Figure 3. Top and side views of geometry-optimized structures of
ExPh and the corresponding natural transition orbitals (NTOs). (a)
Ground state and (b) excited state relaxed geometries.
Figure 4. Fluorescence spectra of (a) ExBT (15 μM), (b) ExBT-OMe
15 μM), and (c) ExPhos (15 μM) recorded upon exposure to
(
Fe(III) (0−300 μM as the FeCl3 salt). (d) Fluorescence spectra of
ExPhos (15 μM) preincubated with 64 U of ALP followed by
exposure to Fe(III) (0−300 μM). (e) Fluorescence changes of
ExPhos (15 μM) with increasing alkaline phosphatase (ALP, 0−64
U). (f) Time-dependent change in the fluorescence-emission intensity
addition of increasing concentrations of Fe(III) (as the FeCl3
salt) to both ExBT (15 μM) and deferasirox (15 μM) resulted
in an increase in their respective UV−vis absorption intensities,
along with a color change from clear to reddish/purple (SI
(
505 nm) of ExPhos (15 μM) when exposed to ALP (two
2
9
Figure S24). No change in UV−vis absorption/color was
seen when Fe(III) was added to ExBT-OMe (SI Figure S24).
We thus conclude that a free phenol is needed to chelate
concentrations, 32 U and 64 U). All measurements were carried
out in deionized water and excited at 320 nm.
2
9
Fe(III). A concentration-dependent quenching of the ExBT
fluorescence was also seen (Figure 4a), as would be expected
for Fe(III) chelation (paramagnetic metal fluorescence
Both Fe(III) concentration and ALP activity are key to the
growth of pathogenic bacteria including biofilm formation and
3
0
13,36−40
quenching).
development.
Biofilms are complex bacterial commun-
Recently, protected versions of known metal chelators (“pro-
chelators”) have been explored in an effort to avoid premature
ities enclosed by extracellular polymeric substances (EPS),
41,42
which provide protection against antibiotics.
biofilms can rapidly adapt to their environments. These
In addition,
41
3
1
metal chelation. We thus prepared the bis-phosphate ester
ExPhos as a phosphatase-responsive fluorescent pro-chelator
that would permit detection of a disease-based biomarker (i.e.,
characteristics result in hard-to-treat infections, promote
43
antibiotic resistance, and lead to patient complications.
32−35
alkaline phosphatase, ALP).
As true for the protected
This provides an incentive to develop fluorescent tools to
image biofilms and visualize biomarkers associated with their
formation and survival.
system, ExBT-OMe, minimal Fe(III) quenching or change in
the UV-Vis absorption was seen for ExPhos (Figure 4c, See SI
Figure S24). However, in the presence of ALP a dose
dependent change in the fluorescence emission was observed
To date, several AIEgens have been reported for bacteria-
44
based imaging applications. For instance, Tang and co-
workers reported an AIEgen that can discriminate between
(
0−64 U), with a final fluorescence emission profile analogous
45
to that of ExBT being seen (Figures 4e and S25). Time-
dependent changes in fluorescence emission intensity were also
seen (see SI Figure S4f). The limit of detection (LoD) was
calculated to be 0.016 75 U (measurements performed after 5
min of incubation; see SI Figure S26). The ratiometric change
in the fluorescence emission was ascribed to the gradual and
stepwise (bis-phosphate ester to monophosphate ester to bis-
phenol) dephosphorylation of ExPhos (as confirmed by LC-
MS analyses; see SI Figures S27−S33). This sequential
conversion leads to a rapid decrease in the blue emission
and a slow increase in the green emission. ExPhos solutions
exposed to ALP (64 U ALP) were subsequently treated with
Gram-positive bacteria, Gram-negative bacteria, and fungi,
while Liu and co-workers detailed a dual-functional TPE-based
antibiotic AIEgen that allows for the intracellular imaging of
46
bacteria in living host cells. However, little attention has been
paid to imaging biofilms. We thus sought to evaluate the
diagnostic and antibiofilm potential of ExBT and ExPhos.
Biofilm experiments were carried out on glass slides with
both Pseudomonas aeruginosa and MRSA. The therapeutic
performance of the broad-spectrum antibiotic cefoperazone-
47
sulbactam (CFP-SUL) was evaluated on its own and in
combination with deferasirox, ExBT, and ExPhos using the
live/dead biofilm caption viability assay (propidium iodide
(PI): dead, red; Syto9: live, green). As shown in the processed
3D images (Figure 5), the use of just CFP-SUL led to a partial
increasing concentrations of Fe(III) (as FeCl ). Quenching of
3
the fluorescence intensity was observed (Figure 4d).
1
280
J. Am. Chem. Soc. 2021, 143, 1278−1283