Photocontrollable fluorogenic probes for visualising near-membrane copper(II) in live cells

Article abstract of DOI:10.1039/c7ra03559d

In this work, we have described the design, synthesis, and characterisation of photocontrollable fluorogenic probe, Mem-5, which is equipped with a photolabile group (nitrobenzyl group), high brightness reporter (fluorescein), and membrane-anchoring unit

Full text of DOI:10.1039/c7ra03559d

RSC Advances
View Article Online
View Journal | View Issue
PAPER
Photocontrollable fluorogenic probes for
visualising near-membrane copper(II) in live cells†
Cite this: RSC Adv., 2017, 7, 31093
Buxiang Chen,a Liulin Wang,a Yanfei Zhao,a Yun Ni,a Chenqi Xin,a Chengwu Zhang,a
a
ac
Jinhua Liu,a Jingyan Ge,b Lin Li
and Wei Huang
*
*
In this work, we have described the design, synthesis, and characterisation of photocontrollable fluorogenic
probe, Mem-5, which is equipped with a photolabile group (nitrobenzyl group), high brightness reporter
(fluorescein), and membrane-anchoring unit (cholesterol or long aliphatic chain). This probe shows an
intense fluorescence enhancement in response to copper(II) without interference from 45 other analytes,
including metal cations, amino acids, and anions, under biological conditions (pH 6–9). The hydrolysis
reaction is second-order, with k ¼ 9.01 ꢀ 10ꢁ5 Mꢁ1
s
ꢁ1. The detection limit is 3.3 ꢀ 10ꢁ7 M in HEPES
Received 27th March 2017
Accepted 31st May 2017
buffer (25 mM). The confocal fluorescence imaging results demonstrated that Mem-5 can visualise near-
membrane copper(II) in live mammalian cells. The clear advantage of our photocontrollable method is
that it successfully avoids the influence of chemical species outside cells during near-membrane specific
detection.
DOI: 10.1039/c7ra03559d
rsc.li/rsc-advances
a range of neurological diseases, such as Wilson's and Alz-
heimer's diseases.17,18 High-affinity copper transporter 1 (Ctr1)
drives the entry of copper(II) into cells,19 while cells must eject
Introduction
Cell plasma membranes are regarded as asymmetric due to
their inner and outer leaets having different compositions,
based on the classic Singer–Nicolson model.1 This prevents
foreign substances from entering the cell barrier freely,
ensuring a relatively stable environment within the cell,2 but
absorbs and digests material inside and outside by endocytosis,
phagocytosis, or exocytosis.3,4 Therefore, this lipid bilayer with
embedded protein membranes is important for cell recogni-
tion, signal transduction, cellulose synthesis, microbril
assembly, and other mechanisms,5 and can regulate cell life
activities as turntables for crucial processes in neurobiology,
muscle contraction, and cell signalling.6,7 Given the pivotal role
of cell plasma membranes, the development of membrane-
specic uorogenic probes to detect near-membrane elements
has attracted increasing attention.8–15
excess copper(II) through the plasma membrane to maintain
a low concentration of cytosolic free copper(II).20 Effective
methods for evaluating free copper(II) near or across the plasma
membrane will aid understanding of the copper(II)
transportation-related biological functions of cells. Regrettably,
tools to precisely detect near-membrane copper(II) in live cells
without interference by external copper(II) have still not been
developed.
With this in mind, herein, we have successfully developed
two new photocontrollable uorogenic probes (Fig. 1) with
different membrane anchors to image near-membrane cop-
per(II) in live mammalian cells with good spatial and
temporal controls using efficiently photolysis by ultraviolet
(UV) light irradiation. Mem-1/2 (structures shown in Fig. 2),
which contain a cholesterol or 12-carbon aliphatic chain,
served as membrane tracers, and their membrane staining
ability was compared using this “piggyback” strategy.21–23 As
shown in Fig. 1, uorescein hydrazide hydrolysed by cop-
per(II) would normally be blocked due to the existence of
a caged unit aer membrane staining,24 facilitating the
complete accumulation of Mem-5/6 with no uorescence.25
Subsequent UV irradiation will force removal of the caged
group, releasing uncaged Mem-3/4 (structures shown in
Fig. 2),26,27 which would then be hydrolysed by copper(II) to
generate a specic localised and strong uorescent signal.28
Notably, the modularity of our probe design should enable
this strategy to be readily applicable to the detection of other
targets near the membrane.
Copper(II) content in the human body is the second most
common trace element, and plays an essential role in human
metabolism.16 Copper(II) overload has been shown to cause
aKey Laboratory of Flexible Electronics (KLOFE), Institute of Advanced Materials (IAM),
Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM),
Nanjing Tech University (Nanjing Tech), 30 South Puzhu Road, Nanjing, 211816, P.
R. China. E-mail: iamlli@njtech.edu.cn; iamwhuang@njtech.edu.cn
bKey Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology
and Bioengineering, Zhejiang University of Technology, Hangzhou 310014, P. R. China
cKey Laboratory for Organic Electronics and Information Displays, Institute of
Advanced Materials (IAM), Nanjing University of Posts & Telecommunications, 9
Wenyuan Road, Nanjing 210023, P. R. China
† Electronic supplementary information (ESI) available: Synthesis and
characterisation, photophysical spectra and background control images. See
DOI: 10.1039/c7ra03559d
This journal is © The Royal Society of Chemistry 2017
RSC Adv., 2017, 7, 31093–31099 | 31093

View Article Online
RSC Advances
Paper
throughout the experiments. Absorption spectra were recorded
using SynergyHTX microplate reader or a Shimadzu UV-3600
UV-Vis-NIR spectrophotometer. Photoluminescence spectra
were recorded using a HITACHI F4600 uorescence spectro-
photometer with excitation slit widths of 5 nm and emission slit
widths of 10 nm. 1H and 13C NMR spectra were collected in
6
ꢂ
CDCl3 or DMSO-d at 25 C using an Avance AV-300 spectrom-
eter. All photochemical reactions were conducted in an ultra-
violet analyser ZF-20D. Ultrapure water was used to prepare all
aqueous solutions. Fluorescein in 0.1 M NaOH (F ¼ 0.85) was
used as the standard for quantum yield measurements. All
spectroscopic measurements were performed in 25 mM HEPES
ꢂ
buffer (pH 7.4) at 37 C. All images were acquired in the same
ꢂ
way at 20 C on Zeiss LSM880 NLO (2 + 1 with BIG) confocal
microscope system equipped with an objective LD C-
Apochromat 63ꢀ/1.15 W Corr M27, 405 nm diode laser, argon
ion laser (458, 488, and 514 nm), HeNe laser (543 and 594 nm),
and 633 nm laser, with 8 AOTF channels for simultaneous
control of 8 laser lines. A PMT detector ranging from 420 nm to
700 nm was used for steady-state uorescence. Internal photo-
multiplier tubes were used to collect the signals in 8 bit
unsigned 1024 ꢀ 1024 pixels at a scan speed of 200 Hz. Images
were processed with Zeiss User PC Advanced for LSM system
(BLUE).
Fig. 1 Overall strategy of our photocontrollable fluorogenic probes,
Mem-5 and Mem-6 (structures shown in box), for imaging membrane
copper(II) in live cells.
Synthetic procedures
All reactions were carried out under a dry nitrogen atmosphere.
Reaction progress was monitored by TLC on pre-coated silica
plates (thickness, 250 mm) and spots were visualised using ceric
ammonium molybdate, basic KMnO4, UV light, or iodine.
Synthesis of Mem-5. In a 50 mL round bottom ask, trie-
thylamine (0.014 mL) was added to a solution of nitrobenzyl-
protected uorescein hydrazide (50 mg, 0.10 mmol) in DMF (2
mL). The mixture was stirred for 5 min under a dry nitrogen
atmosphere. Cholesteryl chloroformate (45.34 mg, 0.10 mmol)
in chloroform (0.8 mL) was added dropwise to the solution. The
reaction was stirred in an ice bath for another 8 h, followed by
the addition of saturated ammonium chloride aqueous solution
(10 mL) and extraction with ethyl acetate (3 ꢀ 30 mL). The
organic layer was washed three times with brine, dried over
anhydrous Na2SO4, and ltered. Aer removing solvent under
reduced pressure, the residue was puried by ash column
chromatography (silica gel, PE/EA ¼ 1 : 1) affording the product
as a pink solid (yield 60%). 1H NMR (300 MHz, CDCl3): d (ppm)
8.03 (d, J ¼ 4.5 Hz, 1H), 7.93 (t, J ¼ 4.5 Hz, 1H), 7.75 (d, J ¼ 3 Hz,
1H), 7.59 (t, J ¼ 7.5 Hz, 1H), 7.45 (m, 3H), 7.09 (s, 1H) 7.02 (d, J ¼
3 Hz, 1H), 6.52 (m, 4H), 6.82 (dd, J1 ¼ 4.5 Hz, J2 ¼ 1.5, 1H), 6.65
(d, J ¼ 4.5 Hz, 2H), 6.52 (d, J ¼ 3 Hz, 2H), 6.05 (dd, J1 ¼ 3 Hz, J2 ¼
1.5 Hz, 1H), 5.42 (s, 1H), 4.60 (dd, J1 ¼ 6 Hz, J2 ¼ 3 Hz, 1H) 3.52
(bs, NH2), 2.48 (s, 2H), 2.00 (m, 5H), 1.56 (m, 10H), 1.33 (m, 4H),
0.91 (m, 22H), 0.68 (s, 3H). 13C NMR (75 MHz, CDCl3): d (ppm)
166.41, 158.49, 152.49, 151.77, 150.66, 150.55, 139.16, 138.63,
134.26, 134.21, 133.05, 128.61, 128.43, 128.37, 128.30, 128.20,
127.50, 124.91, 123.94, 123.42, 123.37, 116.94, 116.51, 112.90,
112.66, 110.04, 103.36, 103.21, 79.31, 71.86, 56.80, 56.27, 50.11,
42.43, 39.83, 39.62, 37.98, 36.92, 36.65, 36.30, 35.87, 32.01,
Fig. 2 Structures of membrane tracers, Mem-1 and Mem-2, with
different anchors, and unphotocontrollable fluorogenic probes, Mem-
3 and Mem-4, for imaging membrane copper(II) in live cells.
Experimental
Materials and instruments
All reagents and solvents were purchased from commercial
suppliers and used without further purication, unless other-
wise noted. N,N-Dimethylformamide (DMF) was distilled over
ꢂ
CaH2. Petroleum ether (PE, 60–90 C), DCM, and ethyl acetate
(EA) were used as eluents for ash column chromatography
with Merck silica gel (0.040–0.063). 1-(1-Bromoethyl)-2-
nitrobenzene was synthesised according to a literature proce-
dure, while uorescein and hydrazine hydrate were purchased
from Adamas.25 Cholesterol chloride and bromododecane were
purchased from Tokyo Chemical Industry. Nuclear (Hoechst
33 342) and membrane (CM orange) trackers were purchased
from Invitrogen. Human hepatocellular carcinoma (HepG2)
cells and fetal bovine serum (FBS) were purchased from ATCC
(HB-8065™) and Biological Industries (BI, 04-001-01ACS),
respectively. Other reagents used in the experiments were
purchased from Sigma-Aldrich and used without further puri-
cation unless otherwise noted. Distilled water was used
31094 | RSC Adv., 2017, 7, 31093–31099
This journal is © The Royal Society of Chemistry 2017

View Article Online
Paper
RSC Advances
31.96, 28.31, 28.09, 27.69, 24.38, 23.93, 23.67, 23.62, 22.90,
22.66, 21.16, 19.36, 18.83, 11.97.
Synthesis of Mem-6. To a stirred solution of nitrobenzyl-
protected uorescein hydrazide (50 mg, 0.10 mmol) in dry
DMF (2 mL) was added Cs2CO3 (114.04 mg, 0.35 mmol) and
bromododecane (25.16 mg, 0.10 mmol) under a dry nitrogen
atmosphere. Aer being stirred for 8 h at 60 ꢂC, the reaction was
stopped with the addition of saturated aqueous ammonium
chloride (10 mL). The mixture was extracted and isolated as
described above in the procedure for Mem-5, to afford Mem-6 as
1
a pink solid (yield 62%). H NMR (300 MHz, CDCl3): d (ppm)
8.04 (d, J ¼ 4.5 Hz, 1H), 7.92 (t, J ¼ 4.5 Hz, 1H), 7.74 (d, J ¼
4.5 Hz, 1H), 7.60 (t, J ¼ 7.5 Hz, 1H), 7.46 (m, 3H), 7.01 (dd, J1 ¼
3 Hz, J2 ¼ 1.5 Hz, 1H), 6.70 (d, J ¼ 4.5 Hz, 1H), 6.52 (m, 4H), 6.04
(dd, J1 ¼ 3 Hz, J2 ¼ 1.5 Hz, 1H), 3.94 (t, J ¼ 7.5 Hz, 2H), 3.63 (bs,
NH2), 1.70 (m, 4H), 1.42 (s, 1H), 1.26 (s, 19H), 0.87 (d, J ¼ 7.5 Hz,
3H). 13C NMR (75 MHz, CDCl3): d (ppm) 166.43, 160.40, 158.41,
153.23, 151.01, 138.79, 134.33, 134.27, 132.94, 129.83, 128.75,
128.60, 128.54, 128.11, 127.57, 124.94, 123.87, 135.35, 112.73,
112.47, 112.31, 110.20, 103.18, 103.00, 101.65, 71.85, 68.45,
63.17, 32.95, 32.02, 29.76, 29.73, 29.70, 29.67, 29.45, 29.22,
26.11, 23.68, 22.79, 14.21.
Fig. 3 Absorption spectra of (a) Mem-1/3/5 and (b) Mem-2/4/6, and
fluorescence emission spectra of (c) Mem-1/3/5 and (d) Mem-2/4/6 in
HEPES buffer (probe concentration, 5.0 mM). Abs ¼ absorbance, RFU ¼
relative fluorescence units.
Live cells uorescence imaging
For organelle-specic imaging experiments, the cells were
incubated with Mem-1 and Mem-2 (1 mM prepared in fresh
ꢂ
medium) for 20 min at 37 C, followed by washing three times
Photouncaging reactions
with PBS. To accurately visualise membrane copper, the cells
were incubated with Mem-3ꢂand Mem-5 (5 mM prepared in fresh
medium) for 20 min at 37 C. Aer washing three times with
PBS, the selected cells were irradiated for 2 min with UV light
through an ultraviolet analyser. The cells were then incubated
with copper(II) (10 equiv.) for a further 60 min at 37 ꢂC. Next, the
cells were washed three times with PBS, then imaged with the
Zeiss LSM880 NLO (2 + 1 with BIG) confocal microscope system
(Fig. 2 and 3). Hoechst 33 342 (250 nM, Ex ¼ 405 nm, PMT range
¼ 420–460 nm) and CM orange (100 nM, Ex ¼ 543 nm, PMT
range ¼ 550–650 nm) were used as nuclear and membrane
trackers, respectively. Meanwhile, another identical set of
samples was treated as described above, but without UV irra-
diation, as the negative control. Background signals of all
images were veried to be nearly zero by imaging the same cells
treated with a negative control.
For in vitro analysis, different concentrations (2.0 mM for
selectivity/kinetic experiments and 10.0 mM for pH titration
experiments) of Mem-5 were placed in quartz glassware and
submitted to UV irradiation (500 mJ cmꢁ2 at 400 nm) for 5 min at
room temperature to generate uncaged Mem-5 in HEPES buffer.
In live cell imaging, cells were incubated with Mem-5 (2.0 mM)
ꢂ
for 20 min at 37 C, followed by further UV irradiation (500 mJ
cmꢁ2, 5 min) at room temperature in cover-free dishes to
generate uncaged Mem-5.
Cell culture and cytotoxicity activity assay
HepG2 cells were cultured in Dulbecco's Modied Eagle's
Medium (DMEM), containing 10% FBS, 100.0 mg Lꢁ1 strep-
tomycin, and 100 IU mLꢁ1 penicillin. HepG2 cells were seeded
in glass-bottom dishes (Mattek) and grown to 70–80% con-
uency. The cytotoxicity activities of the probes were deter-
mined using an XTT colorimetric cell proliferation kit (Roche),
following the manufacturer guidelines. Briey, different cells
Results and discussion
Synthesis and characterisation
were grown to 20–30% conuency (they would reach 80–90% To validate our design, two photocontrollable uorogenic
conuency within 48–72 h in the absence of compounds) in 96- probes (Mem-5/6) and four additional control probes (Mem-1/2/
well plates under the conditions described above. The medium 3/4) were synthesised following modied literature
was aspirated, washed with PBS, and then treated, in dupli- methods,29–31 as summarised in Scheme S1.† Two membrane
cate, with 0.1 mL of the medium containing different tracers (Mem-1/2) with different membrane anchors were ob-
concentrations of Mem-1/2/3/4/5/6 (1–50 mM). Probes were tained from the condensation of uorescein/uorescein esters
applied from DMSO stocks, whereby DMSO never exceeded 1% (reporter,
a dye with high brightness) and cholesteryl
in the nal solution. The same volume of DMSO was used as chloroformate/bromododecane in ꢃ50% yield. Fluorescein
a negative control, while the same volume of staurosporine hydrazide, a copper(II)-responsive unit, was synthesised in one
(STS, 200 nM) was used as a positive control. Aer a total step with an overall yield of 92% from commercially available
treatment time of 24 h, proliferation was assayed using the hydrazine hydrate and uorescein, followed by coupling with
XTT colorimetric cell proliferation kit (Roche), following membrane-targeting moieties to generate Mem-3/4, both in
manufacturer guidelines (Fig. 4a).
50% yield. Nitrobenzyl-protected uorescein hydrazide was
This journal is © The Royal Society of Chemistry 2017
RSC Adv., 2017, 7, 31093–31099 | 31095

View Article Online
RSC Advances
Paper
prepared by the condensation of uorescein hydrazide and 1-(1- than the group a long aliphatic chain at high concentrations.
bromoethyl)-2-nitrobenzene in 54% yield in the presence of Theoretically, a probe/plasma membrane lipid ratio must be
Cs2CO3. Mem-5/6 (ꢃ60% yields) were prepared using a proce- lower than 1/100 for there to be no signicant perturbation of
dure similar to that described above for Mem-3/4. The the biomembrane properties in cell membrane staining.32
compound structures were fully characterised by 1H and 13C Therefore, both Mem-1/3/5 and Mem-2/4/6 were suitable for
NMR, with detailed synthetic procedures and original spectra living cell imaging at low working concentrations, because we
described in the experimental section and ESI.†
incorporated a high brightness reporter in the probe design.
To systemically demonstrate the potential utility of our newly Therefore, to establish that Mem-1/2 was indeed a good
developed probes as good uorogenic systems for live cell membrane tracer for live cell imaging unequivocally, HepG2
imaging, we rst evaluated their chemical, photophysical, and cells were treated with Mem-1/2, followed by one-photon uo-
biological properties. As shown in Fig. 3a and b, under physi- rescence imaging (background control shown in Fig. S5†). We
ological conditions (HEPES buffer, pH 7.4), Mem-1/2, the observed uorescent staining exclusively within the plasma
membrane tracer references of Mem-5/6, had maximum membrane of the cells for Mem-1 (Fig. 4b(2/3), 1 mM treatment
absorptions at 460 nm, with two shoulder peaks at 440 and for 20 min). Otherwise, Mem-2, the probe with a 12-carbon
460 nm, and an emission at 550 nm (Fig. 3c and d, F ¼ ꢃ0.15 aliphatic chain, stained whole cells without specic localisation
with uorescein in 0.1 M NaOH as the reference). Mem-1/2/3/4 on the plasma membrane (Fig. 4b(7/8)). As shown in Fig. 4b(5),
showed nearly no absorption or emission (F ¼ 0.001–0.0015) at the green uorescence signal of Mem-1 overlapped well with the
around the same wavelength, based on the closed-loop struc- red uorescence signal of the commercially available
ture. A similar outcome was observed in organic solvent membrane tracker (CM-orange) with a high contrast ratio.
(ethanol, Fig. S1–S4†). Furthermore, two different membrane Prolonged incubation of the cells with Mem-1 did not lead to
anchors had no effect on the photophysical properties of the
probes, and the caged probes had very low uorescence back-
grounds or high S/N ratios for live cell imaging.
Subsequently, the cytotoxicity assays showed no obvious cell
toxicity at concentrations up to 50 mM for Mem-1/3/5 and 20 mM
for Mem-2/4/6, in human hepatocellular carcinoma (HepG2)
cells (Fig. 4a). The results showed that Mem-1/3/5 with
a cholesterol unit were more suitable for the living cell assay
Fig.
4 (a) XTT assay profiles of Mem-1/2/3/4/5/6, error bars
represent s.e.m. (n ¼ 3). (b) One-photon fluorescence microscopy
imaging of live HepG2 cells by control probes Mem-1 and Mem-2 (1
mM). Live cells were incubated with the probes for 20 min at 37 ꢂC,
followed by addition of a commercially available nuclear stain (NU, 250
nM) and membrane tracker (Mem-tracker, 100 nM), and then further
incubation for 10 min before imaging. The excitation wavelength was
457 nm, while the PMT range was 500–550 nm for probes. Inset in (1)
is the differential interference contrast image (DIC). (3) and (8) are
merged images of (2) and (7) with related DIC, respectively; while (5)
and (10) are merged images of (2)/(4) with the colocalisation analysis (R
¼ 0.80) and (7)/(9) (R ¼ 0.04), respectively. All images were acquired in
the same way. Scale bar ¼ 15 mm.
Fig. 5 (a) Effect of the 45 analytes (10 eq.) on uncaged Mem-5 (2.0 mM,
5 min UV-illumination) in HEPES buffer. (b) Photo of fluorescence with
the effect of analytes (10 eq.) on uncaged Mem-5 (2.0 mM) in HEPES
buffer after 60 min of incubation at 37 ꢂC. (c) Effect of the analytes (50
eq.) on uncaged (5 min of UV radiation) Mem-5 (2.0 mM) with Cu2+ (10
eq.) in HEPES buffer. Red bars represent the fluorescence intensity at
37 ꢂC. Error bars represent s.e.m. (n ¼ 3).
31096 | RSC Adv., 2017, 7, 31093–31099
This journal is © The Royal Society of Chemistry 2017

View Article Online
Paper
RSC Advances
the staining of other lipid-like environments in intracellular
As the pH of the cell cytosol is between 6.8 and 7.4,33 we
organelles, indicating minimal probe internalisation, and con- attempted to determine whether the probe could emit uores-
rming Mem-1 as cell-membrane specic. cence and be stable under these conditions. Therefore, the
We next investigated whether Mem-5 serves as an effective effect of pH on the uorescence response of Mem-5 to copper(II)
photocontrollable uorogenic probe for visualising near- was tested in a pH titration experiment. Mem-5 was found to be
membrane copper(II) in live cells. Upon photolysis with UV stable in a wide pH range of 5–9, and showed a better response
irradiation (500 mJ cmꢁ2 for 5 min), non-uorescent Mem-5 to copper(II) in the region pH 6–9 (Fig. 6a) aer photolysis.
generated non-uorescent uncaged Mem-5, according to When copper(II) (10 equiv.) was added to uncaged Mem-5 (2 mM)
a literature method.24 To evaluate the selectivity of Mem-5 in HEPES buffer, the uorescence intensity increased gradually
toward copper(II) among a series of metal cations, amino acids with time (Fig. 6b). The 1H-NMR titration experiments of Mem-3
and anions, 45 analytes (10 eq.), including K+, Ca2+, Mn2+, Cr2+, with different equivalents of copper(II) indicated that uores-
Co2+, Ni2+, Fe2+, Fe3+, Ba2+, Mg2+, Al3+, Ag+, Cd2+, Na+, Cu2+, cein hydrazide was hydrolysed by copper(II) under these
tyrosine, 2,6-diaminocaproic acid, isoleucine, valine, alanine, conditions (Fig. S6†). Next, the introduction of an increasing
aspartic acid, histidine, cysteine, ornithine, proline, glutamic copper(II) concentration into the solution of uncaged Mem-5 (2
acid, glycine, tryptophan, arginine, serine, threonine, phenyl- mM) induced a gradual increase in the emission at around
2ꢁ
alanine, methionine, cystine, CO32ꢁ, Iꢁ, Clꢁ, Brꢁ, Fꢁ, SO4
,
525 nm (Fig. 6c). The slopes of the plots had a pseudo-rst-order
HPO42ꢁ, HCO3ꢁ, S2ꢁ, and NO3ꢁ, were incubated with uncaged rate constant. A plot of the rate constants vs. copper(II)
Mem-5 in HEPES buffer at 37 C for 1 h, as shown in Fig. 5. A concentrations was a straight line passing through the origin,
ꢂ
signicant uorescence enhancement in Mem-5 was observed indicating that the reaction is second-order overall, with k ¼
when copper(II) was added only, while other species had 9.01 ꢀ 10ꢁ5 Mꢁ1 sꢁ1 (Fig. 6d).34 Fluorescence intensities also
a negligible uorescent intensity (Fig. 5a) with little interference exhibited a linear relationship with the copper(II) concentration,
(Fig. 5c). These results illustrate that Mem-5 had a high selec- as shown in Fig. S7† (R2 ¼ 0.97). A linear regression curve was
tivity for copper(II) recognition.
tted to these normalised uorescent intensity data, and the
Fig. 6 (a) Fluorescence intensities (Ex ¼ 455 nm) at 525 nm of Mem-1/5 (10 mM) and uncaged Mem-5 (10 mM)/Cu2+(10 equiv.) at various pH
values based on pH titration, error bars represent s.e.m. (n ¼ 3). (b) Fluorescence spectra of uncaged Mem-5 (2 mM) in the presence of Cu2+ (10
equiv.) for various periods (0–180 min). (c) Fluorescence intensities of uncaged Mem-5 (2 mM) in the presence of increasing Cu2+ concentration
(0–10 equiv.) (d) Plot of rate constants vs. Cu2+ concentrations. Reaction buffer was 25 mM HEPES (pH ¼ 7.4), and the reaction temperature was
37 ꢂC. (e) One-photon fluorescence microscopy imaging of live HepG2 cells by control probe Mem-3/5 (2 mM). Live cells were incubated with
the probe for 20 min at 37 ꢂC, followed by UV(+) or UV(ꢁ) irradiation (5 min) at room temperature and addition of Cu2+ (10 equiv.) for another
20 min at 37 ꢂC. The cells were then incubated with commercially available nuclear stain (NU, 250 nM) and membrane tracker (Mem-tracker, 100
nM), then further incubated for 10 min at 37 ꢂC before imaging. The excitation wavelength and PMT range were 488 and 500–550 for both
probes. Insets in (1)/(7) are the differential interference contrast images (DIC), while those in (2)/(8) are related mem-tracker channel images. (4)
and (10) are merged images of (3) and (9) with related DIC, respectively, while (6) and (12) are merged images of (3)/(5)/related nuclear stain with
the inset of colocalization analysis of (3)/(5) (R ¼ 0.85) and (9)/(11)/related nuclear stain with the inset of colocalization analysis of (9)/(11) (R ¼
0.87), respectively. All images were acquired in the same way. Scale bar ¼ 15 mm.
This journal is © The Royal Society of Chemistry 2017
RSC Adv., 2017, 7, 31093–31099 | 31097

View Article Online
RSC Advances
Paper
point at which the regression line crossed the axis was consid-
ered the detection limit (3.3 ꢀ 10ꢁ7 M).
3 L. Wu, E. Hamid, W. Shin and H. Chiang, Annu. Rev. Physiol.,
2014, 76, 301–331.
4 G. Meer, D. R. Voelker and G. W. Feigenson, Nat. Rev. Mol.
Cell Biol., 2008, 9, 112–124.
Finally, encouraged by these results, we determined whether
Mem-5 worked as a specic probe for imaging near-membrane
copper(II). As shown in Fig. 6e, HepG2 cells were incubated with
Mem-3/5 in the presence of copper(II). Cells treated with Mem-5
without UV irradiation showed no uorescence, indicating
temporal control of the “caging” strategy (Fig. 6e(2)). However,
signicant uorescent enhancement of Mem-3-treated cells was
observed using the same procedure (Fig. 6e(8)). Upon UV irra-
diation, a gradual increase in green uorescence was observed
both in Mem-5 (Fig. 6e(3)) and Mem-3-treated cells (Fig. 6e(9)).
The same cells were simultaneously treated with commercially
available Mem-tracker to independently verify the localisation
´
5 Z. Darwich, A. S. Klymchenko, D. Dujardin and Y. Mely, RSC
Adv., 2014, 4, 8481–8488.
6 D. Lingwood and K. Simons, Science, 2010, 327, 46–50.
7 E. Sezgin, I. Levental, S. Mayor and C. Eggeling, Nat. Rev. Mol.
Cell Biol., 2017, 18, 361–374.
8 J. L. Pincus, C. Y. Jin, W. J. Huang, H. K. Jacobs,
A. S. Gopalan, Y. J. Song, J. A. Shelnutta and D. Y. Sasaki, J.
Mater. Chem., 2005, 15, 2938–2945.
9 L. Li, X. Q. Shen, Q.-H. Xu and S. Q. Yao, Angew. Chem., Int.
Ed., 2013, 52, 424–428.
results. Both Mem-5 and Mem-3-treated cells showed similar 10 Y. Xia and L. Peng, Chem. Rev., 2013, 113, 7880–7929.
membrane-specic images by comparing the colocalisation 11 Z. Liu, T. P. Yang, X. Li, T. Peng, H. C. Hang and X. D. Li,
analysis in Fig. 6e(6) and (12), indicating the strategy was
Angew. Chem., Int. Ed., 2015, 54, 1149–1152.
amenable to the original design principle. Therefore, our pho- 12 T. Peng and H. C. Hang, J. Am. Chem. Soc., 2015, 137, 556–
tocontrollable method was successfully used to qualitatively
detect near-membrane copper(II).
559.
13 M. D. Molin, Q. Verolet, A. Colom, R. Letrun, E. Derivery,
M. Gonzalez-Gaitan, E. Vauthey, A. Roux, N. Sakai and
S. Matile, J. Am. Chem. Soc., 2015, 137, 568–571.
14 I. A. Karpenko, M. Collot, L. Richert, C. Valencia, P. Villa,
Conclusions
´
In this work, we have described the designed photocontrollable
Y. Mely, M. Hibert, D. Bonnet and A. S. Klymchenko, J. Am.
uorogenic probe, Mem-5, which can visualise near-membrane
Chem. Soc., 2015, 137, 405–412.
copper(II) via uorescence imaging, and is equipped with 15 R. J. Radford, W. Chyan and S. J. Lippard, Chem. Sci., 2013, 4,
a photolabile group, high brightness reporter, and membrane- 3080–3084.
anchoring unit (cholesterol or long aliphatic chain). This 16 D. Strausak, J. F. B. Mercer, H. H. Dieter, W. Stremmel and
probe shows an intense uorescence enhancement in response G. Multhaup, Brain Res. Bull., 2001, 55, 175–185.
to copper(II) without interference from 45 other analytes, 17 A. Robert, Y. Liu, M. Nguyen and B. Meunier, Acc. Chem. Res.,
including metal cations, amino acids and anions, under bio- 2015, 48, 1332–1339.
logical conditions. Live-cell imaging results indicate that the 18 O. Bandmann, K. H. Weiss and S. G. Kaler, Lancet Neurol.,
probe can detect near-membrane copper(II) aer membrane 2015, 14, 103–113.
anchoring using a photo-labile spatial and temporal control 19 B. E. Kim, T. Nevitt and D. J. Thiele, Nat. Chem. Biol., 2008, 4,
releasing method. The probe could be very useful for moni- 176–185.
toring the homeostasis of near-membrane copper(II). A clear 20 B. J. McCranor, H. Szmacinski, H. H. Zeng, A. K. Stoddard,
advantage of our photocontrollable method is that it success-
T. Hurst, C. A. Fierke, J. R. Lakowicz and R. B. Thompson,
fully avoids the inuence of chemical species outside cells
Metallomics, 2014, 6, 1034–1042.
during near-membrane specic detection. An important unre- 21 M. R. Krause and S. L. Regen, Acc. Chem. Res., 2014, 47, 3512–
solved issue from this study is the lack of sensitivity for
recording copper(II) across the membrane.
3521.
22 E. Ikonen, Nat. Rev. Mol. Cell Biol., 2008, 9, 125–138.
23 C. S. Lim, M. Y. Kang, J. H. Han, I. A. Danish and B. R. Cho,
Chem.–Asian J., 2011, 6, 2028–2033.
24 L. Li, J. Y. Ge, H. Wu, Q.-H. Xu and S. Q. Yao, J. Am. Chem.
Soc., 2012, 134, 12153–12160.
Acknowledgements
This work was nancially supported by the National Natural
Science Foundation of China (81672508, 61505076), Natural 25 L. Yuan, W. Y. Lin, Z. M. Cao, L. L. Long and J. Z. Song,
Science Foundation of Jiangsu Province (BK20140951), Natural Chem.–Eur. J., 2011, 17, 689–696.
Science Foundation of Zhejiang Province (LQ16B020003), and 26 Y. R. Zhao, Q. Zheng, K. Dakin, K. Xu, M. L. Martinez and
Key University Science Research Project of Jiangsu Province
(Grant 16KJA180004).
W. H. Li, J. Am. Chem. Soc., 2004, 126, 4653–4663.
27 I. Aparici-Espert, M. C. Cuquerella, C. Paris, V. Lhiaubet-
Vallet and M. A. Miranda, Chem. Commun., 2016, 52,
14215–14218.
28 C. Wu, Q. N. Bian, B.-G. Zhang, X. Cai, S.-D. Zhang, H. Zheng,
S.-Y. Yang and Y.-B. Jiang, Org. Lett., 2012, 14, 4198–4201.
Notes and references
1 S. J. Singer and G. I. Nicolson, Science, 1972, 175, 720–731.
2 H. Lodish, A. Berk, S. L. Zipursky, P. Matsudaira, 29 V. Dujols, F. Ford and A. W. Czarnik, J. Am. Chem. Soc., 1997,
D. Baltimore and J. Darnell, Molecular Cell Biology,
Scientic American Books, New York, 4th edn, 2004.
119, 7386–7387.
31098 | RSC Adv., 2017, 7, 31093–31099
This journal is © The Royal Society of Chemistry 2017

View Article Online
Paper
RSC Advances
30 R. L. Zhang, J. Zhao, G. M. Han, Z. J. Liu, C. Liu, C. Zhang, 33 J. Llopis, J. Michael McCaffery, A. Miyawaki, M. G. Farquhar
B. H. Liu, C. L. Jiang, R. Y. Liu, T. T. Zhao, M. Y. Han and
and R. Y. Tsien, Proc. Natl. Acad. Sci. U. S. A., 1998, 95, 6803–
Z. P. Zhang, J. Am. Chem. Soc., 2016, 138, 3769–3778.
6808.
31 J. F. Hulvat, M. Sofos, K. Tajima and S. I. Stupp, J. Am. Chem. 34 J. H. Lee, C. S. Lim, Y. S. Tian, J. H. Han and B. R. Cho, J. Am.
Soc., 2005, 127, 366–372.
Chem. Soc., 2010, 132, 1216–1217.
32 M. Collot, R. Kreder, A. L. Tatarets, L. D. Patsenker, Y. Melya
and A. S. Klymchenko, Chem. Commun., 2015, 51, 17136–
17139.
This journal is © The Royal Society of Chemistry 2017
RSC Adv., 2017, 7, 31093–31099 | 31099