Lipase sensing by naphthalene diimide based fluorescent organic nanoparticles: a solvent induced manifestation of self-assembly

Article abstract of DOI:10.1039/d0sm02056g

The precise control of supramolecular self-assembly is gaining utmost interest for the demanding applications of manifested nano-architecture across the scientific domain. This study delineates the morphological transformation of naphthalene diimide (NDI) derived amphiphiles with varying water content in dimethyl sulfoxide (DMSO) and the selective sensing of lipase using its aggregation-induced emission (AIE) properties. To this end, NDI-based, benzyl alcohol protected alkyl chain (C1, C5, and C10) linked amphiphilic molecules (NDI-1,2,3) were synthesized. Among the synthesized amphiphiles, benzyl ester linked C5 tailored naphthalene diimide (NDI-2) exhibited AIE with an emission maximum at 490 nm in a DMSO-water binary solvent system fromf_w= 30% and above water content. The fibrous morphology ofNDI-2atf_w= 30% got gradually transformed to spherical aggregated particles along with steady increment in the emission intensity upon increasing the amount of water in DMSO. Atf_w= 99% water in DMSO, complete transformation to fluorescent organic nanoparticles (FONPs) was observed. Microscopic and spectroscopic techniques demonstrated the solvent driven morphological transformation and the AIE property ofNDI-2. Moreover, this AIE ofNDI-2FONPs was employed in the selective turn-off sensing of lipase against many other enzymes including esterase, through hydrolysis of a benzyl ester linkage with a limit of detection 10.0 ± 0.8 μg L^?1. TheNDI-2FONP also exhibited its lipase sensing efficiencyin vitrousing a human serum sample.

Full text of DOI:10.1039/d0sm02056g

Soft Matter
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Lipase sensing by naphthalene diimide based
fluorescent organic nanoparticles: a solvent
induced manifestation of self-assembly†
Cite this: Soft Matter, 2021,
17, 2170
Debayan Chakraborty, Deblina Sarkar, Anup Kumar Ghosh and
Prasanta Kumar Das
*
The precise control of supramolecular self-assembly is gaining utmost interest for the demanding
applications of manifested nano-architecture across the scientific domain. This study delineates the
morphological transformation of naphthalene diimide (NDI) derived amphiphiles with varying water
content in dimethyl sulfoxide (DMSO) and the selective sensing of lipase using its aggregation-induced
emission (AIE) properties. To this end, NDI-based, benzyl alcohol protected alkyl chain (C1, C5, and C10)
linked amphiphilic molecules (NDI-1,2,3) were synthesized. Among the synthesized amphiphiles, benzyl
ester linked C5 tailored naphthalene diimide (NDI-2) exhibited AIE with an emission maximum at
490 nm in a DMSO–water binary solvent system from fw = 30% and above water content. The fibrous
morphology of NDI-2 at fw = 30% got gradually transformed to spherical aggregated particles along with
steady increment in the emission intensity upon increasing the amount of water in DMSO. At fw = 99%
water in DMSO, complete transformation to fluorescent organic nanoparticles (FONPs) was observed.
Microscopic and spectroscopic techniques demonstrated the solvent driven morphological transforma-
tion and the AIE property of NDI-2. Moreover, this AIE of NDI-2 FONPs was employed in the selective
turn-off sensing of lipase against many other enzymes including esterase, through hydrolysis of a benzyl
ester linkage with a limit of detection 10.0 ꢀ 0.8 mg Lꢁ1. The NDI-2 FONP also exhibited its lipase sensing
efficiency in vitro using a human serum sample.
Received 18th November 2020,
Accepted 21st December 2020
DOI: 10.1039/d0sm02056g
rsc.li/soft-matter-journal
because of the strong intermolecular interactions including p–p
stacking. This phenomenon is termed as aggregation-caused
Introduction
Fluorescence sensing has always been ahead in detection techni- quenching (ACQ), which restricts the utility of traditional
ques in comparison to the other methods owing to its rapidness fluorophores.4,5 To this end, the development of fluorescent
and convenient operation.1–3 The conventional luminogens show sensors through self-aggregation and supramolecular approaches
bright emission in the mono-dispersed state but in general this are finding notable significance due to their potential in bioima-
emission gets weakened or quenched at the aggregated state ging, molecular catalysis, selective sensing of environmentally and
biologically relevant molecules and so on.6–10 For example, Tang
et al. introduced new luminophores that show negligible emission
in dilute solution but emit intensely in the solid or aggregated state
School of Biological Sciences, Indian Association for the Cultivation of Science,
Jadavpur, Kolkata – 700032, India. E-mail: bcpkd@iacs.res.in
primarily due to restriction of intramolecular rotation (RIR) and
† Electronic supplementary information (ESI) available: Synthetic schemes of
amphiphiles (NDI-1–3), 1H-NMR, 13C-NMR, mass and IR spectral data of amphi- coined the concept as aggregation-induced emission (AIE).11–13
philes (NDI-1–3), 1H-NMR and mass spectral data of intermediates of NDI-2,
Consequently, several chemosensors have been reported exploiting
Table for comparison of LOD, AFM and bright field images of NDI-2 FONPs, DLS
data of NDI-2 in different binary solvent mixtures, solvent dependent 1H-NMR,
the advantages of AIE like activated radiative transitions, large
Stokes shift, low background noise and high photostability.14–17
normalised UV-Visible spectra of NDI-2, concentration dependent fluorescence,
Fluorescent organic nanoparticles (FONPs) with AIE properties are
TCSPC of NDI-2, stability experiment data, UV-Vis spectra and normalized UV-Vis
spectra of NDI-2 in the presence and absence of lipase, mass spectra of NDI-2 interesting manifestation of such self-aggregation derived from
p-conjugated oligomers, polymers, or chromophores.18–21 Typically,
supramolecular self-assembly is driven by a number of non-
after treating with lipase, fluorescence spectra and Stern–Volmer plot of NDI-2
(10 mM & 50 mM) in the presence of lipase, time dependent fluorescence spectra of
NDI-2 in the presence of CV lipase, fluorescence spectra of NDI-2 FONPs in the
covalent interactions that include hydrogen bonding, p–p stacking,
presence of varying concentration of CV lipase and (NH4)2SO4 solution and
hydrophobic interaction etc.22–25 Tuning of these interactions by
fluorescence spectra of NDI-2 FONPs in the presence of diluted human serum.
See DOI: 10.1039/d0sm02056g
means of changing the structure of building blocks and/or
2170 | Soft Matter, 2021, 17, 2170ꢁ2180
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modifying the polarity of solvent systems can tailor the morphology maximum at 99 vol% water in DMSO. This AIE-active NDI-2
and photophysical properties of the aggregated nanoparticles.26,27 FONP was employed in the selective turn-off sensing of lipase
FONPs can be successfully applied in bio-imaging, stimuli with a limit of detection of 10.0 ꢀ 0.8 mg Lꢁ1
.
responses, therapeutics and biosensing.28–31 In this context,
sensing of biomolecule like enzymes is very crucial both in
industry as well as in health care. Enzymes with a catalytic
activity play an indispensable role in the biological systems and
any kind of irregularity in their efficiency may lead to the
development of major diseases.32,33 Detection and monitoring
of the enzyme activity by a real-time method are crucial for
disease diagnosis and also to understand the metabolic mechanism.
Lipase, one of the main enzymes in the digestive system, can catalyze
reactions like hydrolysis, esterification, aminolysis etc. producing
simplest nutrients for living systems.34–36 Lipase also has notable
technological relevance in pharmaceuticals, agrochemicals,
laundry detergents, biopolymers and biodiesel.37–39 Lipase acts
as a crucial biochemical indicator for the diagnosis of acute
Materials
1,4,5,8-Naphthalenetetracarboxylic dianhydride, 6-aminocaproic
acid, 11-aminoundecanoic acid, Chromobacterium viscosum (CV)
lipase and other enzymes were bought from Sigma-Aldrich. D2O
was supplied by Cambridge Isotope Inc. All other reagents and
solvents required for synthesis, analysis and investigations were
purchased from SRL, India and Spectrochem, India. 1H-NMR
and mass spectra were recorded on AVANCE 500 MHz (Bruker)
and Bruker Ultaflex MALDI-TOF spectrometers using 2,5-
dihydroxy benzoic acid as the matrix, respectively.
Synthesis
pancreatitis (substantial increase in the lipase level), and can Naphthalene diimide (NDI)-based amphiphilic compounds were
also assist in the pancreatic enzyme replacement therapy prepared by following synthetic methods (Scheme S1, ESI†).
(PERT).40–42 Therefore, rapid, convenient and sensitive detection Initially, 6-amino caproic acid was dissolved in a 1,4 dioxane/
of the lipase level is crucial for clinical diagnosis and also for NaOH mixture and 1.0 equiv. di-tert-butyl dicarbonate (BOC
industrial lipase optimization. Although, there are reports on anhydride) was dropwise added to it. The whole solution was
colorimetric, turbidimetric, or fluorescent detection techniques kept for 12 h and then acidified with 1 N HCl. After addition of
using gold nanoparticles,43–45 reports on FONP based lipase brine solution, ethyl acetate was used to extract the organic layer
sensing still remains scarce. AIE-active FONPs can offer a better (yield = 85%). The amine protected acid was coupled with benzyl
lipase sensing method with the advantages of water solubility, alcohol in dry DCM using N,N-dicyclohexylcarbodiimide (DCC,
low cytotoxicity and a high signal to noise ratio.
1.2 equiv.), 4-N,N-dimethylaminopyridine (DMAP, 1.0 equiv.),
Herein, we synthesized naphthalene diimide (NDI)-based, and N-hydroxybenzotriazole (HOBt, 1.0 equiv.) under an N2
benzyl alcohol protected alkyl chain (C1, C5, and C10) linked atmosphere. The reaction mixture was kept stirring for 12 h.
amphiphilic molecules (NDI-1, 2, 3, Fig. 1). Benzyl ester func- Afterwards, the organic part was washed with brine solution and
tionality at the terminal position facilitates lipase catalyzed dried over anhydrous Na2SO4. Column chromatography (60–120
hydrolysis through binding in its hydrophobic pocket. Among mesh silica gel) was used to purify the organic part with 1%
them, only NDI-2 showed self-aggregation with different mor- MeOH/CHCl3 eluent (yield = 82%). The deprotection of the BOC
phology at varying water content in DMSO. At low water content moiety from the purified product was performed by dissolving
(30 vol% water in DMSO), self-aggregated fibre was found to the product in dry DCM and stirring with 1.5 equiv. trifluoroa-
form which transformed to spherical aggregated particles at cetic acid (TFA) for 4 h. The concentrated reaction mixture (by
99 vol% water in DMSO. NDI-2 exhibited AIE at 490 nm (bluish rotary evaporation and vacuum pump drying) was taken in ethyl
green emission) through excimer formation from 30 vol% water acetate and washed with aqueous Na2CO3 (10%) solution twice.
in DMSO and the emission intensity steadily increased being A free N-terminal of the alkyl chain linked benzyl protected ester
was obtained by evaporating the organic solvent (yield = 85%).
Finally, 1,4,5,8-naphthalenetetracarboxylicdianhydride (1.0 equiv.)
and an amine free alkyl chain linked benzyl moiety (2.2 equiv.)
were refluxed at 110 1C in dry DMF for 12 h under an N2
atmosphere. The reaction mixture was taken in DCM after remov-
ing excess DMF and washed with water. Column chromatography
(100–200 mesh silica gel) was employed to purify the concentrate
from DCM using 1% methanol in chloroform as an eluent. Finally,
the C5 tailored benzyl ester linked NDI derivative (NDI-2) was
obtained by precipitating the column purified concentrate in
diethyl ether (yield = 75%). The overall yield of NDI-2 including
all the steps was calculated to be 44%. A similar procedure was
followed with L-glycine (C1) and 11-aminoundecanoic acid (C10) to
synthesize NDI-1 (overall yield = 45%) and NDI-3 (overall yield =
51%), respectively. 1H-NMR, 13C-NMR and MALDI-TOF mass
spectrometry were used to characterize the synthesized NDI
derivatives (Fig. S1–S16, ESI†).
Fig. 1 Structure of synthesized NDI derivatives (NDI-1, n = 1), (NDI-2,
n = 5), and (NDI-3, n = 10).
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Self-assembly of NDI derivatives
In all instances, the samples were drop cast on glass coverslips
and dried under vacuum for 3 h.
All the NDI-based amphiphilic compounds (NDI-1–3) were soluble
in pure DMSO. Stock solution of each amphiphilic compound
(10 mM) was prepared in DMSO. The required volume from the
stock was poured to the different DMSO–water binary mixtures
(0–99% water content) to achieve varying concentration of synthe-
sized amphiphiles for different experiments.
Atomic force microscopy (AFM) study
Solutions of NDI-2 (10 mL, 10 mM) taken in (70 : 30 v/v, 50 : 50 v/v,
and 1 : 99 v/v) DMSO–water were deposited on freshly cleaved mica
(1 cm ꢂ 1 cm) and dried overnight. AFM images were captured on
a Veeco AP0100 instrument in the non-contact mode.
UV-visible spectroscopy
Fluorescence microscopy study
UV-vis spectra of NDI-2 were observed on a PerkinElmer
Lambda 25 spectrophotometer. We have checked the solvent-
dependent UV-vis spectra of NDI-2 (20 mM) in DMSO–water
(1 mL) by varying the water content gradually from 0–99 vol%,
i.e. DMSO (non-self-assembled state) to (1 : 99 v/v) DMSO–water
(self-assembled state). Furthermore, temperature dependent
(25–95 1C) UV-vis spectra of NDI-2 were recorded in 1 : 99 v/v
DMSO–water. Time dependent UV-vis spectroscopic study was
also carried out to know the stability of NDI-2 FONPs. We have
studied the relative absorbance of NDI-2 FONPs for 7 days.
Solutions (10 mL) of NDI-2 (10 mM) in 30 vol%, 50 vol% and
99 vol% water in DMSO were drop cast on glass slides. Images of
air-dried samples were taken under a fluorescence microscope
(Olympus IX-83 used for fw = 99 vol% and Carl Zeiss LSM 880
used for fw = 30 vol%, 50 vol% respectively) at 40ꢂ magnification.
Dynamic light-scattering (DLS) study
The mean hydrodynamic diameter (Dh) of self-assemblies was
determined using a Zen 3690 Nano ZS instrument (Malvern
Instrument Ltd). Dh values of NDI-2 (10 mM) in 30 vol%, 50 vol%
and 99 vol% water in DMSO (fw =30 vol%, 50 vol%, and 99 vol%)
were measured at an angle of 1751. Data analysis was done
using a Cumulant Fit for measuring Dh.
Photoluminescence study
Photoluminescence spectra of DMSO–water solutions of NDI-2
(molecularly dissolved to the self-assembled state) were measured
in a Varian Cary Eclipse spectrometer with excitation wavelength
(lex) = 350 nm. Fluorescence spectra of NDI-2 (50 mM) were taken
in DMSO–water (1 mL) of varying compositions (fw = 0–99 vol%).
Photographs of the cuvette were taken upon irradiation under a
UV lamp at lex = 365 nm of NDI-2 solutions (1 mL) of different
DMSO–water compositions. Concentration dependent photolumi-
nescence study was carried out in 70 vol%, 50 vol%, and 1 vol%
DMSO in water with varying concentrations (20–250 mM) of NDI-2.
Photoluminescence study was also performed to know the stability
of NDI-2 FONPs. We have studied relative fluorescence intensity of
NDI-2 FONPs for 7 days.
Quantum yield (QY) measurement
The QY of the unknown fluorophore NDI derivative (fu) was
measured in comparison to a reference fluorophore with a
known QY (fs) by using the following equation
ꢀ
ꢁ
2
AsFunu
AuFsns2
fu ¼
fs
(1)
where Au and Fu are the absorbance and integrated fluorescence
intensity of the unknown sample and As and Fs are those of the
reference sample at corresponding lex, respectively. nu and ns are
the refractive indices of solvents respectively in which the
unknown sample and known sample were prepared. Solutions
having similar absorbance (o0.01) were used for the measure-
ment of QY. Quinine sulfate in sulfuric acid (0.1 M) with QY (fs) =
54.0% was used as the reference.
1H-NMR study in varying solvents
1H-NMR spectra of NDI-2 (1 mM) were taken in DMSO-d6 and
the solvent mixture of DMSO-d6 and D2O (2: 1) (v/v) and 1 : 1 (v/v),
respectively.
Time-resolved study
Transmission electron microscopy (TEM) study
Time-correlated single photon count (TCSPC) measurement
was carried out in a picosecond diode laser IBH-405. Solutions
(2 mL) of NDI-2 (20 mM) in DMSO, DMSO–water (2 : 3 v/v) and
(1 : 99 v/v) were excited (lex) at 360 nm. Emission (lmon) was
monitored at 390 nm for only DMSO and 470 nm for DMSO–
water mixtures (2 : 3 v/v) and (1 : 99 v/v). Fluorescence decay was
analyzed using the IBH DAS6 software and equation 2 was
followed to analyze the experimental time-resolved fluores-
cence decay p(t)
Solutions (4 mL) of NDI-2 (10 mM each) in 30 vol%, 50 vol% and
99 vol% water in DMSO (fw = 30 vol%, 50 vol%, 99 vol%,
respectively) were deposited on different carbon coated copper
grids having 300 mesh size. Freshly prepared solution (1 mL) of
uranyl acetate (1% w/v) was used for negative staining of the
copper grids. The samples were dried under vacuum for 4–5 h.
TEM images were captured in a JEOL JEM 2010 microscope.
Field-emission scanning electron microscopy (FESEM) study
ꢂ ꢃ
n
X
ꢁt
A ZEISS field emission microscope was used to take FESEM
images of the solutions of NDI-2 (6 mL, 10 mM) in 30 vol% water
in DMSO (fw = 30 vol%) and 99 vol% water in DMSO (fw = 99 vol%).
t
i
pðtÞ ¼ b þ
aie
:
(2)
i
A JEOL-6700F field emission microscope was used to take images where b is the baseline correction (‘‘dc’’ offset), n is the number
of NDI-2 (6 mL, 10 mM) in 50 vol% water in DMSO (fw = 50 vol%). of discrete emissive species, ai is the pre-exponential factors,
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and ti is the excited state fluorescence lifetimes associated with Design of amphiphiles with varying spacer units (NDI-1, 2, 3)
the ith component, respectively. The average lifetime for multi-
exponential decays was calculated using eqn (3)
In this regard, we designed and synthesized naphthalene diimide
(NDI)-based benzyl alcohol protected alkyl chain (C1, C5, and C10)
linked amphiphilic compounds (NDI-1, 2, 3, Fig. 1) where NDI acts
n
X
hti ¼
aiti:
(3)
as an electron accepting core moiety and the appended benzyl
moiety would behave as an electron donating unit.48 We have
included the NDI unit as the core and the benzyl moiety at the
terminal so that the p–p stacking of the aromatic moieties can be
achieved easily during self-aggregation.48,49 To synthesize NDI-1,
NDI-2 and NDI-3, the alkyl chain of varying carbon numbers like
L-glycine acid (C1), 6-amino caproic acid (C5) and 11-amino
undecanoic acid (C10), respectively, were introduced as spacers
linking between the NDI core and benzyl group. Variation in the
HLB in the structural backbone of the amphiphilic compounds by
means of changing the alkyl chain length spacer might influence
its self-aggregation pattern in different solvent systems. Also, the
substituted NDI with different chain lengths may generate differ-
ent orientation and twisting in the molecular backbone due to
restricted intramolecular rotation (RIR). Consequently, it may
exhibit the aggregation induced emission (AIE), which can be
exploited in biomolecular sensing.
All three compounds (NDI-1, 2, 3, Fig. 1) were completely
soluble in DMSO. In the case of NDI-1 and NDI-3, addition of
water (10% and above) to this DMSO solution led to precipita-
tion within a few minutes. In the case of NDI-2, transparent
solution in DMSO became translucent with an increase in the
water content from 30 vol% and above (Fig. 2). This observation
preliminary indicates the initiation of self-assembly by the
NDI-2 amphiphile in the respective solvent systems (Fig. 2a).
Macroscopic translucency of the solution further enhanced up
to 99 vol% possibly due to the formation of higher order
self-aggregates. Based on these findings, it is evident that
the hydrophobic effect of the spacer plays a pivotal role in the
self-assembly of these amphiphilic compounds owing to the
variation in HLB. With increasing water content from 30 vol%
to 99 vol%, the DMSO–water solutions of NDI-2 (50 mM) showed
blue to bluish green transition in the emission colour upon
UV-light irradiation at lex = 365 nm (Fig. 2b). Among three
amphiphilic compounds, all further investigations were carried
out using NDI-2 as it exhibited the physical indication of self-
aggregation.
i¼1
ai
ai
P
where ai ¼
indicates the contribution of a decay component.
CV lipase sensing by NDI-2 FONP
Interaction between NDI-2 FONPs in 99 vol% water in DMSO
with CV lipase was monitored by recording the UV-vis spectra of
CV lipase (0.5 mg Lꢁ1) in the presence of NDI-2 FONP (0–30 mM)
solution. Also, the fluorescence spectra of NDI-2 FONPs (10 mM
and 25 mM) were taken with increasing lipase concentration up
to 300 mg Lꢁ1. Equation: F/F0 = 1 + KSV[Q] was used to calculated
the Stern–Volmer constant (KSV) of the NDI-2 FONP, where F0
and F are the fluorescence intensities of NDI-2 in the absence
and presence of lipase and [Q] denotes the concentration of the
lipase enzyme. The detection limit was calculated from the
linear curve obtained from (F0 ꢁ F)/F0 vs. the concentration of
CV lipase enzyme and we studied the selectivity of the NDI-2
FONP (25 mM) against different enzymes having a concen-
tration of 150 mg Lꢁ1
.
Time dependent photoluminescence spectra were recorded
for NDI-2 FONPs (20 mM) with CV lipase (70 mg Lꢁ1) for 0 to
60 min. Also, human serum samples were collected and cen-
trifuged for 10 min at 8000 rpm. The supernatant was diluted
by 10-fold using Milli-Q water. Photoluminescence study was
performed using 30 mM NDI-2 FONPs after 10 and 60 min
incubation within this diluted serum solution.
Lipase catalyzed ester hydrolysis was investigated by MALDI-
TOF mass spectroscopy. CV lipase (0.5 mg Lꢁ1) in water (500 mL)
and NDI-2 FONP (100 mM, 1 mL of 1 : 99 v/v DMSO–water) were
taken together and kept for 10 min. To this solution, saturated
ammonium sulphate solution and acetone (2 mL) were added
sequentially. The mixture was centrifuged at 12 000 rpm for
30 min and the supernatant was used for the MALDI-TOF mass
spectrometric experiment.
Results and discussion
Evolution of the self-assembly pattern is a contingent process
which is being primarily regulated by the structural design of a
basic building block, amphiphiles. An optimum hydrophilic–
lipophilic balance (HLB) within the amphiphilic molecule can
manifest diverse supramolecular self-assemblies in different
solvent systems.22,46 Changes in the morphology of self-
assemblies due to alteration of the polar head or hydrophobic
tail had been investigated in many instances.26,47 However, the
influence of the spacer unit (between the polar head and
hydrophobic tail) on the morphology of self-assemblies in
varying solvent systems (solvents of varying polarities) has not
received due significance.
Fig. 2 (a) Photograph of the formation of translucent solutions with
increasing water content in DMSO for NDI-2 (50 mM) and (b) photograph
of emission colour of NDI-2 (50 mM) upon UV-light irradiation (lex
=
365 nm).
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Microscopic investigations for the self-assemblies of
amphiphile, NDI-2
of NDI-2 from fibre to sphere upon increasing the water fraction in
DMSO. Only spherical morphologies of the aggregated structures
for the translucent solution of NDI-2 (20 mM) were observed upon
enhancing the water content to 99 vol% in DMSO (fw = 99 vol%).
According to the FESEM, TEM and AFM images, the average
diameter of spherical organic nanoparticles is in the range of
B50–100 nm (Fig. 3c, f and Fig. S17d, ESI†). Similar manifesta-
tions of the self-aggregated structure of NDI-2 in 1 : 99 v/v DMSO–
water were seen in the corresponding fluorescence microscopic
image (Fig. 3i).
Variation in the solvent systems through addition of water in
DMSO showed notable influence in the transformation of the
morphology of NDI-2 self-assemblies. The transition from fibre-
to-spherical morphology with increasing water content presum-
ably progressed through the separation of fibre to spherical
organic nanoparticles due to the increase in the dielectric
constant/polarity of the solvent systems, which has always been
a crucial factor for self-assembly.27,50,51 The fact that the HLB of
the amphiphile also contributes to the aggregation pattern and
morphology of self-assembled structures is also taken into
account.
We investigated the structures of self-aggregates formed by
NDI-2 in varying ratios of DMSO–water binary solvents using
different microscopic techniques. NDI-2 (20 mM, 30 vol% water
in DMSO (fw = 30 vol%)) exhibited fibrous morphology in
FESEM, TEM and AFM images having a length of several
micro-metres and a width in the range of 50–100 nm (Fig. 3a,
d and Fig. S17a, ESI†). Fluorescence microscopic images of the
self-assembly of NDI-2 in DMSO–water (fw = 30 vol%) also
showed green emitting fibre like morphology of 50–100 nm
diameter and several micro-metres in length (excitation filter:
BP 330–385 nm, band absorbance filter covering wavelength
below 405 nm) owing to the light emitting character of the
aggregates (Fig. 3g). Similar morphology was noted in the
corresponding bright field image (Fig. S18a, ESI†) of the
NDI-2 self-aggregate in DMSO–water (fw = 30 vol%). Gradual
enhancement in the amount of water to fw = 50 vol% (50 vol%
water in DMSO) showed the presence of spherical organic
nanoparticles for the self-assembly of NDI-2 (20 mM) along with
the fibrous morphology (Fig. 3 and Fig. S17, S18, ESI†). According
to the FESEM image, the diameter of the newly appeared spherical
self-aggregates was found to be B100 nm (Fig. 3b). In concurrence
with the FESEM images, TEM and AFM (both phase and topology)
images (Fig. 3e and Fig. S17b, c, ESI†) ensured the formation
of spherical-shaped organic nanoparticles by NDI-2 (20 mM, at fw =
50 vol%) having diameters of B100 nm along with the presence of
fibrous aggregates. Similar features were observed also in fluores-
cence and bright filed microscopic images (Fig. 3h and Fig. S18b,
ESI†). The coexistence of spherical organic nanoparticles and fibre
delineates the possible morphological transition of self-aggregates
DLS study
The mean hydrodynamic diameter (Dh) and the size distribu-
tion of the self-aggregates of NDI-2 (20 mM) were measured by
DLS in different DMSO–water compositions. According to the
mean diameter profiles (expressed in number percentage), the
average size of the self-aggregates in 50 : 50 v/v DMSO–water
was found to be B900 nm due to the presence of mostly fibrous
morphology (Fig. 4 and Fig. S18c and S19, ESI†). Dh of self-
aggregates was found to be steadily decreased with enhance-
ment in the amount of water in DMSO. In the case of 20 : 80 v/v
DMSO–water, the Dh of NDI-2 self-aggregates was in the range
of 200–250 nm (Fig. 4) attributed to the possible separation of
self-aggregates (fibre and spherical organic particles). Further
addition of water decreased the average hydrodynamic dia-
meter of self-aggregates to B160 nm at fw = 90 vol%. Finally,
in 1 : 99 v/v DMSO–water, the Dh of self-aggregates reduced to
Fig. 3 FESEM (a–c), TEM (d–f), and fluorescence (g–i) microscopic
images of NDI-2 in (70 : 30, v/v), (50 : 50, v/v), (1 : 99, v/v) DMSO–water
binary solvent mixtures, respectively. ([NDI-2] = 20 mM.)
Fig. 4 DLS measurement of NDI-2 in different DMSO–water binary
solvent mixtures ([NDI-2] = 20 mM).
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B120 nm (Fig. 4 and Fig. S18c, S19, ESI†). The trend in the dependent UV-vis spectra actually showed a transition from the
observed size of NDI-2 self-aggregates measured by DLS in aggregated phase to molecularly dissolved species when the
varying solvent compositions is in accordance with that of the temperature was increased. At temperatures 45 1C and beyond,
microscopic investigations. Generation of small organic particles is the absorption spectra are similar to that of DMSO at room
a spontaneous manifestation of self-assemblies which may take temperature. We have also normalised the solvent dependent
place at low water content even in those cases where no fiber is and temperature dependent UV spectra to understand the spectral
present as an aggregated structure.19,21 The higher value of average pattern as well as the shift of lmax (Fig. S20, ESI†). A red shift of
hydrodynamic diameter at lower fw may be attributed to the 3 nm in the solvent dependent UV spectroscopic study and in the
presence of fibres which reduced with increasing water amount case of temperature dependent UV spectra a blue shift of 4 nm
owing to the generation of spherical organic nanoparticles.
were noted above 35 1C, similar to previously obtained findings.
Although the change is slight, it is distinct and corroborated to the
literature.21,54
Spectroscopic investigation of NDI-2 self-assembly
The aggregation pattern during the formation of self-assemblies
by NDI-2 in varying compositions of DMSO–water was investigated
by UV-vis spectroscopy (Fig. 5a). In pure DMSO (molecularly
dissolved state), NDI-2 (20 mM) exhibited sharp UV bands in the
range of 330–400 nm with absorption maxima (lmax) at 382 nm,
which may be attributed to the p–p* transition polarized along the
long axis of NDI chromophores (Fig. 5a).52,53 Addition of water
within the DMSO solution of NDI-2 resulted in an initial drop in
the absorbance value. Also, with a gradual increase in the water
content, the ratio of the vibronic features I(0–0)/I(0–1) reduced from
1.1 (in only DMSO) to 0.9 at fw = 99% in DMSO (Fig. 5a). These
characteristic changes in the absorption spectra of NDI-2 in the
presence of water hinted toward the self-assembly-induced aggre-
gated structure from its monomeric state in DMSO. Alongside the
gradual decrease in the optical density, the absorption maxima
got slightly red-shifted with the enhancement of water in DMSO.
Finally, at fw = 99 vol%, lmax appeared at 385 nm (Fig. 5a). This
bathochromic shift of lmax indicated that the self-assembly of the
Non-covalent forces in self aggregation
Solvent-dependent 1H-NMR spectra were recorded to investi-
gate the involvement of different non-covalent interactions (e.g.
p–p stacking and hydrophobic interactions) during self-
aggregation of NID-2 (Fig. S21, ESI†). The protons of the
NDI-2 aromatic ring and benzyl group exhibited sharp NMR
signals at d = 8.66 ppm and d = 7.31 ppm, respectively, in
DMSO-d6 in its non-assembled state. With an increase in the
amount of D2O in DMSO-d6 from 0 vol% to 30 vol% and
50 vol%, 1H-NMR signals of the NDI-2 aromatic ring and benzyl
group got shifted to d = 8.47 ppm and d = 7.11 ppm, respectively
(Fig. S21, ESI†). This upfield shift in NMR signals depicts that
p–p stacking between NDI cores as well as the benzyl moiety of
NDI-2 got facilitated with increment in D2O content during self-
aggregation.
Luminescence properties of self-aggregates of NDI-2
amphiphiles took place possibly through J-aggregation (head-to- The observed changes in the UV-vis spectral pattern of NDI-2
tail fashion arrangement) during the transition from the fibrous during self-aggregation intrigued us to study its emission
morphology to the generation of spherical organic nanoparticles. behaviour in DMSO–water solvent mixtures. Very weak emis-
Furthermore, we recorded temperature dependent (25–95 1C) sion intensity was noted in the photoluminescence spectrum of
absorption spectra of NDI-2 (20 mM) in 1 : 99 v/v DMSO–water NDI-2 (50 mM) in DMSO (molecular dissolved state) with emis-
(Fig. 5b). Initially, no major change in the spectral pattern was sion maxima at 410 nm (Fig. 6a). Moreover, it got further
noted with an increase in temperature up to 35 1C. However, the decreased with increasing water content up to 20%. Interest-
lmax got blue shifted by B4 nm for the NDI derivative with further ingly, at 30% water content (fw = 30 vol%), an excimer peak with
elevation in temperature up to 95 1C (Fig. 5b). Hence, the emission maxima at 494 nm was observed (Fig. 6a and b).
aggregated organic nanoparticles of NDI-2 start to lose their self- Further addition of water led to a notable increase in the
assembled structure from 45 1C and above. Temperature emission intensity of the excimer peak with its highest intensity
Fig. 5 (a) UV-Visible spectra of NDI-2 (20 mM) in varying DMSO–water solvent mixtures. (b) Temperature dependent UV-visible spectra of NDI-2 (25 mM)
in 1 : 99 v/v, DMSO–water.
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Fig. 6 (a) Fluorescence spectra (lex = 350 nm) of NDI-2 (50 mM) in DMSO–water solvent mixtures. (b) Shift in the emission maximum peak of NDI-2 with
the increase of the volume fraction of water. (c) Plot of relative emission intensity (I/I0) vs. the fraction of water in DMSO of NDI-2 where I0 = emission
intensity in pure DMSO solution. The inset in panel (c): photographs of NDI-2 in the DMSO–water mixture with fw values of 0 vol%, 30 vol%, and 99 vol%,
respectively, irradiated under 365 nm UV light.
at fw = 99 vol%. Along with the increase in fluorescence NDI-2 (50 mM) did not show any noticeable fluorescence.
intensity, NDI-2 showed a red shift of 84 nm with respect to However, bluish green emission was observed from 30 vol%
the emission peak in pure DMSO (Fig. 6a and b). This behaviour water content which became more intense with increasing
was in concurrence with the solvent dependent absorption water content being maximum at 99 vol% due to the excimer
spectroscopic trend of NDI-2. Gradual increment of water formation (490 nm). Furthermore, concentration-dependent
resulted in the self-aggregation of NDI-2 possibly through stacking fluorescence spectra of NDI-2 were monitored at fw = 30 vol%,
of the chromophoric units that led to the formation of an excimer 50 vol% and 99 vol% water content in DMSO within a concen-
peak at 490 nm due to aggregation-induced emission (AIE). tration range of 60–250 mM, 40–200 mM and 20–150 mM,
Distinct enhancement in the emission intensity was noted from respectively (Fig. S22, ESI†). In all three instances, with an
fw = 50 vol% and above possibly due to the morphological increase in the NDI-2 concentration, the fluorescence intensity
transformation of aggregates from fibre-to-spherical organic nano- of the characteristic excimer band gradually enhanced (lem
particles. The emission intensity of NDI-2 is 8.5-fold higher at fw = 490 nm) and became saturated at and above 200 mM, 150 mM,
99 vol% compared to that in DMSO (Fig. 6a–c). and 125 mM for fw = 30 vol%, 50 vol% and 99 vol%, respectively
=
The quantum yield (QY) of NDI-2 at fw = 99 vol% was 3.57% (Fig. S22, ESI†). This observation clarifies that enhancement in
(with respect to quinine sulfate hydrate), which is comparable the AIE was observed possibly due to the participation of a
to or higher than the earlier reports on NDI derivatives.19,21 The larger number of amphiphilic molecules in self-aggregation
other two amphiphilic compounds i.e. NDI-1 and NDI-3 posses- with an increase in the concentration of NDI-2 and became
sing C1 and C10 alkyl chain spacer connecting NDI units and saturated beyond a specific concentration. It is also noticeable that
benzyl alcohol did not show any AIE in any solvent composi- at higher water content, AIE is significant from a very low concen-
tions due to the absence of any self-aggregating behaviour. tration of amphiphilic compounds. For example, in the case of fw =
NDI-2 in different compositions of DMSO–water (1 mL) was 30 vol%, AIE started from 125 mM (absence of a monomeric peak)
irradiated under a UV lamp at lex = 365 nm for the visual effect whereas AIE is noteworthy from a relatively low concentration of
of the AIE phenomenon (Fig. 2b and 6c). DMSO solution of 40 mM and 20 mM for fw = 50 vol% and fw = 99 vol%, respectively.
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of enzyme.55 Consequently, it may lead to alteration of the
emissive property of the AIE-gens in the absence of an electron
donating benzyl moiety.
Table 1 Lifetime values of NDI-2 in the presence of different fractions of
water in DMSO (lex = 360 nm)
Average
Fraction of lmonitored t1 (ns)
water (%) (nm) (a1)
t2 (ns)
(a2)
t3 (ns)
(a3)
lifetime
hti (ns)
Initially, we tried to know if there is any interaction between
lipase and NDI-2 through UV-vis spectroscopy. Gradual addi-
tion of NDI-2 FONPs (fw = 99 vol%) to 1 mL solution of CV lipase
(0.5 mg Lꢁ1) showed a steady increase in the absorbance value
(Fig. S25a, ESI†). The characteristic peak of NDI-2 FONPs
started to appear at and beyond 5 mM concentration. After
addition of 30 mM lipase, no notable elevation in the absor-
bance value was observed. Moreover, the characteristic peak of
lipase at 280 nm flattened with increasing concentration of
NDI-2 FONPs. We have also performed the control experiment
by recording the UV spectra of NDI-2 in fw = 99 vol% in DMSO
with its increasing concentration in the absence of lipase (Fig.
S23b, ESI†). The peak at 280 nm is obviously not visible due to
the absence of lipase. We have also normalised the UV-vis
spectra of NDI-2 in the presence of CV lipase with respect to
lmax = 385 nm (Fig. S26, ESI†). With an increasing amount of
NDI-2 FONPs in the lipase solution, the peak height of lipase
got gradually reduced indicating the interaction between NDI-2
0
50
99
400
500
500
1.22 (0.70) 2.25 (0.28) 8.70 (0.015) 1.61
7.51 (0.25) 21.38 (0.20) 1.14 (0.55) 6.78
9.34 (0.27) 23.38 (0.33) 1.93 (0.39) 10.99
This indicates that at high water content, initiation of self-
aggregation takes place at a low concentration of amphiphile.
Naturally, the emission intensity of aggregated NDI-2 at a higher
amount of water was also higher than that of a lower amount of
water at the same concentration of the amphiphile.
The AIE behaviour of NDI-2 was further demonstrated by
performing time resolved study of NDI-2 (20 mM) in pure
DMSO, 50 vol% and 99 vol% water in DMSO with picosecond
excitation at 360 nm (Fig. S23, ESI†). A triexponentially fitted
decay curve was obtained in each case. Emission of NDI-2 in
DMSO was monitored at lmon = 400 nm and the average
lifetime (hti) was 1.61 ns having components of 1.22 ns
(70%), 2.25 ns (28%), and 8.70 ns (1.5%). At fw = 50 vol% and
99 vol%, the hti value was found to be 6.78 ns and 10.99 ns,
respectively, monitored at lmon = 500 nm with components of
FONPs and lipase. Also, the ratio of the vibronic features I(0–0)
/
I(0–1) increased from 0.9 to 1.0 (Fig. S26, ESI†). All the UV-vis
spectroscopic evidence supports the interaction between NDI-2
FONPs and lipase.
7.51 ns (25%), 21.38 ns (20%), and 1.14 ns (55%) for fw
=
Next, fluorescence spectra of NDI-2 FONPs (25 mM) were
recorded in fw = 99 vol% DMSO–water solution with increasing
CV lipase concentration (Fig. 7a). The emission intensity of
NDI-2 FONPs at lem = 490 nm gradually got quenched with the
increase in CV lipase concentration starting from 1 mg Lꢁ1. The
quenching effect was steadily continued up to 150 mg Lꢁ1 and
thereafter no significant change in the quenching effect was
noted up to an experimental concentration of 300 mg Lꢁ1 for
lipase (Fig. 7a and b). The decrease in fluorescence is attributed
to the disruption of AIE in the presence of CV lipase due to
enzymatic degradation of NDI-2 FONPs (ester hydrolysis) (Fig.
S27, ESI†). All spectra were taken after 10 min incubation of
NDI-2 FONPs and CV lipase. The Stern–Volmer equation was
used to analyze the quenching of NDI-2 FONPs:
50 vol% and 9.34 ns (27%), 23.38 ns (33%), and 1.93 ns (39%)
for fw = 99 vol%, respectively. Decay times for each solution and
relative fractions of decay are given in Table 1. The long decay
lifetime at higher percentage of water is the manifestation of
pre-associated excimer formation due to AIE. The longer-lived
excited states observed at fw = 50 vol% and 99 vol% arise from
pre associated NDIs in the assemblies. Notably, NDI-2 in pure
DMSO, which does not undergo self-assembly, had the shortest
lifetime.
We also performed time dependent UV-vis spectroscopic
study as well as photoluminescence study to know the stability
of NDI-2 FONPs. The relative absorbance (A/A0) and relative
fluorescence intensity (I/I0) of NDI-2 FONPs were recorded with
time (Fig. S24, ESI†). Here A0 and A were the original absor-
bance and absorbance on different days, respectively. Similarly,
I0 and I represent the original fluorescence intensity and
fluorescence intensity on different days. No significant change
in the relative absorbance and fluorescence intensities was noted
for 7 days. Hence, these FONPs were stable for a considerable
time period.
F/F0 = 1 + KSV[Q]
(4)
In this equation, F0 and F are the fluorescence intensities of
NDI-2 FONPs in the absence and presence of CV lipase; KSV is
the Stern–Volmer constant and [Q] is the quencher concen-
tration. The Stern–Volmer constant (KSV) of 0.00854 mgꢁ1 L was
calculated from the plot F/F0 of NDI-2 FONPs vs. lipase concen-
tration (Fig. 7c) which showed linearity in quenching of NDI-2
Sensing of lipase by NDI-2 FONPs
After successful fabrication of NDI-2 FONPs, we intended to FONP emission by lipase. After ensuring successful detection of
utilize their emissive behaviour in lipase sensing owing to the CV lipase by fluorescence quenching of NDI-2 FONPs, we
presence of benzyl ester functionality in the amphiphilic molecule. measured the efficiency of the quencher in terms of sensitivity
Chromobacterium viscosum (CV) lipase belongs to the esterase by measuring the limit of detection (LOD) value of CV lipase by
family that can catalyze the hydrolysis of the ester group present FONPs. To this end, (F0 ꢁ F)/F0 vs. concentration of lipase was
in the amphiphiles. In this case (NDI-2) ester of benzyl alcohol is plotted (Fig. 7d) for NDI-2 FONPs (fw = 99 vol%) that showed a
more susceptible to enzymatic hydrolysis owing to the binding of linear regression equation: (F0 ꢁ F)/F0 = 0.0176 + 0.0076x, where
the aromatic ring in the hydrophobic pocket close to the active site x is the lipase concentration with a correlation coefficient (R2)
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Fig. 7 (a) Fluorescence spectra of NDI-2 FONPs (25 mM) in the absence and presence of varying concentration of CV lipase (excitation wavelength =
350 nm), (b) fluorescence intensities of NDI-2 FONPs (25 mM) in the absence and presence of varying concentration of CV lipase (excitation wavelength =
350 nm), (inset) (b): photographs of NDI-2 in the DMSO–water mixture at fw = 99 vol% in the absence and presence of CV lipase (150 mg Lꢁ1) irradiated
under 365 nm UV light. (c) Stern–Volmer plot of CV lipase doped in (1 : 99, v/v) DMSO–water solution of NDI-2 (25 mM), and (d) fluorescence response of
NDI-2 FONPs towards CV lipase sensing with varying concentration of lipase in (1 : 99, v/v) DMSO–water ([NDI-2] = 25 mM).
of 0.984. The limit of detection (LOD) was found to be 10.8 mg Lꢁ1 ammonium sulphate solution and acetone (2 mL) were added
calculated based on 3s/S (s is the standard deviation and S is the sequentially. The mixture was centrifuged at 12 000 rpm for
slope of the calibration curve). A similar experiment was carried 30 min to precipitate out the insoluble part. Ammonium
out using 10 mM NDI-2 FONPs at fw = 99 vol% with gradual sulphate was added for salting out excess lipase (if any) from
increment of CV lipase concentration (Fig. S28, ESI†). In this the reaction mixture so that the hydrolysed product (carboxylic
case, the Stern–Volmer constant (KSV) was measured to be acid fragment) remains in the supernatant of the acetone–water
0.009346 mgꢁ1 L and the LOD was found to be in the similar mixture after centrifugation. Since ammonium sulphate was
range (9.2 mg Lꢁ1). With varying concentration of NDI-2 FONPs added after the enzymatic reaction, chances of its influence on
(10–25 mM), the LOD for CV lipase is calculated to be 10.0 ꢀ the enzymatic hydrolysis are negligible. The supernatant com-
0.8 mg Lꢁ1. Comparison of other lipase sensors in terms of prising of a fragmented part of the NDI derivative was investi-
LOD, available in the literature, is given in the Table S1, ESI.† gated by MALDI-TOF mass spectroscopy. Lipase, being an
The limit of detection in the present study is either comparable esterase enzyme, cleaves the ester group of the substrate
to or better than some other reported literature studies. The (NDI-2) i.e. breaks the C–O single bond of –(CO)–O–CH2–Ph
dynamic range of the sensor is represented by the concentration and generates carboxylic acid (Fig. S27, ESI†). The benzyl ester
range of CV lipase up to which the fluorescence intensity functionality at the terminal of the amphiphile might have
quenching is linear. It was found to be 10–100 mg Lꢁ1 based facilitated the binding of the NDI derivative at the hydrophobic
on the Stern–Volmer equation and LOD obtained in different pocket of lipase in the vicinity of the active site resulting in the
concentrations of NDI-2 FONPs.
cleavage of the ester bond. Existence of the fragmented product
We have also carried out time dependent photolumines- in the mass spectrum (obtained mass was 518.60 [M + Na]+ and
cence spectra to investigate the time dependence of the lipase calculated mass was 495) confirmed the lipase catalyzed hydrolysis
assay (Fig. S29, ESI†). NDI-2 FONPs (20 mM) were treated with of benzyl ester. We further carried out the same fluorescence
CV lipase (70 mg Lꢁ1) and fluorescence spectra were taken in a spectroscopic measurements in the presence of ammonium
time span of 0 to 60 min. The fluorescence intensity decreased sulphate to confirm that it does not disturb the reaction. We
rapidly up to 10 min and after that the rate of quenching got have provided the fluorescence spectra of NDI-2 FONPs (25 mM)
significantly slower.
with increasing lipase concentration in the presence of ammo-
Lipase catalysed hydrolysis of NDI-2 FONPs in 99 vol% water nium sulphate solution (25 mM) (Fig. S30, ESI†), which follow the
in DMSO by CV lipase was also investigated by mass spectro- same trend as that in the absence of ammonium sulphate.
metry (Fig. S27, ESI†). CV lipase (0.5 mg Lꢁ1) in water (500 mL)
Selectivity towards the target analyte is a very essential para-
and NDI-2 FONP (100 mM, 1 mL of 1 : 99 v/v DMSO–water) were meter which determines the effectiveness of a new fluorescent
taken together and kept for 10 min. To this solution, saturated sensor. To this end, we investigated the selectivity of NDI-2
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in NDI-2 amphiphilic molecules. This experiment was repeated
thrice and the error bar indicates the standard deviation.
Human serum samples were collected for demonstrating the
feasibility of the sensor in practical application. The samples
were centrifuged for 10 min at 8000 rpm and the supernatant
was diluted by 10-fold using Milli-Q water. Photoluminescence
study was performed with 30 mM NDI-2 FONPs after 10 and
60 min incubation within this diluted serum solution. The
fluorescence intensity of NDI-2 got decreased and it almost
became half after 60 min (Fig. S31, ESI†). This quenching in the
emission of NDI-2 FONPs took place due to the possible
presence of lipase in human serum.
Conclusions
Herein, we have designed and synthesized NDI based amphi-
philic compounds with spacers (C1, C5 and C10) of different
hydrophobicity having terminal benzyl esters. Among the three
amphiphiles, NDI-2 (C5 spacer) only could form self-assemblies
of diverse morphologies in different compositions of the
DMSO–water binary mixture. With an increase in water con-
tent, the fibrous morphology of NDI-2 at fw = 30 vol% slowly
transformed to spherical organic particles at fw = 99 vol%.
Along with the transformation in morphology, formation of
bluish green emitting FONPs took place through AIE with an
emission maximum at 490 nm. The transition from fibre-to-
spherical morphology with increasing water content progressed
through J-type aggregation presumably due to the increase in
the dielectric constant/polarity of the solvent systems. The AIE
property of NDI-2 in fw = 99 vol% DMSO–water was used for CV
lipase sensing by turning off the fluorescence with a LOD of
10.0 ꢀ 0.8 mg Lꢁ1. NDI-2 FONPs showed selective sensing of
lipase against many other enzymes including esterases as well as
exhibited its lipase sensing efficiency in vitro using a human
serum sample. The observation in the present study is expected
to further increase the potential of FONPs in sensing application.
Conflicts of interest
There are no conflicts to declare.
Fig. 8 Enzyme selectivity of NDI-2 FONP (20 mM) against different
enzymes (in each case the enzyme concentration is 150 mg Lꢁ1).
Acknowledgements
P. K. D. is thankful to the Science and Engineering Research
Board (SERB), Department of Science and Technology (DST),
India, for financial assistance (No. EMR/2017/000656). D. C.
acknowledges UGC, India, and D. S. & A. K. G. acknowledge
CSIR, India, for Research Fellowships.
FONPs by treating their solution (fw = 99 vol%) at 25 mM with
different enzymes. Fluorescence spectra of NDI-2 FONPs were
recorded in the presence of pepsin, trypsin, chymotrypsin,
glucose oxidase (GO), and other esterases including DNAase,
RNAase, alkaline phosphatase and esterase from porcine liver
(carboxyl esterase) having an enzyme concentration of 150 mg Lꢁ1
after an incubation for 10 min. Interestingly, except CV lipase, no
other enzyme could exhibit notable quenching of NDI-2 FONP
emission (Fig. 8a–j). Only CV lipase was able to quench the
fluorescence of NDI-2 FONPs significantly up to B3 times among
all of them owing to the cleavage of the benzyl ester bond present
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2180 | Soft Matter, 2021, 17, 2170ꢁ2180
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