Near-infrared fluorescent probe for sensitive detection of Pb(II) ions in living cells

Article abstract of DOI:10.1016/j.ica.2017.06.044

A new near-infrared fluorescent probe (NIR-PbP) for sensitive detection of Pb(II) ions in solution and living cells has been rationally designed and synthesized. The NIR-PbP is inherently non-fluorescent and gains fluorescence in the presence Pb(II) ions. The ion detection is based on Pb(II)-induced unmasking the fluorophore through the opening of the spyrocycle, with more than 500-fold fluorescence enhancement for sub-micromolar Pb(II) concentration. The NIR-PbP has high sensitivity, good photo-stability, low detection limit, and reversible response to Pb(II) ions.

Full text of DOI:10.1016/j.ica.2017.06.044

Accepted Manuscript
Research paper
Near-Infrared Fluorescent Probe for Sensitive Detection of Pb(II) Ions in Living
Cells
Jianheng Bi, Mingxi Fang, Jianbo Wang, Shuai Xia, Yibin Zhang, Jingtuo
Zhang, Giri Vegesna, Shuwei Zhang, Marina Tanasova, Fen-Tair Luo, Haiying
Liu
PII:
S0020-1693(17)30192-5
http://dx.doi.org/10.1016/j.ica.2017.06.044
ICA 17693
DOI:
Reference:
Inorganica Chimica Acta
To appear in:
Received Date:
Revised Date:
Accepted Date:
9 February 2017
16 June 2017
17 June 2017
Please cite this article as: J. Bi, M. Fang, J. Wang, S. Xia, Y. Zhang, J. Zhang, G. Vegesna, S. Zhang, M. Tanasova,
F-T. Luo, H. Liu, Near-Infrared Fluorescent Probe for Sensitive Detection of Pb(II) Ions in Living Cells, Inorganica
Chimica Acta (2017), doi: http://dx.doi.org/10.1016/j.ica.2017.06.044
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Near-Infrared Fluorescent Probe for Sensitive Detection of Pb(II) Ions in Living Cells
Jianheng Bi,a Mingxi Fang,a Jianbo Wangb*, Shuai Xia,a Yibin Zhanga, Jingtuo Zhang, Giri Vegesna,a Shuwei Zhang,a Marina Tanasova, a*
Fen-Tair Luo,c* and Haiying Liua*
aDepartment of Chemistry, Michigan Technological University, Houghton, MI 49931, E-mail: hyliu@mtu.edu, and mtanasov@mtu.edu
bCollege of Biological, Chemical Sciences and Engineering, Jiaxing University, Jiaxing 314001, China E-mail: wjb4207@mail.ustc.edu.cn
cInstitute of Chemistry, Academia Sinica, Taipei, Taiwan 11529, Republic of China, E-mail: luoft@gate.sinica.edu.tw
Abstract
A new near-infrared fluorescent probe (NIR-PbP) for sensitive detection of Pb(II) ions in
solution and living cells has been rationally designed and synthesized. The NIR-PbP is
inherently non-fluorescent and gains fluorescence in the presence Pb(II) ions. The ion detection
is based on Pb(II)-induced unmasking the fluorophore through the opening of the spyrocycle,
with more than 500-fold fluorescence for sub-micromolar Pb(II) concentration. The NIR-PbP
has high sensitivity, good photo-stability, low detection limit, and reversible response to Pb(II)
ions.
Keywords: Pb(II) ions, Fluorescence, Fluorescent probe, Near-infrared emission.
1. Introduction
In this paper, we report a near-infrared fluorescent probe (NIR-
PbP) with the spirocyclic structure which is highly sensitive to the
Lead is one of the most hazardous heavy metal pollutants of the Pb+2 ions in the solutions and in living cells. The NIR-PbP is
air, water, and soil that plays a key role in biological, environmental colorless and non-fluorescence with the intact spirocyclic amide
and chemical fields. [1-3] It is estimated that millions of workers in structure in the absence of Pb+2 ions. The fluorescence of the NIR-
the United States are potentially exposed to lead [4-9], and led PbP is triggered by the presence of Pb(II) ions, inducing significant
exposure is linked with behavioral abnormalities, learning absorbance enhancement at 718 nm and near-infrared fluorescence at
impairment, decreased hearing, high blood pressure, kidney 735 nm. The turn-on fluorescence is based on Pb-catalyzed opening
disorders, and neurological impairment [10-12]. A greater worry is of the spyrocycle and unmasking of the fluorophore.
that children are the most at-risk for lead exposure which can cause
cognitive and neurological impairment [13-15]. Therefore, it is 2. Experimental Section
important to keep lead concentration level in our surrounding
2.1 Instrumentation
environment low and precisely monitored. From the various
1H NMR and 13C NMR spectra were obtained by using a 400
approaches, detecting Pb(II) with fluorescent probes allows for
MHz Varian Unity Inova NMR spectrophotometer. 1H and 13C NMR
efficient monitoring of lead ions with high sensitivity and low
detection limit [16, 17]. Among different fluorescent probes, the
spectra were recorded in CDCl3, chemical shifts (δ) are given in ppm
relative to solvent peaks (1H: δ 7.26; 13C: δ 77.36) as internal
standard. Absorption spectra were obtained on a Perkin Elmer
turn-on probes allow to detect low concentrations of lead in aqueous
media and in living cells because of the Pb+2-induced activation of
fluorescence [18-22].
Lambda 35 UV/VIS spectrometer. Fluorescence spectra were
Recently, near-infrared (NIR) fluorescent probes have received
obtained on a Jobin Yvon Fluoromax-4 spectrofluorometer. Each of
much attention due to its advantages in deep tissue penetration, low
the samples was scanned with increments of 1 nm. Fluorescent
background interference and minimal damage to cells and tissues
images were collected with Olympus IX 81 confocal laser scanning
[23-26]. While significant advances have been made towards
microscope.
detecting cellular targets or small molecules with near-IR probes
[24-28], no such probes for Pb(II) detection in living cells were
reported.
2.2 Reagents
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All reagents and solvents were purchased from commercial every three days. Cells were seeded into the 35 mm glass-bottom
suppliers and used without further purification. Air- and culture dishes (F 20 mm) for 1–2 days to reach 70–90% confluence.
moisture-sensitive reactions were conducted in oven-dried The cells were washed with DMEM (x3) and then incubated with 2
glassware using a standard Schlenk line or dry box techniques mL DMEM containing the NIR-PbP (10 µM) in 5% CO2 and 95%
under the inert atmosphere of dry nitrogen.
air atmosphere for 30 min at 37 °C. Cells were then washed with
PBS (2 x 1 mL) at room temperature, and 1 ml was added for
imaging. Images were taken with the Olympus IX 81 confocal laser
scanning microscope using HC×PLAPO 60X oil objective, with
excitation by 635 nm laser, and 675–775 nm emission. For led-
measurements, cells were first treated with Pb(II) (20 µM) in 5%
CO2 and 95% air atmosphere for 30 min at 37 °C. Cell were washed
with PBS (3 x 1 ml), NIR-PbP (10 µM in DMEM, 1 mL) was added,
and cell were incubated for another 30 min. The cells were then
washed with PBS (2 x 1 ml) at room temperature, and after addition
of 1 mL PBS were imaged with a confocal microscope (635 nm
excitation, 675–775 nm emission).
2.3 Synthesis
Synthesis of NIR-PbP: To the compound 1 (0.25 g, 0.44 mmol) in
anhydrous dichloromethane (20 mL) were added N-
hydroxysuccinimide (0.06 g, 0.58 mmol), DCC (0.09 g, 0.44 mmol)
were added. The resulting reaction mixture was stirred at room
temperature for 30 min under nitrogen atmosphere. To this mixture,
compound 2 (0.14 g, 0.58 mmol) and triethylamine (0.1 mL) were
added, and the mixture was further stirred overnight at room
temperature. After the mixture was washed with water (2 × 30 mL)
and brine (2 × 30 mL), the organic layer was separated, dried over
anhydrous sodium sulfate, filtered, and concentrated under vacuum.
The crude compound was purified by column chromatography using
2.6 Cytotoxicity assay
DCM/EtOH (40:1 to 20:1, v/v) to yield fluorescent probe A as a The cytotoxicity of a probe against HeLa cells was measured by
brown solid (0.14 g, 40%). 1H NMR (400 MHz, CDCl3): δ 8.44-8.43 using the standard MTS assay. The cells were seeded into 96-well
(d, J = 4.0 Hz, 2H), 7.82-7.80 (d, J = 8.0 Hz, 1H), 7.58-7.54 (m, 2H), cell culture plate at 5×103/well in complete growth medium. Cells
7.45-7.36 (m, 5H), 7.17-7.02 (m, 5H), 6.85-6.81 (t, J = 8.0 Hz, 1H), were incubated for 24 h at 37 °C under 5% CO2 atmosphere to
6.61-6.59 (d, J = 8.0 Hz, 1H), 6.24-6.15(m, 3H), 5.36-5.33 (d, J = attach. After removal of the medium and washing with PBS for three
12.0 Hz, 1H), 3.82-3.73 (m, 4H), 3.43-3.28 (m, 4H), 3.15 (s, 3H), times, cells were incubated with fresh medium containing various
2.66-2.31 (m, 4H), 1.75-1.71 (d, J = 16.0 Hz, 6H), 1.53-1.24 (m, concentrations of the NIR-PbP for 48 h, respectively. The probe-
6H), 1.17-1.13 (t, J = 8.0 Hz, 6H). 13C NMR (100 MHz, CDCl3): δ containing media was then replaced with fresh growth media and
168.84, 157.85, 152.94, 151.82, 148.20, 145.57, 139.02, 136.83, CellTiter 96® Aqueous (2 µL/well) was added. After incubating for
132.31, 132.03, 128.27, 127.92, 123.61, 123.34, 122.97, 122.20, 2h, the cell viability was determined by measuring the light
121.74, 120.41, 119.71, 119.48, 108.55, 105.15, 103.47, 103.42, absorbance at 490 nm with a microplate reader. The data was
97.96, 92.29, 67.08, 60.09, 52.57, 45.64, 44.56, 38.03, 29.90, 29.36, collected in triplicates, and each data point was averaged. Data
28.69, 28.50, 25.46, 23.12, 22.20, 12.79. IR (cm-1): 3051.99, analysis was carried out with respect to measurements for untreated
2970.16, 2929.33, 1687.18, 1623.32, 1593.55, 1517.02, 1468.37, cells (negative control). Cell viability (%) was calculated by
1376.75, 1318.29, 1265.76, 1215.64, 1194.79, 1126.58, 735.03, comparing the absorbance of the control cells to that of treated cells.
701.52. HRMS (FAB) calcd for C51H55N6O2 [M+H]+, 783.4386;
found, 783.4404.
3. Results and Discussion
2.4 Optical measurements
3.1 Design, synthesis, and turn-on properties of NIR-PbP
All absorption and emission spectra were recorded using a
standard 1 cm path length quartz fluorescence cuvette at room
temperature. NIR-PbP solution (10 µM) was used each time in the
presence of 5 equivalents of different metal ions to determine the
selectivity of the probe at room temperature. Pb(ClO4)2 was used as
a source of Pb+2 for all solution and cell studies. Standard lead
nitrate solution (1 M) was prepared freshly for each measurement.
For each measurement, the calculated amount of metal ions solution
was added to a solution containing 0.5 µM NIR-PbP in an
acetonitrile/water (99/1, v/v, organic medium) or 10 % citric
acid/ethanol (80/20, v/v, aqueous medium) co-solvent system. After
lead ions were added to the solution and mixed homogenously, its
UV-vis absorption and fluorescence spectra were obtained after 10
min. The slit widths of excitation and emission were both set to 5
nm, and the excitation wavelength was set at 620 nm for
fluorescence spectroscopies.
2.5 Fluorescent detection of lead ions in living cells
HeLa cells were grown according to ATCC protocol in DMEM
(high glucose) medium supplemented with 10% FBS, and 1%
strep/pen as a complete growth medium in a 5% CO2 humidified
incubator at 37 °C. Cells were passaged with sub-cultivation of 1:3
Scheme 1. Synthesis and Pb-induced fluorescence mechanism for NIR-PbP
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For designing NIR-PbP - a near-infrared emissive probe for
We investigated the fluorescence responses of NIR-PbP to Pb.
Pb(II) - we chose a compound 1 (Scheme 1) as a unique dye Figure 2a shows the fluorescence spectra of NIR-PbP in the absence
combining large extinction coefficient, high quantum yield, near- and presence of Pb(II) ions. NIR-PbP does not exhibit any emission
infrared emissions (720 nm), photostability, and the controlled band in the absence of lead due to its non-fluorescent property with
fluorescence on–off switching mechanism [29, 30]. Compunds 1 was the intact spirocycle. The fluorescence emission appears upon
synthesized following reported procedures [29, 30], and coupled addition of Pb(II) at 735 nm. Gradual increase of Pb(II) ion
with
dipicolylamine
derivative
(N1,N1-bis(pyridin-2- concentration to 2.3 µM induces more than 500 folds enhancement
ylmethyl)ethane-1,2-diamine) (2) [31] as a strong electron-donating of the fluorescence intensity. The fluorescence response is in the
group that selectively chelates to Pb+2 ions via an amidation reaction linear relationship with concentrations (Figure 2b), indicating that
[24]. This reaction triggers the cyclization that forms the closed NIR-PbP is potentially applicable for quantitative detection of Pb(II)
spirolactam structure and yields the NIR-PbP (Scheme 1). NIR-PbP ions using fluorometric assay. We also investigated pH effect on
is colorless, non-fluorescent, and does not absorb in visible and near- fluorescent NIR-PbP and demonstrated that the probe displays
infrared regions.
selective responses to Pb+2 at neutral and basic pH values (Figure S2,
With designing NIR-PbP, we envisioned that Pb(II) ions could be SI).
specifically chelated by the dipicolylamine moiety of the probe.
Upon Pb(II) binding, the cooperative binding to the carbonyl of the
amide moiety would be expected to promote the opening of the
spirolactam, and unmasking the fluorophore through the stabilization
of the enhanced π-conjugation. We tested our hypothesis by
investigating the absorbance responses of NIR-PbP to different
concentration of Pb ions in the organic or aqueous medium. Upon
addition of Pb(II) ions in to the probe in the organic medium, strong
characteristic absorption bands with peaks at 479 nm, 663 nm
(shoulder peak) and 718 nm was observed (Figure 1). Gradual
increases of Pb(II) ion concentration from 0.4 µM to 2.3 µM resulted
in significant increase in absorbance at 718 nm accompanied with a
shoulder peak at 663 nm (Figure 1a). As a result, the solution of the
fluorescent probe displays distinct color changes from colorless to
green. The absorbance at 718 nm of NIR-PbP shows a linear
response to Pb ion concentration with up to 500-fold enhancement
reaching 2.3 µM Pb(II) indicating a significant sensitivity of NIR-
PbP (Figure 1b). The observed alterations in the UV-spectra support
the opening of the spirolactam and formation of extended π-
conjugation. NIR-PbP was also able to sense Pb(II) in aqueous
medium with low percentage of an organic co-solvent (Figure S1,
SI).
Figure 2. Fluorescence responses of fluorescent NIR-PbP to
different concentrations of Pb(II) ions. a) Increase in fluorescence
for 0.5 µM NIR-PbP upon titration with Pb(II) ions in
acetonitrile:water (99:1); b) Plot of fluorescence intensity at 735 nm
against Pb(II) ion concentrations.
3.2 NIR-PbP exhibits selectivity towards Pb(II) ions.
Considering the possibility for fluorescent probes to interaction
with other metal ions, we have investigated whether NIR-PbP can
selectively sense Pb(II) ions. We studied the absorption and
emission responses of NIR-PbP to other metal ions including Zn2+,
Fe2+, Hg2+, Cd2+, Ca2+, Co2+, Cu2+, Mg2+, Ni2+, Na+, and K+ (Figure
3a) As before, the addition of 2.5 µM Pb(II) ions to the solution
containing 0.5 µM of NIR-PbP led to the appearance of a strong
absorption peak at 718 nm. In contrast, under the same condition,
other metal ions did not generate this phenomenon, except for Fe2+.
However, the absorbance at 718 nm of the solution containing Pb+2
was 9-times higher than that of Fe2+, and more than 30-times higher
than for other metal ions (Figure 3a). The fluorescence responses of
Figure 1. Responses of NIR-PbP to varied concentrations of Pb(II)
ions. a) Absorption (UV) spectra for 0.5 µM NIR-PbP upon titration
with 0 – 2.3 µM of Pb(II) in acetonitrile:water (99:1). b) Plot of
absorbance at 718 nm against Pb(II) concentration.
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NIR-PbP to different metal ions showed the similar trend. Only Pb(II) equivalents, only a slight decrease in the fluorescence intensity of
ions were able to induce the significant enhancement of the the probe was observed (Figure S4b). This reveals that NIR-PbP
fluorescence for NIR-PbP in the near-infrared region. The exhibits high reproducibility, which is crucial for designing reusable
fluorescent intensity of NIR-PbP−Pb complex is more than 150-fold Pb sensor devices.
higher than that of Zn2+, Fe2+ and any of all other metal complexes
(Figure 3b). These results indicate that NIR-PbP has a high binding
affinity to Pb+2 and can generate selective responses in absorption
and fluorescence spectra toward Pb+2 over other metal ions. Pb-
selectivity of NIR-PbP was also confirmed through co-treatment of
the probe with metal ion mixtures (Figure S3), showing that near-IR
and fluorescence signals are turned-on only in the presence of lead.
3.6 NIR-PbP as Pb(II) sensor in live cells
NIR-PbP was further used to detect lead in living cells. First,
cytotoxicity of NIR-PbP was established by the MTS assay with
HeLa cell line. The cellular viability was estimated to be greater than
85% after 48 h, indicating the lack of cytotoxicity for NIR-PbP at
concentrations below 10 µM (Figure S5).
For Pb+2 detection in live cells, HeLa cells were treated with NIR-
PbP, and Pb+2 and fluorescence of the probe was recorded with the
confocal fluorescence microscope (Figure 4). When HeLa cells were
incubated with NIR-PbP (10 µM), no fluorescence was observed.
After additional incubation with Pb+2, red fluorescence appears. An
overlay of bright-field and fluorescence images indicates that the
fluorescence signals are localized in the intracellular area. These
results show that NIR-PbP can be applied to visualize Pb ions within
the living cell.
Figure 3: Selective responses of NIR-PbP to Pb+2 over other metal
ions. NIR-PbP (0.5. µM) was treated with Pb(II) (2.5 µM) and other
metal ions (10 µM). The absorption (a) and fluorescence (b) spectra
of the mixture solution were then measured.
Figure 4. Confocal fluorescence images of HeLa cells with NIR-
PbP in the absence and presence of Pb(II). a) Bright field with NIR-
PbP; b) fluorescence recording for NIR-PbP without Pb(II) ions; c)
a/b overlay; d) Bright field for NIR-PbP and Pb(II) ions; e)
fluorescence image of NIR-PbP and Pb(II) ions, f) d/e overlay.
We also examined the photostability of NIR-PbP−Pb complex
using a time-dependent fluorescence measurement. For this part, the
solution containing fluorescent NIR-PbP and Pb ions was excited
continuously at 680 nm for 70 minutes, and fluorescence intensity
was measured every 5 minutes. The resulting time-dependent
fluorescence (Figure 4a) showed that there was no significant
decrease of the fluorescence intensity under 70 minutes excitation
for NIR-PbP. We also evaluated the reproducibility of NIR-PbP for
sensing Pb ions by using EDTA as a strong Pb+2 chelator capable of
dissociating the Pb from the probe. After adding equimolar
quantities of EDTA to NIR-PbP−Pb complex (Figure S4a), the
fluorescence was immediately quenched, indicating the dissociation
of the complex and the cyclization of the spyrolactam. However,
further addition of Pb+2 to this solution restored the strong
fluorescence, clearly indicating the reusability of the probe. Also,
after six cycles of alternative addition of the equal EDTA and Pb
4. SUMMARY
In summary, a new near-infrared fluorescent probe for Pb+2
detection was synthesized, and its chemosensing properties were
established by absorption and fluorescence spectroscopies. The
probe exhibited high selectivity to Pb(II) ions over a wide variety of
other metal. Upon addition of Pb(II), NIR-PbP probe gains
absorption in near-IR region of the spectra and changes from
colorless to green. It also shows strong “off–on” fluorescence
accompanied by a color change from colorless to fluorescent pink in
acetonitrile. The spectral changes were observed due to reversible
spirocyclic ring-opening mechanism upon biding of Pb(II) ions. The
lead-induced absorbance at 718 nm and the fluorescence intensity at
735 nm are proportional to the concentration of Pb(II) ions,
indicating that colorimetric or fluorometric method can be used for
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Acknowledgement
This research reported in this publication was partially supported by
the National Institute of General Medical Sciences of the National
Institutes of Health under Award Number R15GM114751 (to H.Y.
Liu), the National Science Foundation (award number 1048655) (to
Haiying Liu) and the National Science Foundation of China (No.
21606102) (to Haiying Liu).
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Journal Name
ARTICLE
• The probe shows not any absorption
peaks in visible and near-infrared
region in the absence of Pb(II) ions.
• Near-infrared fluorescent probe
shows no fluorescence in the
absence of Pb(II) ion.
• The
probe
shows
significant
absorption peak at 718 nm in the
presence of Pb(II) ions.
• The probe shows highly fluorescence
at 735 nm in the presence of Pb(II)
ions.
• The probe responds to Pb(II)
reversibly
in absorption and
fluorescence changes.
• The probe can detect Pb(II) in living
cells.
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