an enhanced NIR fluorescent emission with the high signal-to-background ratio in
mice. In the same year, Han et al. [16] reported a fluorescent turn-on probe termed
AcGQCy7 (6) to detect β-gal activity in living cells. Because of its disrupted
p-conjugated system, the prepared probe 6 was nonfluorescent in the NIR region,
but after the enzymatic cleavage of β-galactose by β-gal under physiological conditions, the probe emitted strong fluorescence. The probe (300 μM) was dissolved in
HBSS was tested for the presence or absence of β-gal (15 U mL
À1 ) at 37
C for
30 min. The color of the solution rapidly turned into dark blue when it was in contact
with β-gal, and a broad absorbance band covering of 400–580 nm was detected. The
fluorescence intensity of the β-gal-treated solution was 110-fold higher than that of
the solution without it. The cellular imaging capability of the probe was studied in
two cell lines, rat glial tumor C6 (control, no β-galactosidase expression) and its
derivative, C6/LacZ, which constitutively expresses β-gal. C6/LacZ or C6 cells were
incubated in HBSS buffer containing probe 6 (1 mM) at 37
C for 40 min. As
expected, a bright fluorescence signal was detected only in the β-gal-positive
C6/LacZ cells but not in the β-gal-negative C6 cells. Co-localization studies indicated that the cleaved product that specifically targeted mitochondria as the red
fluorescence from 6 was overlapped with the MTG signal. This finding agrees with
the author’s another research of QCy7 as an excellent mitochondria mark.
In 2016, Gu et al. [17] reported a ratiometric near-infrared probe (DCM-β-gal)
which was able to be activated by β-gal for the real-time fluorescent quantification
in vivo and in situ. They designed 7 by grafting a β-gal activatable unit onto an NIR
fluorophore DCM-OH. Upon excitation of the new absorption peak at 535 nm, a
strong NIR fluorescence enhancement was observed with a peak at 685 nm. The
color changed from faint yellow to rose red, which allows the colorimetric detection
of β-gal using the naked eye. Furthermore, owing to the alteration in concentrationdependent ratio, the detection limit of DCM-β-gal was 1.7 Â 10
À4 U mL
À1 . In order
to get living cells generating endogenous β-gal, lacZ gene was introduced into 293T
cells through a gene transfection method to overexpress β-gal. After incubation with
7 (10 μM) at 37
C for 30 min, the lacZ-(+) 293T cells successfully overexpressed
β-gal and were observed to exhibit a decreased fluorescence in the green channel
as well as a concomitant increase in the red channel. These results suggested
that 7 could be specifically activated in β-gal-expressing cells, hence offering a
ratiometric and light-up NIR readout for the in situ quantitative tracking and
visualization of endogenous β-gal in living cells (Figs. 2 and 3).
2.3 ESIPT and AIE Fluorescent Probes
Besides ICT mentioned above, the β-gal could also be detected through varieties of
other mechanisms, such as excited-state intramolecular proton transfer (ESIPT),
the aggregation-induced emission (AIE), and so on. ESIPT is a fast photochemical
process in which a proton transfers from a hydroxyl (or amino) unit to a carbonyl
oxygen (or imine nitrogen) atom in the excited state of a fluorophore [18, 19]. ESIPT
Fluorescent Probes for Diagnostics of β-Galactosidase: From Micro to Macro
189
mice. In the same year, Han et al. [16] reported a fluorescent turn-on probe termed
AcGQCy7 (6) to detect β-gal activity in living cells. Because of its disrupted
p-conjugated system, the prepared probe 6 was nonfluorescent in the NIR region,
but after the enzymatic cleavage of β-galactose by β-gal under physiological conditions, the probe emitted strong fluorescence. The probe (300 μM) was dissolved in
HBSS was tested for the presence or absence of β-gal (15 U mL
À1 ) at 37
C for
30 min. The color of the solution rapidly turned into dark blue when it was in contact
with β-gal, and a broad absorbance band covering of 400–580 nm was detected. The
fluorescence intensity of the β-gal-treated solution was 110-fold higher than that of
the solution without it. The cellular imaging capability of the probe was studied in
two cell lines, rat glial tumor C6 (control, no β-galactosidase expression) and its
derivative, C6/LacZ, which constitutively expresses β-gal. C6/LacZ or C6 cells were
incubated in HBSS buffer containing probe 6 (1 mM) at 37
C for 40 min. As
expected, a bright fluorescence signal was detected only in the β-gal-positive
C6/LacZ cells but not in the β-gal-negative C6 cells. Co-localization studies indicated that the cleaved product that specifically targeted mitochondria as the red
fluorescence from 6 was overlapped with the MTG signal. This finding agrees with
the author’s another research of QCy7 as an excellent mitochondria mark.
In 2016, Gu et al. [17] reported a ratiometric near-infrared probe (DCM-β-gal)
which was able to be activated by β-gal for the real-time fluorescent quantification
in vivo and in situ. They designed 7 by grafting a β-gal activatable unit onto an NIR
fluorophore DCM-OH. Upon excitation of the new absorption peak at 535 nm, a
strong NIR fluorescence enhancement was observed with a peak at 685 nm. The
color changed from faint yellow to rose red, which allows the colorimetric detection
of β-gal using the naked eye. Furthermore, owing to the alteration in concentrationdependent ratio, the detection limit of DCM-β-gal was 1.7 Â 10
À4 U mL
À1 . In order
to get living cells generating endogenous β-gal, lacZ gene was introduced into 293T
cells through a gene transfection method to overexpress β-gal. After incubation with
7 (10 μM) at 37
C for 30 min, the lacZ-(+) 293T cells successfully overexpressed
β-gal and were observed to exhibit a decreased fluorescence in the green channel
as well as a concomitant increase in the red channel. These results suggested
that 7 could be specifically activated in β-gal-expressing cells, hence offering a
ratiometric and light-up NIR readout for the in situ quantitative tracking and
visualization of endogenous β-gal in living cells (Figs. 2 and 3).
2.3 ESIPT and AIE Fluorescent Probes
Besides ICT mentioned above, the β-gal could also be detected through varieties of
other mechanisms, such as excited-state intramolecular proton transfer (ESIPT),
the aggregation-induced emission (AIE), and so on. ESIPT is a fast photochemical
process in which a proton transfers from a hydroxyl (or amino) unit to a carbonyl
oxygen (or imine nitrogen) atom in the excited state of a fluorophore [18, 19]. ESIPT
Fluorescent Probes for Diagnostics of β-Galactosidase: From Micro to Macro
189