A thermo-responsive supramolecular hydrogel that senses cholera toxin: Via color-changing response

Article abstract of DOI:10.1039/d0cc00839g

A Pyrene-based amphiphile with C4-alkanoyl spacer and lactose (PyLac) self-assembles in the aqueous media to form an injectable hydrogel. It shows preferential binding with Cholera Toxin (CT) via its terminal galactose residue, and hence can be employed for the selective detection of CT via color-changing response.

Full text of DOI:10.1039/d0cc00839g

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DOI: 10.1039/D0CC00839G
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A Thermo-responsive Supramolecular Hydrogel that Senses
Cholera Toxin via Color-Changing Response
Dipen Biswakarma,a Nilanjan Dey a and Santanu Bhattacharya *a,b
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
A Pyrene-based amphiphile with C4-alkanoyl spacer and lactose maltose-appended compound (PyMal) showed no detectable
(PyLac) self-assembles in the aqueous media to form an injectable aggregation in aqueous media, while the lactose analogue
hydrogel. It shows preferential binding with Cholera Toxin (CT) via (PyLac) formed injectable hydrogel under similar conditions
its terminal galactose residue, and hence can be employed for the (Fig. 1b). Since PyLac possesses terminal galactose residue, we
selective detection of CT via color-changing response.
anticipated that it might interact with CT. Such binding event
can not only alter the fluorescence response of sensor gelator
molecule, but also their arrangements in the self-assembled
state. Upon encapsulation, the hydrophilic lactose residue
would no longer be available for hydrogen bonding. Thus, one
could expect that molecular-level interaction with CT might
influence the macroscopic property like gel-to-sol transition
process.
As mentioned earlier, PyLac above a certain concentration
[Critical Gelator Concentration (CGC): 1.52 mM], readily
formed an opaque gel in pure water. The hydrogel so formed
was thermo-reversible as confirmed by the repetitive freeze-
thaw cycles (Fig. 1d). Moreover, hydrogel samples remain
stable over an extended period without undergoing noticeable
decomposition. The gel melting temperature (Tgel) of the
hydrogel was found to be elevated with the increasing
concentration of the gelator indicating the formation of more
compact 3-D arrangement (Fig. S1a). Also, the hydrogel rapidly
Hydrogels belong to the class of functional soft materials due
to their easily tuneable optoelectronic properties and large
structural diversity. Particularly, hydrogels formed by the self-
or co-assembly of biocompatible interactive units, such as
peptides, amino acids, urea, steroids or carbohydrates have
recently come into focus due to their non-toxic nature and
‘highly specific’ on-demand response.1 Among them,
carbohydrate-based hydrogels provide chiral 3D-template in
addition to high water affinity and extensive hydrogen bonded
networks.2 Such hydrogels have indeed been used for diverse
purposes, such as optical sensing, wound healing, cell
encapsulation and sustained drug-release etc.3 On the other
hand, cholera is one of the most acute infectious diseases
known to mankind, characterized by profuse watery diarrhoea
and vomiting. Cholera Toxin (CT), secreted by Vibrio Cholerae,
is responsible for this fatal disease. The crystal structure of CT
indicates the presence of a hexameric AB5 type architecture,
where the five identical B-subunits specifically bind to the
glycolipid gangliosides via galactose units.4 Thus, galactose-
bearing synthetic multivalent systems, such as dendrimers and
nanoparticles (Table S1) can show specific interactions with
CT.5 However, till date no attempt has been made to utilize
supramolecular hydrogels for bio-sensing of Cholera Toxin.
Considering these, we report herein, the design and synthesis
of pyrene-based amphiphilic molecules with C4-alkanoyl
spacer and disaccharides, such as lactose and maltose as
hydrophilic units (Fig. 1a). Interestingly, the conformations, as
well as the 3D-arrangements of the sugar hydroxyl groups,
showed marked influence on their self-assembly process. The
a. ADepartment of Organic Chemistry, Indian Institute of Science, Bangalore,
Karnataka 560012, India
Fig.1. (a) Pictorial representation of PyLac amphiphiles in water. Gel picture of PyLac
(2.5 mM) under (b) daylight and UV lamp (> 365 nm). (c) Sol formation of PyMal at high
concentration of 10 mM. (d) Gel-to-sol transition in presence of heat/cool and
shake/rest. (e) AFM, (f) SEM, (g) TEM images of the solution of PyLac (0.09 mM). (h)
Fluorescence microscopy images of the solution of PyLac (0.18 mM).
b. School of Applied & Interdisciplinary Sciences, Indian Association for the
Cultivation of Science, Kolkata 700032, India
E-mail: sb@iisc.ac.in, director@iacs.res.in; Tel: +91 (0)33 24734688.;
+918022932664
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converted to ‘sol’ under mechanical stress but reverted
spontaneously to gel upon resting, thus confirming the
thixotropic property of the hydrogel (Fig. 1d). On contrary,
even at sufficiently high concentration (~10 mM), PyMal does
not gelate, rather formed a viscous solution (Fig. 1c). The
different behavior of PyMal may be attributed to the higher
water solubility of the maltose residue compared to that of the
lactose, in addition to the configuration, as well as 3-D
orientations of the OH groups.
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DOI: 10.1039/D0CC00839G
The morphology of the gel was examined using wide range
of microscopic imaging. Both SEM (Fig. 1f) and AFM (Fig. 1e)
revealed the formation of spherical nanostructures with a
diameter of 180 ± 22 nm. On the other hand, TEM indicated
the presence of well-defined vesicle-like structures (Fig. 1g).
The vesicle-like nanostructures of the hydrogel were also
observed under the fluorescence microscopy (Fig. 1h). In order
to get the sizes of the vesicle-like aggregates in the solution
phase, diluted hydrogel samples were examined by dynamic
light scattering (DLS), which indicated the presence of particles
with the average hydrodynamic diameter of 303 ± 19 nm (Fig.
S1b). Further, the mechanical properties of the hydrogel
samples were evaluated via viscoelastic measurements. The
oscillatory amplitude sweep experiments (Fig. S2a) revealed
that the magnitude of the storage modulus (G’) value
(viscoelasticity of the hydrogel) increased with increase in
gelator concentration. Similarly, the frequency sweep
experiment indicated that the viscoelastic behavior of the
hydrogel was maintained throughout the frequency range of
1-100 Hz under the applied strain of 0.01% and G’ was always
greater than that of G” (Fig. 2c). Further, the thixotropic
property of the hydrogel was established using a hysteresis
loop test. The magnitude of G’ dropped from 1751 Pa to 409
Pa when a 30% strain was applied, while the mechanical
stiffness of the native hydrogel was restored to almost 97% of
the original value within a few secs upon release of the
induced strain (Fig.S2b). Such a characteristic of the hydrogel
allowed injection of them through a syringe needle of narrow
aperture and could be tuned into various alphabetical shapes
(Fig. 2b).6
In order to examine the mode of self-assembly, the FT-IR
spectrum of solid PyLac was compared with that of the dried
gel (Fig. 2a). The stretching frequencies of N-H and O-H groups
in solid PyLac were observed at 3414 and 3298 cm-1
respectively, which shifted to 3391 and 3290 cm-1 in case of
the dried hydrogel. This suggests the presence of H-bonding
interaction during the gelation. IR spectra also showed the
shifting of C=O (amide group) from 1650 cm-1 to 1642 cm-1,
confirming the participation of hydrazide units in
intermolecular H-bonding. In addition, the presence of the
pyrene unit allowed us investigation of the assembling nature
of the gelator molecules by conventional spectrophotometric
methods. Variable concentration UV/Vis spectra of PyLac
showed the presence of well-resolved vibronic peaks of
‘monomeric’ pyrene species at relatively low concentrations,
whereas, on increasing concentration (> 0.05 mM), one could
witness broad spectrum with residual absorbance at a longer
Fig.2 (a) FT-IR spectra of solid and dried hydrogel of PyLac. (b) Injectability
property of PyLac hydrogel. (c) Concentration dependent oscillatory frequency
sweep rheology data of PyLac. (d) Variable temperature fluorescence of PyLac
(0.80 mM) solution.
wavelength range, suggesting the formation of larger
aggregates (Fig. S3).7 Similarly, the concentration-dependent
emission spectra (Fig. S4a) revealed the presence of two
distinct bands at 378 and 396 nm at lower concentrations,
resembling the vibronic bands of the ‘monomeric’ pyrene
moiety with critical aggregation concentration (CAC) of 0.02
mM (Fig. S4b). However, formation of broad emission band at
470 nm was observed at high gelator concentration. This red-
shifted emission band may be attributed to a combination of
aggregation, π-π stacking and H-bonding interactions. The
relatively high average decay constant (τav = 1.27) observed at
470 nm band further confirmed our proposition.8 Variable
temperature emission studies show gradual diminution of the
fluorescence intensity at 470 nm, suggesting heat-mediated
‘breaking’ of the ‘slipped’ π-π stacking structures (Fig. 2d).
The supramolecular chirality of PyLac hydrogel was
established by circular dichroism (CD) studies, where the
presence of a positive bisignated signal was observed at high
concentration, indicating that the chiral information of the
sugar unit is transferred to the pyrenyl moiety in the self-
assembled state, leading to the formation of P-helical (right-
handed helical) aggregates (Fig. S5a). Further, the unperturbed
positive cotton signal during variable-concentration studies
suggests that concentration of monomer units have no affect
on the nature of the chiral assembly. Moreover, intensity of
the CD signal was found to be quenched at elevated
temperature and almost no detectable CD signal was observed
beyond 65 °C (Fig.3a, S5b) indicating heat-mediated
disassembly of the ordered arrangement. To understand the
packing of molecules in the self-assembled state, the powder
X-Ray diffraction (p-XRD) of freeze-dried gel samples were
performed, which showed the presence of Bragg reflection
peaks at 2θ = 3.29°, 8.69°, 12.84° indicating the corresponding
d-spacings at 2.06, 1.02, 0.69 nm respectively (Fig. 3b). The
reciprocal ratio of these reflection peaks was found to be
1:1/2:1/3 suggesting lamellar arrangements of gelator
molecules with a repetitive distance of 2.06 nm. XRD studies
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multivalent CT protein. Moreover, titration studies (Fig.4a)
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indicate that the present system is quDitOeI:s1e0n.1s0i3t9iv/De0iCnC0n0a8t3u9rGe
and could detect CT at as low as 0.4 µM in aqueous media. A
closer look at the emission profile showed an increase in the
I1/I3 value (I1 = intensity at 377 nm band, I3 = intensity at 396
nm band) with a rising concentration of CT. The smaller I1/I3
value at lower concentration of CT indicates hydrophobic
environment around the pyrene moiety of the hydrogelator
due to its aggregated form in the native state, while the larger
I1/I3 value at higher CT concentration suggests an increase in
the local polarity during the formation of hydrogelator-CT
assembly (Fig. S6a).11 To verify the formation of such hetero-
aggregates in the presence of CT, we repeated the titration of
PyLac with CT (Fig.4b) at a lower concentration of the
hydrogelator (5 µM). At this concentration, no distinguishable
emission peak was observed at 470 nm. However, upon
Fig. 2 (a) Variable temperature CD spectra of PyLac (0.20 mM) solution. (b) p-XRD titration with CT, emission intensity at 455 nm band enhanced
spectra of freeze-dried hydrogel of PyLac. (c) Proposed nano-structural model for the
formation of PyLac hydrogel.
with concomitant quenching at both 377 and 396 nm bands.
Thus, at low concentration of PyLac, ratiometric probing of CT
is possible. Moreover, the UV-Visible spectra of a PyLac (5 µM)
further revealed the presence of reflection peaks at 25.1° with
in the presence of CT (8 µM) showed a loss of fine-structured
a d-spacing value of 0.35 nm and this is presumably due to the
profile with an increase in the absorbance value at the higher
characteristic π-π interaction among the pyrenyl units. On the
wavelength region (Fig. S6b).
other hand, the molecular length, calculated from energy
Both observations reinforce the idea of toxin-mediated
optimized structure, was found to be 1.90 nm (Fig. 3c).
hetero-assembly formation. Importantly, the specificity of
Thus, from the above shreds of evidences, one can
PyLac towards CT was retained even in the presence of a large
speculate the arrangement of the PyLac molecules in the 3-D
number of competitive analytes (Fig. 4c). As expected, a
hydrogel network. Since the molecular length obtained from
drastic increase in hydrodynamic diameter from ~50 ± 10 nm
the computational studies is found to be shorter than that of
to 440 ± 25 nm was observed when PyLac at 5 µM
the XRD studies, one can assume significant interdigitation of
concentration was treated with CT (Fig. S7a-b). Similarly, time-
the pyrene residues in hydrogel.9 As expected, due to
dependent emission studies of PyLac showed multi-
hydrophobic nature, the polyaromatic pyrenyl units stay away
exponential decay (at 470 nm) with a large average time-
from the bulk water while lactose moieties direct towards
constant (τav = 1.3) in the presence of CT (Fig. 4d). This
water owing to the presence of free -OH units. The formation
enhanced average lifetime of PyLac can be correlated with the
of extensive H-bonding network via both hydrazone (donor-
slower dynamics of the dye molecules upon binding with
acceptor type) and lactose units is feasible.10 Most
CT.12The transmission electron microscopic images also show
importantly, at high concentration (above CGC value), these
aggregation of the PyLac molecules in the presence of CT
molecular level interactions help propagation of the self-
(Fig.S7c-d). Further, to prove the importance of the terminal
assemblies into three-dimensional structures, which ultimately
leads to the formation of vesicle-like morphology (Fig.3c).
Since PyLac amphiphiles possess galactose units at the
terminal position, the current system was employed for optical
sensing of CT. For all subsequent spectroscopic studies, we
have used PyLac concentration as 0.8 mM, which is lower than
its CGC value but higher than CAC. This suggests that PyLac
exists essentially in the aggregated state at this concentration
in water. As expected, PyLac showed cyan colored
fluorescence at this condition with a broad emission band at
~470 nm, along with sharp peaks at 380-440 nm region.
Addition of CT to the aqueous solution of PyLac resulted in a
~5.3-fold quenching of the emission intensity at 470 nm band
with ~14 nm blue-shift in the emission maximum (Fig. 4a).
Thus, the color of the solution turned from cyan to blue in the
presence of CT. The CT-triggered ‘dissociation’ of the
performed molecular assembly might be the probable reason
for this fluorescence quenching, while blue-shift indicates
hetero-assembly formation between gelator molecules and
Fig. 3 Titration of (a) PyLac (0.8 mM), (b) PyLac 5µM with increasing CT concentration.
(c) Selectivity studies for the interaction PyLac (0.8 mM) with different analyte. (d)
Fluorescence Decay profile of PyLac in presence and absence of CT.
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OH groups of the sugar moiety showed profound effect in
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DOI: 10.1039/D0CC00839G
controlling the self-assembly process. The viscoelastic
measurement indicates thixotropic nature of the gel, whereas,
morphological analysis showed formation of vesicle-like
nanostructures. Importantly this lactose-bearing pyrenyl
system exhibited color changing response (ratiometric
biosensing) in the presence of cholera toxin (CT). Addition of
CT induced dissociation of the preformed self-assembled
structure of PyLac by concurrent formation of a ‘more stable’
hetero-aggregate, further led to gel-to-sol transition.
Fig. 4 (a) Linear change in the emission intensity of PyLac (5 μM, λex = 345 nm) with
increasing CT (0-8 μM) in the presence of different analyte present in stool samples. (b)
Gel-to-sol transition in the presence of CT (0.6 mM). (c) Images captured (UV lamp >
365 nm after addition of CT onto pre-coated TLC plate of PyLac hydrogel. (d) Pictorial
representation for the interaction of PyLac with CT.
Conflicts of interest
There are no conflicts to declare.
Notes and references
galactose unit, the interaction of CT was also monitored in the
presence of PyMal compound (Fig. S7e). In spite of high
structural similarity, this compound showed no interaction
with CT, indicating the indispensable role of the terminal
galactose unit in the sensing process.
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Further, PyLac was utilized for detecting CT in the presence
of various ionic analytes, commonly present in the watery
stool samples (diarrhoea).13 Since, no interaction was seen
from any of these analytes, it is anticipated that PyLac can
even be used for clinical diagnosis (Fig. S8). Further, a dose-
dependent linear change in the emission intensity was
observed with CT (0-5 µM) (Fig.5a). Also, the present system
was engaged in the estimation of CT in different types of
natural water samples. For this, water samples were collected
from different places were incubated with various amounts of
CT (0-4 µM) prior to spectral analysis. Recovery analysis
indicates that in most cases the proportional error was less
than 5%, which indeed is an exciting observation (Fig.S9).
The fact that CT interacts with PyLac at molecular level,
intrigued us to check its effect on preformed supramolecular
assembly. Interestingly, the addition of CT (0.6 mM) in sub-
stoichiometric amount into a preformed gel sample (3 mM)
immediately induced a gel-to-sol transition (Fig. 5b). The
binding of CT to galactose units possibly disrupted the H-
bonding network, leading to the breakdown of self-assembled
structure and the consequent induction into sols (Fig. 5d).
Further, to check the relevance of both the pyrene and lactose
units in CT sensing, two more hydrogel systems were
examined. Though in this case, both maltose (1) and lactose (2)
appended probes form hydrogel, selective gel to sol transition
was observed only in case of 1. Also, such observations ruled
out the direct involvement of pyrene in the sensing process
(Table S10). To employ the sensory systems even in remote
areas, we developed low-cost paper strips for rapid detection
of Cholera Toxin. The PyLac (0.8 mM) gel coated paper strips
showed quenching of cyan fluorescence in the presence of CT.
Remarkably, even on such paper strips, we could see dose-
dependent changes in the emission intensity (Fig. 5c).14
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In conclusion, we present here a thermoreversible
injectable hydrogel based on a pyrene-disaccharide (PyLac) in
which both configurations, as well as 3-D orientations of the
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