Rational design of NIR fluorescence probes for sensitive detection of viscosity in living cells

Article abstract of DOI:10.1016/j.saa.2019.02.068

Developing near-infrared (NIR) fluorescence probes for detection of intracellular viscosity is still sufficiently challenging. In this work, three kinds of D-A-D type naphthyl and 2,1,3?benzoxadiazol hybrid NIR dyes functionalized with amino (NY1), N?methylamino (NY2) and N,N?dimethylamino (NY3) groups for intracellular micro-viscosity detection were designed and synthesized. All the probes exhibited very weak NIR emission in low viscosity environment and obvious fluorescence enhancement with the increased viscosity. Different substituent groups had a high impact on the photophysical properties and response sensitive of the probes to viscosity. The structure-property relationships were systematic investigated. The results showed that stronger electron-donating ability and larger steric effect of N,N?dimethylamino led to a narrower energy gap and more sensitive to viscosity environment. Therefore, NY3 exhibited higher signal noise ratio for viscosity detection and was successfully applied for imaging the changes of intracellular micro-viscosity. This work provides an efficient way to design powerful NIR fluorescence probes for viscosity detection.

Full text of DOI:10.1016/j.saa.2019.02.068

Accepted Manuscript
Rational design of NIR fluorescence probes for sensitive detection
of viscosity in living cells
Gaobin Zhang, Yun Ni, Duoteng Zhang, Hao Li, Nanxiang Wang,
Changmin Yu, Lin Li, Wei Huang
PII:
DOI:
Reference:
S1386-1425(19)30190-8
https://doi.org/10.1016/j.saa.2019.02.068
SAA 16844
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
To appear in:
Received date: 21 October 2018
Revised date:
Accepted
date:
21 December 2018
17 February 2019
Please cite this article as: G. Zhang, Y. Ni, D. Zhang, et al., Rational design of NIR
fluorescence probes for sensitive detection of viscosity in living cells, Spectrochimica
Acta Part A: Molecular and Biomolecular Spectroscopy, https://doi.org/10.1016/
j.saa.2019.02.068
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ACCEPTED MANUSCRIPT
Rational design of NIR fluorescence probes for sensitive detection of
viscosity in living cells
a,†
a,†
Gaobin Zhang , Yun Ni , Duoteng Zhanga, Hao Lia, Nanxiang Wanga, Changmin Yua,*, Lin Lia,*,
Wei Huanga,b,
*
aKey Laboratory of Flexible Electronics (KLOFE) & Institute of Advanced Materials (IAM),
Jiangsu National Synergetic In-novation Center for Advanced Materials (SICAM), Nanjing
Tech University (NanjingTech), 30 South Puzhu Road, Nanjing, 211816, P. R. China;
bShaanxi Institute of Flexible Electronics (SIFE), Northwestern Polytechnical University
(NPU), 127 West Youyi Road, Xi’an 710072, P. R. China
*Corresponding authors. E-mail address: iamcmyu@njtech.edu.cn; iamlli@njtech.edu.cn;
iamwhuang@njtech.edu.cn
†These authors contributed equally to this work.
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ABSTRACT: Developing near-infrared (NIR) fluorescence probes for detection of intracellular
viscosity is still sufficiently challenging. In this work, three kinds of D-A-D type naphthyl and
2,1,3-benzoxadiazol hybrid NIR dyes functionalized with amino (NY1), N-methylamino (NY2)
and N,N-dimethylamino (NY3) groups for intracellular micro-viscosity detection were designed
and synthesized. All the probes exhibited very weak NIR emission in low viscosity environment
and obvious fluorescence enhancement with the increased viscosity. Different substituent groups
had a high impact on the photophysical properties and response sensitive of the probes to viscosity.
The structure-property relationships were systematic investigated. The results showed that
stronger electron-donating ability and larger steric effect of N,N-dimethylamino led to a narrower
energy gap and more sensitive to viscosity environment. Therefore, NY3 exhibited higher signal
noise ratio for viscosity detection and was successfully applied for imaging the changes of
intracellular micro-viscosity. This work provides an efficient way to design powerful NIR
fluorescence probes for viscosity detection.
Keywords: Fluorescence; Chemosensor; viscosity; NIR; Cell imaging
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1. Introduction
Intracellular viscosity is an important environmental factor to maintain the cellular diffusion
processing and has a deep influence on a number of physiological activities [1-3], including the
biochemical reactions, molecules and proteins transporting and cellular membranes structural state
[4, 5]. The changes of micro-viscosity are related to many diseases and pathologies, and have been
regarded as a contributor or indicator to inflammation, atherosclerosis, diabetes, and Alzheimer’s
disease [6-8]. Therefore, it is important to develop efficient methods with high resolution to
monitor the change of intracellular micro-viscosity [9-12]. Recently, with the development of
fluorescence technologies, small molecule fluorophores have been designed as probes to detect the
native intracellular micro-viscosity [13-19]. The fluorophores are termed as “molecular rotors”
that are constructed with twist donor and acceptor (D-A) structures [20-24]. The fluorescence
intensity of these fluorophores will be changed with the increase of viscosity by the mechanism of
twisted intramolecular charge transfer (TICT), molecular planarization or Förster resonance
energy transfer (FRET) [25-30]. However, most of reported fluorescence-based viscosity probes
are green or yellow dyes with short fluorescence emission (<600 nm). For cell imaging, the short
absorption and emission could cause damage to cells and also be easily affected by background
fluorescence. Near-infrared (NIR) dyes with long absorption and emission wavelength have the
advantages of good tissue penetration ability, minimum photon damage to biological samples and
low auto fluorescence, which have been considered as ideal candidates for intracellular viscosity
imaging [31-35]. To obtain NIR emission, the dyes are often designed with large planar
π-conjugate system to decrease the energy gap. However, the π-π stacking always quenches the
emission of NIR dyes. D-A or D-π-A type molecules are also used to design NIR dyes [36-39].
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Obviously, the weak interactions between donor and acceptor always cannot achieve the long
wavelength emission. However, although the strong D-A interactions could lead to the narrow gap
and result in the NIR emission, the limited HOMO and LUMO overlaps would make these D-A
compounds exhibit unexpectedly low fluorescence. On the other hand, some D-π-A type NIR dyes
could react with biothiols, hydrazine or anions by Michael reaction [40-42]. Moreover, the
extending π electron system usually leads to the low photostability of the NIR dyes that along with
photobleaching at short time under photoexcitation, which also limits their application in
bioimaging. Thus, it is important to select the suitable donor and acceptor in the design of NIR
probes. Recently, NIR probes for intracellular viscosity detection have been reported. Lin et al.
reported a D-π-A type NIR probe using malononitrile as acceptor for detection of viscosity in
cellular lipid droplets, zebrafishes and living mice [31]. Peng and co-works reported a NIR probe
based on cyanine derivatives that displayed ratiometric fluorescence response in viscous
environments [30]. However, the relationships between photophysical properties of NIR dyes and
response ability to micro-viscosity are still unclear. In addition, there is much room needed to be
improved in terms of stability, sensitivity and specially enlarging Stocks shift, which could
obviously avoid self-absorption in the intracellular viscosity imaging.
Herein, three kinds of D-A type NIR probes (NY1, NY2 and NY3) for intracellular
micro-viscosity detection have been designed and synthesized. As shown in Scheme 1, these
probes were constructed by using 2,1,3-benzoxadiazol as the acceptor and naphthalene derivative
as the donors. The straightforward coupling by Suzuki reaction could omit the double bond, which
is contributed to increase the molecular rigid to avoid the addition reaction and photobleaching.
Furthermore, in order to regulate the emission and sensitivity to viscosity, we synthesized three
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probes with amino (NY1), methylamino (NY2) and dimethylamino (NY3) groups, respectively.
By optimizing the D-A structure, we expected that, with the increase in viscosity, the restriction of
intramolecular motions and the intramolecular planarization would occur, and subsequently
impeding the TICT process to induce the emission enhancement. In this study, the responses of
three probes to viscosity and the structure-property relationships were systematic studied.
Furthermore, due to possess higher steric hindrance and stronger electron donor effect, NY3
modified with dimethylamino was demonstrated to be more sensitive to viscosity changes. Finally,
we have also successfully used it to monitor the viscosity changes in living cells with fluorescence
microscopy.
Scheme 1.
2. Experimental
2.1. Materials and instrumentation
All reactions were carried out under a dry nitrogen protection. Silica gel 60 (200-300 mesh,
Silicycle) was used for column chromatography. All reagents and solvents were purchased from
commercial suppliers and used without further purification, unless otherwise noted. N,
N-Dimethylformamide (DMF) and dichloromethane (CH2Cl2, DCM) were distilled over CaH2.
Petroleum ether (PE, 60-90°C), DCM, ethyl acetate (EA) and methanol (MeOH) were used as
eluents for flash column chromatography with Merck silica gel (0.040-0.063). Reaction progress
was monitored by TLC on pre-coated silica plates (250 μm thickness) and spots were visualized
by ceric ammonium molybdate, basic KMnO4, UV light or iodine. 1H and 13C NMR spectra were
collected in CDCl3 or DMSO-d6 at 25°C by using an Avance AV-300 spectrometer. Chemical
shifts were reported in parts per million relative to internal standard tetramethylsilane (Si(CH3)4 =
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0.00 ppm) or residual solvent peaks (CDCl3 = 7.26 ppm, DMSO-d6 = 2.50 ppm, MeOD = 3.31
ppm). 1H NMR coupling constants (J) were reported in Hertz (Hz) and multiplicity was indicated
as follows: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet). Mass spectra were
recorded on a Finnigan LCQ mass spectrometer, a Shimadzu LC-IT-TOF spectrometer. Analytical
HPLC was carried out on Shimadzu LC-IT-TOF or LC-ESI system equipped with an autosampler,
using reverse-phase Phenomenex Luna 5 μm C18 100 Å 50 × 3.0 mm columns, and the flow rate
was 0.6 mL/min. Absorption spectra were recorded using SynergyHTX microplate reader or a
Shimadzu UV-3600 UV-Vis-NIR spectrophotometer. Photoluminescent spectra were recorded
using a HITACHI F4600 fluorescence spectrophotometer with excitation slit widths of 5 nm and
emission slit widths of 10 nm. All the measurements were performed at room temperature. All
images were acquired on Zeiss LSM880 NLO (2+1 with BIG) Confocal Microscope System
equipped with objective LD C-Apochromat 63×/1.15 W Corr M27, cell incubator with
temperature control resolution ± 0.1oC, 405 nm Diode laser, Argon ion laser (458, 488 and 514
nm), HeNe laser (543 and 594 nm), and a Rack LSM 880 incl. 633 nm laser, with 8 channels
AOTF for simultaneous control of 8 laser lines. A PMT detector ranging from 420 nm to 700 nm
for steady-state fluorescence was used. Internal photomultiplier 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).
2.2. Synthesis and Characterization
2.2.1. N,N-dimethyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)naphthalen-2-amine (1) In a
100 mL three-neck flask, 6-bromo-N,N-dimethylnaphthalen-2-amine (252.0 mg, 1.0 mmol),
4,4,4',4',5,5,5',5'-octamethyl-2,2'-bi(1,3,2-dioxaborolane) (304.7 mg, 1.2 mmol) and CH3COOK
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(186.3 mg, 2.0 mmol) were added in 5 mL dioxane solution. After adding catalytic amount of
palladium chloride, the mixture was stirred at 80°C for 12 h under N2. Then, the solution was
extracted into EtOAc (3×75 mL). The organic layer was combined and the solvent was evaporated
in vacuo. The residue was purified by silica column (PE/DCM = 4/1) to afford 1 as a white
1
powder (190.0 mg, yield : 64 %). H NMR (500 MHz, DMSO-d6): δ 8.23 (s, 1H), 7.76-7.73 (m,
2H), 7.63 (d, J = 8.2 Hz 1H), 7.15 (dd, J1 = 2.2, J2 = 9.0 Hz, 1H), 6.91 (s, 1H), 3.07 (s, 6H), 1.39
(s, 12H).
2.2.2. 7-chlorobenzo[c][1,2,5]oxadiazol-4-amine (2) On the basis of a previously reported
procedure [43]. The acetic acid (9 mL) was heated to 70°C. Then, NBD-Cl (0.22 g, 1.0 mmol, 1.0
equiv.) and Fe powder (0.18 g, 3.0 mmol, 3.0 equiv.) were added. The mixture was stirred at 70°C
for 30 min under N2. The reaction was cooled to room temperature and concentrated under
reduced pressure. The residue was dissolved in ethyl acetate and washed with NaHCO3 (3×50 mL).
The organic layer was combined and dried with anhydrous Na2SO4. The residue was purified by
silica column (EA/PE = 1/10) to give compound 2 as a yellow powder (146 mg, yield: 86 %). 1H
NMR (500 MHz, DMSO-d6): δ 7.40 (d, J = 10 Hz, 1H), 6.75 (s, 2H), 6.28 (d, J = 10 Hz, 1H).
2.2.3. 7-chloro-N-methylbenzo[c][1,2,5]oxadiazol-4-amine (3) To a solution of compound 2 (0.05
g, 0.3 mmol) in acetonitrile (5 mL) was successively added CH3I (0.018 mL, 0.29 mmol, 1.0
equiv.) and Cs2CO3 (300 mg, 0.87 mmol, 3 equiv.). Then the reaction was stirred 6 h at 60°C. The
product was extracted with EA (3×75 mL). The organic layer was combined and the solvent was
evaporated in vacuo. The residue was purified by silica column (PE/EA = 10/1) to afford
compound 3 as an orange powder (18 mg, yield: 33 %). 1H NMR (500 MHz, MeOD): δ 7.24 (d, J
= 7.8 Hz, 1H), 5.99 (d, J = 7.8 Hz, 1H), 3.01 (s, 3H).
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2.2.4. 7-chloro-N,N-dimethylbenzo[c][1,2,5]oxadiazol-4-amine (4) To a solution of compound 2
(0.05 g, 0.29 mmol) in acetonitrile (5 mL) was successively added CH3I (0.072 mL, 1.16 mmol, 4
equiv.) and Cs2CO3 (300 mg, 0.87 mmol, 3 equiv.). Then the reaction was stirred 12 h at 85°C.
The product was extracted with EA (3×75 mL). The organic layer was combined and the solvent
was evaporated in vacuo. The residue was purified by silica column (PE/EA = 20/1) to afford 4 as
1
an orange red powder (32 mg, yield: 56 %). H NMR (500 MHz, CDCl3): δ 7.19 (d, J = 8.1 Hz,
1H), 5.94 (d, J = 8.1 Hz, 1H), 3.30 (s, 6H).
2.2.5. 7-(6-(dimethylamino)naphthalen-2-yl)benzo[c][1,2,5]oxadiazol-4-amine (NY1) In a
100 mL three-neck flask, the compound 2 (50 mg, 0.3 mmol), compound 1 (100 mg, 0.36 mmol,
1.2 equiv.), Na2CO3 (65 mg, 0.6 mmol, 2 equiv.) and catalytic amounts of palladium chloride were
added in 5 mL dioxane. The mixture was stirred at 100°C for 12 h under N2. The product was
extracted with EA (3×75 mL). The organic layer was combined and the solvent was evaporated in
vacuo. The residue was purified by silica column (PE/EA = 10/1) to afford NY1 as a red powder
1
1
(50.2 mg, yield: 55 %). H NMR (500 MHz, MeOD): δ H NMR (500 MHz, MeOD) δ 8.33 (s,
1H), 7.89 (dd, J1 = 2.0 Hz, J2= 8.7, 1H), 7.76 (d, J = 9.0 Hz, 1H), 7.71 (d, J = 8.7 Hz, 1H), 7.60 (d,
J = 7.6 Hz, 1H), 7.24 (d, J = 9.0 Hz, 1H), 6.99 (d, J = 2.0 Hz, 1H), 6.50 (d, J = 7.6 Hz, 1H), 3.05
(s, 6H). 13C NMR (125 MHz, MeOD): δ 149.33, 148.80, 145.38, 134.31, 134.31, 133.49, 129.68,
129.37, 126.87, 126.63, 126.44, 125.38, 118.78, 116.65, 107.45, 106.12, 40.83. ESI-MS (m/z):
Calcd for [M+H]+ = 305.14, found: 305.05.
2.2.6. 7-(6-(dimethylamino)naphthalen-2-yl)-N-methylbenzo[c][1,2,5]oxadiazol-4-amine (NY2) In
a 100 mL three-neck flask, the compound 3 (50 mg, 0.3 mmol), compound 1 (100 mg, 0.36 mmol,
1.2 equiv.), Na2CO3 (65 mg, 0.6 mmol, 2 equiv.) and catalytic amounts of palladium chloride were
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added in 5 mL dioxane. The mixture was stirred at 100°C for 12 h under N2. Then the solution
was extracted with EA (3×75 mL). The organic layer was combined and the solvent was
evaporated in vacuo. The residue was purified by silica column (PE/EA = 10/1) to afford NY2 as a
1
red powder (35.3 mg, yield: 37 %). H NMR (500 MHz, CDCl3): δ 8.37 (s, 1H), 7.92 (dd, J = 8.7,
1.7 Hz, 1H), 7.82 (d, J = 9.0 Hz, 1H), 7.75 (d, J = 8.7 Hz, 1H), 7.59 (d, J = 7.6 Hz, 1H), 7.22 (d, J = 7.6
Hz, 1H), 7.02 (s, 1H), 6.21 (d, J = 7.6 Hz, 1H), 5.06 (s, 1H), 3.09 (s, 6H), 3.08 (s, 3H). 13C NMR (125
MHz, CDCl3): δ 149.23, 145.34, 136.03, 134.24, 130.37, 129.67, 129.27, 126.93, 126.55, 126.12,
125.32, 119.17, 116.63, 116.44, 106.17, 101.79, 40.90, 30.19. ESI-MS (m/z): Calcd for [M+H]+ =
319.15, found: 319.15.
2.2.7.
7-(6-(dimethylamino)naphthalen-2-yl)-N,N-dimethylbenzo[c][1,2,5]oxadiazol-4-amine
(NY3) In a 100 mL three-neck flask, the compound 4 (50 mg, 0.3 mmol), compound 1 (100 mg,
0.36 mmol, 1.2 equiv.), Na2CO3 (65 mg, 0.6 mmol, 2 equiv.) and catalytic amounts of palladium
chloride were added in 5 mL dioxane solution. The mixture was stirred at 100°C for 12 hours
under N2. After cool down to temperature, the reaction mixture was extracted with EA (3×75 mL).
The organic layer was combined and the solvent was evaporated in vacuo. The residue was
purified by silica column (PE/EA = 10/1) to afford pure NY3 as a red powder (47.8 mg, yield:
1
48%). H NMR (500 MHz, CDCl3): δ 8.35 (s, 1H), 7.90 (dd, J1 = 1.5 Hz, J2 = 8.6, Hz, 1H),
7.81-7.71 (m, 3H), 7.54 (d, J = 7.8 Hz, 1H), 7.19 (d, J = 7.8 Hz, 1H), 6.21 (d, J = 7.8 Hz, 1H),
3.34 (s, 6H), 3.07 (s, 6H). 13C NMR (125 MHz, CDCl3): δ 149.97, 146.16, 138.51, 134.22, 129.90,
129.33, 127.19, 127.07, 126.96, 126.54, 126.29, 125.54, 116.62, 108.95, 106.28, 106.18, 42.04,
41.86, 40.92, 40.90. ESI-MS (m/z): Calcd for [M+H]+ = 333.17, found: 333.13.
2.3. Absorption and fluorescence spectra
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The solvents with different viscosity were obtained by mixing methanol and glycerol in
different proportions. Viscosity value was recorded by NDJ-8 rotational viscometer. The solutions
of the probes with different viscosity were prepared by adding 10 µL of stock solution (1.0 mM)
to 1.0 mL of solvent mixture to obtain the final concentration of the probe (10 µM). These
solutions were sonicated for 5 min to eliminate air bubbles. The spectra of the probes with
different viscosity were measured in a UV spectrophotometer and a fluorescence
spectrophotometer.
2.4 Cytotoxicity determined by MTT method
HeLa cells were cultured in Dullbecco’s modified Eagle medium (DMEM), containing 10%
FBS (Fetal Bovine Serum) and 1% Antibiotic-Antimycotic at 37°C in a 5% CO2 and 95% air
incubator. Toxicity toward HeLa cells was determined by using the MTT Cell Proliferation and
Cytotoxicity Assay Kit (Beyotime) following manufacturer’s guidelines.
2.5. Cell imaging
HeLa cells were seeded in Φ35 mm confocal laser dishes at a density of 5 × 105 cells per dish
and grown for 24 h. Cell cultures were incubated with the probes at the concentration of 10 μM
and maintained at 37°C in an atmosphere of 5% CO2 and 95% air for 30 min, and then cells were
washed with PBS (3 × 2 mL per well). Before cell imaging, the organelle trackers were performed
according to manufacturer’s guidelines. The cells were imaged using confocal laser scanning
microscopy (ZEISS LSM 880) with one and two-photon lasers (with a 63× water-immersion
objective lens). (λex = 543 nm, λem = 560-620 nm for NY3 channel; λex = 405 nm, λem = 425-500
nm for Hoechst channel; λex = 633 nm, λem = 653-710 nm for Mitochondria tracker channel; λex =
633 nm, λem = 653-720 nm for Lysosome tracker channel)
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3. Results and discussion
3.1. Design and synthesis
As shown in Scheme 1, NY1, NY2 and NY3 were obtained by the Suzuki reaction with the
straightforward coupling of donor and acceptor groups to increase the stability of the probes in the
bioimaging. The donor intermediate of N,N-dimethylaminonaphthalene boronic acid pinacol ester
(compound 1) was firstly prepared by 6-bromo-N,N-dimethylnaphthalen-2-amine and
bis(pinacolato)diboron under palladium catalysis at N2 atmosphere. 4-Chloro-7-nitrobenzofurazan
(NBD-Cl) was restored by Fe powder to give the aromatic amine compound 2, which further
reacted with CH3I to get the mono-methylamino and dimethylamino substituted intermediate
compounds 3 and 4. Finally, compound 1 was coupled with compounds 2, 3 and 4, respectively,
under the condition of palladium catalysis at N2 atmosphere and purified by silica column
chromatography using PE/EA mixture as eluent, yielding red powder solid NY1, NY2 and NY3.
1
All the compounds were successfully characterized by MS, H and 13C NMR in the Supporting
Information.
3.2. Photophysical properties
The substituent effects on photophysical properties of the compounds were firstly investigated.
Absorption and fluorescence spectra of three probes were measured in THF to evaluate the effect
of different N-substituted groups at acceptor moiety. As shown in Fig. 1a, normalized absorption
spectra clearly indicated the probes from NY1 to NY3 showed red-shifted in THF with the
maximum absorption at 456 nm, 466 nm and 476 nm, respectively. This red-shifted absorption
could be well explained by the different D-A interactions that gradually increased from amino,
mono-methylamino to dimethylamino. However, the similar trend was not observed for emission
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spectra (Fig. 1b). The fluorescence peaks of NY2 and NY3 were at 670 and 690 nm, while, NY1
exhibited a longer emission peak compared with NY2 in THF and was almost the same with the
maximum emission peak of NY3 at 690 nm. This phenomenon may associate with the interactions
between the probe and solvent molecules. The stronger interactions between excited NY1 and
solvent molecules would promote the non-radiative transition to result in the red-shift of emission.
On the other hand, our probes also exhibited a large Stokes shift more than 180 nm, which are
beneficial for applications over a longer period and can avoid interference of background
fluorescence in bioimaging and detection.
Fig 1.
Generally, the D-A type molecules are well recognized for their intramolecular charge transfer
(ICT) characteristics. The photophysical properties of such type fluorophores are easily affected
by the polarity of the surrounding environment. In order to understand the solvatochromic
behavior, absorption and emission spectra of the probes in different solvents with varying polarity
were carried out. As displayed in Fig. S1, with the increasing of solvent polarity, the absorption
wavelength of all the dyes was gradually red-shifted. In addition, the emission intensity was
decreased with the increase in solvents polarity, which is due to the presence of charge transfer
characteristics [44, 45]. These results indicated that all the probes showed significant
solvatochromism significant ICT characteristics. The dipolements of ICT molecule in excited state
is rapidly enhanced as the positive and negative charges separation, which subsequently induces
the strong dipole-dipole interactions between the probes and solvent molecules. To response the
interactions, the reorientation of the polar solvent molecules around the excited dye would occur
and accompany by the dye molecular structural relaxation. It would give rise to non-radiative
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transition strengthen that induced emission from a lower energy state, resulting in the fluorescence
red-shift and decrease Meanwhile, with the increase of solvent polarity from toluene to DMF,
Stokes shifts were changed from 5742 to 6628 cm−1, 5459 to 6049 cm−1, and 5378 to 5956 cm−1
for NY1, NY2 and NY3, respectively. The smaller changes of NY3 in Stokes shifts than that of
NY1 and NY2 suggested that the probe NY3 was more stable in polar solvents. Generally, the
relations of Stokes shift with varying dielectric constant of different solvents by Lippert-Mataga
plot are used to estimation of dipole moment changes from ground to excited state [46, 47]. As
shown in Fig S2, it was found that all the probes showed linear increase of Stokes shift with the
solvent polarity increasing, which suggested the positive solvatochromism. The slopes of
Lippert-Mataga plot of the probes were 1153, 1288 and 1625 cm−1 for NY1, NY2 and NY3,
respectively. The relative larger slope of NY3 indicated the strong transition dipole moment,
which suggested more charge separation and stronger ICT properties in NY3.
3.3. Response to viscosity
Fig 2.
The above results have demonstrated that the probes possessed ICT properties and the
substituents had an obvious influence on the photophysical properties due to the steric effects and
electron-donating ability. To evaluate the response of the probes to the viscosity, the absorption
and fluorescence spectra of the probes with different volume fractions of glycerol in methanol
were recorded. As shown in Fig S3, the absorption wavelength of the probes had negligible
changes with the increase of viscosity. It is noted that the fluorescence intensities in MeOH
solution were quite weak, especially for the NY1. However, the fluorescence intensities around
690 nm of the probes dramatically enhanced with proportions of glycerol increasing in the mixture
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solvent (Fig 2). 22, 31 and 130-folds increases of the fluorescence intensities for NY1, NY2 and
NY3 were achieved when the fraction of glycerol was increased from 0 to 90% in
methanol/glycerol mixture solution. Meanwhile, the fluorescence quantum yields of the NY1,
NY2 and NY3 were also increased from 0.02, 0.10 and 0.18 in MeOH to 0.27, 0.42 and 0.67 in fg
= 90% methanol/glycerol mixture solution. Hence, the enhanced emission of the probes with the
glycerol fractions increasing is ascribed to the changes of environmental viscosity. Meanwhile,
due to the substituent effects on the sensitivity of the probes to viscosity, the fluorescence intensity
of NY3 showed higher enhancement in methanol/glycerol mixture solutions with 90% fraction of
glycerol compared to that of NY1 and NY2. In addition, the plot exhibited a good linear
relationship between the fluorescence intensity (logI690) and the viscosity value (logη) by using
Förster-Hoffmann equation (R2 = 0.98). The slop values of NY1, NY2 and NY3, which could
assess the sensitivity of the probes to the viscosity, were calculated to be 0.255, 0.428 and 0.636,
respectively [48]. The enhanced slop value of NY3 clearly indicated the sensitivity was enhanced
with the number of methyl substituent in amino growing. These results suggested that the
substituent played a key role in the fluorescence response of the probes to viscosity and three of
them could be served as excellent NIR probes for detection of viscosity changes. In addition, a
large Stokes shift more than 170 nm for these three probes was obtained, which could significantly
reduce the interference of background fluorescence in bioimaging and detection.
Fig 3.
Considering the applications of the probes in the complex biochemical environment, their
fluorescence may be influenced by local pH, anions, cations as well as amino acids in living cells.
To confirm whether the probes were influenced by the various interferents or not, the selectivity of
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the probes was next investigated. The fluorescence spectral profiles of the probes in different pH
solution were firstly performed (Fig. S4). The fluorescence intensities of the probes had no
obvious changes in the range of pH = 4-10. However, when the viscosity of the fluid was
increased, the fluorescence intensities of these probes were rapidly enhanced. These results
indicated that our probes could be used to monitor the viscosity changes in the physiological
environment. Moreover, the effects of more than thirty biological relevant species, including
various normal anions, cations and amino acids, on the fluorescence of the probes were also
investigated (Fig. 3). Obviously, the addition of various ions and amino acids to the solution of the
probes in MeOH and methanol/glycerol mixture solution (fg = 90%) did not induce any obvious
change in the fluorescence intensity. These results further confirmed that the fluorescence
intensities of the probes were not affected by the various biological relevant species and could be
used for the detection of viscosity change in a complex physiological environment.
3.4 Theoretical calculation and mechanism studies
Fig 4.
In order to investigate the mechanism of these three probes in the detection of viscosity change,
the quantum chemistry theory calculations were performed. The molecular structures of these
probes in the gas phase were optimized using dependent density functional theory (DFT)
calculations with the Gaussian 09 program at the B3LYP/6-31G* level [49-52]. Three rotatable
parts existed in each molecule of the probes. In the ground state, the amino and methylamino
groups were almost on the same plane with the naphthalene ring and benzofurazan. However, all
the probes possessed twist structure due to the steric hindrance, leading to an about 25° dihedral
angle between the naphthalene ring and benzofurazan. The absorption spectra and molecular
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structures of the probes in the excited state were also calculated using the time-dependent
functional theory (TD-DFT) method based on the optimized structure of the ground state. The
calculated absorption peaks of NY1, NY2 and NY3 were 445, 449 and 454 nm, respectively,
which were close to the experimental data. In addition, the energy gap was gradually decreased
with the number of N-methyl group increasing, which was also consistent with the experimental
results. These results indicated that the quantum chemistry theory was quite useful for
understanding the structure-property relationships. To further study the substituent effect on the
photophysical properties, the molecular orbital distributions and charge transfer processes were
analyzed. The max absorption of the probes was from the transitions of S0 to S4 (HOMO to
LUMO+1). As shown in Fig 4, the HOMO electron cloud was homogeneously distributed in the
whole molecule, which indicated the naphthalene and benzofurazan ring were well conjugated,
although there was a slight twist angle between them. Meanwhile, the charges of the amino,
methylamino and dimethylamino of the probes was decreased in sequence with the transition from
the HOMO to the LUMO+1, indicating a prominent ICT process was occurred. The electron cloud
of naphthalene group had negligible changes when the probes transferred from ground to excited
state. The previous studies showed that the rotation of N,N-dimethylamino group could induce to
form the TICT state, which subsequently resulted in the better sensitivity of the NY3 to the
viscous microenvironments in comparison with that of NY1 and NY2 [53]. Thus, NY3 was next
chosen to monitor the viscosity changes in living cells.
3.5 Cell imaging
Intracellular viscosity imaging experiments were conducted in HeLa cells. Before fluorescence
imaging, the standard MTT assay was carried out to evaluate the cytotoxicity. As shown in Fig S5,
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the cell viability was more than 90% after incubating with NY3 (10 µM) for 12 h. The result
showed that NY3 had negligible cytotoxicity to the cells after a long period at low micromolar
concentrations. To examine the subcellular location of NY3, the commercially available nucleus,
lysosome and mitochondria trackers were employed for colocalization study. As shown in Fig S6,
the one- and two-photon confocal microscopic cell images indicated that NY3 could enter the cells
within 30 min. The merged images between NY3 channel and the above organelle trackers
showed that the staining of NY3 did not overlay with the organelle trackers. This result indicated
that the probe NY3 was not localized in the single organelle, such as mitochondria, lysosome and
nucleus after it entered into the cytoplasm.
Fig 5.
The ability of the probe NY3 for the detection of intracellular viscosity change was further
evaluated by fluorescence imaging. The HeLa cells were firstly incubated with NY3 at 37°C,
25°C and 4°C for 30 min, respectively. Previous studies showed that the decrease of culture
temperature could increase the intracellular viscosity [54, 55]. As shown in Fig. 5, an enhanced
fluorescence signal from NY3 channel in the cell images with the incubation temperature
decreasing. Furthermore, we used the nystatin and carbonyl cyanide 3-chlorophenylhydrazone
(CCCP) as inductive agent to regulate the viscosity in cells. Nystatin is well-known as an
ionophore to induce mitochondrial structural changes or swelling through interruption of the ionic
balance [34,53,56]. CCCP is a membrane-potential uncoupler, which could disrupt mitochondrial
membrane potential to change the viscosity [57]. Thus, both of them could enhance the
intracellular viscosity by inducing the changes of mitochondrial structure. As shown in Fig. 5, the
HeLa cells incubated with only the probe NY3 showed weak fluorescence. However, when the
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cells were pretreated with CCCP and nystatin for 30 min, almost 3-folds enhancement of
fluorescence intensity were observed after incubating with the probe NY3. Besides the
upregulation of the intracellular viscosity by decreasing temperature or using CCCP/nystatin
treatment, the cells immobilization with 4% paraformaldehyde (PFA) also could significantly
increase the viscosity, which has been demonstrated by incubating the probe NY3 with PFA-fixed
cells. These above results have clearly shown that NY3 could be employed for monitoring the
changes of intracellular viscosity.
Fig. 6
Moreover, to further clarify the capacity of the probe for detecting the intracellular viscosity,
time-dependent imaging of the cells with nystatin treatment was performed. HeLa cells were
firstly pretreated with nystatin for different time (0 h, 2 h, 4 h and 6 h) and subsequently incubated
with probe NY3 for another 30 min. As showed in Fig. 6, the weak fluorescence was observed
when the cells treated with nystatin at the beginning. With the incubation time increasing, the
fluorescence signal was gradually enhanced and a high corresponding relation between the
incubation time and fluorescence intensity was also observed. These results further demonstrated
that the probe NY3 could be served as an excellent viscosity probe for the detection of
intracellular viscosity.
4. Conclusion
In conclusion, we have successfully developed a series of D-A-D type NIR dyes functionalized
with amino, N-methylamino and N,N-dimethylamino groups for intracellular micro-viscosity
detection. All of the probes exhibited typical ICT properties and sensitivity to viscosity changes.
The fluorescence signals of the three probes were weak in the non-viscous media, but obvious
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enhanced in viscous environments. These probes showed NIR emission and large Stokes shift
more than 170 nm, which enable them quite suitable for the detection of viscosity change in living
cells and in vivo. NY3 modified with N,N-dimethylamino has been proved to be more sensitive to
viscosity due to stronger electron-donating ability and larger steric effect, which was successfully
applied to imaging the intracellular micro-viscosity changes. This work provides an efficient way
for design powerful NIR fluorescence probe for intracellular micro-viscosity detection.
Acknowledgements
This work was financially supported by the National Natural Science Foundation of China
(81672508 and 61505076), Jiangsu Provincial Foundation for Distinguished Young Scholars
(BK20170041), China-Sweden Joint Mobility Project (51811530018), National Postdoctoral
Program for Innovative Talents (BX201700119), China Postdoctoral Science Foundation Funded
Project (2018M632293), China-Sweden Joint Mobility Project (51661145021), Natural Science
Foundation of Guangdong Province (2017A030313299). and Key University Science Research
Project of Jiangsu Province (Grant 16KJA180004).
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Scheme 1.
Fig. 1
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Fig. 2
Fig. 3
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Fig. 4
Fig. 5
Fig. 6
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Scheme 1. Synthesis route of the probes NY1, NY2 and NY3 and their response to viscosity
change.
Fig. 1 (a) Absorption and (b) emission spectra of the probes in THF.
Fig. 2 Fluorescence spectra of NY1 (a), NY2 (b) and NY3 (c) in methanol (M)/glycerol (G)
mixtures with different fractions of glycerol. Forster-Hoffmann plot of the NY1 (d), NY2 (e) and
NY3 (f).
Fig. 3 The fluorescence response of NY1 (a), NY2 (b) and NY3 (c) in methanol and
-
methanol/glycerol mixtures with 90% fractions of glycerol to various analytes; 1-46: Br-, HCO3 ,
2-
2-
2-
CO3 , Cl-, SO3 , F-, SO4 , SCN-, I-, Fe2+, Ba2+, Fe3+, Pd2+, Ca2+, Zn2+, Ni2+, Cd2+, Al3+, Mg2+,
Mn2+, Cu2+, Ag+, glycine, L-cystine, L-ornithine, L-valine, L-arginine, L-histidine, L-proline,
L-alanine, L-tyrosine, L-aspartic acid, L-glutamic acid, L-threonine, L-methionine, glutathione,
homocystine, L-cysteine, tryptophan, isoleucine, serine, lysine, DL-dithiothreitol, guanidine
hydrochloride, blank.
Fig. 4 The calculated electron cloud distributions and energy gap of the probes.
Fig. 5 (a) Confocal images of HeLa cells incubated with NY3 (10 μM) for 30 min at different
temperature or with pretreatment of CCCP, nystatin and 4% PFA; (b) Normalized intensity of the
images of (a) in HeLa cells. Scale bar = 20 μm.
Fig. 6 (a) Confocal images of HeLa cells pretreated with nystatin for 0 h, 2 h, 4 h and 6 h,
afterwards incubating with NY3 for 30 min; (b) Normalized intensity of the images of (a) in HeLa
cells. Scale bar = 20 μm.
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Graphical abstract
Rational design of NIR fluorescence probes for sensitive detection of
viscosity in living cells
A series of D-A-D type naphthyl and 2,1,3-benzoxadiazol hybrid NIR
dyes functionalized with amino (NY1), N-methylamino (NY2) and
N,N-dimethylamino (NY3) groups for intracellular micro-viscosity
detection have been designed and synthesized.
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Highlights
Rational design of NIR fluorescence probes for sensitive detection of
viscosity in living cells
1. A series of NIR probes with large Stokes shift were designed and synthesized.
2. The dyes were sensitive and selective to the change of viscosity.
3. Structure-property relationships are systematic investigated in detail.
4. The probe was successfully applied for imaging the changes in intracellular micro-viscosity.
29