
emission quenching was observed at 400 nm band, while at high
concentration (30-80 mg/L), fluorescence enhancement was
noticed at 454 nm (Figure 3b). Thus we could observe a dose-
dependent linear change in the emission intensity over a wide
range of HSA concentration (0-80 mg/L) (Figure 3c). This linear
ratiometric response is very crucial for accurate estimations of
HSA level in unknown samples with minimum background
interference. Further, the limit of detection (using blank variation
30
constant/ slower decay). The temperature variation studies
suggested that the aggregation phenomenon was highly thermo-
reversible in nature, even up to multiple numbers of the cycle. A
clear shift from aggregate to monomer emission was observed
when the temperature was raised from 15 to 85 °C (Figure 2c &
o
S3). Reversal of solution temperature back to 15 C,
regenerated the aggregate emission band. Changes in pH of the
medium indicated that the maximum inter-chromophore
association was observed at pH range 4.5–6.5 due to
protonation at the benzimidazole sites (Figure S4). Also, the
addition of conventional assembly–promoting electrolytes, such
as KCl was found to escalate the agglomeration process (Figure
31
S5). When emission spectrum of 1 was recorded in a highly
viscous medium (30% glycerol in water), a similar type of
emission enhancement was observed at 454 nm band due to
restricted motions of the individual interactive units (Motion-
induced change in emission) (Figure 2d). From the above shred
of observations, we can conclude that the red-shifted emission
band in case of nanoassembly also arises as the moleucles
adopt rigid molecular structure in the aggregated state (AIE
32
effect). The stimuli-response nature of the aggregates will also
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method) of 1 for HSA in PBS (pH 7.4 at 25 °C) was determined
to be 0.27 mg L .
the embedded molecule (from flexible to restricted motion) and a
‘turn-on’ emission signal can be observed at 454 nm band
-1 37-39
4
4,45
8
6
4
2
0
0
0
0
0
(Figure 4 & S8, Scheme S1).
Though the excited state
(a)
properties (increase in fluorescence intensity at 454 nm) of
FONs of 1 (formed at pH 6.0) is quite similar to that of 1 + HSA
complex, their ground state characteristics are entirely different.
In the former case, intramolecular motions of the monomer units
become in molecular aggregates (AIE effect), whereas in the
later situation, the single probe experiences motion changes
upon complexation with an analyte, here HSA (MICE effect). In
spite of striking similarity in the output emission signal, no
aggregate formation was observed in the presence of HSA.
As the albumins from different species mostly possess highly
homologous primary structure, differentiation among them is
1
2
3
4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
-
-
20
40
(
b)
Analytes added (80 g/mL)
c)
(
80 mg/L
2.0
1.5
1.0
0.5
1
50
46
difficult. However, when emission spectra of 1 were recorded
HSA
in presence of other serum albumins (like HSA, BSA, RSA etc.),
comparatively larger emission enhancement was observed with
HSA at 454 nm band (Figure 5a). However, no such preference
was witnessed when changes in emission intensity of 1 were
recorded at 400 nm band (Figure S9). This might be expected,
as at 400 nm, the emission response mostly dominated by the
nonspecific electrostatic interaction. Surfactants can destroy the
native structure of albumin protein and preferentially bind to its
1
00
50
0
mg/L
0
3
50
400
450
500
550
600
0
20
40
60
80
Wavelength (nm)
HSA added (mg/L)
Figure 3. (a) Changes in emission intensity of 1 (ex  350 nm, 10 µM) at 454
nm in presence of various bio-analytes at pH 7.4 (Images show changes in
emission color of 1 in presence of analytes, the sequence is same as
mentioned in the bar plot). (b) Emission titration of 1 (ex  350 nm, 10 µM)
with HSA (0‒80 mg/L) at pH 7.4 in PBS buffer. (c) Ratiometric change in
emission intensity of 1 (ex  350 nm, 10 µM) upon addition of HSA at pH 7.4
PBS buffer.
47,48
hydrophobic pockets (exposed during denaturation).
Thus,
when the emission response of 1 towards HSA was monitored in
presence of CTAB, no red-shifted emission band could be
observed (Figure S10). This again indicated the red-shifted
emission band at 454 nm appeared during the encapsulation of
dye molecules inside hydrophobic pockets of the protein.
However, there was barely any change in the extent of emission
quenching at 400 nm band.
The analysis of thermodynamic parameters (H and S) as
calculated from variable temperature emission titration studies
indicated that compound 1 with HSA involves a combinational
40
effect of electrostatic and hydrophobic interactions (Table S1).
A negative value of H with positive entropy change (S) at 400
nm, suggested that the spectral changes at 400 nm were
governed by the electrostatic interactions. At the same time,
positive changes in both enthalpy and entropy values were
observed at 454 nm, which ensured the role of hydrophobic
State-A
H
N
Turn On
at 454 nm
State-B
O
O
Turn Off
at 400 nm
Hydrophobic
cavity
HSA
4
1-43
interaction in the recognition process.
From the above
HSA
1
:1 encapsulated complex of 1
observations, it can be assumed that there may be two
independent binding sites available for compound 1 in HSA.
Some molecules (due to the positively charged backbone) will
nonspecifically bind to the negatively charged surface of the
protein via electrostatic interaction, mostly with surface-exposed
negatively charged functional groups, like carboxylates or
hydroxyl groups etc. Since the surface exposed negatively
charge in case of HSA will be pH dependent, the extent of
quenching (at 400 nm) will also be affected by pH of the medium.
Since electrostatic complexation can enhance the micropolarity
around the probe molecules, an emission quenching is observed
due to dipole-dipole interaction. It is important to note that
quenching of emission intensity at 400 nm was also observed in
presence of other proteins we well, especially upon addition of
proteins with pI value < 6.0, such as casein, -lactalbumin etc
Nonspecific electrostatic interaction
1
80
150
120
State-B
1
50
em  400 nm
State-A
120
9
0
90
60
60
Monomer
emission
3
0
30
1 + HSA
1 alone
0
0
3
50
420
490
560
630
350
420
490
560
630
Wavelength (nm)
Wavelength (nm)
Figure 4. A schematic diagram shows two different modes of interaction of 1
with HSA at pH 7.4, nonspecific electrostatic interaction (State A) and site-
specific hydrophobic encapsulation (State B).
The identification of the binding site (site of encapsulation) of 1
in HSA is very crucial for a deep understanding of its working
principle. In order to do so, we first determined the stoichiometry
of interaction of 1 with protein at 454 nm band using continuous
(
Figure S7). This confirmed the nonspecific nature of the
electrostatic interaction. On the other hand, owing to their
amphiphilic nature, some of the probe molecules may go inside
the hydrophobic interior of the HSA (highly selective for HSA as
observed in figure 3a). This may significantly alter the motions of
4
9,50
variation method.
An inflection point at ~0.5 mol-fraction
suggested there might be a 1:1 interaction of 1 with HSA (Figure
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S11). The site-specific nature of 1 was verified by conventional
dye displacement assays in presence of three different albumin
binding drugs, including warfarin (domain IIA), ibuprofen
As the compound showed a prominent influence of pH on its
assembly formation behavior, we investigated the effect of pH
on its albumin sensing property. The emission spectrum of 1 at
pH 6.0 clearly showed aggregate emission with a maximum at
454 nm. Titration with HSA at this condition induced dissociation
of the preformed molecular self-assembly at lower concentration
range (0-10 µg/mL), which was perceptible from the blue shift
(~45 nm) in the emission maximum. Further increase in HSA
concentration (10-90 µg/mL) showed a similar type of spectral
change as observed at pH 7.4, i.e. first quenching of the
emission intensity at 400 nm band as HSA concentration varied
from 10-40 µg/mL (electrostatic interaction) and then a ‘turn-on’
emission response at 454 nm from 40 to 90 µg/mL (MICE effect)
(Figure 5d & S15). However, the extent of emission quenching
at 400 nm was found to be inferior in this case due to poor
electrostatic binding of the dye molecules with HSA. The
probable reason for this might be the lowering of surface-
exposed negative charge of HSA due to pH-driven shift in the
protonation equilibrium.
51
(
domain IIIA), and digitoxin (domain IIIB) (Figure 5b). The
selective displacement of warfarin (~50-53 %) suggested that
the dye might have a preference towards the site-IIA of HSA.
The shallow binding pocket of HSA at domain IIA (in comparison
to BSA) can explain the higher degree of RIR in case of HSA
52
with expected larger emission enhancement at 454 nm band.
The time-dependent emission studies indicated an increase in
average decay time of 1 in presence of HSA (Figure S12). This
is quite expected, since in the encapsulated state, probe
molecule adopts rather rigid molecular structure and thus
fluorescence dominates the energy transfer process, not the
53,54
non-radiative relaxation.
Along with conformational rigidity,
the relatively hydrophobic environment around the dye
molecules also diminishes the possibility of water-mediated
emission quenching upon encapsulation.This was evident by the
higher quantum yield (at 454 nm) of the dye in presence of HSA
(
Φ  0.18) as compared to its nanoaggregate form (Φ  0.1).
Besides this, changes in circular dichroism signals of the protein
upon incubated with different amount of 1 showed a perturbation
in the -helical structure of the protein template (Figure 5c). This
suggested that the probe molecule is embedded in the α-helices
Effect of terminal functional groups on HSA sensing
To investigate the role of aliphatic chain and the end functional
groups on the protein recognition ability of the probe, we
involved additional compounds in studies with variation in the
55,56
from subdomain IIA of HSA.
The FT-IR analysis also
4 8
chain lengths (C vs. C aliphatic chain) and end-functional units
revealed that FT-IR stretching frequencies (amide I band, -OH
(tertiary nitrogen ends vs. quaternary ammonium center) (Figure
S16). Surprisingly, all these compounds showed prominent
nano-assembly formation in water (at pH 6.0) despite their
structural diversity (Figure S17). Variable temperature studies
ensured the thermo-reversible nature of aggregates remained
unfazed even after modifying the lengths of the aliphatic chain or
the nature of the terminal functional group (Figure S18). Besides
that, pH and ionic strength dependent changes in emission
signals were also observed similar to that of compound 1 (Figure
S19). However, interaction with HSA clearly indicated that the
compounds having the hard positive charge at the nitrogen end
(2 and 4) experienced stronger dipole-dipole interaction with the
surface-exposed negatively charged functional groups of HSA.
This led to a higher degree of emission quenching at 400 nm
band in presence of HSA (higher Stern-Volmer quenching
constant values) at pH 7.4 (Figure 6a & Table S2).
and –NH) related to α-helices of protein got affected during
57,58
interaction with 1 (Figure S13 & S14).
a) 80
(
Without any drug
(
b) 1.8
With Ibuprofen
With Warfarin
With Digitoxin
60
1
.6
.4
40
1
2
0
1.2
.0
1
0
2
0
40
60
80
HSA added (mg/L)
Proteins added (80 g/mL)
(
c)
1
(d) 100
00
1
at pH 6.0
8
6
4
2
0
0
0
0
0
HSA 10 mg/L
HSA 30 mg/L
HSA 80 mg/L
50
Wavelength (nm)
210
240
0
1
alone
10 µM
-
50
100
150
On the other hand, compounds with longer aliphatic chains (1
and 2) will have higher tendency to go into the hydrophobic
cavity of the protein molecule (type 2 interaction). Thus a higher
degree of emission enhancement (population of molecule bound
to the hydrophobic cavity is more) could be observed at 454 nm
HSA
-
0
µM
350 400 450 500 550 600 650
Wavelength (nm)
-
Figure 5. (a) Change in emission intensity of 1 (ex  350 nm, 10 µM) at 454
nm upon addition of different kinds of albumin at pH 7.4 in PBS buffer. (b)
Drug-displacement assay of 1 (ex  350 nm, 10 µM) for identifying site-
specific interaction of 1 with HSA at pH 7.4 in PBS. (c) Circular dichroism
spectra of HSA (80 mg/L) in presence of 1 (0-10 µM) at pH 7.4. (d) Interaction
of compound 1 (ex  350 nm, 10 µM) with HSA at pH 6.0 in PBS buffer.
(
Figure 6b & Figure S20). Nevertheless, in all the cases, we
could observe the distribution of compounds in both interactive
sites. Thus irrespective of their extent of interaction, one can
observe emission quenching at 400 nm and fluorescence turn-
on at 454 nm band.
Effect of pH on the extent of HSA sensing
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Chemistry - An Asian Journal
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(
a) 2.1
Compound 1
Compound 2
Compound 3
(b) 2.1
Compound 1
Compound 2
Compound 3
Compound 4
(hypoalbuminemia)
gastrointestinal protein loss, while high level (hyperalbuminemia)
suggests dehydration and increase the body weight.
Similarly, the reference range of albumin in urine is 2.2–25 mg/L.
Higher concentration of albumin in human urine normally
indicates
diseases
like
nephrosis,
1
1
1
.8
.5
.2
1.8
Compound 4
63-65
1
.5
.2
1
0
5
10
15
20
25
3
0
40
50
HSA added (mg/L)
at 50 mg/L trypsin
60
70
80
90
suggests high permeability of renal glomeruli due to diabetes or
HSA added (mg/L)
66,67
renal damage.
On the other hand, the normal concentration
(
c)
1
80
50
20
(
d) 1.8
of HSA in saliva is generally less than 0.5 g/L. An increase in
concentration, in this case, indicates severe health problems
at 40 mg/L trypsin
1
1
0 min
1
.6
1.4
.2
.0
at 30 mg/L trypsin
at 20 mg/L trypsin
68
such as type-2 diabetes mellitus or stomatitis etc. In a typical
Time
9
6
3
0
0
0
experimental procedure, we have monitored the change in
emission intensities of 1 both at 400 and 454 nm band in
presence of serum/urine/saliva samples appropriately diluted in
buffer medium (pH 7.4) (Figure 7a, c, e). The HSA
concentrations in these matrices were then estimated using
standard calibration plot as obtained from the titration studies in
normal buffered medium (pH 7.4). To our relief, the HSA levels
estimated by the present method were found to be close to the
results determined by using the BCG method (Figure 7b, d, f).
The relative variations between these two methods are less than
at 10 mg/L trypsin
at 0 mg/L trypsin
120 min
1
1
0
3
50 400 450 500 550 600 650
Wavelength (nm)
0
30
60
90
120
Time (min)
Figure 6. Change in emission intensities of compounds (1-4) upon addition of
HSA (a) monitored at 400 nm, (b) monitored at 454 nm. (c) Time-dependent
changes in emission spectra of 1 + HSA upon addition of trypsin (50 mg/L) in
water (pH 7.4). (d) Time-dependent changes in emission intensity of 1 + HSA
at 454 nm with various amounts of trypsin (0-50 mg/L).
15% in all cases (Table S3-S5). Notably, the plasma, urine, and
saliva samples need to be diluted by 50, 20 and 1000 fold
respectively for the present assay, which means that only 4 µL
of plasma, 10 µL of urine and 0.2 µL of saliva will be sufficient
for HSA quantification by 1. These findings suggested that
quantification of HSA by this newly developed protocol was quite
sensitive and highly reliable.
Detection of Trypsin through digestion of HSA template
The design of optical probes for trypsin is highly desirable for
clinical diagnosis purpose as its abnormal level in the human
body could serve as the marker for many cardiovascular and
59-61
neurodegenerative diseases.
Thus we asked if the
preformed probe-HSA conjugate can be repurposed as the
template for trypsin sensing. Even though trypsin does not
directly interact with the probe molecule, trypsin-mediated
digestion of protein should release the encapsulated probe
molecule into the bulk medium. As a result, probe molecule can
regain its conformational flexibility (change in motion from
restricted to flexble). Thus the addition of trypsin could induce
quenching in the emission intensity of 1 + HSA ensemble at 454
nm. A 2 h incubation time was needed to observe the saturation
in the emission signal of 1 + HSA in presence of trypsin (Figure
25
(a)
(b)
0.36
0.34
0.32
0.30
0.28
20
15
10
5
0
1
2
3
4
5
Urine Sample Analysis (x 20)
Urine Sample Analysis
60
40
20
0
(c)
0
0
0
.34
.32
.30
(d)
6c). Further, time-dependent changes in the emission intensity
of 1 + HSA (at 454 nm) were monitored at different trypsin
concentrations, varied from 0 to 50 mg/L at pH 7.4 (Figure
0.28
0.26
1
2
3
4
5
6
d).The minimum detectable concentration of trypsin was
Serum Sample Analysis (x 100)
Serum Sample Analysis
(e)
800
00
400
determined to be 0.15 mg/L. As biological matrices may contain
other hydrolytic enzymes as well, we studied the sensitivity of
the system against other enzymes, such as lipase, apyrase,
glucosidase etc. To our relief, none of these enzymes produced
detectable alternation in emission signal of 1 + HSA (Figure
S21a). As EDTA is known for inhibiting the activity of trypsin at
biological pH, we repeated the emission studies of 1 + HSA with
trypsin in presence of 10 mM EDTA. As expected, here we
could observe the lesser extent of interaction with trypsin (Figure
S21b).
(f)
0.36
0.34
0.32
0.30
0.28
6
200
0
1
2
3
4
5
Saliva Sample Analysis (x 1000)
Saliva Sample Analysis
62
Figure 7. (a, c, e) Change in emission intensities of 1 (ex  350 nm, 10 µM)
upon addition of diluted urine, serum and saliva extracts at pH 7.4 in water (b,
d, f) Estimation of HSA contents of urine, serum and saliva samples collected
from healthy individuals.
Estimation of HSA in different biological fluids
The variation of HSA levels in human serum, urine or saliva can
serve as the biomarker for various diseases. We exploited the
rapid response of 1 towards HSA in estimating albumin contents
of these complex biofluids. The normal concentration of a range
of HSA in blood serum is 35–50 mg/L. However, its low level
Conclusions
In summary, we have developed a series of rapidly scalable
carbazole-based amphipathic dyes, capable of forming thermo-
reversible, pH-sensitive nanoaggregates at pH 6.0 in water.
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Though the compounds did not show any assembly formation at
pH 7.4, they could exhibit a change in emission signal from blue
to cyan upon binding to HSA. The mechanistic investigation
suggested the distribution of compounds at two independent
interaction sites. Nonspecific electrostatic binding of the
compound onto the negatively charged protein surface led
Dynamic Light Scattering Studies (DLS): DLS measurements were
done using
a Malvern Zetasizer NanoZS particle sizer (Malvern
Instruments Inc., MA) instrument. Samples were prepared and examined
under dust-free conditions. Mean hydrodynamic diameters reported were
obtained from Gaussian analysis of the intensity-weighted particle size
distributions.
to
effect), while encapsulation of dye into the hydrophobic cavity of
site-IIA induced turn-on response at 454 nm (cyan emission,
҅t urn-off҆ response at 400 nm band (blue emission, electrostatic
Atomic Force Microscopy (AFM): Solutions of the samples were drop-
cast on freshly cleaved mica sheets and then carefully air-dried 48 h. The
AFM images of the samples were recorded using a JPK 00901 AFM
instrument and analyzed using a Nano-Wizard software: Tapping mode,
10 nm tip radius, silicon tip, 292 kHz resonant frequency, 0.7-1 Hz scan
speed, 256x256, and 512x512–pixels. Finally, the images were analyzed
using Pico View 1.8.6 software.
҅
҆
MICE effect). Thus we could achieve ratiometric sensing of HSA
solely by exploiting noncovalent interactions. Most notably, the
probes also showed noticeably large interaction with HSA
compared to other serum albumins, which makes them even
more attractive for pathological applications. Further,
quantification of HSA level was performed in a wide range of
biofluids, including human urine, blood plasma, and saliva etc. In
all cases, the estimated values were further verified by the well-
established BCG method without notable variation (<15%). In
addition, selective detection of trypsin (a digestive enzyme) was
also achieved at a sub-nanomolar concentration by engaging
preformed probe-HSA complex as the detection template. Thus
we anticipate that the newly developed system can be converted
into a diagonestic probe for ratiometric estimation of both HSA
and trypsin in a wide range of biological fluids.
Circular Dichroism (CD): All the CD spectra were recorded on a
JASCO instrument, Model J-815-150S. Experiments were performed by
purging dry N2 gas continuously. Data were collected in a quartz cuvette
of 1 mm path length.
Fluorescence Microscopy: Diluted solutions of the samples are drop-
coated on pre-cleaned glass slides and left overnight for drying in a dust
free environment and finally evacuated. The fluorescence microscopic
image was taken on an Olympus IX-71 microscope.
Fluorescence Decay Experiment: Fluorescence lifetime values were
measured by using a time-correlated single photon counting fluorimeter
(
Horiba Jobin Yvon). The system was excited with nano LED of Horiba -
Jobin Yvon with a pulse duration of 1.2 ns. Average fluorescence
lifetimes (τav) for the exponential iterative fitting were calculated from the
decay times (τi) and the relative amplitudes (ai) using the following
relation (1).
Experimental Section
Material and methods: All reagents and starting materials were
obtained from the best known commercial sources and were used
without further purification. Solvents were distilled and dried prior to use.
FTIR spectra were recorded on a PerkinElmer FTIR Spectrum BX
2
2
2
τav=(a1τ1 +a2τ2 +a3τ3 )/(a1τ1 +a2τ2 +a3τ3)
(1)
Where a , a and a3 are the relative amplitudes and τ , τ , and τ are the
1
2
1
2
3
lifetime values, respectively. For data fitting, a DAS6 analysis software
1
13
71,72
system. H and C NMR spectra were recorded on a Bruker Advance
version 6.2 was used.
1
13
DRX 400 spectrometer operating at 400 and 100 MHz for H and
C
NMR spectroscopy, respectively.
Estimation of thermodynamic parameters during ligand-protein
interaction: The binding constants are first determined at a different
temperature. Then the values of Hº and Sº are calculated from the
slope and intercept of the Van’t Hoff plot (2) of ln K against 1/T. The
Gibbs energy change Gº is estimated according to Eq. (3).
Design and Synthesis of compounds 1-4: All the compounds involved
in the present study were synthesized based on methods reported in the
69,70
literature.
(Please see ESI)
Ln K = (-Hº/RT) + Sº/R
Gº = Hº - TSº
(2)
(3)

UV-visible and fluorescence Experiment: The UV−vis and
fluorescence spectra were recorded on a Shimadzu model 2100 UV-vis
spectrometer and Cary Eclipse spectrofluorimeter respectively. In the
emission experiments, the slit widths (for both the excitation and
emission channel) and the excitation wavelengths were chosen based on
the absorption maxima of the probe molecules. To monitored the effect of
pH, sensing experiment was performed in buffered media of different pH
According to Ross and Subramanian, a positive H value is usually taken
as typical evidence for hydrophobic interactions. Furthermore, specific
electrostatic interactions between ionic species in aqueous solution are
characterized by a positive value of S and a negative H value,
whereas negative H value and negative S value changes arise from
7
3
van der Waals forces and hydrogen bond formation.
(
HCO2Na/ HCl buffer for pH 2, Tris/HCl for pH
7
and
Analysis of human urine, saliva, and serum samples: Human blood
serum, saliva or urine samples of healthy volunteers were collected from
Indian Institute of Science (IISc) Health Centre with written consent from
the donors. The samples were stored at -80ºC for future use. During the
experiment, the samples were first kept at room temperature for 0.5 h
and then tested without any pre-treatment except required dilution in PBS.
Na2B4O7·10H2O/NaOH for pH 12). Again during temperature-dependent
experiments, 5 min of incubation was given in each case for thermal
equilibrium.
Transmission Electron Microscopy (TEM): The samples were made
under the dust-free condition and onto a Formvar-coated, 400 mesh
copper grid and allowed to remain for 15 min. After that, the grid was air-
dried and the last traces of solvent were removed under high vacuum.
Then the changes in morphologies were observed under TEM (TECNAI
T20) operating at an acceleration voltage (DC voltage) of 100 keV.
Micrographs were recorded at a magnification of 10000-80000 X.
Acknowledgements
S.B. thanks, DST (J. C. Bose Fellowship) for the financial
support of this work. N.D. and BM thanks IISc for research
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associate fellowship and the Indian Association for the
Cultivation of Science, Kolkata for the financial support of this
work presented in this manuscript.
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Keywords:
Human
Serum
Albumin
•
Fluorescent
10.1021/acs.analchem.7b03520.
Nanoaggregates • Ratiometric Sensing • Biological Fluids •
Trypsin
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Table of Contents
FULL PAPER
Nilanjan Dey, Basudeb Maji and Santanu
Bhattacharya*
Page No. – Page No.
Title: Motion Induced Change in Emission as an
Effective Strategy for Ratiometric Probing of
Human Serum Albumin and Trypsin in Biological
Fluids
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