Direct micro-scale monitoring of molecular aggregation, its growth and diffusion: Via aggregation-induced emission

Article abstract of DOI:10.1039/d0sm00108b

Time-dependent monitoring of aggregation-induced fluorescence of a model compound namely, (Z)-3-(3′,5′-bis(trifluoromethyl)-[1,1′-biphenyl]-4-yl)-2-(4-bromophenyl)acrylonitrile unearth hitherto unknown molecular level events such as onset of molecular aggregation, their growth, size, and diffusion dynamics. The presented generalized approach can also be extended to in situ monitoring and controlling of various biological aggregation processes down to a single-cell level and all aspects of materials chemistry, as well.

Full text of DOI:10.1039/d0sm00108b

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Karsten Baumgarten and Brian P. Tighe
Viscous forces and bulk viscoelasticity near jamming
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Received 00th January 20xx,
Accepted 00th January 20xx
Direct micro-scale monitoring of molecular aggregation, its growth
and diffusion via aggregation-induced emission†
DOI: 10.1039/x0xx00000x
www.rsc.org/
Mane Jyothi, Mari Annadhasan, Vinay Pradeep Vuppu, and Rajadurai Chandrasekar *
Time-dependent monitoring of aggregation-induced fluorescence
of a model compound namely, (Z)-3-(3',5'-bis(trifluoromethyl)- control of the solution state dynamics of molecular-aggregation
[1,1'-biphenyl]-4-yl)-2-(4-bromophenyl)acrylonitrile unearth processes such as the formation of (detectable) microscale
However, to our knowledge, concurrent investigation and
hitherto unknown molecular level events such as onset of aggregates, their growth, concentration, and diffusion, are not
molecular aggregation, their growth, size, and diffusion dynamics. studied in the literature. Hence, we envisioned exploiting an AIE-
The presented generalized approach can also be extended to in- molecule to study the molecular-aggregation process. The
situ monitoring and controlling of various biological aggregation investigations required time-correlated monitoring of the AIE
processes down to a single-cell level and all aspects of materials intensity in dilute solution-, supersaturated solution- and solid-
chemistry, as well.
states during aggregation down to microsecond timescale with FL
Molecular aggregation plays a pivotal role in many vital processes micro-spectroscopy.
in nature and human-made materials. According to IUPAC
For our studies we prepared a model AIE-molecule, namely, (Z)-
definition, the term aggregation defines a “process whereby 3-(3',5'-bis(trifluoromethyl)-[1,1'-biphenyl]-4-yl)-2-(4-
otherwise discrete molecules or particles form aggregates.”1 Though bromophenyl)acrylonitrile (1) shown in Scheme 1, which is
aggregation is primarily an important process, the growth of structurally rather similar to 1-cyano-trans-1,2-bis-(3′,5′-bis-
aggregates in solution-phase is not well understood. At times, in trifluoromethyl-biphenyl) ethylene (CN-TFMBE).10 Both CN-TFMBE
and 1 are structurally close as they possess a dominant electron-
withdrawing cyano functionality anchored to the vinylene unit. In
the solution state, due to bulky cyano-substitution, the CN-TFMBE
experiences a non-planar conformation (twisted geometry and
intramolecular rotation) and thereby annihilate the excitons non-
radiatively rendering no or feeble emission.10 However, in the
crystalline-state, the C-N···H hydrogen bonding interaction of the
cyano group with phenyl hydrogens and π-π interactions limits the
intramolecular rotation and provide rigidity, thus enhancing the
optical emission.10-12 As expected from the molecular structure, 1
emit cyan fluorescence (FL) via AIE mechanism. Here, we report the
results on the time-correlated monitoring of polymorphic aggregate
dilute solutions, some -molecular systems which are optically non-
emissive reveal their emission at very high concentrations due to
aggregation. The optical emission from the molecular aggregate
arises because of the intramolecular conformation changes, which
overpower the non-radiative pathway via the restriction of
intramolecular rotations – known as aggregation-induced emission
(AIE).2,3 The fluorescence (FL) spectroscopy snap-shots of AIE
molecule aggregation process as a function of time could be
extended to understand and regulate molecular or macromolecular
level aggregation events in solution-phase. Investigation of
molecular aggregation process is essential as it is unequivocally
associated with (i) over human 50 diseases,4-7 including Alzheimer’s-
and Parkinson’s-diseases and type II diabetes, and (ii) organic and
polymer semiconductors device properties, such charge carrier
mobility and electronic coupling8,9 and, so on. Importantly, apart
from understanding different types of aggregate
growth, their size, and diffusion dynamics.
CF3
Br
CF3
OH
B
Pd(PPh3)4, THF
OH
+
CF3
O
K2CO3, 85 oC, 24h
O
CF3
4
growth, it also essential to develop a technique that would allow
2
3
+
NaOEt, EtOH
40 oC, 40 min
Br
CF3
CN
Br
School of Chemistry, University of Hyderabad
Prof. C. R. Rao Road, Gachibowli, Hyderabad-500046
India.
CN
CF3
1
5
E-mail - r.chandrasekar@uohyd.ac.in
Scheme 1 Synthetic scheme for the preparation of AIE molecule 1.
† Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
monitoring of the growth process.
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Fig. 2 A and B) Optical and FL microscopy images of microcrystals of 1, respectively. C
and D) FESEM images of microcrystals of 1, E) Solid-state absorbance and F) FL spectra
of 1 with two different laser excitations (355 and 405 nm), respectively.
shaped active optical waveguiding13 bright cyan emitting micro-
crystals (Figure 2A,B and Figure S5). Field emission scanning
microscope (FESEM) images revealed the square-shaped cross-
section of the one-dimensional micro-crystals (Figure 2C, D and S4).
Micro-crystals of 1 showed an extended absorption band up to 450
nm (Figure 2F). Optical excitation of micro-crystals with a 355 nm
laser displayed FL spectra with two distinctive bands with multiple
shoulders (Figure 2F). However, excitation of the same micro-
crystals with 405 nm laser resolved the spectral features very well
and revealed the shoulder peak positions at 420, 447, 477 and 523
nm (Figure 2F). This laser-light-dependent spectral transformation
also indicates the sensitivity of the molecular crystals for different
polarization directions of the laser source. The origin of greenish-
blue (cyan) colour is due to the appearance of intense peaks at 477,
479 and 525 nm with the spectral tail extended up to 675 nm
(Figure 1C). The cyan AIE originate as a result of restricted
intramolecular rotations and near planar conformation, both enable
an efficient -conjugation providing AIE for compound 1.14 The
solid-state absolute FL quantum yield of the aggregate 1 is 11%.
Generally, the morphology of evolving molecular aggregate
during self-assembly is concentration-dependent, which fluctuates
due to random flow of evaporating solvent. Therefore, the
dynamics of the molecular aggregation process was probed in-situ
with confocal FL micro-spectroscopy (camera speed at 25 fps)
shown in the inset of Fig. 3A. The sample was excited using a 405
nm laser (reflection mode; objective 20×; numerical aperture = 0.4).
Initially, by placing an acetonitrile solution of 1 (c = 2 mM; volume =
50 L) on a coverslip the onset of AIE intensity during solvent
evaporation (within 200 s) was collected from multiple areas. For
every 0.3 s the AIE (if any) was collected by a CCD detector for the
duration of up to 0-300 s. Each experiment (see Figure 3A) revealed
random time-dependent AIE signal counts (from several thousand
to ~3 million). Significantly, the dynamic AIE counts provide a
qualitative estimate of the time-dependent (fluctuating)
aggregate(s) size (concentration) in a given micro-volume during
solvent evaporation.
Fig. 1 A) ORTEP (50% probability thermal ellipsoids) views of 1. All hydrogen atoms are
omitted for clarity. B) Solid-state molecular packing of 1. C) Photographs of crystal 1
under ambient and UV light, respectively.
The synthesis of 1 required precursor compounds 4 and 5. At
first, compound 4 was prepared via Suzuki cross-coupling by
reacting 1-bromo-3,5-bis(trifluoromethyl)benzene 2 with the 4-
formyl phenyl boronic acid 3 in 89% yield. Condensation of 4 and 2-
(4-bromophenyl)acetonitrile 5 provided target AIE molecule 1 with
94% (Scheme 1). Crystallization of molecule 1 in chloroform
produced needle-shaped crystals with a square-shaped cross-wise
geometry. The X-ray analysis [CCDC: 1971747] of single crystal
revealed a near planar conformation of 1 (Figure 1A). The torsion
angles between bromophenyl and the vinyl units and
trifluoromethyl substituted biphenyl units were 7 and 1,
respectively. Molecule 1 possesses intermolecular C−F···H (2.656 Å)
and C−N···H (2.482 Å) H-bonding interactions and also -
interactions (3.713 Å) (Figure 1B and Fig. S9).
In the solution-state, compound 1 showed subtle luminescence
upon excitation with UV light (365 nm). The emission spectrum
exhibited peaks around 370, 411 and 433 nm with a meagre
quantum yield of 1.38% (see Figure S3). The steady-state FL spectra
of 1 in acetonitrile showed increased AIE intensity upon addition of
(poor solvent) water or viscous solvents (Figure S6 and S8). Self-
assembly of compound 1 in acetonitrile or chloroform formed rod-
To comprehend the reason behind time-dependent fluctuating AIE
counts, six peculiar-looking plots (viz., I to IX) were identified for
scrutiny (Figure 3 B-J). Plot-I, presented in Figure 3B showed no AIE
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Fig. 3 A) Time-dependent AIE intensities of hundreds of experiments. The inset shows the cartoon of the FL-micro-spectrometer. B-J) AIE intensity versus time plots of six
representative experiments. The insets show the confocal optical images taken immediately after the experiment. The yellow circles of the insets show the laser excitation area. K)
Pictorial representation of the various process involved during the aggregate growth.
signal until 40 s, indicating no recordable molecular aggregation. sharp rise of AIE intensity signifies the onset of the
Beyond 40 s, the AIE signal quickly appeared, and its intensity spectroscopically observable aggregate growing to reach a specific
increased swiftly (vertically) to about 55 k counts within 1 s. The
size. Afterwards, the AIE counts persisted till complete solvent
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vaporisation indicating cessation of the aggregate growth. The imply complex (multiple) diffusion dynamics of the micro-crystal(s)
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DOI: 10.1039/D0SM00108B
micro-area image revealed the fibrous morphology of the grown after its(their) growth in the evaporating solvent medium.
aggregates (see Figure 3B inset). The course of AIE counts as a
In conclusion, we demonstrated a robust technique to follow the
function of time in Plot-II (Figure 3C) was slightly different from molecular aggregates process using confocal micro-FL spectroscopy.
Plot-I. In the beginning, a typical fast-growth (within 2 s) from the From the time-correlated monitoring of AIE intensity, the following
onset of detectable aggregate occurred with AIE counts reaching key characteristics of molecular aggregates (Figure 3K) can be
40k at 20 s; subsequently, after initial slow growth, the aggregate inferred.
size remained constant till 132 s. Surprisingly, over again, the AIE
intensity vertically soared to 55 k at 133 s specifying further growth
of the aggregate or sudden diffusion of another aggregate towards
the growing aggregate.
 A range of detectable aggregation time indicates a varying
growth rate of molecular aggregate in different areas of the
sample.
 The variation of the AIE intensity correlates directly with the
size and concentration of the aggregates in a given area.
Higher AIE counts revealed the existence of bigger-sized and
tightly-packed aggregates in contrary to smaller-sized and
dispersed aggregate with lower AIE counts.
 The decrease of the AIE intensity at varying rates indicates
diffusion of the aggregates caught in the solvent convection
current.
 The diffusion dynamics of bigger-sized aggregate in the
crowded regions are more prominent than the smaller-sized
ones.
In Plot-III (Figure 3D), the AIE counts revealed that the growth of
visible aggregate started after 42 s. Later, after a nearly 31 s the AIE
counts reached a transitorily stable state with 450 k, and was stable
till 176 s. The growth track of the aggregate involved three steps;
initially steep progress, then sluggish, and later a slight back-and-
forth diffusion (see v-shaped dip) from the growth site. The shape
of the curve and the AIE counts reveal the slow growth rate and
larger size of the aggregate, respectively, compared to the previous
two cases. After 125 s, surprisingly, two peaks appeared at 176 and
214 s with a sudden surge of the AIE intensity up to 550 k. The rise
and fall (about 100 k) of AIE intensity twice imply jiggle movements
of the smaller aggregate towards the bigger one. The path of the
curve between the two peaks (time gap: 86 s) indicates an abrupt
(<1 s) diffusion of a smaller aggregate towards (with a 100 k AIE
count) the bigger one, followed by its gentle diffusion away (~86 s)
from it.
 The horizontal lines or a constant AIE intensity point to stable
aggregate(s) without growth and diffusion.
Our original findings provide new insights and illustrate practical
experimental protocols which can be applied to (i) probe the
concentration change in a specific site down to the single-cell level,
and (ii) monitor aggregation dynamics and motion of
biomacromolecules tagged with AIE luminogens.
Interestingly, the curve displayed in Figure 3E (Plot-IV) exhibited
two steep growth steps with AIE counts reaching 8 and 18 k,
respectively. In between the two vertical growth lines, a slight
decline of counts point towards slow diffusion (~17 s) of a fast-
growing aggregate from the observation area. Beyond 40 s, the
aggregate stabilised its position without any substantial growth and
diffusion. Here, the meagre counts and the signal-to-noise ratio hint
towards a much smaller size of the aggregate. Additionally, the
optical image recorded immediately after the experiment (inset of
Figure 3E) and the video revealed a cluster of small fibres excluding
the area irradiated with the laser beam indicating diffusion of fibres
away from the light beam during their growth. Remarkably, the
shape of the curve shown as Plot-V in Figure 3F revealed a less
complicated path taken by the particular aggregate. Here, the
aggregate slowly and steadily grew for nearly 15 s and then
gradually diffused away from the observation area in 2 min.
On the contrary, in Plot-VI, after 40 s, with an initial lag of 9 s,
the aggregate grew much bigger quickly reaching a colossal AIE
count of 1.1 M in 3 s and later, it diffused away rather fast but not
entirely from the observation area (Figure 3G). The inset image of
Fig. 4G, captured immediately after the experiment showed aligned
fibres and also revealed a local disturbance in the fibre organisation
around the laser illumination area (see the yellow circle). In case-VII
presented in Figure 3H, the shape of the curve (with two slope
values) between 23 s and 67 s indicates that after rapid growth, the
aggregate diffusion is initially slow and later quick. An intricate
pattern of the curves shown in Figure 3I and J (Plot-VIII and -IX)
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
This work was financially supported by DST (New Delhi; Grant No.
INT/RUS/RSF/P-05) and SERB (CRG-2018/001551). M. J. and V. V. P.
thank CSIR for JRFs.
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